key: cord- -kbpvt on authors: atherstone, c.; galiwango, r. g.; grace, d.; alonso, s.; dhand, n. k.; ward, m. p.; mor, s. m. title: analysis of pig trading networks and practices in uganda date: - - journal: trop anim health prod doi: . /s - - - sha: doc_id: cord_uid: kbpvt on east africa is undergoing rapid expansion of pig rearing, driven by increasing pork consumption. introduction and expansion of pig production systems in this biodiverse landscape may create new risks, including zoonotic pathogen transmission. historically, biosecurity measures have primarily been focused at farm level, ignoring the important function pig traders fulfill between farmers and consumers. this study interviewed pig traders operating at uganda’s only registered pork abattoir to describe their characteristics, business practices, biosecurity practices, and pig health management and reporting practices. all the traders were male, and nearly all ( . %) relied on pig trading as their primary source of income. most of the pigs brought for processing at the slaughterhouse were purchased from smallholder farms ( . %). in addition, there was a significant difference in the high price paid per kilogram at farm gate by region (p = . ). high prices paid at farm gate were associated with holiday periods (p < . ), harvest season (p < . ), and drought (p < . ). traders preferred buying live pigs from male farmers ( . %) because they were considered the final decision makers and owned the pigs being sold. all pig traders were aware of clinical signs indicating a pig was sick. this study has provided baseline information on pig trader practices in uganda. improvements in local pork slaughterhouses and markets will benefit not only pig traders in accessing consistent customers but also individual pig farmers by increasing their market access. finally, given their role as a link between farmers and consumers, traders would benefit from targeted inclusion in disease control and prevention strategies. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the domestic pig population in uganda, currently estimated at . million, plays an essential economic and social role in a country, in which % of households derive some or all of their livelihoods directly from livestock (uganda bureau of statistics ). pig keeping has grown in popularity as a livelihood activity due to their high reproduction rates, rapid weight gain, potential to provide quick financial returns, and rising demand for pork. in the past years, pork consumption has increased more than -fold, from an estimated annual per capita consumption of . kg in to . kg in . pork currently accounts for more than a third of the annual per capita meat consumption (food and agriculture organization (fao) ) . further, total pork consumption is projected to increase by % between and in uganda due to human population growth (food and agriculture organization ) . the increase in pork consumption, while exceptionally rapid in uganda, is not unique in the region. the democratic republic of congo's total consumption of pork is expected to increase by %, tanzania by %, and kenya by % between and (food and agriculture organization ) . introduction and expansion of pig production systems in these biodiverse landscapes may create new risks, including pathogen transfer from pigs to humans (ocaido et al. ; wilson ; food and agriculture organization (fao) ; atherstone et al. ; hamill et al. ; food and electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. agriculture organization ). of particular public health interest is the role of pigs in the zoonotic transmission of emerging pathogens to people (atherstone et al. ; vergara-alert et al. ; middleton and westbury ; ma et al. ; kobinger et al. ; conlan et al. ; mccormack and allworth ; abubakar et al. ; marsh et al. ) . as pig traders form an important link between pig farms and pork customers, research informing their knowledge, attitudes, and practices is essential. african swine fever (asf) is considered the major infectious disease constraint to pig production in africa (penrith et al. ) , and as such, research to date has focused on this infection. penrith and vosloo reported that outbreaks of asf in new areas of africa have almost all been associated with movement of domestic pigs and pig products (penrith and vosloo ). in uganda, several studies to characterize practices associated with the occurrence and spread of asf identified the collection of pigs and pig products from farms (kabuuka et al. ) , distribution of infected pork by traders , pig movements due to restocking and trade (kalenzi atuhaire et al. ; nantima et al. ) , free range movement of pigs on farms (nantima et al. ) and trade of live pigs and pig products (tejler and teijler ) as risk factors. these studies focused on pig farmers and their perception of trading and pig movements. a limited number of studies have targeted pig traders, but again, these were restricted to knowledge and practices related to asf transmission and control (dione et al. ; chenais et al. ; muhangi et al. ) . despite the link between disease spread and pig movement, little is known about broader trading practices, motivations for buying, and patterns of purchases and sales of pigs in uganda. given pig traders' important role in supplying pork for a rapidly expanding consumer base and linking farmers with consistent markets, a better understanding of their practices and motivations around purchasing, transportation, and pig health management is needed. this would assist in developing policies that specifically support traders and the important functions they serve while identifying suitable interventions to ensure a safe, reliable pork supply. therefore, the objectives of this study were to ( ) describe pig trader characteristics, trading practices, biosecurity practices, pig health management, and reporting practices and ( ) map source locations of pigs purchased to supply pork through the major abattoir in uganda. wambizzi cooperative society limited is located in nalukolongo, southwestern kampala, uganda's capital city. wambizzi was selected as it is the only registered pig abattoir in uganda and has many pig traders supplying live pigs to meet the urban demand for pork. as a registered slaughterhouse, carcasses processed at wambizzi are visually inspected by kampala city council authority (kcaa) meat inspectors and stamped bfit for human consumption.^pork processed at the abattoir is sold in the greater kampala area to pubs, pork joints, hotels, butchers, supermarkets, and private organizations (non-governmental organizations, missions, and private individuals). the slaughterhouse has a capacity of pigs/ day, but the supply of pigs fluctuates substantially throughout the year (roesel et al. ) . according to slaughterhouse records, an average of , kg of pork was processed monthly from april to may . using the estimated annual per capita consumption of . kg of pork (food and agriculture organization (fao) ) and the kampala population of . million (uganda bureau of statistics ), wambizzi produced roughly . % of the pork consumed in kampala during this time frame. pig slaughtering also occurs in backyards and slaughter slabs to supply informal, roadside pork joints and butcheries. pig traders privately operating from wambizzi use the abattoir facility to slaughter their pigs and have their pork inspected and stamped to meet requirements in the formal marketplace. traders buy live pigs from farms/markets and aggregate them into groups for transport to the abattoir. during this interim period (farm gate to abattoir), pigs are under the ownership and care of traders. once processed, traders sell the pork to their own customers in the sales building adjacent to the evisceration and inspection building. while membership with the abattoir is not required, traders pay a fee per pig ( ugx, i.e., . usd in ) to use the abattoir facilities (roesel et al. ). pig traders were interviewed between october and october , during periods corresponding with national holidays when the sale and consumption of pork increases (roesel et al. ; ouma et al. ) . there is no formal register of pig traders operating at wambizzi. in preparation for this research, a member of the research team with prior experience working with pig traders informally questioned traders on site over several days to develop a more recent estimate of the number of traders operating from wambizzi. based on this, the total number of traders operating at wambizzi was estimated at . thus, we aimed to interview traders over the course of the study. a variety of methods were used to recruit participants, including direct approach and active-snowballing. traders arriving at the abattoir were approached by a local member of the research team and initially asked about their interest in learning about the study. if the trader expressed interest, information on the scope and purpose of the research was provided orally. traders who indicated that they were willing to participate in the research study were asked to give written consent to be interviewed. if the trader was unable to give written consent due to physical impairment or illiteracy, their thumbprint was provided in place of signature. additional traders were identified by asking participants who had completed the interview for the name and contact information of other pig traders operating from wambizzi. furthermore, we observed trader brands on pigs at slaughter (e.g., number or letter carved on the animal at the time of purchase) and asked participants who had completed the interview if they could identify the trader who supplied the pig. the enumerator then contacted these newly identified pig traders to invite their participation in the research study. a local enumerator with previous experience working with pig traders in uganda was recruited and trained for data collection. a structured questionnaire was adapted from previous research conducted with pig traders by the international livestock research institute (ilri) in uganda under the smallholder pig value chains development project (cgiar livestock and fish research program ) . the questionnaire captured information on pig trader characteristics, live pig buying practices, transportation practices, and pig health management. traders also reported the sub-counties where they had purchased live pigs over the months prior to the interview date. the questionnaire was developed in english and translated into the local language (luganda). the questionnaire comprised primarily closed-ended questions to keep the interview to a maximum of min. openended questions regarding buying practices and clinical signs observed in pigs were included, with answers recorded exactly as the interviewee stated. the full questionnaire is included in the supplementary materials. the slaughter process started at am each morning and preceded the selling of pork from am to am. the enumerator was on site by am each morning to identify pig traders previously not interviewed. however, to ensure that the interview did not conflict with pig trader's business, most interviews took place between am and noon each day. data from the questionnaires was entered into epi info . (centers for disease control, atlanta, ga, usa). following data cleaning, data was exported to spss . (ibm corp., armonk, ny, usa) for analysis. standard descriptive analysis was performed for categorical and quantitative variables describing pig trader characteristics, live pig purchasing practices, transportation practices, and pig health management. population pyramid style graphs were prepared to compare pork demand and pork farm gate prices by months. source locations (reported to the sub-county level) were entered into microsoft excel and checked for spelling accuracy. the subcounties were then joined to the centroid of each sub-county polygon in the global administrative unit layers for uganda (food and agriculture organization, rome, italy) using arcgis . (environmental systems research institute, redlands, ca, usa). the number of pig traders operating in each sub-county was mapped using graduated symbols. two binary outcome variables of interest were explored further, namely high price paid at farm gate over the last year ( / ) and low price paid at farm gate over the last year ( / ). reasons given for prices paid were recoded into binary variables (yes/no) and used as explanatory variables. univariable binomial logistic regression analyses were conducted to evaluate the associations of the binary explanatory variables with both the outcome variables. explanatory variables with a p value < . were included in two multivariate regression models for high or low price paid to evaluate associations after adjusting for other variables in the model. in addition, non-parametric analyses were conducted for four quantitative variables (high price paid per kilogram, low price paid per kilogram, number of live pigs bought during high demand weeks, and number of live pigs bought during low demand weeks) to identify significant differences by operating region (kruskal-wallis test), number of districts (mann-whitney u test), and number of regions (mann-whitney u test) traders purchased live pigs from. operating region was identified based on the location pig traders reported purchasing live pigs in. because price and number of pigs purchased were not identified by individual districts and many pig traders operated in multiple regions, operating region was binned into three categories: central only, eastern only, and all other regions (including western, northern, and responses that covered multiple regions). number of regions and number of districts a pig trader operated in were recoded into two responses: above median and below median. independent variables with significant differences (p < . ) were subject to post-hoc pairwise comparisons to identify which specific responses(s) were significantly different from each other. a total of interviews were conducted with pig traders operating from wambizzi between october and october . no traders declined participation in the study. pig trader characteristics are shown in table . all pig traders interviewed were male and ranged in age from to years (median years; first quartile (q ) = , third quartile (q ) = ). the median number of years working as a pig trader was years (range months- years; q = , q = . ). a large proportion ( . %; / ) of participants had not completed primary school. most pig traders were engaged in trading as their primary source of income ( . %; / ) and described their business operation as fixed ( . %; / ), meaning that they had established locations for buying live pigs and selling pork. proximity to pork customers was the primary reason for having a fixed business operation ( . %; / ) with most traders supplying pigs solely to wambizzi ( . %; / ). when asked about other pig traders operating in their areas, almost all the traders had competition for live pigs in their buying areas ( . %; / ). almost two thirds of the pig traders were not members of a trading group or cooperative ( . %; / ). buying and transportation practices are shown in table . most traders sourced their pigs directly from smallholder farms ( . %; / ). only one trader reported purchasing pigs at a livestock market. when pig traders were asked about whom they prefer to purchase pigs from at the farm, . % ( / ) preferred buying from men rather than from women. reasons offered by traders who preferred to purchase from men included that men were the following: the decision makers on the farm, faster decision makers, and owned the pigs being sold. further analysis to understand this gender preference was not possible because the number of responses for women were all less than . lorries (trucks) were the most common type of vehicle used to transport pigs to the abattoir ( . %; / ). the majority of traders rented the vehicles they used to transport pigs ( . %; / ). vehicles were cleaned after each use ( . %; / ) using both water and laundry washing powder ( . %; / ). none of the pig traders reported using bleach or any other type of disinfectant to clean their vehicles. the pig waste (feces, urine, bedding) left in the vehicle after transporting the pigs was most commonly heaped at wambizzi for crop farmers to collect and use for compost in their gardens ( %; / ). june and december were frequently identified as months with high customer demand for pork, whereas february and september were associated with low customer demand (fig. ) . during months when demand for pork was low and high, respectively, traders bought a median of . pigs (range - pigs/week; q = ; q = ) and . pigs (range - pigs/week; q = ; q = ) per week. figure shows the months traders associated with paying high or low farm gate prices to purchase pigs. the median reported high prices at farm gate was ugx/kg (range − ugx/kg; ugx = usd as of january ). when low prices were paid at farm gate, the median was ugx/kg (range - ugx/kg). reasons for paying high and low prices at farm gate are shown in table . in multivariate logistic regression, holiday period, crop/coffee harvesting season, and drought were significantly associated with high price paid, whereas drought, school fees due time, and sick pigs were significantly associated with low price paid. figure shows the source locations of pigs purchased on the day of interview and preceding months. pig traders reported buying live pigs in one to eight districts (median ) across one to three regions (median ) in uganda. thirty-six percent of traders purchased live pigs only in the central region (n = ) and % of traders purchased live pigs only in the eastern region (n = ). farm gate prices by operating region as well as the number of districts/regions that a pig trader operates in are outlined in table . pig traders operating in one region paid significantly higher prices per kilogram at farm gate than traders operating in two to three regions (p = . ). the number of live pigs purchased per week by operating region and number of districts/regions that a trader operates in is shown in table . during months when demand for pork was low, the region(s) a pig trader operated in to purchase live pigs was significantly associated with the number of pigs purchased (p = . ). pig traders operating in only central region purchased a significantly higher number of pigs during low demand months than traders operating in only eastern region (p < . ). traders operating in only the eastern region purchased a significantly lower number of pigs during low demand months than traders operating in all other regions (p = . ). all the pig traders reported recognizing clinical signs indicating a pig was sick. when asked to list these signs, the most commonly stated signs were dropping of ears ( %; / ), reddening of ears ( . %; / ), straightening of the tail ( . %; / ), and weakness or difficulty standing ( . %; / ). traders typically did not report pigs considered to be sick to anyone ( . %; / ). if there was reporting, the trader informed a meat inspector on site at wambizzi ( %; / ) or a veterinary officer ( %; / ). if sick pigs were observed while under the traders' care, . % of the traders did nothing to care for the sick pigs ( / ). if action was taken, the sick pig was slaughtered at wambizzi and the meat sold ( . %; / ). this is the first study to describe ugandan pig trader characteristics and business practices around live pig buying, transportation, and health management. the prices paid to farmers for their pigs were associated with the number of regions and districts a pig trader operates in. in addition, pig traders reported paying higher prices during holiday periods and harvest season (crops/coffee). traders preferred buying live pigs from male farmers because they considered them the final decision makers and owned the pigs being sold. finally, we found that all pig traders checked for clinical signs in pigs that indicated the animal was sick. pig traders who operate in only one region paid, on average, higher prices per kilogram to farmers for their pigs. considering that such traders are likely to travel shorter distances to source their pigs, compared to those traders that work in more regions, this may suggest that the distance travel outcome variables with less than five responses were not including in the univariable analysis b p value = . . explanatory variable removed from multivariable logistic regression model fig. source locations of live pigs on the day of interview and preceding months, as reported by pig traders interviewed at wambizzi cooperative society slaughterhouse, kampala, uganda, to source pigs has an impact on the price paid for the pigs, with traders traveling less offering higher prices to farmers. however, another possibility may be that traders who operate in multiple regions are large-scale traders, buying pigs in bulk, and therefore paying lower prices. nevertheless, given the importance of pigs as an asset within smallholder farming households, it is advantageous when pig farmers can secure more income from the sale of their pigs. given that almost half the traders in this study operated in four or more districts in uganda, pigs are traveling large distances from farm to slaughterhouse. reducing the distance pigs' travel for processing is both an animal welfare and a disease mitigating practice, especially for limiting the dissemination of asf (tejler ) . thus, there is a need for locally regulated slaughter facilities and/or improved transport infrastructure throughout the country to reduce the distance traveled from farm to slaughterhouse. centralized slaughter facilities would also help address the lack of consistent market access, a commonly cited constraint among pig farmers (ouma et al. n.d.; wabacha et al. ; kagira et al. ; muhanguzi et al. a ). moreover, traders in this study reported that the primary reason for maintaining a fixed business operation was proximity to pork customers. local slaughter facilities would provide a centralized location for traders to access customers. this study also found that holiday periods, harvest season, and drought were the most commonly cited reasons trader paid high prices at farm gate. other studies have noted that smallholder farmers keep pigs as a source of cash in times of need (dione et al. ; deka et al. ; gichohi et al. ) . when pig traders need to source live pigs around harvest season, they pay more for these pigs because farmers have recently sold their crops and, therefore, do not need the additional cash generated from the sale of a pig. the situation is a little different around the holidays. an increase in pig sales and pork consumption during festive seasons is well documented (roesel et al. ; dione et al. ; adams et al. ; kambashi et al. ) . it is possible that traders have a harder time sourcing the number of pigs they need at the holiday period or that farmers know of the increased holiday demand and raise their prices. the higher prices paid for pigs around holidays are inducements for farmers to time their pig rearing activities to take advantage of the economic benefit of having pigs ready for sale according to holiday periods. free ranging, tethering, and feeding of crop residues and grasses to pigs are common practices among smallholder pig farmers in east africa chenais et al. ; tejler ; kagira et al. ; dione et al. ; muhanguzi et al. b; nantima et al. ) . in all these production systems, drought would reduce the amount and quality of feed available for pigs and, therefore, would reduce the number of pigs suitable for sale. it is also possible that farmers intentionally chose not to rear grower and fattener pigs during known seasons of drought. it will be important to address live pig supply issues, whether at holiday periods, drought, or from other causes, to ensure a consistent pork supply so that consumers are able to access the quality and quantity of pork they demand. at the farm level, this research found that pig traders have a strong preference to buy live pigs from male pig farmers. a study in kenya found that the decision to keep pigs was made by men (simiyu and foeken ) . because of the large financial investment, technical knowledge required to raise pigs and role as sole decision makers in their home, men maintained control of pigs and leveraged this control for any financial decisions made over the animals (simiyu and foeken ) . these subtle cultural values around livestock ownership and household financial decision making come into play in accessing markets for livestock and livestock products. women tend to face more challenges than men in accessing and benefiting from markets, especially more formal markets (kristjanson et al. ) . furthermore, given women's traditional responsibility for household food security, their level of control over decisions about whether to sell or consume the family's animal products, as well as over how to use any income obtained from the sale of animal foods, could greatly determine the nutritional well-being of household members (kristjanson et al. ) . while there is clearly a need for support of women at the household level in accessing markets for their livestock, this research shows that there is also a need to work with pig traders to enable female pig farmers to access consistent markets for their pigs. in this study, all the traders interviewed observed clinical signs they described as indicating a pig was sick. the frequently observed clinical signs such as reddening of the ears, dropping of ears, and weakness or affected movements are consistent with clinical signs of asf (chenais et al. ; tejler ; dione et al. ) . despite recognition of these signs as indicators of sickness, traders failed to report these suspected cases to the proper authorities. similar findings have been reported in previous research in uganda chenais et al. ; muhangi et al. ; nantima et al. ) and are consistent with studies conducted in indonesia (leslie et al. ) . given that traders play an essential role in transporting pigs, there is a need to develop policies and strategies to integrate pig traders into disease reporting and disease mitigating strategies without fear of recrimination or detriment to their business. in addition, given the pressures pig traders are under to meet quality standards of pork customers, pig traders would benefit from training on disease mitigating strategies including safe and hygienic slaughter practices, perhaps through an industry association or group. this would also address the gap between traders admitting that they are responsible for conducting their business in support of disease prevention but do not perceive themselves as key actors in the control of disease (dione et al. ). there are several limitations to this study. first, responses to the questionnaire are subject to recall bias. this is especially true of answers around the number and location of pigs purchased over the months prior to the interview. however, the number of pigs for which traders provided a source location (n = ) was considerably less than the number of pigs processed at the slaughterhouse (n = , for july -june ) (roesel et al. ). it appears that when the pig traders were unsure of actual numbers, they underreported, or only reported the location they sourced the pigs without any accompanying number of pigs purchased. self-reported locations are likely to be reliable as the traders used communitybased scouts to identify pigs for sale. we purposely interviewed pig traders during periods when demand for pork was historically high and theorized that this would mean that more pig traders would be bringing pigs in for processing. however, it is possible that there are pig traders that only operate sporadically and would have been missed in this study. we worked with the pig traders at wambizzi to identify other pig traders to interview. we also cataloged the brands on each pig being processed, as each trader has a unique symbol to identify their pigs once they have been processed. every effort was made to identify all potential research participants. previous research, undertaken with the management at wambizzi, stated that there were pig traders regularly operating from the premises (roesel et al. ) . given this, our study team managed to identify three times the number of pig traders. further analysis beyond descriptive analyses was hindered by the low number of responses for certain variables. for example, we were unable to analyze why pig traders prefer buying live pigs from men rather than women. given the priority of gender empowerment in uganda and the significance of livestock in alleviating poverty for women and children, it is important to identify ways to support women in accessing markets for their pigs. more detailed interviews and focus groups may shed additional light on trader buying decisions. this study has provided baseline information on pig trader practices in uganda. the prices paid at farm gate for pigs are affected by the number of regions and districts a trader operates in to procure pigs. given the animal welfare and disease transmission implications of pigs traveling over multiple districts and regions from farm to slaughterhouse, consideration should be given to establishment of local pork slaughterhouses and markets and improvements to transport infrastructure. furthermore, pig traders prefer buying live pigs from male farmers. for women to overcome the challenges of accessing formal livestock markets, there is a need for additional research to identify how women can access pork markets in uganda, particularly if pig traders are involved. finally, this research shows that pig traders are observing sick pigs but fail to report these sick pigs. historically, disease control interventions have been focused 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gulu district outbreaks of african swine fever in domestic pigs in gulu district the national livestock census report livestock susceptibility to infection with middle east respiratory syndrome coronavirus characterisation of smallholder pig production in kikuyu division, central kenya zoonoses and antimicrobial resistance in kenya and malawi acknowledgements the authors would like to thank the management of wambizzi cooperative society who facilitated this research and the pig traders operating at wambizzi for their involvement in this study. this research benefited from the technical input and tools developed by ilri uganda colleagues ben lukuyu, emily ouma, michel dione, and kristina roesel.funding funding for this study was provided by the cgiar research program on agriculture for nutrition and health, led by the international food policy research institute. statement of animal rights this human study has been approved by the appropriate ethics committees and has therefore been performed in accordance with the ethical standards laid down in the declaration of helsinki and its later amendments. the authors declare that there is no conflict of interest. key: cord- -fi s x authors: andries, k.; pensaert, m.; callebaut, p. title: pathogenicity of hemagglutinating encephalomyelitis (vomiting and wasting disease) virus of pigs, using different routes of inoculation date: - - journal: j vet med b infect dis vet public health doi: . /j. - . .tb .x sha: doc_id: cord_uid: fi s x summary: forty‐eight pigs were inoculated by different routes with the vw isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oral — nasal route ( ) or into the infraorbital nerve ( ) became sick after an incubation period of days. six of the pigs inoculated into the stomach wall, of the pigs inoculated intramuscularly and of the pigs inoculated intracerebrally became ill after an incubation period of – . days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. zusammenfassung: die pathogenität von hämagglutinierendem enzephalomyelitis‐virus (kümmern und erbrechen) bei shweinen nach infektion über vershiedene inokulationswege achtundvierzig schweine wurden übegr verschiedene inokulationswege mit dem vw ‐isolat des hämagglutinierendtn enzephalomyelitis‐virus (kümmern und erbrechen) infiziert. alle schweine, die entweder kombiniert oro‐nasal ( ) oder über den infraorbitalnerv ( ) infiziert wurden, erkrankten nach einer inkubationszeit von tagen. sechs der sieben über die magenwand inokulierten, oder intramuskulär und der intrazerebral infizierten tiere wurden nach einer inkubationszeit von – , tagen krank. bei den schweinen, die intravenös oder in die bauchhöhle bzw. direkt in den magen inokuliert wurden, kamen erkrankungsfälle nicht vor. alle erkrankten schweine zeigten das syndrom des kümmerns und erbrechens. von oro‐nasal infizierten schweinen, die während des frühstadiums der erkrankung getötet wurden, konnte das virus regelmäßig von den tonsillen und dem respirationstrakt und gelegentlich vom gewebe des pons‐medulla‐bereiches sowie aus der magenwand reisoliert werden. von schweinen, die nach infektion über andere routen erkrankten, ließ sich immer virus isolieren. alle tiere, die nicht erkrankten (mit ausnahme eines ferkels) bildeten jedoch antikörper. der ort der virusvermehrung, mit dem das erbrechen zusammenhängt, ließ sich nicht ermitteln. die ergebnisse der vorgelegten untersuchungen lassen den schluß zu, daß die virusausbreitung im körper über die nervenbahnen erfolgt. rÉsumÉ: pathogénicité du virus hémagglutinant de l'encéphalomyélite du porc (dépérissement et vomissement) après infection par différents modes d'inoculation porcs ont été infectés selon différents procédés d'inoculation avec l'isolement vw du virus hémagglutinant de l'encéphalomyélite (dépérissement et vomissement). tous les porcs infectés soit par la voie combinée oronasale ( ) soit par le nerf infraorbital ( ) tombèrent malades après une incubation de jours. des animaux infectés par la paroi stomacale, des par voie intramusculaire et des intracérébralement tombèrent malades après un temps d'incubation de – , jours. il n'y a pas eu de maladie chez les porcs inoculés par voie intraveineuse, dans l'abdomen ou directement dans l'estomac. tous les porcs malades ont présenté le syndrome de dépérissement et de vomissement. chez les animaux infectés par voie oro‐nasale et sacrifiés au début de la maladie, on a pu régulièrement réisoler le virus à partir des amygdales et de l'appareil respiratoire, parfois du tissu de la région «pons‐medulla» et de la paroi stomacale. le virus a toujours été isolé chez les porcs tombés malades après un mode d'infection différent. tous les animaux qui ne furent pas malades (à l'exception d'un porcelet) formèrent des anticorps. l'endroit de multiplication du virus lié au syndrome de vomissement n'a pas été déterminé. les résultats de ces essais permettent de conclure que la propagation du virus dans le corps se fait par voie nerveuse. resumen: la patogeneidad del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos) en el cerdo tras infección a través de vías diversas de inoculación se infectaron cuarenta y ocho cerdos a través de diferentes vías de inoculación con el aislamiento vw del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos). todos los cerdos infectados bien con arreglo al procedimiento combinado buco‐nasal ( ) o bien a través del nervio infraorbitario ( ) enfermaron tras un tiempo de incubación de días. seis de siete animales inoculados a través de la pared gástrica, de por vía intramuscular y de por vía intracerebral enfermaron tras un tiempo de incubación de – , días. no se registraron casos de enfermedad en los cerdos inoculados por vía intravenosa o en la cavidad abdominal resp. directamente en el estómago. todos los cerdos que enfermaron presentaban el síndrome de hipotrepsia y vómitos. de los cerdos infectados por vía buco‐nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tracto respiratorio y, en ocasiones, del tejido correspondiente al ámbito puente de varolio‐medula, así como de la pared gástrica. se logró siempre aislar virus de cerdos que enfermaron tras infección por otras vías. sin embargo, todos los animales que no enfermaron (excepción hecha por un lechón) produjeron anticuerpos. no se logró descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con los vómitos. los resultados de los estudios presentados permiten llegar a la conclusión de que la propagación del virus en el organismo acontece a través de las vías nerviosas. in , an infectious encephalomyelitis of viral origin was observed in pigs in canada ( ) . the clinical illness was characterised by a prodromal stage with anorexia, depression, retching and vomiting, followed by motor disorders ( ) . the virus responsible for this disease was later found to be a porcine coronavirus ( ; ) which, because of its hemagglutinating properties, was called hemagglutinating encephalomyelitis virus or hev ( ) . an infectious disease of suckling pigs, characterised by vomiting and wasting, was described in england in ( ) and later observed in other european countries ( ; ; ; ; ) . the disease, called vomiting and wasting disease, was shown to be caused by a virus antigenically and structurally similar if not identical to the hev from canada ( ; ; ). a similar coronavirus, which caused mainly vomiting and wasting symptoms in hysterectomy-derived colostrum-deprived pigs was also reported from the u.s.a. ( ). using the same field isolates mengeling was able to reproduce experimentally the two clinical entities, the encephalitic form and the vomiting and wasting form ( ) . it was, therefore, concluded that both types of the disease, are caused by the same virus. few studies have been performed on the pathogenesis of this viral infection. transmission experiments have proved that reproducible results could be obtained only if colostrum-deprived pigs were used ( ; ). clinical disease has been succesfully reproduced after inoculation of the virus by oral, intranasal, intramuscular and intracerebral routes ( ) . in other studies, the virus was reisolated with regularity from brain, respiratory tract and oronasal swabs, but only if the pigs were examined during the early stages of the disease ( ; ) . hemagglutination inhibiting and seroneutralizing antibodies were demonstrable starting at and days after inoculation respectively ( ). these antibodies are suspected to preclude virus isolation at later stages of the disease ( ; ; ). the present studies were primarily designed to determine whether a virus isolate, obtained from pigs with the vomiting and wasting syndrome only, could produce clinical signs after inoculation by different routes. at the same time, some information was obtained on the optimal conditions for virus reisolation and on the distribution of the virus in different organs particularly after oronasal inoculation. . . virus the virus strain, designated vw , was isolated in belgium in as previously described ( ) . the virus stock used represented the loth passage in primary pig kidney (ppk) cells and contained "' tcid,, per ml. the studies were performed in hysterectomy-derived-colostrum-deprived (hdcd) pigs which were individually housed in horsfall type units. they were inoculated at the age of to days using different routes of inoculation. a blood sample was withdrawn prior to inoculation to assure the absence of specific viral antibodies. sixteen piglets were inoculated by combined oral and nasal (on) route, using ml. of stock virus. in seven piglets, . to ml. of stock virus was inoculated directly into the stomach wall after laparotomy around the pyloric sphincter at four inoculation sites. five pigs were inoculated with ml. of stock virus directly into the gastric lumen after laparotomy by inserting a needle through a canula which perforated the stomach wall. in two pigs, ml. of stock virus was brought into the abdominal cavity after laparatomy. two pigs were inoculated with ml. of stock virus intravenously into the ear vein. five pigs were inoculated in the cerebral cortex, using ml. of stock virus, by perforating the skull with a needle. four pigs were inoculated into the infraorbital nerve with . ml. of stock virus by insertion of the needle into the infraorbital canal. eight pigs were inoculated with ml. of stock virus intramuscularly either in the neck muscles or in the foreleg muscles or in the hindleg muscles. all pigs were observed three times a day for clinical signs. when sick, pigs were killed at time intervals varying from one to five days after the appearance of clinical signs and different tissues were collected for virus isolation. from the on inoculated pigs, serum was collected at the time of killing for detection of hemagglutination inhibiting (hi) and seroneutralizing (sn) antibodies. the pigs which did not become sick during the present study were followed clinically until weeks after the inoculation. during the last week of the observation period, a serum sample was collected and examined for the presence of hi-antibodies. from the pigs killed at different time intervals after inoculation, the following tissues were collected for virus isolation : nasal mucosa, tonsils, lungs (apical and cardiac lobes), pyloric region of the stomach, pons and medulla combined, cerebrum, cerebellum and blood clot. from each organ, a per cent suspension (w/v) was prepared in phosphate buffered saline by homogenisation. after centrifugation at x g., the supernatant fluid was collected and . ml. inoculated in each of tubes with fully sheeted primary pig kidney (ppk) cells. after four days, hemadsorption with chicken erythrocytes was attempted on tubes. the remaining tubes were used for making a second passage. the cells and culture medium were subjected to two cycles of freezing and thawing and, after centrifugation a t x g., the supernatant was inoculated in tubes with fresh monolayers of ppk-cells. hemadsorption was performed on all tubes days later. the procedures used for the hemagglutination inhibition and seroneutralisation test were previously described ( ) . in the presentation of the results, differentiation will be made between the on inoculated pigs and the pigs inoculated by other routes. the number of pigs in the on inoculated group was sufficiently high to allow a more complete study since animals could be killed at several time intervals after the appearance of clinical signs. for the other routes of inoculation, it was the main purpose to see whether clinical illness could be obtained or not. the overall results obtained after on inoculation of the pigs are presented in table i . the individual results of these pigs are presented in table . all pigs were free of hiand sn-antibodies at the time of inoculation. it can be seen from the tables that all on inoculated pigs became ill. the incubation period varied from to days with an average of days. the earliest clinical signs were characterised by anorexia and vomition which suddenly appeared. the vomition usually started after the uptake of milk. the vomit first consisted of milk curds and changed, after a few hours, to a yellow foamy fluid. as soon as the piglets took up more milk, curds were seen again. vomition and retching movements were very regularly present during the first hours. afterwards, the frequency of vomition decreased but the appetite remained markedly reduced. the smaller pigs became emaciated and moribund after to days. the course of the disease was somewhat slower in the stronger pigs. virus could be isolated from of the pigs. as shown in table , the virus was isolated most consistently from the lungs and tonsils. it was sometimes isolated from the pons + medulla and nasal mucosa, and only in pigs from the stomach wall. virus was not isolated from the cerebrum, the cerebel- lum and the blood clot. all pigs which were killed days or later after inoculation had hi-antibodies, whereas sn-antibodies were detected starting days after inoculation. the pons + medul li a of these pigs was positive by immunofluorescence. the overall results obtained in the pigs inoculated by other routes are presented in table . none of the pigs inoculated into the stomach lumen, the abdominal cavity or the ear vein had become sick at weeks after inoculation. all these animals, except one of the pigs inoculated into the gastric lumen, had hi-antibodies at that time. a variable number of pigs became sick after inoculation by other routes. the clinical disease was not different from that observed after on inoculation. in table , the sick pigs were considered to be positive for virus when it was isolated from one or more of the collected organs, the inoculated organ not included. six of the seven pigs inoculated in the pyloric region of the stomach became sick after an average incubation period of days. from two of the six pigs, the virus was reisolated from the stomach wall only whereas, in the remaining four, the virus could be detected in other tissues also. hi-antibodies (titer ) were present in the serum of the pig without clinical signs at the end of the observation period. of the intracerebrally and intramuscularly inoculated pigs, three of the five and six of the eight respectively became sick after an average incubation period of * / days, all the diseased pigs were positive for virus. the pigs which remained healthy had built up hi-antibodies at titers varying from to at the end of the experiment. all three pigs inoculated into the infraorbital nerve became sick after an average incubation period of days and were positive for virus. the clinical disease and the results of virus isolation obtained upon combined oral and nasal (on) inoculation were similar to those previously described ( ) . the virus was most consistently reisolated from the respiratory tract which is probably the natural way of infection. seroneutralizing antibodies appear soon after the beginning of the clinical symptoms and may be a limiting factor for the further succesful isolation of the virus from organs. for the diagnosis of hemagglutinating encephalomyelitis by virus isolation, pigs should therefore be examined preferably within two days after the start of clinical signs. the negative results obtained in pigs and . , killed within this period of time, are probably due to the fact that tonsils and pons + medulla were not collected for virus isolation. the pons + medulla of these pigs was later found to be positive by immunofluorescence. the irregularity with which virus was isolated from the stomach wall and pons + medulla does not allow any conclusions to be drawn about the exact target organ, if it is supposed that virus replication in one of these organs is responsible for clinical signs. this irregularity may be due to the lack of sensitivity of the virus isolation technique. recently, it has been found that centrifugation of tissue suspensions at x g. instead of x g. can remove up to log,, tcid,, of hemagglutinating encephalomyelitis virus. in this way, virus isolation from a tissue suspension with little infectious virus could give a false negative result. it is known from electron microscopic studies that hev during intracellular replication is mainly present in cytoplasmic vesicles with a large amount of membranous material ( ). the removal of virus by centrifugation may, therefore, be due to sedimentation of particles associated with subcellular fragments. upon inoculation into the stomach lumen, clinical signs were not observed. this finding indicates that virus reaching the gastric lumen after swallowing does not initiate clinical disease. however, the seroconversion observed in of the pigs indicates that a subclinical infection has occurred. the ability to reproduce typical disease after a very short incubation period in of the pigs inoculated in the stomach wall may be significant in the light of the pathogenesis. in two of these piglets, virus was isolated from the stomach wall only. this points to the stomach wall as a possible target organ for the virus. it is, however, not excluded that the isolated virus is a residue of the inoculum rather than a result of virus replication. the first possibility is rather improbable since these pigs were killed and days after inoculation respectively. in the present experiments, not all the pigs became sick after intracerebral inoculation and the disease consisted of the vomiting and wasting syndrome. these results are somewhat different from those described by greig, who obtained clinical signs consisting of motor central nervous disorders in all inoculated pigs ( ) . this may be related to the observation that, with the present virus isolate, only the vomiting and wasting syndrome has been observed, not only in the original outbreak but also in experimental pigs. it should be mentioned, however, that mengeling was able to reproduce both clinical entities with the same virus isolate ( ) . the inability to find the virus in the cerebral cortex of on inoculated pigs may indicate that the present isolate has little or no affinity for this part of the central nervous system. it is not impossible that, in intracerebrally inoculated pigs, the appear-ance of clinical signs is determined by the ability of the virus to reach the pons + medulla, another possible target organ, from the deposition site. the findings that none of the pigs became sick after intravenous inoculation and that the virus was never isolated from the blood clot of pigs killed at different time intervals after on inoculation indicate that the target organ is not reached through viraemia. on the other hand, the ability to obtain typical disease after inoculation into the infraorbital nerve suggests that virus spread within the body occurs via nerve pathways. whether clinical signs are obtained in pigs after intramuscular inoculation may be dependent on the ability of the virus to enter into nerves. from the present studies, it was impossible to determine the exact target organ of the vw virus. the results indicate that virus replication, either in the stomach wall or in the pons + medulla or possibly in both, may be responsible for causing the vomiting and wasting syndrome in pigs. forty-eight pigs were inoculated by different routes with the vw isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oralnasal route ( ) or into the infraorbital nerve ( ) became sick after an incubation period of days. six of the pigs inoculated into the stomach wall, of the pigs inoculated intramuscularly and of the pigs inoculated intracerebrally became ill after an incubation period of - . days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. the study was supported by the belgian research institute for industry and agriculture (instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw), brussels, belgium. the technical assistance of gerry van ( ) la patogeneidad del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y vbmitos) en el cerdo tras infeccibn a travds de vias diversas de inoculacibn se infectaron cuarenta y ocho cerdos a travks de diferentes vias de inoculacibn con el aislamiento vw del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y v mitos). todos s cerdos infectados bien con arreglo a procedimiento combinado buco-nasal ( ) o bien a travks del nervio infraorbitario ( ) enfermaron tras un tiempo de incubacidn de dias. seis de siete animales inoculados a travks de la pared glstrica, de por via intramuscular y de por via intracerebral enfermaron tras un tiempo de incuba-ci n de - , dias. no se registraron casos de enfermedad en s cerdos inoculados por via intravenosa o en la cavidad abdominal resp. directamente en el estbmago. todos s cerdos que enfermaron presentaban el sindrome de hipotrepsia y v mitos. de s cerdos infectados por via buco-nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tract respiratorio y, en ocasiones, del tejido correspondiente a lmbito puente de varolio-medula, asi como de la pared gistrica. se logr siempre aislar virus de cerdos que enfermaron tras infecci n por otras vias. sin embargo, todos s animales que no enfermaron (excep-ci n hecha por un lech n) produjeron anticuerpos. no se logr descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con s v mitos. los resultados de s estudios presentados permiten llegar a la conclusi n de que la propagaci n del virus en el organism acontece a travds de las vias nerviosas. vomiting and wasting disease in piglets isolement en france et identification du virus de la maladie du vomissement et du dcpcrissement des porcelets (coronalike virus) lesions induced by hemagglutinating enccphalomyelitis virus strain n in pigs vomiting and wasting disease in piglets a hemagglutinating virus producing encephalomyelitis in baby pigs characteristics of a coronavirus (strain n) of pigs experimentally induced infection of newborn pigs with hemagglutinating encephalomyelitis virus strain n pathogenicity of field isolants of hemagglutinating encephalomyelitis virus from neonatal pigs characteristics of a coronavirus causing vomition and wasting in pigs the size and morphology of tge and vomiting and wasting disease viruses of pifs a viral encepha omyelitis of pigs. further studies on the transmissibility of the disease in ontario vomiting and wasting disease of piglets key: cord- -c f l s authors: sauter, kristin a.; waddell, lindsey a.; lisowski, zofia m.; young, rachel; lefevre, lucas; davis, gemma m.; clohisey, sara m.; mcculloch, mary; magowan, elizabeth; mabbott, neil a.; summers, kim m.; hume, david a. title: macrophage colony-stimulating factor (csf ) controls monocyte production and maturation and the steady-state size of the liver in pigs date: - - journal: am j physiol gastrointest liver physiol doi: . /ajpgi. . sha: doc_id: cord_uid: c f l s macrophage colony-stimulating factor (csf ) is an essential growth and differentiation factor for cells of the macrophage lineage. to explore the role of csf in steady-state control of monocyte production and differentiation and tissue repair, we previously developed a bioactive protein with a longer half-life in circulation by fusing pig csf with the fc region of pig igg a. csf -fc administration to pigs expanded progenitor pools in the marrow and selectively increased monocyte numbers and their expression of the maturation marker cd . there was a rapid increase in the size of the liver, and extensive proliferation of hepatocytes associated with increased macrophage infiltration. despite the large influx of macrophages, there was no evidence of liver injury and no increase in circulating liver enzymes. microarray expression profiling of livers identified increased expression of macrophage markers, i.e., cytokines such as tnf, il , and il known to influence hepatocyte proliferation, alongside cell cycle genes. the analysis also revealed selective enrichment of genes associated with portal, as opposed to centrilobular regions, as seen in hepatic regeneration. combined with earlier data from the mouse, this study supports the existence of a csf -dependent feedback loop, linking macrophages of the liver with bone marrow and blood monocytes, to mediate homeostatic control of the size of the liver. the results also provide evidence of safety and efficacy for possible clinical applications of csf -fc. lations in mice ( ) , there were a number of clinical trials of applications in cancer and other indications ( ) . the interest in csf as a therapeutic agent has been reinvigorated by evidence of the requirement for macrophages in tissue regeneration in multiple organs ( ) and the finding that macrophages generated in response to csf have trophic roles ( , ) . csf treatment has been shown to promote regeneration and repair in injury models in the kidney ( ), brain ( , ) , and bone ( ) . the pleiotropic impacts of csf mutations in mice ( ) suggest that repair in most tissues is reliant on macrophages that depend on this growth factor. applications of csf therapy were constrained by the very short half-life of the -amino acid active form of csf . we developed a bioactive protein with a longer half-life in the circulation. we fused pig csf , which is equally active in all mammalian species tested ( ) , with the fc region of pig igg a (csf -fc) ( ) . the expected increase in half-life was confirmed, and csf -fc administration to mice produced substantial increases in circulating monocyte and tissue macrophage numbers, at much lower doses than the native protein. an unexpected effect of the treatment was a substantial increase in the size of the liver, associated with extensive hepatocyte proliferation ( ) . this observation was consistent with previous data implicating csf -dependent macrophages in hepatic regeneration. acute liver failure in human patients, for example due to paracetamol toxicity, is associated with the loss of clearance functions that protect the body against the contents of the portal blood. circulating csf levels in human patients upon admission to hospital with paracetamol poisoning were found to be predictive of subsequent prognosis ( ) . administration of csf -fc in mouse models produced both accelerated regeneration of the liver and, perhaps more importantly, very rapid restoration of clearance functions ( , , ) . pigs have been used increasingly as models of human disease ( ) . the gene expression profiles of stimulated mouse and human macrophages differ greatly, and those of pig macrophages are much more human-like ( ) . aside from the possible applications as human disease models, pigs are a major livestock species. early weaning in pigs, when the mucosal barrier and innate immune systems are immature, is associated with susceptibility to a very wide range of mucosal and systemic bacterial and viral infections that produce significant losses ( ) . the results obtained in mice cannot necessarily be extrapolated to large animals, including humans, in which the profiles of macrophage activation by both csf and microbial stimuli are very different ( ) . our results confirm that, as in the mouse, csf -fc can drive hepatocyte proliferation and modulate the size of the liver in a large animal model. the data support applications of csf -fc in liver regeneration and provide further evidence for the role of csf in monocyte/ macrophage maturation. in combination with earlier data, they reinforce the conclusion that circulating csf is a central contributor to the homeostatic control of the size of the liver. animals. approval was obtained from protocols and ethics committees of roslin institute or agri-food and biosciences institute (afbi) for the trials. the experiments were carried out under the authority of a uk home office project license, under the regulations of animals (scientific procedures) act . csf -fc was made as previously described ( ) and provided by zoetis. large white pigs ϳ . wk of age from one litter were used. four days prior to the first injection each pig was weighed and had blood collected into an edta tube. pigs were injected subcutaneously once a day for a total of days with the appropriate volume of csf -fc ( . mg/kg; n ϭ ) or pbs vehicle (n ϭ ). pbs injection was used to control for the possible impact of stress and restraint associated with treatment. in experiments on weaners, large white ϫ landrace pigs ϳ wk of age from three litters were used. six days prior to the first injection each pig was weighed and an estimated weight was extrapolated for each for the first injection day. pigs were injected intramuscularly once a day for days with the appropriate volume of csf -fc ( . mg/kg; n ϭ ) or pbs (n ϭ ). on the second injection day the pigs were weaned. all pigs were sedated with ketamine and azaperone before being euthanized by captive bolt. neither subcutaneous nor intramuscular injection produced any side effects. isolation of pbmc and bmc. blood was collected into blood collection bags containing acid citrate dextrose (acd) (sarstedt) or into beakers containing acd (sigma). the buffy coat was layered onto lymphoprep (axis-shield) and centrifuged for min at , g with no brake. peripheral blood mononuclear cells (pbmc) were retrieved and red cells were removed with cell lysis buffer (biolegend). pig bone marrow cells (bmc) were obtained by flushing the bone marrow from ribs with rpmi/ mm edta followed by removal of red cells with cell lysis buffer. all isolated cells were suspended in pbs prior to counting and cryopreservation. flow cytometry analysis. cells were washed, pelleted, resuspended in blocking buffer (pbs/ % heat inactivated fcs), transferred to a -well plate (v-bottom), and incubated on ice for - min. the plate was centrifuged for min at g followed by removal of supernatant. cells were resuspended in l of pbs containing the appropriate antibody or isotype control (table ) . samples were incubated at °c in the dark for min before being washed two times with l pbs. cells were resuspended in l pbs with . % sytox blue (invitrogen) immediately prior to analysis using a bd fortessa lsr flow cytometer (becton dickinson). analysis was performed using flowjo software (flowjo). complete blood count analysis. an aliquot of blood from acd blood collection bags was analyzed for complete blood cell counts. total white blood cell (wbc) was measured on the siemens advia analyzer. wbc differential counts were performed by making a blood smear counterstained with giemsa stain prior to cells of each cell type being counted. the absolute value for each wbc type was determined by using the total wbc and % leukocytes. manual platelets counts were carried out using a hemocytometer slide (by the r(d)svs clinical pathology laboratory, university of edinburgh). plasma analysis. plasma was analyzed by r(d)svs clinical pathology laboratory for cortisol (performed on siemens immulite analyzer) as well as a large animal liver damage profile (performed on the il analyzer from instrumentation laboratories). tissue processing. tissues were dissected, weighed (liver, spleen, and kidney), and placed in % neutral buffered formalin or rnalater (ambion). for histology, tissues were processed overnight using an excelsior tissue processor (thermo fisher scientific). sections were embedded in paraffin wax prior to -m sections cut and mounted onto slides (superfrost plus, thermo fisher scientific). slides were dried overnight at °c before °c for min. sections were stained with h&e or immunohistochemistry was performed by r(d)svs pathology department. immunohistochemistry for cd . antigen retrieval was performed with proteinase k (dako s ) for min. nonspecific protein binding was blocked using . % goat serum (vector laboratories) for min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s ) for min. sections were incubated for min using mouse anti-pig cd (serotec mca ga) diluted / . visualization using secondary reagent dako envision mouse hrp (dako k ) for min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app ) for min and dab enhancer for min (newmarket scientific dab concentrate cat. no. co - ) was performed by the r(d)svs pathology department. the staining was analyzed using image j (fiji). immunohistochemistry for ki and pcna. antigen retrieval was performed by boiling in mm sodium citrate buffer. nonspecific protein binding was blocked using . % goat serum (vector laboratories) for min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s ) for min. sections were incubated for min using rabbit anti-human ki (abcam ab ) diluted / , . visualization was performed with secondary reagent immpress hrp anti-rabbit igg (peroxidase polymer; vectormp- ) for min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app ) for min and dab enhancer for min (newmarket scientific dab concentrate cat. no. co - ). statistical analysis. data were analyzed by t-tests. results are presented as treatment group means Ϯ se. all analyses were performed using graphpad prism . (graphpad software). a p value Ͻ . was considered statistically significant. microarray. total rna was prepared from liver samples using trizol, prepared for hybridization using the ambion wt expression kit (life technologies), following the manufacturer's instructions, except for the input amount of rna ( ng input instead of ng) and hybridized in a random order to the affymetrix porcine gene . st array (performed by edinburgh genomics, university of edinburgh). statistical analysis of the array data utilized partek genomic suite (partek). for network analysis, the normalized array data were uploaded to the software biolayout express d (http://www.biolayout.org/) as described previously ( , ) . the data from the microarray are available at gene expression omnibus ncbi (http://www.ncbi.nlm.nih.gov/geo/) accession code gse . we first examined -wk-old pigs of both sexes from the same litter, with control and treated groups weight matched in pairs. based on mouse data ( ) we used a dose of . mg/kg for three daily treatments followed by cull h after final injection. in previous studies, we have compared csf -fc with the native, non-fc conjugate, form of csf as a control protein. native csf has a much shorter half-life and at the same dose had no effect on monocyte-macrophage numbers ( ) . we have not compared csf -fc directly with an irrelevant fusion protein, or with an isotype control for the fc component. however, an equivalent dose of igg ( - mg depending on the size of the pig) would have no impact on the plasma igg concentration ( - mg/ml). csf -fc treatment of macrophages in vitro did not induce proinflammatory cytokines ( ) and, in keeping with the lack of intrinsic proinflammatory activity, there was no evidence of any reaction at the sites of injection in any treated animals. the most obvious effect of csf -fc administration was hepatosplenomegaly. csf -fc doubled the spleen/body weight ratio and increased the liver/body weight ratio by % after only days (fig. a ). the total wbc count was significantly increased, mainly due to lymphocytosis in addition to the expected monocytosis (fig. b) . csf -fc accelerates the maturation of macrophage populations in peripheral blood monocytes. csf has been implicated in the maturation of blood monocytes in both mice and humans, driving the formation of the nonclassical (cd low , cd high ) subset in humans (ly c low in mice) ( , ) . pig blood monocytes can also be separated into subsets based on expression of surface markers, although the distinctions are not as clear as in other species ( ) . expression of various markers by peripheral blood monocytes was assessed by flow cytometry staining (fig. ). in addition to the increase in total wbc seen in fig. , the proportion of monocytes, detected by cd a (sirpa) was increased around twofold (fig. b ). the proportion of cells expressing cd was also increased ( fig. a) . no increase was seen in the percentage of cd ϩ lymphocytes ( fig. c ) the best-characterized monocyte maturation marker in pigs is the haptoglobin receptor, cd ( ) , which has also been implicated as a receptor for the major pig viral pathogen, porcine reproductive and respiratory syndrome (prrsv) ( ) , and which varies inversely with cd expression. double staining with the two markers indicated that csf -fc shifted the profiles of both markers, favoring expansion of the cd ϩ cells (fig. d ). the results are consistent with an impact of csf -fc to promote both monocyte production/ release and maturation. impact of csf -fc treatment on the bone marrow. we next investigated whether the ability of csf -fc to promote monocytosis was associated with expansion of progenitor pools in the marrow (fig. ) . figure , a and b, demonstrates a substantial increase in large cd ϩ monocytes, and even greater increase in cd ϩ cells in the marrow of treated animals, consistent with the pattern seen in blood. aside from monocyte progenitors, a key population of macrophages in bone marrow forms the center of hemopoietic islands. in mice, these cells express sialoadhesin (cd , which provides a receptor for immature erythrocytes) and are critical for suc-cessful engraftment in bone marrow transplantation ( ) . as shown in fig. c , the csf -fc treatment produced a substantial increase in the cd ϩ population in pigs. the csf -fc treatment did not expand the small percentage of cells that express cd (kit), a marker of the stem cell population (fig. d) , suggesting that csf -fc acts primarily to promote proliferation/expansion of committed progenitors. in bone marrow of pig, neither cd a nor cd provides a useful marker of monocyte lineage cells, being detected on the large majority of the cells (fig. , a and b) and only marginally increased by csf -fc. we also examined the expression of the csf r (cd ) using either a recently described monoclonal antibody ( ) or labeled csf -fc. there was some evidence of expansion of the positive cell populations in each case, but the levels of labeling were very low (fig. , c and d) . we suggest that the receptor may be downregulated by csf -fc. counts. pigs ( -wk-old males and females) were injected with pbs or . mg/kg csf -fc for days prior to euthanasia on day . blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show means Ϯ se. **p Ͻ . , ***p Ͻ . , ****p Ͻ . by t-test; n ϭ - pigs per treatment. a: liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. b: total wbc count, lymphocyte number, neutrophil number, monocyte number, and platelet number. origin of the increase in liver and spleen weight. csf -fc treatment caused a substantial increase in macrophage numbers in both organs, detectable by immunolocalization of cd . in immunostained sections of liver csf -fc treatment increased cd ϩ area (quantified with imagej) from an average of less than . % to an average of over % (fig. a) . in spleen csf -fc treatment had an even greater effect, causing an increase of cd ϩ area from an average from ϳ % to % (fig. b ). as in mice ( ) , in the spleen the majority of the increase in size could be attributed to increased red pulp macrophages and also to expansion of the marginal zones. by contrast, the increase in the area apparently occupied by macrophages is not sufficient to explain the substantial increase in the size of the liver. sections of liver were stained for the figure shows images of the liver from two control and two csf -fc-treated pigs. the pigs are relatively young, and still growing, and accordingly there is significant ongoing proliferation evident from ki staining. the vast majority of ki ϩ nuclei in both control and csf- -fc-treated pig livers were large and round, consistent with identity as hepatocytes. macrophage nuclei are more difficult to visualize in histological sections, because the cells and nuclei are much smaller and ramified in the sinusoids. very occasional smaller ki ϩ nuclei visible in the sinusoids suggested that some infiltrating monocyte-macrophages were also proliferative, as shown directly in the mouse system ( ) . the images in fig. also show an obvious increase in cellularity in response to csf -fc. we counted the total nuclei and the proportion stained with anti-ki in representative large fields from each animal. as shown in fig. , csf -fc treatment almost doubled the total number of nuclei in each field and produced a threefold increase in the percentage of those nuclei stained with anti-ki . essentially the same findings were made with staining for proliferating cell nuclear antigen (pcna) (not shown). the sections in fig. show no evidence of pathology in the liver; notably, there are no pyknotic nuclei and granulocytes are absent. granulocyte infiltration is the hallmark of tissue injury, including injury to the liver ( ) . changes in the liver might occur secondary to alterations in the gut. csf has been attributed indirect functions in control of proliferation and differentiation of gastrointestinal epithelium ( , ) . treatment with csf -fc in pigs produced a small but significant increase in the mean villus length of the mid jejunum but had no detectable effect in the ileum, cecal base, or ascending colon (fig. ) . there was also no significant change in goblet cell number. effect of csf -fc on liver function. a panel of biochemical tests to measure serum enzymes, bile acid, bilirubin, and protein concentrations was performed to assess hepatic function. the only change was a small increase in bile acids and bilirubin in serum from csf -fc-treated pigs (fig. ) , only marginally outside the normal range ( ) . since standard enzymic indicators of liver injury (alkaline phosphatase, alanine aminotransferase, ␥-glutamyl transpeptidase) were unchanged, the increase in bile acids probably reflects the increased size of the liver. to examine the impact of csf -fc on liver function in more detail, we profiled the transcriptome. the expression results were filtered to remove genes detected below an arbitrary relative intensity threshold and also genes that did not differ by more than . -fold between the highest and lowest value in the nine samples. the second criterion removed around % of probes on the microarray, including many hepatocyte-specific gene products. figure c shows that the relative abundance of representative examples of these known hepatocyte gene products, albumin (alb), cd , fetuin, and transferrin (tf), within the total liver rna pool was unchanged in response to csf -fc. in other words, the infiltration of the liver by macrophages was insufficient to dilute the contribution of hepatocyte mrna to the total mrna pool. that finding is consistent with the histological observation above, that even in the csf -fc-stimulated state the infiltrating macrophages appear to make up no more than % of the total area of the liver. hence, the % increase in total liver weight can be attributed primarily to an increase in using imagej software the total cd ϩ area was calculated from representative images per pig per organ. graphs show means Ϯ se. *p Ͻ . , **p Ͻ . by t-test; n ϭ - pigs per treatment. g csf controls the steady-state size of the liver hepatocytes, consistent with the extensive proliferation and increased cellularity shown in fig. . we clustered the included probe sets based on expression pattern and displayed them using biolayout express d . the advantage of using the clustering approach is that genes that might appear regulated, but in only a subset of animals, appeared in separate smaller clusters. these may reflect the interanimal variation in macrophage-inducible gene expression that we documented previously in a study of pig breeds ( ) . the gene lists of specific clusters are provided in supplemental table s . (supplemental material for this article is available online at the journal website.) functional annotations of the two large clusters were tested using david (supplemental table s ). cluster , the set of genes elevated in all csf -fctreated pigs, was clearly enriched for genes involved in the cell cycle and innate immunity, whereas cluster , the set that was reduced in the csf -fc-treated pigs, was enriched in genes involved in metabolism. importantly, there is no evidence among the induced genes of expression of apoptosis-associated genes, no induction of acute phase genes, and no appearance of classical granulocyte marker genes such as s a /s a or mpo. figure shows the expression profiles of a number of genes that highlight the biological processes involved. cluster (fig. a ) includes many genes that were shown previously ( ) to be restricted to macrophages, such as the transcription factor spi ; surface markers sirpa, emr , and itgam; known csf -inducible genes plau, mmp , chi l , and c q; endocytic receptors marco, msr , and fcgr a; and pattern recognition receptors tlr , , , , , , and . on average, the relative contribution of cluster , macrophagespecific genes, to the liver total rna increased by three-to fivefold in response to csf -fc, again consistent with the fig. . in mice, the recruited monocytes express the chemokine receptor ccr and apparently respond to ccl ( ) . by contrast, ccr was enriched in the liver mrna of treated pigs, alongside three of its known ligands, ccl , ccl , and ccl l . as previously observed in mice ( ) , monocytes recruited to the treated livers apparently responded to proinflammatory signals, since cluster contained numerous known lps-inducible genes ( ) such as inflammatory cytokines tnf, il a, and il b; interferon targets ido ; multiple type interferon targets irf , irf , ifitm , and ifit ; tgfb ; and costimulators of t cell activation cd , cd , and cd . cluster also contains numerous cell cycle-associated genes, including pcna; the key transcription factors foxm , e f , e f , and e f ; and several cyclin genes ccna , ccna , ccnb , ccnb , ccnd , and ccnd . the increased expression of enzymes of glycolysis hk , hk , hk pfkp, pgk , pgd, pkm, gpi, gapdh, and ldha also reflects the requirement for aerobic glycolysis in proliferating cells ( ) . cluster (fig. b) , the set of genes reduced in the csf -fc-treated pigs, most likely reflects the functional zonation of the liver between periportal and perivenous regions of liver lobules ( , , ) and the selective proliferation of cells derived from portal progenitors that has been observed in regenerating liver ( , , ) . it includes genes involved in xenobiotic metabolism and detoxification, notably p family (e.g., cyp a , cyp e ), glutathione s-transferases (e.g., gstaa ) and aldo-ketoreductases (e.g., akr c ), and the gluconeogenic enzyme pck , that are known to be enriched in perivenous locations. the cluster contains the gene for the regulator of hepatocyte stem cells, sox ( ), indicating that these cells are not expanded in the csf -fc-treated livers. unexpected members of this cluster are genes for the growth hormone receptor (ghr) and the target, igf , and both estrogen (esr ) and androgen (ar) receptors. also unexpected is the inclusion of the receptor for hepatocyte growth factor, met, which is implicated in regeneration ( ), but this might reflect autoregulation in response to its ligand ( ) . although models of acute liver failure in pigs have been described ( , ) , and may be one path to clinical development of csf -fc as a treatment, it is challenging to perform sufficient replicates to test a clinical intervention. we therefore considered an alternative in production pigs. commercial pigs are normally weaned at wk, when the gut is immature. diarrhea and disseminated infections with organisms such as escherichia coli and streptococcus suis are relatively common ( ) . in this respect, the pig has been studied as a model of early-life stress ( ) . the biology of early weaning in pigs may also be relevant to intestinal failure-associated liver disease in neonates and children ( ) . weaner pigs were treated with a higher dose of . mg/kg csf -fc for two daily injections, immediately prior to weaning and on the day of weaning followed by euthanasia h following the final injection. at this early time point, there was already a significant increase in the spleen/body weight ratio and a trend toward increased liver/body weight ratio (fig. a ). the number of cd ϩ cells was more than tripled in the bone marrow, from ϳ % to over % (fig. b) , and increased numbers of cd ϩ macrophages were confirmed by immunostaining in liver and spleen (fig. c ). at this time point, there was not a significant monocytosis, indicating that both marrow expansion and tissue macrophage proliferation precede monocyte expansion and are likely direct effects of csf -fc. we repeated the treatment in a larger cohort of weaned pigs. this study was conducted in a high health status research unit, which reflected commercial practice. we explicitly removed zinc from the feed, which is usually added to reduce weaningassociated infections. given the production of inflammatory cytokines and reduction in igf- in the liver of treated pigs, we measured weight gain daily in all animals. csf -fc ( . mg/kg) was administered to pigs for two daily intramuscular injections on the day before and the day of weaning, and pigs were killed days after the second injection. although some pigs showed evidence of mild postweaning diarrhea, all the animals in both groups continued to gain weight rapidly (fig. a) . the treated pigs, like the treated mice left for longer after the final injection, demonstrated hepatosplenomegaly (fig. b) , and the increased numbers of cd ϩ macrophages in the liver remained evident after days (fig. c ). in this study we have extended previous studies in mice ( ) to examine the impact of a sustained increase in csf activity on monocyte-macrophage homeostasis. all of the impacts we have observed are consistent with the known biological activity of csf . in mice, the same impacts on monocyte-macrophage numbers and maturation can be generated by injection of very much higher doses of native csf ( ) or injection of a much larger native form of human csf ( ) . the doses of native csf required are prohibitive in a large animal. although we cannot entirely eliminate other functional contributions of the fc component, the increase in circulating half-life is the most obvious explanation for the increased efficacy compared with native csf . the nature of the so-called hepatostat, which determines that the liver returns to a size that is strictly proportional to body size, has continued to be something of a mystery ( , ) . although there are many candidates, including growth factors and inhibitors, extracellular matrix proteins and metabolites, and circulating hormones that can regulate hepatic regeneration, it is unclear how any of them functions as a sensor. in a previous study, we made the striking observation that csf treatment of mice (using an fc conjugate with an increased circulating half-life) was able to increase the size of the liver as well as the number of kupffer cells. this ability is quite unique. in the present study, we have extended the finding to the domestic pig, an animal that is considerably more humanlike in size and vascular biology. the data in fig. show that a major impact of csf -fc treatment in pigs is to increase the number of hepatocytes through extensive proliferation, so that the total cellularity of the liver is increased even more than the increase in total liver weight. hepatocyte proliferation, as opposed to hypertrophy, is also a feature of liver regeneration in response to partial hepatectomy ( - ) . we have made the reciprocal observation in mice; namely, that prolonged depletion of kupffer cells with anti-csf r treatment leads to a reduction in the size of the liver ( ) . others have shown that liver regeneration is greatly impaired in csf -deficient or anti-csf r-treated mice ( ) and in mice depleted of blood monocytes ( ) and have promoted liver repair by infusing csf -stimulated macrophages into the portal vein ( ) . the impact of csf -fc on hepatic growth in mice was dependent on monocyte recruitment, as evident from the impact of knockout of ccr ( ) . the role of monocyte-macrophage products, including the inflammatory cytokines tnf, il , and il , in hepatocyte proliferation has been well recognized ( , ) . csf -fc action in mice was partly dependent on il ( ) , which was also induced in all of the csf -fc-treated pigs. the effect of csf -fc treatment demonstrates that csf -dependent monocyte recruitment is both necessary and sufficient to drive hepatic proliferation and can drive it beyond the homeostatic limits even in a large animal. the effect of csf treatment supports other evidence of the existence of a homeostatic feedback loop. macrophages, notably those of the liver ( ) and blood monocytes ( ) , together regulate the level of circulating csf via receptor-mediated endocytosis. this mechanism is evident from the massive increase in circulating csf seen in animals treated with anti-csf r ( ), and the importance of the liver is evident in patients following partial resection ( ) . the csf -fc treatment reveals that elevated csf can provoke expansion of the committed monocyte pool in the marrow (fig. ) , maturation of the monocytes toward a resident phenotype (fig. ) , and infiltration of the liver (fig. ) . hence, the physiological hepatostat ( - ) may actually be a "macrostat." of course, the increased macrophage numbers in the liver elicited by csf produce secondary impacts, not only removing potential toxins from the portal blood, but altering the balance of metabolism in the liver between portal and venous-associated functions. interestingly, resident kupffer cells are selectively located toward the portal vein in mouse liver lobules ( ) , which might also serve to localize macrophage-derived hepatocyte proliferative signals. the expression profiling of the livers of the csf -fc-treated pigs (fig. ) closely parallels results obtained previously in the mouse system ( ) . the newly recruited monocytes clearly respond to tlr-mediated and other signals to express the large majority of transcripts seen when csf -stimulated bone mar- fig. . effect of a short course of csf -fc in weaning pigs. pigs ( -wk-old males and females) were injected with pbs or mg/kg csf -fc for days prior to euthanasia on day . graphs show means Ϯ se. *p Ͻ . , **p Ͻ . , ***p Ͻ . by t-test; n ϭ pigs per treatment. a: blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and monocyte number. b: bm from ribs was collected as described in materials and methods. bm cells were analyzed via flow cytometry for expression of cd with exclusion of dead cells using sytox blue. representative flow cytometry plots are shown. c: formalin-fixed liver and spleen tissue was prepared and stained for cd . representative images are shown. fig. . long-lasting effect of csf -fc in weaning pigs. pigs ( -wk-old males and females) were injected with pbs or . mg/kg csf -fc for days prior to euthanasia days after the final injection; n ϭ pigs per treatment. a: body weight was recorded at each of the time points shown and total body weight change over the duration of the experiment was graphed for pbs treated pigs (black) and csf -fc-treated pigs (red). b: bar graphs show means Ϯ se. ***p Ͻ . , ****p Ͻ . by t-test. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. c: formalin-fixed liver tissue was prepared and stained for cd . representative images are shown. scale bar ϭ m. csf controls the steady-state size of the liver row-derived macrophages respond to lps ( ) . however, classical neutrophil chemoattractants such as il were not detected; we found no evidence of neutrophil infiltration, no induction of classical acute phase response or apoptosis genes, and no evidence of damage to liver cells. furthermore, the pigs showed no adverse impacts, and in weaners the treatment did not impair their rapid growth (fig. ) . this is consistent with earlier data, in which recombinant csf has previously been administered in phase clinical trials by continuous infusion to humans and was well-tolerated ( , ) , and indicates that the fc fusion protein does not produce any additional toxicity. the lack of severe consequences may be attributed in part to induction of the anti-inflammatory cytokines il and tgfb in the liver of the treated animals. whatever the mechanism, the outcome suggests that csf -fc specifically promotes a proregenerative cellular response in liver, as it does in other organs ( ) . in the final set of experiments (fig. ) , we progressed toward the clinical application of csf -fc in a pig model. the model may also reflect a practical application in pig production to bolster the innate immune system at weaning. we showed that only two doses, administered intramuscularly, were sufficient to produce a sustained increase in monocyte count, liver size, and liver macrophage numbers and produced no adverse local reaction. we propose that csf -fc could provide protection against disseminated infections arising from the immature gut of early-weaned animals. similarly, clearance functions of the macrophages of the liver are crucial to prevent sepsis in acute liver failure in humans, and csf -fc rapidly promoted clearance functions in mouse disease models ( ) . the fact that administration of csf -fc to pigs increased the size of the liver and the liver macrophage population (i.e., the clearance capacity, noting also the increased expression of clearance receptors in the 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animal studies. we thank colleagues at zoetis for providing the csf -fc, specifically the authors listed in our previous publication ( ) . we also show our gratitude to iveta gazova, anna raper, and jun hu for helping to process samples and deliver them to their appropriate locations.present address for g. m. davis: the university of manchester, oxford road, manchester m pl, united kingdom. key: cord- -tbkitz m authors: sookhoo, jamie r. v.; brown-jordan, arianne; blake, lemar; holder, ridley b.; brookes, sharon m.; essen, stephen; carrington, christine v. f.; brown, ian h.; oura, christopher a. l. title: seroprevalence of economically important viral pathogens in swine populations of trinidad and tobago, west indies date: - - journal: trop anim health prod doi: . /s - - - sha: doc_id: cord_uid: tbkitz m the objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (swiv), as well as the seroprevalence of porcine parvovirus (ppv), transmissible gastroenteritis virus (tgev), porcine reproductive and respiratory syndrome virus (prrsv), porcine respiratory coronavirus (prcv), porcine circovirus type (pcv- ), and classical swine fever virus (csfv) in pigs in trinidad and tobago (t&t). blood samples ( ) were randomly collected from pigs at farms throughout t&t. serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial elisa kits, and the circulating strains of swiv were identified by the hemagglutination inhibition test (hit). antibodies against swiv were detected in out of the samples ( %). out of a total of farms, tested positive for swiv antibodies. hi testing revealed high titers against the a/sw/minnesota/ / h n strain and the ph n pandemic strain. antibodies against ppv were detected in out of the samples ( %), with out of farms testing positive for ppv antibodies. antibodies against pcv- were detected in out of the samples tested ( %), with out of the farms testing positive for pcv- antibodies. no antibodies were detected in any of the tested pigs to prrsv, tgev, prcv, or csfv. monitoring and surveillance for viruses that affect swine are crucial in the prevention and control of diseases that can cause severe economic losses to the swine populations of trinidad and tobago (t&t) and the wider caribbean region. to date, there have been limited or no published studies investigating the prevalence of viruses circulating in t&t's swine populations. in late , according to the world animal health information system (wahis) database, columbia reported to have experienced classical swine fever (csf) limited to one or more zones and porcine reproductive and respiratory syndrome (prrs) was reported to be present in columbia and suriname. viruses such as classical swine fever virus (csfv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus type (pcv ), and swine influenza virus (swiv) have been reported to be circulating in venezuelan swine herds (kwiecien ) . the close proximity ( km) of trinidad to venezuela, and the known illegal trade of animals and animal products between the two countries, renders trinidad a high-risk location for the introduction of these viruses. the objectives of this study were to assess the prevalence of selected viruses, namely porcine parvovirus (ppv), transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), csfv, pcv- , prrsv, and swiv in the pig populations of t&t and to identify the strains of swiv that are circulating in t&t pigs. understanding the background levels of these viruses in swine populations in t&t will enable the development of science-based risk assessments, thus aiding the successful prevention, management, and control of future viral outbreaks within t&t and the caribbean region. this will improve the health status and productivity of swine and pork production in t&t. the sample size (n = ) was estimated by using an anticipated true herd-level prevalence of %, tolerance of . %, a % confidence level, and herd-level specificity and sensitivity of and % respectively. this sample size was achieved as a modification of the cannon and roe ( ) formula for livestock disease surveys as modified by humphry et al. . sampling was conducted on pig farms throughout t&t. sampling was carried out from october to february , and samples were taken from large pig farms and small pig farms. three of the small farms had animals slaughtered at public abattoirs. a map showing the location of the farms and abattoirs sampled is shown in fig. . small farms were classified as operations with - pigs and large farms as operations with - pigs. none of the pigs sampled were vaccinated for any of the viruses under investigation. two large farms were sampled on slaughter days. twentyfive pigs were randomly selected and these samples were used. on the large farms where slaughtering was not being carried out, a randomization process was used. each finisher, breeding sow, and boar were designated a number, the numbers were then placed into a bag, and numbers were drawn at random. these pigs were sampled for antibody testing. in total, samples were collected from large farms. ten samples were taken at random from each of the small farms that slaughtered their pigs in public abattoirs. these samples were taken at slaughter. the remaining small farms were visited, and samples were taken at random. on farms with fewer than pigs, all of the pigs on the farm were sampled. in total, samples were collected from small farms. blood was taken from the right anterior vena cava of pigs using g . -in. needles for nursery pigs and -in. needles for adults. samples were collected in red-topped (no anticoagulant) tubes and centrifuged in a beckman model tj- centrifuge at rpm for min to separate serum. the samples were collected from finisher pigs, and where possible, samples from older animals were taken. -large farms -small farms serum antibodies to influenza were detected by the hi test according to standard methods (organization for animal health ). to reduce non-specific reactions, the sera were treated with u/ml of receptor-destroying enzyme at °c for h, inactivated at °c for min, and adsorbed with % (v/v) chicken red blood cells (crbcs) overnight at °c. following treatment, the starting dilution of serum was / , and doubling dilutions were prepared, prior to the addition of an equal volume of both four hemagglutinating units of virus and % crbcs. following incubation at °c for min, the plates were examined for hemagglutination of crbcs. hi titers were recorded as the reciprocal of the highest initial dilution of serum which completely inhibited hemagglutination. lf lf lf lf sf sf sf sf sf sf sf sf sf sf sf tf tf tf tf tf tf tf tf tf lf lf lf sf sf sf sf sf sf sf sf sf sf sf tf tf tf tf tf tf tf tf tf lf lf lf lf sf sf sf sf sf sf sf sf sf sf sf tf tf tf tf tf tf tf tf tf to % (fig. a) . the pigs sampled in tobago were all negative for swiv antibodies. hi testing on a selection of of the elisa positive samples revealed high titers against the a/sw/minnesota/ / h n strain of swiv as well as the ph n pandemic strain ( table ) . one of the pigs also showed a high titer against the a/perth/ / h n strain. antibodies to ppv were detected in % ( out of ) of sampled pigs and on % ( out of ) of the farms sampled, including large farms in central and eastern trinidad. approximately % of ppv positives corresponded to an hi titer of , to , according to the test kit insert (lsivet™ porcine parvovirosis-serum, lissieu, france), suggesting that field infections of ppv were occurring in these swine. the percentage of pigs testing positive for antibodies to ppv on each farm ranged from to % (fig. b ). the pigs sampled in tobago were all negative for ppv antibodies. antibodies to pcv- were detected in % ( out of ) of sampled pigs from t&t and on % ( out of ) of the farms sampled on both islands, including the major farms in trinidad. of the total number of pigs sampled from trinidad, % ( out of ) tested positive for pcv- antibodies which were found on . % ( out of ) of trinidad farms. of the total number of pigs sampled from tobago, % ( out of ) were positive for antibodies to pcv- . of the farms sampled from tobago, all tested positive for pcv- antibodies. approximately % of the pcv- positives corresponded to an antibody titer of or higher according to the test kit insert (biochek pcv- antibody test kit-holland) suggesting that field infections of pcv- were occurring in these swine. the percentage of pigs testing positive for antibodies to pcv- on each farm ranged from to % (fig. c ). ten farms were positive for antibodies to swiv, ppv, and pcv , and three farms were positive for antibodies to both swiv and pcv , but not ppv. one small farm (sf ) was positive for antibodies to swiv and ppv but not pcv , and two small farms (sf and sf ) as well as all of the tobago farms were positive for only pcv . no antibodies were detected in any of the sampled pigs against prrsv, tgev/prcv, or csfv. out of approximately , domestic pigs in t&t, (~ %) were sampled and tested for the presence of antibodies to the viruses under investigation. the sample size (n = ) was estimated by using an anticipated true herd-level prevalence of %, tolerance of . %, a % confidence level, and herd-level specificity and sensitivity of and % respectively (cannon and roe ) . the amount of pigs sampled in this study was constrained by issues of free access to farms. many farmers were reluctant to allow their pigs to be sampled due to the invasive nature of the blood sampling procedure and the resulting high levels of stress caused to the animals. the more common approach to determining sample size in this type of study uses the formula developed by cannon and roe ( ) where p is the anticipated population proportion which is assumed at % and z is . which is the approximate value of the . percentile point of a normal distribution curve used to construct approximate % confidence intervals. this value is found using a z α/ table where the confidence level used is % and α is . . using the canon and roe formula, the sample size n was calculated to be samples; however, to reduce the number of pigs to be sampled further, the confidence can be relaxed (decreased) and the tolerance, increased (humphry et al. ) allowing for a sample size of . swiv can cause severe respiratory signs in pigs, with morbidity rates sometimes reaching %. the primary economic impact is related to retarded weight gain resulting in an increase in the number of days to reach market weight (organization for animal health ). in the uk, the financial loss resulting from reduced weight gain in finishing pigs alone due to swiv has been estimated at approximately £ per pig (morilla et al. ) . in this study, antibodies against (rajao et al. ) . the % seroprevalence found in pigs of t&t is therefore consistent with similar studies carried out in central and north american countries and suggests that swiv is endemic in the trinidadian domestic swine population, but not tobago. in order to detect the likely strains of swiv circulating in the pigs, a selection of elisa positive samples were further tested using hit. a combination of h n strains was selected to try to understand the relative incidence of h n in pigs within the country. due to significant antigenic diversity, strains were selected from both european pigs, which may be representative of global strains but also more recent human strains, given the frequency of transmission from humans to pigs which may result in the establishment of a stable lineage of virus. the pandemic strain ph n was also included, which is known to be present in pigs worldwide at present. the h n serology results (table ) show different reactivity patterns. a/sw/minnesota/ / is most significant since this is the strain of h n virus that has undergone reassortment in north american pigs through multiple generations over several years (anderson et al. ) . interestingly, it is well known that many of the pig populations of trinidad originated from minnesota farms indicating a potential introduction pathway. high titers of this strain were observed in the samples tested which would be indicative of quite recent exposure/active circulation of such viruses. one of the samples showed quite a high titer against the a/perth/ / h n strain which is a contemporary human virus that does not have a stable lineage in pigs. as expected, reactivity to the european viruses was low as there is very little transfer of swine between europe and trinidad. the pandemic ph n strain showed strong reactivity in all of the samples tested which indicates probable circulation of virus or recent exposure. in the case of the small farms sampled in this study, all of the owners lived either on the farm or within km of their farm and several other houses were present in close proximity to the pig farms. this close proximity of people and pigs could enhance both zoonotic and reverse zoonotic transmission of influenza viruses from humans to pigs, thus maintaining influenza virus circulation in the swine herds (australian pork industry biosecurity programme, apibp ). these factors are akin to those which defined traditional influenza epicenters in southeast asia (ma et al. ). personal hygiene, the granting of sick leave to pig handlers showing flu-like symptoms, as well as biosecurity, should therefore be encouraged on both small-and large-scale farms. such measures should include risk assessment checks for visitors, the use of protective clothing, respiratory protection for people to reduce viral dissemination (zoonotic and reverse zoonotic), handwashing before and after handling animals, restriction on sharing of equipment and tools between farms, and controls relating to the movement of animals and vehicles in and out of the farm. training of workers on pig farms to recognize influenza-like symptoms in humans and pigs should also be carried out (adeola et al. ) . porcine parvovirus (ppv) causes severe economically devastating reproductive symptoms in breeding sows. it is known to cause abortion and is associated with stillbirths, mummifications, embryonic death, and infertility (smedi). ppv infection is one of the most common and important causes of infectious infertility in swine. ppv is ubiquitous and worldwide in its distribution; it is therefore an infection which needs to be carefully managed (porcine parvovirus, the pig site ). this study identified out of the serum samples ( %) as antibody positive for ppv. interestingly, markedly different levels of seroprevalence for antibodies to ppv were observed across the different swine farms in t&t with seroprevalence levels on farms ranging from to % (fig. b) . antibodies were not observed in any of the pigs sampled on two of the largest farms in trinidad, suggesting that the biosecurity and husbandry measures practiced on these two farms may be effective at stopping this introduction of ppv onto the farms. interestingly, despite the relatively high seroprevalence for ppv observed in trinidadian domestic pigs, there are limited reports of reproductive problems in the pigs. this could be due to farmers simply not reporting reproductive issues on their farms when they occur or alternatively could be due to a state on endemic stability on the affected farms, meaning that all the breeding sows are being infected with ppv prior to their first pregnancy. on the farms that contained serologically positive pigs, it was found that all the breeding sows that were sampled on these farms had antibodies to the virus prior to their first pregnancy, which would explain the lack of ppvrelated reproductive clinical signs reported to the veterinary services. it is, however, very important to note that some pig farms in trinidad, and all the pigs sampled on tobago, tested negative for ppv antibodies. these ppv-negative farms should be particularly careful when bringing pigs into their farms, ensuring that they avoid the introduction of ppvpositive animals. these farmers should ensure that they test all pigs for ppv antibodies and antigen prior to introduction, or alternatively, they should vaccinate their breeding sows prior to their first pregnancy. all pig farmers from ppvnegative farms should be advised of the importance of maintaining high levels of biosecurity in order to keep their farms free of ppv. all of the samples taken from pigs on the island of tobago (n = ) tested negative for antibodies to both swiv and ppv. in light of this, tobago pig farmers should be encouraged to maintain a closed system. tobago has approximately small backyard pig farms throughout the island, of which were sampled. these tobago pig farmers usually breed and rear their pigs on their farms and may occasionally buy replacement sows from neighboring farms in tobago to reduce excessive inbreeding. pig farmers from tobago should therefore be advised to avoid importing pigs from trinidad and should maintain their current husbandry practices in order to ensure that their pigs remain seronegative for swiv and ppv. any pigs, including boars, coming into the country should be quarantined and tested for the presence of these viruses. farmers from tobago should be educated on the serological situation of their pigs and be made aware of the risks they face in the event of their naïve pigs becoming exposed to these viruses. the farmers should also be educated on biosecurity measures to be implemented to ensure the prevention of ppv and siv being introduced into their pig population. interestingly, trinidad has at least three pig farms which, on occasions, have imported semen from the usa. this may possibly be the source of ppv infection in the country. pcv- is one of the top three most economically important swine pathogens, behind prrsv and mycoplasma pneumoniae (thacker ) . if pigs are left unvaccinated for pcv- , producers could see up to a us$ loss per pig (thacker ) . high seroprevalences ( . %) of pigs for pcv- antibodies have been reported in canada (liu et al. ) , the usa ( %) (nawagitgul et al. ) , taiwan ( . %) (wang et al. ) , northern ireland (> %) (walker et al. ) , and columbia ( . %) (monroy et al. ). in the present study, the overall seroprevalence for pcv- antibodies in t&t pigs was found to be %. there is, however, little to no evidence for pcv- -associated diseases in t&t pigs. given the high seroprevalence for pcv- observed in the t&t pigs, there is clearly a need to closely monitor pig herds for evidence of pcv- -associated diseases. routine surveillance for clinical signs and the regular examination of specimens from abattoirs should be adopted. farmers should also be encouraged to adopt sound husbandry practices such as age segregation, good sanitation as well as measures to minimize stress and avoid overcrowding. the application of these measures is essential to avoid the development of diseases in pigs that are associated with pcv- infection such as post-weaning multisystemic wasting syndrome (pmws). interestingly, some farms showed very high seroprevalence levels for certain viruses, whereas other farms showed low seroprevalence levels. it is possible that this was due to ongoing infection at the time of sampling. alternatively, this could have been due to the different management practices and stocking densities on the farms. high population densities are known to facilitate the rapid spread of pathogens throughout livestock populations. a study in belgium found that the number of pigs per pen was positively associated with swine influenza h n seropositivity (maes et al. ) . ewald et al. ( ) also found that a high pig density was a risk factor for herds to become infected with influenza h n and h n viruses and furthermore that a large number of pigs per pen creates physiological stress, which in turn can alter the immune system and predispose pigs to infection. the reduced seroprevalence levels for swiv observed on the small compared to the large farms may have been associated with the lower stocking densities, as well as the lower overall numbers of pigs, on the small farms. it is known that respiratory viruses are less efficiently maintained on small as opposed to large pig farms (maes et al. ; poljak et al. ; simon-grife et al. ) . the two small farms (sf and sf ), on which over % of the pigs were seropositive for swiv, were the two largest of the small farms sampled (with close to pigs). the pigs on these farms were kept at a very high stocking density, which may have been the reason for the high seroprevalence for swivobserved on those farms. economic losses resulting from viruses such as siv and pcv are usually related to a decrease in average daily gain (adg) and reduced feed conversion efficiency in affected pigs. these viruses may also result in increased carcass condemnation at slaughter and treatment costs for ill pigs (van alstine ). for siv, seropositive and virus-positive pigs have been found to have significantly decreased growth performance compared to seronegative pigs, even though feed intake was not decreased. reduced feed conversion efficiency led to lower average daily growth, additional feed requirements, and longer time needed to reach the kg body weight (er et al. ). pigs vaccinated for pcv have been shown to deliver a sizable return on investment of up to approximately us$ per pig over unvaccinated pigs (gillespie et al. ). also, in the case of ppv, it has been suggested that the cost of an epidemic could result in losses of up to us$ per sow (cutler and gardner ) . losses of between us$ and $ per pig would be devastating to the pig industry in t&t and could result in the closure of affected farms. this study highlights the importance of carrying out regular serological monitoring for economically important viruses of swine that are circulating in the region. although antibodies to csf, prrs, tge, and prcv were not observed in pigs from t&t, it is important to continue monitoring for these viruses, as outbreaks of prrs and csf have recently been reported in south american countries neighboring trinidad, including venezuela, ecuador, suriname, and columbia (world animal health information system, wahis interface ). it is well known that domestic species, including pigs, are illegally imported into trinidad from the south american mainland, especially venezuela. although the porcine coronaviruses (tge and prcv) have not previously been reported to be present in trinidad, the presence of these viruses has been suspected for many years, possibly due to previous importation of pigs and pig semen from the usa and canada. this study has revealed that these viruses are not present in domestic pigs in t&t. in conclusion, this study shows that swiv and ppv are present and circulating in trinidadian domestic pig populations; however, these viruses were not observed to be present in pigs sampled from tobago. strains of swiv confirmed as likely to be circulating in pigs on the island of trinidad include a north american h n strain and the ph n pandemic strain. pcv- , however, was observed to be circulating in domestic pigs from the islands of both trinidad and tobago. pigs on both islands of trinidad and tobago did not have antibodies to csf, prrs, tge, and prcv. detection of pandemic strain of influenza virus (a/h n /pdm ) in pigs, west africa: implications and considerations for prevention of future influenza pandemics at the source characterization of co-circulating swine influenza a viruses in north america and the identification of a novel h genetic clade with antigenic 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development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type characterization of porcine circovirus type in taiwan world animal health information system (wahis interface) -version ; disease information, disease distribution maps acknowledgements we thank the pig farmers of trinidad and tobago for providing access to their pigs for sampling. we are grateful to the academic and technical staff of the uwi school of veterinary medicine especially mr. roger malcolm and dr. marc driscoll who were particularly instrumental in the sampling collection stages. we owe our gratitude to the director of veterinary public health, dr. saed rahaman, and the livestock and livestock products board of trinidad and tobago. we also thank the uwi trinidad and tobago research and development impact fund (rdi fund) for funding the study. we thank natalie mcginn at the animal and plant health agency-weybridge for technical support. conflict of interest the authors declare that they have no competing interests.ethical approval all procedures performed in studies involving animals were in accordance with the ethical standards of the university of the west indies (uwi) research and development impact fund and the uwi ethics committee. key: cord- -psnec qp authors: mbareche, hamza; veillette, marc; pilote, jonathan; létourneau, valérie; duchaine, caroline title: bioaerosols play a major role in the nasopharyngeal microbiota content in agricultural environment date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: psnec qp background: bioaerosols are a major concern for public health and sampling for exposure assessment purposes is challenging. the nasopharyngeal region could be a potent carrier of long-term bioaerosol exposure agents. this study aimed to evaluate the correlation between nasopharyngeal bacterial flora of swine workers and the swine barns bioaerosol biodiversity. methods: air samples from eight swine barns as well as nasopharyngeal swabs from pig workers (n = ) and from a non-exposed control group (n = ) were sequenced using s rrna gene high-throughput sequencing. wastewater treatment plants were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. results: a multivariate analysis showed air samples and nasopharyngeal flora of pig workers cluster together, compared to the non-exposed control group. the significance was confirmed with the permanova statistical test (p-value of . ). unlike the farm environment, nasopharynx samples from wastewater workers did not cluster with air samples from wastewater treatment plants. the difference in the microbial community of nasopharynx of swine workers and a control group suggest that swine workers are carriers of germs found in bioaerosols. conclusion: nasopharynx sampling and microbiota could be used as a proxy of air sampling for exposure assessment studies or for the determination of exposure markers in highly contaminated agricultural environments. the microbial flora of aerosols, referred to as bioaerosols, consists of a combination of viable and non-viable microorganisms (e.g., bacteria, fungi and viruses) and derived compounds of biological origin (e.g., animal and plant debris, endotoxins, exotoxins, and other microbial metabolites) [ ] [ ] [ ] . bioaerosols are ubiquitous in indoor and outdoor environments and are generated from various natural and/or anthropogenic sources. composed of particles ranging in size from a few nanometers to µm in diameter, bioaerosols remain suspended in the air for long periods of time and may travel many kilometers depending on the size of the particle [ , [ ] [ ] [ ] [ ] [ ] . therefore, the dispersal of bioaerosols may impact the air quality of extensive areas that are far from the source and can create public health issues, due to the presence of highly diverse and dynamic microbial communities. beyond affecting the time that particles remain suspended and the distances they travel, particle size plays a role in human diseases, as it dictates which pathway the particle follows in the respiratory tract after inhalation [ ] . for example, particles with a size of µm to µm tend to get deposited in the upper airways, while larger particles may remain in the nasal cavity [ , ] . bioaerosols can be a transmission vector for infectious diseases and are responsible for a variety of health problems, principally through inhalation [ , [ ] [ ] [ ] [ ] . human exposure to bioaerosols is associated with a wide variety of acute and chronic diseases ranging from allergies, asthma, rhinitis, sinusitis and bronchitis, mostly due to occupational exposure [ , [ ] [ ] [ ] [ ] . however, health risks from bioaerosols also exist just from living in close proximity to an intensive source of airborne biological particles [ , ] . additionally, other health problems linked to bioaerosols include fatigue, headache, mucous membrane irritation syndrome, nasal congestion, sore throat, and irritation of the nose and eyes [ , , ] . the industrial environment is the main source of occupational health issues, due to the presence of raw organic materials, the prevalence of operations releasing harmful bioaerosols (e.g., mechanical operations such as wood planning, straw chopping, animal bedding, hay handling, and compost pile turning) and the eventual large amounts of bioaerosols present in confined spaces. for example, biowaste facilities are characterized by notable concentrations of bioaerosols, due to the intense microbial activity involved in waste degradation and the activities performed by workers [ ] [ ] [ ] [ ] . wastewater treatment plants (wwtps) represent another environment where workers are subject to bioaerosol exposure, due to the steps required for the treatment of discharged municipal and industrial effluents [ , ] . intensive animal farming practices in confined buildings that hold a large number of animals (e.g., pigs, poultry, cattle) are also associated with extreme exposure to airborne microbes. the variety of possible sources (e.g., animals, feces, feed, litter) present at farms leads to the emission of complex mixtures of biological particles [ ] [ ] [ ] [ ] [ ] [ ] . moreover, the dynamic nature of the microbial composition makes health risk evaluation complicated for farm workers and nearby residents [ , ] . environmental hygienists continue to insist that insufficient exposure assessments are a primary reason for the absence of bioaerosol exposure limits and strategies to mitigate risk [ , ] . there are several challenges and limitations to measuring bioaerosol exposure. the type of sampler, time and duration of sampling, meteorological conditions [ ] , and geographical positions all affect bioaerosol sampling efficiency, making it difficult to compare studies and limiting collaboration efforts in bioaerosol exposure studies. an added challenge in terms of measuring microbial diversity is that culture-dependent analytical approaches recuperate the cultural/viable portion of bioaerosols exclusively. in contrast, high-throughput sequencing (hts) methods are associated with a more in-depth characterization of the microbial content of a sample [ , , [ ] [ ] [ ] [ ] . to overcome the aforementioned challenges, the scientific community that studies aerosols has identified the need to explore new alternatives and complementary approaches for assessing bioaerosol exposure, such as identifying markers that can help classify environments based on human health risks [ ] [ ] [ ] . the upper respiratory tract, which includes the nasal cavity and the nasopharynx and is a primary pathway for inhaled air, is an important niche for transient environmental bacteria and for colonization. previous studies have already used nasopharynx or nasal cavity samples to look for specific microorganisms, using culture-based approaches [ , ] , or revealed the presence of bacterial resistance genes, using molecular biology approaches [ ] . recently, an hts approach was used on samples from the anterior and posterior cavity of the nose to study the bacterial diversity of individuals [ ] . however, because the microflora in the nasal cavity is dynamic and fluctuant [ ] , a region like the nasopharynx may represent a more long-term reservoir of inhaled bioaerosols. to the best of our knowledge, no study has ever used a s rrna amplicon-based hts approach to investigate the microbial diversity of nasopharyngeal flora, in order to assess occupational exposure. microbiome studies that use upper respiratory tract tissue and focus on specific pathologies (such as asthma, chronic obstructive pulmonary disease and chronic rhinosinusitis) may underestimate the effect of occupational exposure on the microbiomes of the subjects examined in those studies. we hypothesis that the microbiome of patients with respiratory disease is most likely affected by exposure to their work environment in addition to disease, rather than to disease alone. this study aims to contribute to the development of a new alternative method for assessing bioaerosol exposure, by linking the nasopharyngeal flora of pig farm workers to the bioaerosol microbial composition of their occupational environment, and to explore the role of bioaerosols in the nasopharyngeal microbial content. the s rrna amplicon-based hts approach was used to determine bacterial diversity, while qpcr was used to evaluate the presence of human pathogenic agents and bacterial resistance genes in bioaerosols and in nasopharyngeal samples from pig workers. additionally, wwtps were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. although the experiment focused on pig farmers and the buildings that they work in, the results may have implications for a wider population of agricultural workers. consequently, nasopharynx samples could be used as proxies for air samples in exposure assessment studies and for the determination of exposure markers in agricultural settings. in addition, the study advocates for the need of systematic bioaerosol exposure study when evaluating the nasopharyngeal microbiota. air samples were collected from eight confined pig buildings in eastern canada during fall/winter . inside each farm building, a sampling site was designated based on worker activities and bioaerosol exposure. the buildings visited were mechanically ventilated and contained between - pigs each weighing between - kg. there were no obvious signs of illness affecting the animals. air samples were collected from eight wwtps in the province of quebec, located in eastern canada, the during summer and winter seasons. summer visits occurred between september and july , and winter visits occurred between february and march . four sampling sites were chosen depending on the wastewater treatment process (screening, de-gritting/degreasing, settling tank, and bio-filtration), workers' daily tasks, and the level of confinement of the space. a liquid cyclonic impactor coriolis µ biological air sampler (bertin corp., rockville, md, usa) was used for collecting air samples. the samplers were set at l/min for min ( m of air per sample), placed within - m of the bioaerosol source and away from any turbulent air flow (e.g., away from building exhaust fans). fifteen milliliters of a phosphate buffer saline (pbs) solution ( . m, ph . , lonza, walkersville, md, usa) were used as the collecting solution. nasopharyngeal samples were taken from controls (university students and staff never exposed to animal farms), pig farmers and wwtp workers between the fall of and spring . all controls, pig farmers, and wwtp workers were non-smokers and none were taking antibiotics. the protocol was approved by the ethics committee of the institut universitaire de cardiologie et de pneumologie de québec (cer ). nasopharyngeal samples were collected by a nurse using swabs (puritan ® hydraflock ® collection devices, guilford, ma, usa) with a dry secure transport system. briefly, a swab was inserted into the nose until a resistance was felt and then turned a few times before it was removed. samples were transported to the laboratory at • c. from the ml collecting solution of the coriolis µ biological air sampler (bertin corp.), a . ml aliquot was centrifuged for min at , × g (j. pilote protocol = ml aliquot, min, , × g). the supernatant was discarded and the pellets were kept at − • c until dna extraction. likewise, the tips of the swabs were cut and vortexed thoroughly in ml pbs (lonza) and discarded. suspensions were then centrifuged for min at , × g, the supernatants were discarded and the pellets were stored at − • c until dna extraction. total genomic dna from air and nasopharyngeal samples was extracted with a powerlyser ® powersoil isolation dna kit (mo bio laboratories, carlsbad, ca, usa) following the manufacturer's instructions. dna samples were stored at − • c until subsequent analyses. the amplification of targeted genes, equimolar pooling, and sequencing were performed at the plateforme d'analyses génomiques (ibis, université laval, québec, qc, canada). the s rrna v -v region was amplified using the sequence-specific regions described in comeau et al. using a two-step dual-indexed pcr approach, specifically designed for illumina ® instruments, san diego, ca, usa [ ] . the gene-specific sequence was first fused to the illumina ® truseq sequencing primers and pcr was carried out in a total volume of µl containing × q buffer (neb, ipswish, ma, usa), . µm of each primer, µm of each of the dntps, u of q high-fidelity dna polymerase (neb) and µl of template dna. pcr thermoprotocol began with an initial denaturation at • c for s followed by cycles of denaturation at • c for s, annealing at • c for s, extension at • c for s and a final extension at • c for min. the pcr reaction was purified using the axygen pcr cleanup kit (axygen ® , waltham, ma, usa). the quality of the purified pcr product was checked on a % agarose gel. a fifty to -fold dilution of the purified product was used as a template for a second round of pcr in order to add barcodes (dual-indexed) and for missing sequences required for illumina sequencing. the thermoprotocol for the second pcr was identical to the first one but with cycles. pcr reactions were purified again in the same way as above, checked for quality on a dna bioanalyzer chip (agilent ® , santa clara, ca, usa) and then quantified spectrophotometrically with the nanodrop ® (thermo fisher scientific, waltham, ma, usa). barcoded amplicons were pooled in equimolar concentrations for sequencing on the illumina ® miseq machine. the oligonucleotide sequences that were used for pcr amplification are presented in table . briefly, after de-multiplexing the raw fastq files, the reads generated from the paired end sequencing were combined using the make.contigs script from mothur [ ] . quality filtering was also performed with mothur, using the screen.seqs script to discard homopolymers, reads with ambiguous sequences, and reads with suspiciously short lengths. similar sequences were gathered together in order to reduce the computational burden, and the number of copies of the same sequence was displayed to monitor the abundance of each sequence. this de-replication step was performed with vsearch [ ] . the sequences were then aligned with the bacterial reference silva core alignment using the qiime script align_seqs.py [ ] . operational taxonomic units (otus), with a % similarity cut-off, were clustered using the uparse method implemented in vsearch. uchime was used to identify and remove the chimeric sequences [ ] . qiime was used to assign taxonomy to otus based on the silva database reference training dataset for taxonomic assignment and to generate an otu table. a metadata-mapping file was produced that includes information about air and nasopharyngeal samples. the microbial diversity analyses, including statistical analyses, conducted in this study, were achieved using qiime plugins in version . . as described in qiime scripts (http://qiime.org/scripts/). the names of the scripts used are mentioned in the results section of each analysis. pcr was performed with cfx- and cfx- touch™ real-time pcr detection systems (bio-rad laboratories, mississauga, on, canada) to evaluate the presence of six human pathogens (clostridium difficile, listeria monocytogenes, mycobacterium avium, salmonella spp., staphylococcus aureus, and methicillin-resistant staphylococcus aureus (mrsa)), and antibiotic and metal resistance genes (cephalosporin, colistin, zinc). the pcr mixture contained µl of dna template, - nm for each primer, - nm probe and µl of × iq™ supermix or iq™ sybr ® green supermix (bio-rad laboratories) in a µl reaction mixture. the results were analyzed using bio-rad cfx manager software, version . (bio-rad laboratories). positive control and standard curves ranging from × to one copy of the targeted genes were used for each protocol using genomic dna or synthetic genes as templates (integrated dna technologies, coralville, ia, usa). negative controls were included in the plates as ntc (non-template controls). all primers and probes were purchased from integrated dna technologies. the primers, probes, hybridization temperatures, amplicon sizes and original references for all targeted genes are listed in pilote et al. [ ] . for alpha diversity measures, the normality was verified using the d'agostino and pearson omnibus normality test. the assumption of data normality was not fulfilled. non-parametric mann-whitney u tests (two-tailed) were performed to highlight that there are significant differences in diversity measures between the groups of samples. a p-value ≤ . was considered statistically significant. all of the results were analyzed using the software graphpad prism . (graphpad software, inc., san diego, ca, usa). to determine the statistical significance of the variation in the observed microbial community composition with multivariate analyses (pcoa), a permanova test was performed on the unweighted unifrac matrix. the compare_categories.py qiime script was used to generate the statistical results. because permanova is a non-parametric test, significance is determined through permutations. in this case, permutations were used. a p-value ≤ . was considered to be statistically significant. detailed information about the performance of the test is presented in the multivariate section of the results. the non-parametric mann-whitney u test was used to ascertain whether or not differences in otu abundances were statistically significant between the controls and pig farmers. to test otu differential abundance, the null hypothesis was that the populations that the two groups of samples were collected from have equal means. the range of p-values obtained for the most differentially abundant otus between the control samples and the pig farmer samples are presented in the differential abundance section of the results. this study used both positive and negative controls. the negative controls include unused swabs that underwent the same extraction protocol as the swabs collected from the subjects of this study. a pcr amplification targeting the s rrna genes allowed us to confirm the very low biomass of the negative controls compared to the nasopharyngeal swabs from the pig workers and non-exposed controls. for this reason, negative controls did not pass the next step of illumina hts. additional negative controls consisted of outdoor air samples that were taken outside the pig buildings sampled in this study. these samples showed enough concentration of bacterial biomass with the pcr amplification. thus, outdoor negative controls were sequenced. however, the number of reads and subsequent otu clustering was low compared to the indoor air samples. during the rarefaction step, the negative controls were not included in the analyses due to a low number of sequences. the goal is to have a number of sequences per sample deep enough to cover most of the bacterial diversity. positive controls consisted of a mock community containing equal concentrations of bacteria purchased from atcc ( strain even mix genomic material atcc ® msa- tm ). sequencing of the mock community showed a taxonomic profile resembling the expected microbial community, but with different relative abundances. in total, air samples from pig buildings, nasopharynx samples from farmers and nasopharynx samples from the non-exposed control group resulted in , , sequences (air samples = , ; farmers = , , , controls = , , ). following quality filtering and the discarding of singletons, , , unique sequences clustered into otus. representing the non-agricultural control environment (wwtps), , , sequences came from air samples ( , ) and nasopharynx samples ( , , ) from plant workers. after quality filtering and the removal of singletons, , unique sequences clustered into otus. a rarefaction analysis was performed to validate the sequencing depth and to confirm the effective sampling of the microbial diversity using the alpha_rarefaction.py qiime script. the lowest-depth sample parameter was used for the rarefaction analyses, allowing equal numbers of sequences for all samples. therefore, the samples with a sequencing depth lower than the reference sample were excluded from the analyses. the higher the sequencing depth, the more likely that the full diversity coverage is attained. the sequencing depth was , sequences for all the groups of samples: the air samples from pig buildings, the nasopharyngeal samples of pig farmers and non-exposed controls. the points shown in figure were calculated as follows: ten values from to , analyzed sequences were randomly selected. for each of these values, the corresponding number of otus observed, was noted for all of the samples. then, the average number of otus observed, plus or minus one standard deviation, were calculated for each of the ten values. the samples were divided into three groups: air from pig buildings, pig farmers and non-exposed controls. the slope of the curves shows sufficient sequencing depth and good bacterial coverage in all samples. moreover, pig farmers and air samples showed the highest average number of otus compared to non-exposed controls. four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, nonexposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, non-exposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b) . the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary material). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less figure . taxonomic profile showing the relative abundance of each bacterial class across nasopharyngeal flora samples from pig farmers, non-exposed controls and air samples from pig buildings. taxa written in bold type were specific to farmers and air from pig buildings. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary materials). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in nonexposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in non-exposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. listeria monocytogenes was detected in % of non-exposed controls and in % of pig worker samples. mycobacterium avium was not detected in the nasopharynx samples of either group. likewise, antibiotic and zinc resistance genes were present at a higher frequency among pig farmers compared to non-exposed controls. moreover, cephalosporin, and colistin resistance genes were exclusively detected in the nasopharyngeal flora of pig farmers. table . human pathogens and antibiotic and zinc resistance genes in the nasopharyngeal flora of pig workers compared to non-exposed controls. given the many potential microbial sources, animal farmers inhale a variety of aerosolized bacteria that impact their health [ , , ] . in this study, the bacterial populations in bioaerosols from pig buildings were compared to those of the nasopharyngeal flora of farmers using bioinformatics tools to determine if nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. systemic microbial ecology analyses led to unequivocal results with identical conclusions throughout the analyses. the alpha diversity of bacterial species in the air from pig buildings and the nasopharyngeal flora of farmers were not statistically different. the evaluation of species diversity was introduced by whittaker and defined as the number of species and their proportional abundance within one sampling site [ ] . there are different ways to measure alpha diversity and an extensive list of indexes has been presented by magurran and mcgill [ ] . in addition to the usual chao richness estimates and shannon/simpson diversity measures [ ] [ ] [ ] [ ] , pd whole tree was also used to analyze the alpha diversity in samples in this study. pd stands for phylogenetic diversity and is defined as the minimum length of all phylogenetic branches required to span a given set of taxa on the phylogenetic tree [ ] . all four of the alpha diversity measures revealed greater bacterial richness and diversity in the nasopharyngeal samples from pig farmers compared to non-exposed individuals. the observed similarity between bioaerosols from pig buildings and the nasopharyngeal flora from farmers is indicative of occupational exposure and, consequently, a transient presence and/or possible colonization of the upper respiratory tract regions by environmental bacteria. these findings are even more interesting given that the majority of pig farmers recruited for this study do not work in the eight pig buildings selected for air analysis. this suggests that airborne bacteria associated with pig buildings can take over the microbiota in farmers' nasopharynxes. the establishment of this "new" microbial community could represent a microbial signature for the nasopharynx of pig farmers. also, a higher prevalence of viruses in the nasopharynxes of farmers compared to the non-exposed control group could play a role in the increased alpha-diversity [ ] . beta diversity analyses revealed that long-term exposure, such as occupational exposure to bioaerosols in the air of pig buildings, appeared to modify the nasopharyngeal microbiota of farmers. common approaches to evaluating changes in the community composition (beta diversity) rely on the creation of a (dis)similarity matrix to calculate the distance between samples. dis(similarity) matrices may be calculated using different methods depending on the type of dataset, analyses, and the objectives of the study, as some metrics are more suitable than others [ ] [ ] [ ] [ ] . the unifrac distance metric was used as efficacy was proven with s rrna bacterial genes [ ] . in addition, pcoa coupled with permanova offers a robust statistical significance of sample grouping using distance matrices. this non-parametric multivariate analysis of variance separates the distance matrix into sources of variation to describe the robustness and significance of a variable in explaining the variations observed between samples. it is based on the anova experimental design but analyzes the variance and determines the significance by permutations, as it is a non-parametric test [ ] . whereas, anova/manova assumes normal distributions and a euclidean distance, permanova can be used with any distance measure. the two analyses led to the same conclusions for this study. therefore their usefulness when used together as a tool to visualize and evaluate sample clustering was confirmed. the distinct clusters formed between the combination of pig farmers, and the air from pig buildings, and non-exposed individuals, is clearly linked to a strong divergence in the nasopharyngeal microbiota of farmers compared to other non-exposed individuals. mechanical deposition of < µm diameter inhaled particles on nasopharyngeal surfaces by inertial impaction [ ] , represents a continuous source of environmental bacteria to the nasopharynx. this continuous source of bacterial exposure may therefore be responsible for the establishment of a reservoir of bacteria reflecting long-term exposure (e.g., occupational exposure). a thorough understanding of the established bacterial community may then lead to a better evaluation of the risks associated with an environment. for example, domestic animals share some of their microbiota with their human cohabitants, supposedly through frequent and direct contact [ , ] . song et al., ( ) mention that airborne microbiota plays an important role in microbial transfer to the human upper respiratory tract. ten thousand litres of air are inhaled daily and the bioaerosols in the air may have an effect on the human nasal microbial community [ ] . finally, as different farm animals are associated with different microbiota, the normal nasopharyngeal flora of farmers may be differently disturbed. in other words, farmers working with different animals may have a different disturbances of their natural nasopharyngeal flora. therefore, the microbial fingerprint of the nasopharynx may be directly linked to a specific type of farming environment and the potential long-term health effects on farmers. the high abundance of firmicutes and bacteriotedes in pig buildings has been shown [ ] [ ] [ ] . interestingly, the results of this study are consistent with the literature, but with the added information indicating that, these same phyla colonize the nasopharynxes of farmers. more particularly, other studies have previously shown clostridia to be the most abundant class of bacteria in bioaerosols from pig buildings [ ] . this study not only confirmed the abundance of clostridia in air samples, but also that this class was the dominant class found in nasopharynx samples, while it was practically absent in non-exposed individuals. clostridium spp., identified as differentially abundant in the farmer nasopharyngeal samples in the present study, comprise potentially pathogenic species [ ] . specifically, clostridium butyricum, also differentially abundant in samples from farmers, has been identified as an emerging pathogen by public health authorities. some c. butyricum pathogenic strains were associated with the occurrence of necrotizing enterocolitis, a bowel disease [ ] . in addition, prevotella spp., which was strikingly more abundant in nasopharynx samples, is a well-known agent involved in upper respiratory tract infections [ ] [ ] [ ] . moraxella is another genus identified by the differential abundance analyses as being predominant in the nasopharynxes of farmers. like for other taxa identified in this study, strains of moraxella spp. were previously detected as airborne bacteria in pig buildings [ ] . the rate of colonization of moraxella spp. in healthy adult populations is around % [ ] . species of this genus are opportunistic pathogens responsible for upper and lower respiratory tract infections [ ] . taxa identified exclusively in nasopharyngeal samples and air samples from pig buildings could be candidates for new markers to assess exposure to bioaerosols in pig farming environments. although, actinobacteria are most abundant in the non-exposed controls, no pathogen was identified in the most abundant taxa presented in figure (except haemophilus influenza). an example of the most abundant actinobacteria in non-exposed control is micrococcus luteus that was differentially more abundant in controls compared to farmers. another example of firmicutes is staphylococcus epidermis that was more abundant in the controls compared to the farmers. the respiratory health of farmers has been of great interest for testing the hygiene hypothesis that stipulates that exposure to microbes from intensive farming during early life could be beneficial to health in adulthood [ , ] . however, the acceptance of the hygiene hypothesis is not unanimous in the scientific community [ ] . in this study, taxa identified as differentially abundant among farmers could hypothetically play a role in the prevention of allergy and the development of atopic diseases. indeed, some bacteria identified through this investigation (e.g., pedobacter, pelomonas, and megasphaera) have been linked to healthy respiratory conditions [ ] . a recent study, conducted by kraemer and colleagues, also found a distinct clustering between samples from the nasal cavities of pig farmers and air samples from their workplace, when compared to the nasal cavities of non-exposed individuals [ ] . moreover, samples from the nasal cavities of cow farmers clustered separately from pig worker samples and air samples from pig buildings [ ] . although the nasal cavity is a more transient environment for environmental bacteria than the nasopharynx, it confirms the hypothesis of a microbial fingerprint specific to the farming environment. it supports the idea of creating a worldwide database, that lists potential markers specific to certain environments, and to the nasopharynxes of the people working in them. this database could represent an important asset for associating bioaerosol exposure with health problems [ ] . the lack of a correlation between the nasopharynx of wastewater workers and bioaerosols from wwtps could be explained by the nature and duration of exposure. workers wear personal protective devices (e.g., masks) at certain working sites (e.g., biofiltration), which may affect the establishment of a 'new' environmental microflora. supporting this idea, the most abundant bacteria, shared by non-exposed controls and wastewater workers, are naturally occurring skin bacteria like propionibacterium, corynebacterium, staphylococcus, streptococcus, and cutibacterium. these taxa were not present (relative abundance less than %) in air samples from wwtps (data not shown). farmers do not usually wear respiratory protection and, moreover, they often live in the farming environment (e.g., in a house located near farms) and are consequently continuously exposed to the microbes generated from farming activities (occupational and residential exposures). therefore, the exposure that farmers are subjected to is more likely to modify their natural nasopharyngeal flora. the agricultural/non-agricultural hypothesis presented in this work regarding the nasopharyngeal flora of exposed workers should be validated in other agricultural and industrial environments. recent studies of the microbiome of the upper respiratory tract may have underestimated the influence of occupational exposure when considering the effect of a particular disease on the natural flora of upper respiratory tract tissue [ ] [ ] [ ] . the fact that the work environment may affect the natural flora of an exposed person on a long-term scale is a crucial consideration when he/she becomes a patient whose upper respiratory tract microbiota is the target of the disease. the results obtained in this work emphasize the importance of considering the environment of the nasopharyngeal flora of exposed workers, who are or could become patients suffering from chronic respiratory diseases. in the same way that recent advances in methods for identifying microbes has helped implicate the upper respiratory tract microbiome in inflammatory respiratory diseases, evaluating bioaerosol exposure can help us support the roles of resident microbes in both healthy and diseased tissues. specific human pathogens and antibiotic and zinc resistance genes were detected in the nasopharynxes of pig farm workers as well as in bioaerosols of pig buildings [ ] . for bacterial diversity analyses, the qpcr approach supports the use of the nasopharynx as an alternative to air sampling. the presence of zinc and antibiotic resistance genes, in the nasopharynxes of farmers, is explained by the use of zinc and antibiotics in animal farming (for therapeutic or sub-therapeutic use, such as for growth promotion) and implies possible human health risks. extensive use of zinc in pig feed is responsible for the proliferation of zinc-resistant bacterial communities at farms [ ] . cephalosporin is a commonly used antimicrobial drug in human infections and the spread of its resistance constitutes part of the antibiotic resistance crisis [ ] . finally, the mcr- gene was found in the nasopharynxes of half of the pig farmers in this study, although the use of colistin is extremely regulated in north america and limited to multi-drug resistant microbes [ ] . future studies should include detailed health information on the sampled individuals to investigate the nasopharynx microbiota associated with certain occupational health problems. additionally, a longitudinal study of the bacterial diversity, in the nasopharynxes of farmers and in bioaerosols from pig buildings, could unveil a long-term variation in microbial content. finally, information about the diet of exposed human (or animal) and antibiotic use could be added to the analyses as important factors influencing the microbiota. this is the first study to link the nasopharyngeal flora of exposed humans with the source of the exposure in an agricultural setting, using bacterial diversity analyses and the detection of specific pathogens and resistance genes. the results suggest that workers are carriers of bioaerosol-associated bacteria and that nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. furthermore, pig farmers are also carriers of specific human pathogens and resistance genes 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open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -nsq z authors: denner, joachim; mueller, nicolas j. title: preventing transfer of infectious agents date: - - journal: int j surg doi: . /j.ijsu. . . sha: doc_id: cord_uid: nsq z xenotransplantation using pig cells, tissues and organs may be associated with the transfer of porcine infectious agents, which may infect the human recipient and in the worst case induce a disease (zoonosis). to prevent this, a broad screening program of the donor animals for putative zoonotic microorganisms, including bacteria, viruses, fungi and others, using sensitive and specific detection methods has to be performed. as long as it is still unknown, which microorganism represents a real risk for the recipient, experience from allotransplantation should be brought in. due to the fact that pigs can be screened long before the date of transplantation, xenotransplantation will become eventually safer compared with allotransplantation. screening and selection of animals free of potential zoonotic microorganisms, caesarean section, vaccination and/or treatment with chemotherapeutics are the strategies of choice to obtain donor animals not transmitting microorganisms. in the case of porcine endogenous retroviruses (pervs) which are integrated in the genome of all pigs and which cannot be eliminated this way, selection of animals with low virus expression and generation of genetically modified pigs suppressing perv expressions may be performed. xenotransplantation using pig cells, tissues and organs has to overcome three hurdles before being applied in the clinic for the treatment of organ failure: immunological rejection, physiological incompatibility and transfer of infectious agents. the microbiological safety of xenotransplantation is an important issue, however it can be managed easily. the risk of infection is also known in allotransplantation. numerous infectious agents have been transmitted together with human donor transplants, including human cytomegalovirus (hcmv), human immunodeficiency virus- (hiv- ) and rabies virus [ ] . since xenotransplantation allows screening the donor animals beforehand, most risks can be excluded by careful testing and xenotransplantation finally will be a microbiologically safer technology compared with allotransplantation. like all animals, pigs carry numerous microorganisms in their digestive tract and on their skin, and therefore cells, tissues and organs to be used for transplantation should be removed under aseptic conditions. the number of microorganisms present in the tissues and organs of interest should be zero [ ] . in some reviews concerning the microbiological safety of xenotransplantation numerous microorganisms are listed which were thought to induce zoonoses when transmitted to the human recipient [ ] . zoonosis means that the microorganisms not only infect the new host, but cause a disease. in general, bacteria, fungi, parasites and viruses may be transmitted. however, at present it is rather difficult to classify most of the porcine microorganisms into pathogenic and non-pathogenic for human recipients. in addition, when a microorganism is pathogenic in the pig it does not mean that it is also pathogenic in humans and vice versa. the risk of transmission is certainly higher when pharmaceutical immunosuppression to prevent immunological rejection of the transplant will be applied. therefore it is still unclear which microorganisms should be monitored. the auckland island pigs which had been used in the first clinical trials performed by the new zealand company lct were screened regularly for bacteria, viruses and toxoplasma (table ) [ ] . the g€ ottingen minipigs which are used for numerous biomedical investigations are screened regularly for bacteria, viruses, three fungi and four parasites (http//www.minipigs.dk/). an additional screening of the g€ ottingen minipigs involved perv [ ] , hepatitis e and other microorganisms [ , ] . in general, most microorganisms found in pigs to be used for xenotransplantation may be eliminated by specified pathogen free (spf) or designated pathogen-free (dpf) breeding of the animals. in the case there is a bacterial or fungal infection in the donor pig, treatment with antibiotics or chemotherapeutics may be successful. at the moment hepatitis e virus (hev), porcine cytomegalovirus (pcmv), porcine circoviruses (pcv), porcine lymphotropic herpes viruses (plhv), and porcine endogenous retroviruses (pervs) are thought to pose the main risk for reasons to be discussed below and therefore these microorganisms will be analysed in the next chapters in more details. in most cases hev causes self-limiting hepatitis in humans. whereas hev of the genotype (gt) and gt are found in people, are transmitted mainly by contaminated water and are causing a high mortality during pregnancy, hev gt and gt are swine viruses and do not cause a disease in pigs, however, when they infect humans they may cause in rare cases a zoonotic disease (for review see [ , ] ). a severe hepatitis after infection with hev gt and gt was observed only in the case of other underlying liver diseases. importantly, neurological disorders have also been described for hev gt and gt . note, that only hev gt / may pose a risk when xenotransplantation using pig cells, tissues and organs is performed, not gt or gt . usually hev gt / are transmitted by contaminated meat or direct contact with infected pigs. hev gt rna was detected in pig liver at grocery stores and infectious virus could be isolated [ , ] . hev transmission by shellfish and vegetables possibly contaminated by pig manure as well as by blood transfusion and allotransplantion was also reported. a chronic infection was more likely to develop in immunosuppressed patients, including hiv- infected individuals [ , ] . sensitive pcr-based methods have been developed to determine a hev infection and to genotype the virus. detection of hev and its elimination from pigs seems not to be easy. first, the virus is heterogeneous, e.g., subtypes of genotype exist, what makes it difficult to design efficient pcr or real-time pcr. second, the virus load seems to be very low so that even highly sensitive pcrs may be unable to detect the virus. although hev gt / are widely distributed, the prevalence in pigs, especially in multitransgenic pigs generated for usage in xenotransplantation, is not well studied. in contrast, the non-transgenic auckland island pigs, generated by living cell technologies (lct) in new zealand are better table microorganisms tested in auckland island pigs used for islet cell transplantation [ ] . [ ] . although hev is very common in pigs in new zealand, the auckland island pigs were free of all hev [ ] . another well investigated breed is the g€ ottingen minipig produced by ellegaard in denmark. these animals are used worldwide for numerous biomedical investigations. the herd was established by entry of animals obtained by caesarean sectioning and colostrum deprivation. despite this, hev was found in one study in % of the animals ( of ) [ ] , in another, using real-time pcr and western blot analysis detecting antibodies against hev, hev was found in only very few animals [ ] . the result suggested a transplacental mother-to-piglet transmission of the virus. this observation may help to explain how the virus entered the pig herd despite caesarean sectioning and other precautions. it remains unclear, whether the absence of the virus in all older g€ ottingen minipigs is due to the elimination of the virus possibly by the immune system or due to the limits of the detection methods. in order to eliminate hev from a herd, a hev elimination program was proposed ( fig. ) [ ] . elimination should include selection of hev negative animals using highly sensitive real-time pcr. since it is not clear, whether the animals are truly negative, or carry hev in concentrations below the sensitivity of the detection method, a treatment step should be included using ribavirin, a guanosine analogue used to stop viral rna synthesis. although there are no data on the treatment of hev infection with ribavirin in pigs, ribavirin has been successfully used for the treatment of other virus infections in pigs [ ] . another strategy may include a vaccination step, e.g. using a vaccine based on a recombinant orf- fragment of hev gt that has been approved by the chinese fda [ ] . since hev gt - represents only one serotype, the induced antibodies should be protecting from infection with all genotypes [ ] . immunisation of pigs with orf- of pig hev gt resulted in effective protection [ ] , indicating that pigs can be immunized and mount an effective antiviral immune response. as human cytomegalovirus (hcmv) causes severe transplant rejection in allotransplantation [ , ] , considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (pcmv) in the setting of xenotransplantation. pcmv is endemic in the world pig population, it is acquired early in life and pcmv infection results in seroconversion and lifelong latent infection [ ] . pcmv spreads by both vertical and horizontal transmission [ , ] . active infection causes fatal systemic failure in piglets less than weeks of age. the clinical symptoms of infected piglets include pneumonia and inclusion body rhinitis with a high mortality rate. pcmv-infected sows are prone to abortion, with pathological changes including edema in the heart and other organs [ ] . pcmv can remain latent in adult pigs. the ubiquitous nature of herpesviruses, including pcmv, means that these viruses should be a major focus in the development of xenotransplantation. herpesviruses are able to infect other species. porcine cells can be infected by hcmv [ ] , indicating that the pig transplants may be infected when the recipient is hcmv positive, and pcmv can infect human cells [ ] . entry of hcmv into porcine endothelial cells depends on both the cellular vascular origin and the viral strain [ ] . when pcmv was transmitted by the pig transplant into baboons, the baboon cmv was activated causing invasive disease and consumptive coagulopathy, the pcmv was mainly replicating in the pig transplant causing ureteric necrosis in one transplant [ e ]. when baboons received pig kidneys from pcmv-infected pigs, the survival time was . days in comparison to . or days when organs from uninfected animals were transplanted [ ] . in a similar experiment with cynomolgus monkeys the difference was . days versus . days [ ] . alone the presence of the virus had such an important influence on transplant survival. when cd -transgenic large white pigs were analyzed, all animals were found positive for pcmv, however under specified pathogen free (spf) or designated pathogen-free (dpf) conditions pcmv-free animals were obtained [ ] , indicating that selection of pcmv-free animals by caesarean delivery and spf breeding is possible. on the other hand, due to reactivation of the baboon cmv after pig cardiac xenotransplantation with pharmaceutical immunosuppression a lethal outcome in some cases was observed despite prophylactic treatment with the antiviral drugs ganciclovir or valganciclovir [ ] . the auckland island pigs, already used in clinical islet cell transplantation were shown to be free of pcmv [ ] . as mentioned above, pcmv can be eliminated easily by caesarean delivery and dpf or spf breeding of the herd [ , ] . in addition, early weaning of the piglet from the sow can eradicate cmv [ ] . to be on the safer site, a treatment with the antiviral drugs ganciclovir, cidofovir, foscarnet, acyclovir, valaciclovir, a prodrug of acyclovir, or valganciclovir can be included into the elimination protocol ( fig. ) [ ] . concerning vaccination against hcmv, despite the urgent need for allotransplant recipients, no success was reported, although first attempts to use the major envelope glycoprotein gb have demonstrated efficacy against hcmv infection and on hcmv-induced disease [ e ]. immunization studies with the gb protein of pcmv should be performed in pigs. porcine lymphotropic herpesvirus (plhv) , , and are common porcine viruses, however the prevalence and importance of these viruses for xenotransplantation is not well studied [ ] . phylogenetic analyses showed that all three phlv clustered together with ruminant gammaherpesviruses, but the plhv- is more distantly related to plhv- and plhv- [ ] . the transmission of phlv in pigs is not well understood. plhv may be fig . . schematic presentation of the proposed virus elimination program. the original herd was screened for the presence of a putative zoonotic virus (grey, positive animals; pink, negative animals). negative animals (that may be actually positive, but below the detection limit) were selected and using caesarean delivery, and treatment with antiviral drugs and/or vaccines, truly negative animals could be obtained and used for further breeding and xenotransplantation. transmitted by pre-partum cross-placental vertical transfer and post-partum horizontal transmission, however, cross-placental transfer is not the common way [ ] . between % up to % of animals in different herds in germany, ireland, france, spain and the united states were infected with one of the plhv [ e ]. in contrast to pcmv, early weaning cannot eradicate phlv [ ] . circoviruses belong to the smallest viruses replicating autonomously in mammalian cells [ ] . porcine circovirus (pcv ) has not been linked with any disease, whereas pcv is the causing agent of post-weaning multisystemic wasting syndrome (pmws), a multifactorial disease in pigs [ ] . this means that the presence of the virus is necessary for the disease but requires additional factors. the onset of the disease and the severity of the symptoms are influenced by the status of the immune system and genetic predisposition [ ] . characteristic clinical signs of pmws are wasting, respiratory dysfunction, enlargement of inguinal lymph nodes, diarrhoea and a generalised depletion of lymphocytes. although humans ingest pcv-contaminated foods and are exposed to pcv through other sources, serological evidence indicated that no transmission of pcv to humans took place [ ] . also, a contamination of a human vaccine with pcv was shown not to transmit pcv to humans [ , ] . retrospective testing confirmed the presence of pcv dna in rotarix, an oral live-attenuated human rotavirus vaccine for children, beginning with the initial stages of its development and in vaccine lots used in clinical studies conducted preand post-licensure [ ] . when human cell lines have been infected with pcv and pcv , pcv persisted in most cell lines without causing any visible changes, while pcv -transfected cells showed a cytopathogenic effect [ ] . most importantly, the infection was non-productive [ , ] . porcine endogenous retroviruses are the result of a transspecies transmission of a retrovirus and integration into the genome of all pigs. perv-a and perv-b are polytropic viruses, infecting also human cells (for review see [ ] ). perv-c infects only pig cells, it is present in most, but not all pigs. whereas transformed human cells lacking some intracellular restrictions factors such as abopec can be infected easily, human primary cells can be infected only by high-titre perv-a and recombinant perv-a/c after adaptation on human cells leading to an increased number of transcription factor binding sites in their long terminal repeats [ e ]. since perv-c are present in many pigs, but not all, the selection of pigs not carrying perv-c proviruses automatically prevents generation of high-titre recombinant perv-a/ c. until now, no transmission of pervs was observed, neither in first clinical trials enrolling more than patients, nor in preclinical pig to non-human primate transplantation, nor in infection experiments in small animals or non-human primates with or without pharmaceutical immunosuppression (for review see [ ] ). however, most of the patients in the clinical trials were not exposed for a long time to the xenotransplant and with some exceptions associated with parallel kidney allotransplantations, no pharmaceutical immunosuppression was applied. in addition, no transmission of perv was observed in numerous pig to nonhuman primate transplantations [ ] . however, it is meanwhile clear that non-human primates are not a suitable model to study this question since non-human primates carry e in contrast to humans e a mutated receptor for perv allowing only infection at a low efficiency [ ] . therefore, the question, whether pervs may be transmitted during xenotransplantation is still open and an elimination of infectious proviruses is advised, since retroviruses may induce tumours and immunodeficiencies. pervs are closely related to other gammaretroviruses such as the feline leukaemia virus (felv), the murine leukaemia virus (mulv) and the koala retrovirus (korv) [ ] . all these related viruses induce severe immunodeficiencies and tumours in the infected host. gammaretroviruses used as vectors in gene therapy for the treatment of a severe combined immunodeficiency in children were shown to induce leukaemia in the patients [ ] , indicating that transspecies transmission of gammaretroviruses into human may induce tumour development. highly sensitive and specific methods have been developed to screen for the prevalence and expression of perv in donor pigs. whereas expression of perv in german landrace pigs is relatively low, expression in yucatan minipigs is high [ ] . in these animals viral protein may be detected in different tissues of the animals [ ] and infectious virus was found released from stimulated peripheral blood mononuclear cells (pbmcs) [ ] . crossing of landrace pigs with minipigs increased the expression rate of perv [ ] . when g€ ottingen minipigs were screened, a relatively low expression and no release of virus particles were observed [ ] . the elimination programs for hev and pcmv were described in detail above [ , ] . the elimination programs for other microorganisms (with exception of perv, see below) will be similarly, including screening of the donor pigs using specific and sensitive methods, treatment with antiviral drugs and immunisation (fig. ) . for some porcine microorganisms effective vaccines are available, for example for circoviruses (ingelvac circoflex ® ) and mycoplasma (ingelvac circoflex ® ). several other vaccines are available to treat pigs and prevent transmission of the infectious agents (table ) [ ]. however, vaccination is only necessary, when the infectious agent is present in the pig herd and pose a risk for the transplant recipient. in the case of bacteria it has to be considered whether the donor pig (and in some cases also the recipient) should be treated with antibiotics. since pervs are present in the genome of all pigs and cannot be eliminated by the way other microorganisms can be eliminated easily, other strategies were developed to prevent transmission of these infectious agents. first, animals with a low copy number of perv and a low expression rate should be selected. second, animals not carrying perv-c should be selected in order to prevent perv-a/ c recombination. third, transgenic pigs have been generated expressing small interfering rna, specifically inhibiting the expression of perv and therefore lowering the probability of perv transmission [ e ].since in the genome of pigs multiple perv proviruses were found, sometimes more than , an elimination of all perv copies by genome editing using zinc finger nucleases (zfns) may be difficult. we selected zfn specific for a sequence of the highly conserved pol region of the virus. however first attempts to eliminate pervs in the pig genome failed, obviously due to the strong toxic effect when zfns were cutting the genome at multiple sites and therefore destabilising the genome [ ] . a lower concentration of the zfn and a gradual elimination may be alternative strategies. in addition, new attempts using crispr/cas (clustered regularly interspaced short palindromic repeats/crisprassociated) for gene editing should be undertaken with the goal to eliminate at least the replication competent proviruses. fourth, and last but not least, transmission of pervs may be prevented by a vaccine. when immunising different animal species (goats, rats, mice, hamster, guinea pigs) with the recombinant transmembrane envelope protein p e and the surface envelope protein gp of perv, always effective neutralising antibodies were induced [ e ]. the results of the 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targeting the n-terminal helical region of the transmembrane envelope protein p e of the porcine endogenous retrovirus (perv) neutralising antibodies against the transmembrane protein of feline leukaemia virus (felv) increased neutralizing antibody response after simultaneous immunization with leucogen and the feline leukemia virus transmembrane protein immunization with the transmembrane protein of a retrovirus, feline leukemia virus: absence of antigenemia following challenge supplementary data related to this article can be found at http:// dx.doi.org/ . /j.ijsu. . . . key: cord- - oyzg tq authors: duffy, mark a; chen, qi; zhang, jianqiang; halbur, patrick g; opriessnig, tanja title: impact of dietary spray-dried bovine plasma addition on pigs infected with porcine epidemic diarrhea virus date: - - journal: transl anim sci doi: . /tas/txy sha: doc_id: cord_uid: oyzg tq experimental data suggest that the addition of spray-dried plasma (sdp) to pig feed may enhance antibody responses against certain pathogens and negatively impact virus survival. the benefit of sdp on escherichia coli infection is well documented. the aim of this study was to determine the effect of bovine sdp (bovsdp) in the pig diet on acute porcine epidemic diarrhea virus (pedv) infection. a total of -wk-old conventional crossbred pigs were used and divided into three groups. treatments included ) a negative control group fed a commercial diet and sham inoculated with commercial liquid porcine plasma (n = ), ) a positive control group fed a commercial diet and inoculated with pedv-spiked porcine plasma (pedv; n = ), and ) a third group of pigs fed the commercial diet with inclusion of % spray-dried bovine plasma and inoculated with pedv-spiked porcine plasma (bovsdp; n = ). although clinical signs associated with pedv infection were mild in the bovsdp group, two of eight pigs in the pedv group developed moderate clinical disease and had to be euthanized. the pedv igg and iga antibody levels and prevalence rates were significantly (p < . ) higher in the pedv–bovsdp group compared with the pedv group at d postinoculation. the average fecal pedv rna shedding time was . ± . d for the pedv–bovsdp group and . ± . d for the pedv group with an overall time to clearance of pedv shedding of d for pedv–bovsdp pigs and at least d for pedv pigs, which was not different (p = . ). the results indicate that addition of bovsdp induced an earlier anti-pedv antibody response in pigs experimentally infected with pedv thereby reducing clinical disease and the amount and duration of viral shedding during acute pedv infection. coronaviridae. pedv isolates can be divided into two genogroups: g , which encompasses a majority of strains isolated before , and g , which contains more recently identified strains (huang et al., ) . pedv was first discovered in north america in april and since its emergence has caused substantial losses to the u.s. pork industry. the clinical disease associated with pedv is characterized by watery diarrhea and vomiting in all ages of pigs and high mortality rates in young pigs (pensaert and debouck, ) . an intact intestinal mucosa, which prevents the entrance of agents across the epithelium, is a first line of protection with great importance in weaned pigs that, due to separation from their dam and comingling with other pigs, are under high stress and exposed to numerous pathogens they have not encountered before (pitman and blumberg, ; boudry et al., ; pie et al., ; bailey et al., ; moretó and pérez-bosque, ). producers commonly add spraydried plasma (sdp) of porcine or bovine origin to weaned pig diets as it has been shown to promote growth (grinstead et al., ) and aids in combating common postweaning pathogens such as escherichia coli (torrallardona et al., ; bosi et al., ) . it is well recognized that the addition of sdp to pig feed enhances the immune response and decreases pathogen loads compared with pigs without plasma access through feed. for instance, plasma protein supplements modulated the mucosal immune response in organized and diffuse gut-associated lymphoid tissue, which is accompanied by a reduction of proinflammatory cytokine production (bosi et al., ; nofrarias et al., ; pérez-bosque et al., ) . a canadian case-control study investigating factors associated with mortality due to porcine circovirus type (pcv ) found that nursery rations were more likely to contain sdp in control herds compared with clinically affected case herds (dewey et al., ) ; however, alternatively the observed differences may be completely unrelated to the sdp inclusion in the diets. in addition, the biological neutralization activity of antibodies against common pig pathogens present in sdp was conserved and may contribute to the biosafety of commercially available sdp (polo et al., ) . previously, it has also been shown that sdp has an intrinsic effect reducing survival of pedv under in vitro conditions (quist-rybachuk et al., ) . after the introduction of pedv into the united states, concerns on possible contribution of porcine-based sdp in rapid farm-to-farm transmission were raised (pasick et al., ) despite studies clearly demonstrating that pedv is inactivated during the spray-drying process (opriessnig et al., ; pujols and segales, ; gerber et al., b) . to eliminate any possible risk of pig pathogen spread via sdp while retaining its benefits as part of a pig diet, pork producers may switch from porcine-origin sdp toward bovine-origin sdp (bovsdp). the purpose of this study was to determine whether there is any benefit of adding bovsdp to a diet of pigs during acute pedv infection. the experiment was approved by the iowa state university institutional animal care and use committee (iacuc approval number - - -s). sixteen, -wk-old, crossbred, colostrum-fed pigs were selected from a commercial pedv-free herd. the farm of origin was tested on a monthly basis for pedv rna on representative fecal samples and for pedv antibodies on selected serum samples and had no history of clinical diarrhea. the farm of origin was also free of porcine reproductive and respiratory syndrome virus (prrsv), influenza a virus (iav), and mycoplasma hyopneumoniae. before shipment to the research facility, the pigs received a one dose commercial pcv vaccine (merck animal health, inc.). the pigs were transported to the research facility at iowa state university, randomly assigned to one of three groups of three to eight pigs, and housed in separate rooms on solid concrete floors (table ) . upon arrival of the pigs in the research facility, they were tested negative for pedv antibodies in serum and pedv rna in fecal samples. the experimental design is summarized in figure . briefly, the pigs were inoculated with pedv at wk of age (day postinoculation or dpi ), and all pigs were necropsied at dpi . starting with arrival in the research facility and for the duration of the study, all pigs were fed the same standard commercial corn-soybean meal-dried whey-based diet ( table ) except for the diet of the pedv-bovsdp group, which was supplemented with % spray-dried commercial bovine plasma replacing soy protein concentrate on an equal total lysine basis ( figure ). the commercial spray-dried bovine plasma (lot #s ) was produced in a manufacturing plant located in kansas, united states, and submitted to commercial spray-drying conditions including a minimum outlet temperature of °c throughout substance (throughout the entire particle's mass). the control diet did not contain any bovsdp. for the pedv inoculation, the passage pedv g b isolate - e (chen et al., ) at viral concentration of . % tissue culture infectious dose per milliliter was used. in brief, pedv was propagated on vero cells (atcc ccl- ) with minimal essential medium supplemented with tryptose phosphate broth ( . %), yeast extract ( . %), trypsin ( µg/ml), gentamicin ( . mg/ml), penicillin ( unit/ml), streptomycin ( µg/ ml), and amphotericin ( . µg/ml) as previously described (chen et al., ) . commercial liquid porcine plasma negative for pedv rna was used as diluent to adjust the pedv inoculum (pedv and pedv-bovsdp groups) or saline (negative control [neg] group) to a total volume of ml for each pig. inoculation was performed orally by the same team for all pigs. the pigs were held in vertical recumbence between the legs of the holder while the second person administered the material by slowly dripping the inoculum into the mouth of the pig using a syringe on dpi when the pigs were wk old. after pedv inoculation, all pigs were monitored daily for signs of illness such as lethargy, vomiting, or diarrhea. rectal temperatures were taken every other day on each pig, and the fecal consistency score ranging from = normal, = semisolid, = pasty, and = liquid was determined (gerber et al., ) . all pigs were weighed at dpi − and again at dpi , and the adg was calculated. to verify the presence of pedv iga and igg antibodies, blood samples were collected in serum separator tubes on dpi , , and and centrifuged at , rpm for min at °c. all serum samples were tested for the presence of anti-pedv igg or iga by an "in-house" indirect elisa based on the spike gene of a prototype u.s. pedv strain similar to the one used as challenge virus in this study (gerber et al., a; gerber and opriessnig, ) . a sample with a sample-to-positive (s/p) ratio equal to or greater than . was considered positive for pedv igg, and a sample with an s/p ratio equal to or greater than . was considered positive for pedv iga. to determine fecal pedv shedding, rectal swabs were collected from all pigs on dpi and from dpi to using polyester swabs and stored in -ml plastic tubes containing -ml sterile saline. total nucleic acids were extracted from fecal swab suspensions using the magmax pathogen rna/dna kit and an automated nucleic acid extraction system (thermo scientific kingfisher flex, thermo fisher scientific, pittsburgh, pa) according to the instructions of the manufacturer figure . experimental design. blood was collected at arrival and dpi , , and , and rectal swabs were collected d before challenge, on dpi and daily thereafter. rectal temperatures were taken every other day starting with d before pedv inoculation. translate basic science to industry innovation (opriessnig et al., ) . presence of pedv rna was determined by using a quantitative real time rt pcr that was set up using path-id multiplex one-step rt-pcr kit (thermo fisher scientific), and amplifications were performed on the applied biosystems fast real-time pcr system thermocycler and the accompanying software (opriessnig et al., ) . on dpi , all pigs were killed by intravenous pentobarbital overdose (fatal-plus, vortech pharmaceuticals ltd, dearborn, mi) and necropsied. tissues collected from pigs at necropsy included eight sections of small intestines, three sections of large intestine, and one section of mesenteric lymph node. tissues were immediately put in % buffered formalin and routinely processed for histopathology and assessed for lesions by a veterinary pathologist blinded to the treatment status. presence and degree of atrophic enteritis were scored ranging from = normal to = severe. to determine presence and amount of pedv antigen in tissue sections, a pedv immunohistochemical stain was performed on intestines from all pigs as described (stevenson et al., ) . the amount of pedv antigen was scored by a veterinary pathologist blinded to the treatment status with = no signal, = % to % staining, = % to % staining, and = greater than % staining. the statistical software used was jmp pro . analysis of variance (anova) was used for cross-sectional assessment of the average daily weight gain and continuous measures including viral shedding, iga, and igg antibody levels. the pedv genomic copy numbers per milliliter of fecal suspension were log transformed before statistical analysis. if a significant (p < . ) difference was detected, pairwise testing using the tukey-kramer adjustment was performed to determine which groups were different. daily rectal temperature data were analyzed with multivariate anova. non-repeated measures of necropsy and histopathology data were assessed using nonparametric kruskal-wallis anova. if a nonparametric anova test was significant (p < . ), then wilcoxon tests were used to assess the differences between pairs of groups. differences in incidence of clinical scores were evaluated by using the fisher's exact test. for fecal shedding of pedv rna, an area under the curve (auc) was calculated for each animal individually and differences between groups were assessed by unpaired t-test (data passed shapiro-wilks normality test). there was no difference in rectal temperatures among the groups over time, and none of the pigs developed elevated rectal temperatures during the experiment. diarrhea was not observed in the neg group while individual pigs in the pedv group and pedv-bovsdp group had fluid-to-pasty-to-semisolid feces between dpi and with no significant differences among groups. in addition, vomiting was occasionally observed in single pigs during this time. by dpi , two pedv pigs became lethargic and had reduced appetite. one of these pigs developed mild pasty diarrhea. due to welfare concerns, these pigs were euthanized between dpi and . the adg is summarized in table . overall, there were numerical differences among groups for the adg: neg pigs had the highest adg, and pedv pigs had the lowest adg; however, the differences were not significant. all pigs were negative for pedv iga and igg antibodies at arrival, and the neg pigs remained seronegative for the duration of the study (figures and ) . three of five pedv-bovsdp pigs had detectable anti-pedv iga antibodies in serum by dpi , and all pigs in this group and five per eight pedv pigs were positive by dpi . group mean iga levels were higher (p < . ) for pedv-bovsdp pigs compared with pedv pigs at dpi ( figure ). group mean igg levels were also higher (p < . ) in pedv-bovsdp pigs at dpi compared with the pedv pigs ( figure ). all pedv-infected pigs in both groups had anti-igg antibodies by dpi (figure ). all pigs were pedv rna negative at dpi − , and pedv rna was not detected in any of the neg pigs throughout the study. fecal shedding of pedv rna was first detected in a pedv pig at dpi and shedding in this group lasted until termination of the study at dpi (figures and ) . pigs in the pedv-bovsdp group shed pedv from dpi to (figures and ) . the prevalence of pcr positive pedv-bovsdp pigs compared with the pedv group was higher (p = . ) at dpi , while it was lower (p = . ) at dpi . the average shedding period was . ± . for pedv pigs and . ± . for pedv-bovsdp pigs, which was not different (p = . ). similarly, the cumulative fecal viral rna shedding was not different (p = . ) between pedv pigs and pedv-bovsdp pigs. in the two pigs that were euthanized due to welfare reasons, in addition to the standard set of enteric tissues, sections of liver, lung, spleen, tonsil, heart, and kidney were also collected and assessed to rule out any concurrent systemic infection. one of these two pigs had moderate diffuse atrophic enteritis associated with pedv antigen as determined by immunohistochemical stains (score ). this pig also had the highest amount of pedv rna measured (log . genomic copies per fecal swab at dpi and figure . mean group anti-pedv iga elisa s/p ratios ± sem at days , , and after pedv inoculation (dpi) in the different treatment groups and number of positive pigs/total number of pigs in the group for each day. the results of the two pigs that were killed on dpi and were included at dpi . an s/p ratio greater than . was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < . . the statistical software used was jmp pro . the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. log . at dpi ) when it was killed. the other pig had its peak pedv shedding at dpi (log . pedv genomic copies), but its shedding decreased to log . genomic copies by dpi . no other lesions were seen in these two pigs. the remaining pigs were necropsied at the scheduled time at dpi , and lesions or pedv antigen were not seen in any of these pigs suggesting that the pedv infection and associated lesions had resolved. pedv has become a major economic concern for north american pig producers since it was first . mean group log amount of pedv rna fecal samples in pedv and pedv-bovsdp pigs at different days after pedv infection and number of positive pigs per total number of pigs in the group for each day. an "a" on a given day indicates significant (p < . ) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < . . the statistical software used was jmp pro . . mean group anti-pedv igg elisa s/p ratios ± sem at days , , and after pedv inoculation in the different treatment groups and number of positive pigs per total number of pigs in the group for each day. the results of the two pigs that were killed on dpi and were included at dpi . an s/p ratio greater than . was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. identified in the united states in (stevenson et al., ; chen et al., ) . the lack of highly effective vaccines and the relative ineffectiveness of common treatment methods have led to a search for alternative methods of treating and preventing outbreaks. the inclusion of sdp into the diets of young pigs has been shown to have health benefits for other diseases. this study was designed to determine whether there is any benefit to adding bovsdp to the diet during pedv infection. supplementing feed rations of pigs, fish, poultry, cats, and dogs with sdp as protein source is performed on a regular basis. sdp is a protein-rich product obtained from blood from healthy animals (cattle or pigs) at slaughter (torrallardona, ; pérez-bosque et al., ) . in pet food, sdp is a preferred binder in canned food products due to its high-protein content and its physicochemical properties (polo et al., ; rodriguez et al., ) . porcine sdp was first introduced as protein source for pigs during the early s (cole and sprent, ) and since has been used widely in the diet of weaned pigs (torrallardona, ) . benefits of adding porcine sdp include improvement of weight gain mainly due to increased feed intake and reduction of incidence and severity of diarrhea after weaning (adewole et al., ) . comprehensive information on the effect of sdp obtained in trials involving over , pigs has been summarized (torrallardona, ) . in this study, the pedv challenge was performed after an acclimation period of wk to minimize stress from weaning and transport to the new facility. successful pedv challenge was confirmed by detecting pedv rna in fecal swabs. all pigs ( %) from the bovsdp-pedv group shed pedv in fecal samples from dpi to , whereas between % and approximately % of the animals in pedv group shed the virus during that time. these results highlight that a higher number of animals excreted pedv during the early stages of infection in bovsdp-pedv group. differences in the challenge dose or challenge process can be ruled out. the pedv inoculum was prepared in a similar manner and at the same time for both groups by a single person and stored on ice until challenge of each group. the challenge was conducted by the same personnel for both groups with approximately min between the two groups. the obtained results could have been by chance due to the group sizes. alternatively, the bovsdp-pedv animals could be more prone to excrete pedv in the early stages in the infection. unlike in suckling pigs that are very susceptible to pedv infection, only per pedv-infected pigs (both from the pedv challenged groups) had to be killed due to the severity of clinical signs. this is expected and comparable with other trials infecting -wk-old pigs (crawford et al., ) . in the current study, the pedv igg and iga antibody responses were more rapid in the bovsdp group compared with the pedv group. specifically, by dpi , % of the bovsdp pigs were anti-igg and figure . mean group log amount of pedv rna in pcr positive pigs in fecal samples at different days after pedv infection. an "a" at a given day indicates significant (p < . ) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < . . the statistical software used was jmp pro . iga positive compared with . % of the pedv pigs. of note, while systemic iga antibodies were measured, mucosal iga levels were not determined, and it is therefore unknown how the addition of the bovsdp affected the gut immunity. in a previous study, a good correlation of iga levels in serum and feces was found , and as fecal samples and gut mucosa are more difficult to process during routine lab work, serum iga levels are commonly tested. explanations for the earlier humoral immune response in the bovsdp group may include acceleration of the clinical course by the dietary supplement; however, a more rapid immune response due to earlier replication of the virus in more pigs unrelated to the diet modification is also possible. serum anti-pedv ig in pig serum has been demonstrated to neutralize infectivity of pedv (hofmann and wyler, ) , and bovine plasma could have a similar neutralizing activity, which was not further assessed in this study. besides the presence of possible neutralizing antibodies in sdp, other plasma compounds such as peptides (anderson and anderson, ) could contribute to the benefits seen with sdp addition to a diet. virus shedding in the bovsdp group was . d shorter than in the pedv group. the fecal pedv rna shedding in this group was terminated by dpi . a previous study has shown that pedvinfected pigs shed infectious pedv capable of horizontal transmission for to d after infection (crawford et al., ) . this is similar to what was observed in the pedv-infected pigs without bovsdp in the diet in this study. the data from this trial indicate a beneficial effect of bovsdp on acute pedv infection, which is similar to previous reports using the prrsv infection model (pujols et al., ) . specifically, pigs fed bovsdp were able to clear virus shedding . d sooner than non-bovsdp pigs. the addition of bovsdp to the diet resulted in faster and stronger pedv antibody responses and reduced pedv shedding time compared with pigs with no bovsdp in the diet. limitations of this study include the usage of one single virus titer and the low numbers of pigs tested. in addition, two pedv pigs had to be removed early from the study, which could have impacted the outcomes. a larger study with higher numbers of pigs per group comparing porcine and bovine-derived sdp with multiple necropsy days should be conducted to further confirm the possible benefits of sdp in pedv-infected pigs. gut health of pigs: challenge models and response criteria with a critical analysis of the effectiveness of selected feed additives-a review the human plasma proteome: history, character, and diagnostic prospects the development of the mucosal immune system pre-and post-weaning: balancing regulatory and effector function spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged with enterotoxigenic escherichia coli k weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine isolation and 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pet food recipe emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences spray dried animal plasma as an alternative to antibiotics in weanling pigs-a review. asian-aust effect of fishmeal replacement with spray-dried animal plasma and colistin on intestinal structure, intestinal microbiology, and performance of weanling pigs challenged with escherichia coli k this study was funded by the apc, inc. additional support was provided by the biotechnology and biological sciences research council (bbsrc) institute strategic programme grant awarded to the roslin institute (bb/j / ; bbs/e/d/ ). we thank dr. huigang shen for assistance with sample testing and dr. priscilla gerber for assistance with the statistical analysis. conflict of interest statement. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -jgbjxgh authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid- vaccine candidate chadox ncov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: jgbjxgh clinical development of the covid- vaccine candidate chadox ncov- , a replication-deficient simian adenoviral vector expressing the full-length sars-cov- spike (s) protein was initiated in april following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox ncov- in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov- neutralising titres. as sars-cov- began to spread around the world at the beginning of several vaccine platform technologies were employed to generate candidate vaccines. several use replicationdeficient adenoviral (ad) vector technology and express the sars-cov- spike (s) protein. the first phase i clinical study of an ad -vectored vaccine has been reported , chadox ncov- (azd ) phase i trials (nct ) began in april with phase ii and iii trials (nct ) started soon thereafter, and an ad -vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens - . rhesus macaques immunised with a single dose of chadox ncov- were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study . here, we set out to test the immunogenicity of either one or two doses of chadox ncov- in mice and pigs, to further inform clinical development. 'prime-boost' vaccinated inbred (balb/c) and outbred (cd ) mice were immunised on and days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox ncov- on day . spleens and serum were harvested from all mice on day ( weeks after boost or prime vaccination). analysis of sars-cov- s protein-specific murine splenocyte responses by ifnγ elispot assay showed no statistically significant difference between the prime-only and primeboost vaccination regimens, in either strain of mouse ( figure a ). intracellular cytokine staining (ics) of splenocytes ( figure b) showed, in both mouse strains, that the response was principally driven by cd + t cells. the predominant cytokine response of both cd + and cd + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il- + and il- + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th response , . there were no signficant differences in cd + and cd + t cell cytokine responses between prime-only and primeboost mice. prime-only and prime-boost pigs were immunised on dpv and prime-boost pigs received a second immunisation on dpv. blood samples were collected weekly until dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on dpv ( weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < . ; figure c ). the prime-boost dpv responses were greater than responses observed in either group on dpv, but inter-animal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th -type cytokines (similar to the murine response) but with a higher frequency of s-specific cd + t cells compared to cd + t cells ( figure d ). however, cd + and cd + t cell cytokine responses did not differ significantly between vaccine groups or timepoints ( vs. dpv). sars-cov- s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/c mice compared to their prime-only counterparts (p < . ), however, the difference between vaccine groups for cd mice was not significant (figure a ). antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to pre-vaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from and dpv, respectively (p < . ; figure b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of > log (p < . ). rbd-specific antibody titres showed a similar profile with significant titres in both groups from dpv (p < . ) and a further significant increase in the prime-boost pigs from dpv onwards which was greater than the prime-only pigs (p < . ; figure c ). sars-cov- neutralising antibody responses were assessed using a virus neutralisation test (vnt; figure d ) and pseudovirus-based neutralisation test (pvnt; figure e ). after the prime immunisation, sars-cov- neutralising antibody titres were detected by vnt in and dpv sera from / prime-boost and / prime-only pigs. two weeks after the boost ( dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < . ). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in dpv prime-boost pig sera were significantly greater than earlier timepoints and the prime-only group (p < . ). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = . ; p < . ). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid- vaccine candidate, chadox ncov- (now known as azd ). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [ ] [ ] [ ] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [ ] [ ] [ ] and nipah virus , .the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox ncov- and other covid- vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day , whilst comparing responses days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox ncov- immunisation induced robust th -like cd + and cd + t cell responses in both pigs and mice. this has important implications for covid- vaccine development as virus-specific t cells are thought to play an important role in sars-cov- infection [ ] [ ] [ ] [ ] [ ] . while no correlate of protection has been defined for covid- , recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models , . a single dose of chadox ncov- induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination , . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after prime-boost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals (scientific procedures) act and with approval from the relevant local animal welfare and ethical review body (mice -project license p b f , and pigs -project license pp ). the principles of the r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e cells were grown in dmem containing sodium pyruvate and l-glutamine (sigma-aldrich, poole, uk), % fbs (gibco, thermo fisher, loughborough, uk), . % penicillin/streptomycin ( , u/ml; gibco) (maintenance media) at °c and % co . sars-cov- isolate england- stocks were grown in vero e cells using a multiplicity of infection (moi) of . for days at °c in propagation media (maintenance media containing % fbs). sars-cov- stocks were titrated on vero e cells using mem (gibco), % fcs (labtech, heathfield, uk), . % avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using . % toluidine blue (sigma-aldrich). all work with live sars-cov- virus was performed in acdp hg laboratories by trained personnel. the propagation, purification and assessment of chadox ncov- titres were as described previously . a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids - ) of sars-cov- (genbank mn ), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his tag. recombinant rbd was transiently expressed in expi ™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphate-buffered saline (pbs) ph . buffer. a soluble trimeric s (fl-s) protein construct encoding residues - with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k p, v p) was expressed as described . the endogenous viral signal peptide was retained at the nterminus (residues - ), a c-terminal t -foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi ™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph . buffer. for analysis of t cell responses in pigs, overlapping mer peptides offset by residues based on the predicted amino acid sequence of the entire s protein from sars-cov- wuhan-hu- isolate (ncbi reference sequence: nc_ . ) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile % acetonitrile (sigma-aldrich) at a concentration of mg/ml. three pools of synthetic peptides representing residues - (pool ), - (pool ) and - (pool ) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping mer peptides offset by residues were designed and synthesised (mimotopes) and reconstituted in sterile % dmso (sigma-aldrich) at a concentration of mg/ml. two peptide pools spanning s region (pool : to and - , pool : - ) and peptide pools spanning s region (pool : to , pool : to ) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s (pool and pool ) and s (pool and pool ) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) (envigo) and outbred crl:cd (cd ) (charles river) of at least weeks of age were randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n= and cd n= ). prime-boost mice were immunised intramuscularly with infectious units (iu) ( . x virus particles; vp) chadox ncov- and boosted intramuscularly four weeks later with × iu chadox ncov- . prime-only mice received a single dose of iu chadox ncov- at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further weeks later. pigs: six - -week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = ): 'prime-only' and 'prime-boost'. both groups were immunised on day with × iu ( . × vp) chadox ncov- in ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day . blood samples were taken from all pigs on a weekly basis at , , , , , and dpv by venepuncture of the external jugular vein: ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice: antibodies to sars-cov- fl-s protein were determined by performing a standardised elisa on serum collected -weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with ng/well fl-s protein overnight at °c, prior to washing in pbs/tween ( . % v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for hour at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for hours at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted / in casein, for hour at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a -parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs: serum was isolated by centrifugation of sst tubes at × g for minutes at rt and stored at - °c. sars-cov- rbd and fl-s specific antibodies in serum were assessed as detailed previously with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at / , dilution in pbs with . % tween and % non-fat milk. in addition, after the last wash, a µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for minutes at rt. a µl of biofx nmstop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at nm. end-point antibody titres (mean of duplicates) were calculated as follows: the log od was plotted against the log sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of dpv sera. the ability of pig sera to neutralise sars-cov- was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heat-inactivated (hi) by incubation at °c for hours. virus neutralization test (vnt): starting at a in dilution, two-fold serial dilutions of sera were prepared in well round-bottom plates using dmem containing % fbs and % antibiotic-antimycotic (gibco) (dilution media). μl of diluted pig serum was mixed with μl dilution media containing approximately plaque-forming units (pfu) sars-cov- for hour at °c. vero e cells were seeded in -well flat-bottom plates at a density of × cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by µl of dmem containing % fcs and % antibiotic-antimycotic, before µl of the virus-sera mixture was added to the vero e cells and incubated for six days at °c. cytopathic effect (cpe) was investigated by brightfield microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov- virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in % of the wells (nd ). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov- pseudoviruses were generated in hek t cells incubated at °c, % co . cells were seeded at a density of . x in well dishes, before being transfected with plasmids as follows: ng of sars-cov- spike, ng p . (encoding for hiv- gag-pol), ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem (gibco) along with µl pei ( µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna . ) instead of the sars-cov- s expression plasmid. the following day, the transfection mix was replaced with ml dmem with % fbs (dmem- %) and incubated at °c. at both and hours post transfection, supernatants containing pseudotyped sars-cov- (sars-cov- pps) were harvested, pooled and centrifuged at , x g for minutes at °c to remove cellular debris. target hek t cells, previously transfected with ng of a human ace expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of × in µl dmem- % in a white flat-bottomed -well plate one day prior to harvesting of sars-cov- pps. the following day, sars-cov- pps were titrated -fold on target cells, with the remainder stored at - °c. for pvnts, pig sera were diluted : in serum-free media and µl was added to a -well plate in quadruplicate and titrated fold. a fixed titred volume of sars-cov- pps was added at a dilution equivalent to signal luciferase units in µl dmem- % and incubated with sera for hour at °c, % co . target cells expressing human ace were then added at a density of x in µl and incubated at °c, % co for hours. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by % (ic ). mice: single cell suspension of mouse spleens were prepared by passing cells through μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (mem supplemented with % fcs, pen-step, l-glut and -mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of g/ml on ipvh-membrane plates (millipore) coated with g/ml anti-mouse ifn-γ (clone an ; mabtech). after - hours of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab ( µg/ml; clone r a , mabtech) followed by streptavidin-alkaline phosphatase ( µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with μg/ml golgiplug (bd biosciences) and μl/ml cd a-alexa for hours in a -well u-bottom plate, prior to placing at o c overnight. following surface staining with cd -buv , cd -percp-cy . , cd l-bv , cd -bv , cd -apc-cy and live/dead aqua (thermo fisher), cells were fixed with % neutral buffered formalin (containing % paraformaldehyde) and stained intracellularly with tnf--af , il- -pe-cy , il- -bv , il- -pe and ifn-γ-e diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v (treestar). an acquisition threshold was set at a minimum of events in the live cd + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs fsc-a), size (fsc-h vs ssc), cd + , cd + or cd + cells and cytokine positive. total sars-cov- s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s and s specific cells. pigs: pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold % dmso (sigma-aldrich) in hi fbs . resuscitated pbmc were suspended in rpmi medium, glutamax supplement, hepes (gibco) supplemented with % hi fbs (new zealand origin, life science production, bedford, uk), % penicillin-streptomycin and . % -mercaptoethanol ( mm; gibco) (crpmi). to determine the frequency of sars-cov- s specific ifn-γ producing cells, an elispot assay was performed on pbmc from , , and dpv. multiscreen -well plates (mahas ; millipore, fisher scientific) were pre-coated with µg/ml anti-porcine ifn-γ mab (clone p g , bd biosciences) and incubated overnight at °c. after washing and blocking with crpmi, pbmcs were plated at × cells/well in crpmi in a volume of µl/well. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools at a final concentration of µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after hours incubation at °c with % co , plates were developed as described previously . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool , and data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per x pbmc. to assess intracellular cytokine expression pbmc from and dpv were suspended in crpmi at a density of × cells/ml and added to µl/well to -well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov- s peptide pools ( µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after hours incubation at °c, % co , cytokine secretion was blocked by addition : , bd golgiplug (bd biosciences) and cells were further incubated for hours. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd -percp-cy . mab (clone - - , bd bioscience) and cd β-fitc mab (clone ppt , bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af mab (clone cc , bio-rad antibodies, kidlington, uk), tnf-α-bv mab (clone mab , biolegend), il- mab (clone a d f h , invitrogen, thermo fisher scientific) and il- bv mab (clone mp - d , biolegend) followed by staining with anti-mouse igg a-pe-cy (clone rmg a- , biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. total sars-cov- s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools - specific cells. graphpad prism . . (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the results. neutralising antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < . were considered statistically significant. were immunised on day and with chadox ncov (prime-boost) or chadox ncov on day (prime-only); pigs (n= ) were immunised with chadox ncov- on days and (primeboost), or only on day (prime-only). to analyse sars-cov- s-specific t cell responses, all mice were sacrificed on day for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov- s-peptides, responses of murine splenocytes (a) and porcine pbmc (c) were assessed by ifn-γ elispot assays. using flow cytometry, cd + and cd + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il- , il- and il- (mice; b) and ifn-γ, tnf-α, il- and il- (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. : sars-cov- s protein-specific antibody responses following chadox ncov- primeonly and prime-boost vaccination regimens in mice and pigs. inbred balb/c (n= ) and outbred cd (n= ) were immunised on day and with chadox ncov (prime-boost) or chadox ncov on day (prime-only), whereas, pigs were immunised with chadox ncov- on days and (prime-boost), or only on day (prime-only). to analyse sars-cov- s protein-specific antibodies in serum, all mice were sacrificed on day and pigs were blood sampled weekly until day . antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov- fl-s for both mice (a) and pigs (b), and recombinant s protein rbd for pigs (c). sars-cov- neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in % of the wells (nd ; d) , and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by % (ic ; e). each data point represents an individual mouse/pig sera with bars denoting the median titre per group. a single dose of chadox mers provides broad protective immunity against a variety of mers-cov strains. biorxiv chadox ncov- vaccination prevents sars-cov- pneumonia in rhesus macaques. biorxiv a single dose of chadox mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype is preferentially presented to cd + t lymphocytes by the cd α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag a-mtb effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h n virus vaccine development for nipah virus infection in pigs bovine herpesvirus- -vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs. vaccines (basel) targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid- patients clinical and immunological features of severe and moderate coronavirus disease transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients presence of sars-cov- reactive t cells in covid- patients and healthy donors. medrxiv sars-cov- infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov- in rhesus macaques vaccination with viral vectors expressing np, m and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov- seroconversion in humans this study was supported by engineering sarah gilbert and teresa lambe are named on a patent application covering chadox ncov- . the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. correspondence and material requests to professor simon p. graham (simon.graham@pirbright.ac.uk) and professor teresa lambe (teresa.lambe@ndm.ox.ac.uk). key: cord- -whg w w authors: bhatta, tarka raj; ryt-hansen, pia; nielsen, jens peter; larsen, lars erik; larsen, inge; chamings, anthony; goecke, nicole b.; alexandersen, soren title: infection dynamics of swine influenza virus in a danish pig herd reveals recurrent infections with different variants of the h n swine influenza a virus subtype date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: whg w w influenza a virus (iav) in swine, so-called swine influenza a virus (swiav), causes respiratory illness in pigs around the globe. in danish pig herds, a h n subtype named h n dk is one of the main circulating swiav. in this cohort study, the infection dynamic of swiav was evaluated in a danish pig herd by sampling and pcr testing of pigs from two weeks of age until slaughter at weeks of age. in addition, next generation sequencing (ngs) was used to identify and characterize the complete genome of swiav circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (ha) and neuraminidase (na) proteins. overall, . % of the pigs became pcr positive for swiav during the study, with the highest prevalence at four weeks of age. detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the ha and na proteins of the virus. at least two different h n variants were found to be circulating in the herd; one h n variant was circulating at the sow and nursery sites, while another h n variant was circulating at the finisher site. furthermore, it was demonstrated that individual pigs had recurrent swiav infections with the two different h n variants, but re-infection with the same h n variant was also observed. better understandings of the epidemiology, genetic and antigenic diversity of swiav may help to design better health interventions for the prevention and control of swiav infections in the herds. the influenza a virus (iav) is a negative-sense, single-stranded, eight-segmented rna virus belonging to the family orthomyxoviridae [ ] . the main antigenic proteins are encoded by the surface gene segments hemagglutinin (ha) and neuraminidase (na). the six internal gene segments encode for polymerase b (pb ), polymerase b (pb ), polymerase a (pa), nucleoprotein (np), matrix (m and m ), and non-structural protein (nep-ns ) respectively [ , ] . there are different ha (h to h ) and different na (n to n ) subtypes. most of these subtypes can be found in aquatic birds (h -h and n -n ), whereas only a few subtypes are found in mammals [ , ] . in pigs, circulation of iav, so-called swine influenza a virus (swiav), is currently mainly limited to three different subtypes including h n , h n and h n [ ] [ ] [ ] . avian-like swine h n swiav, was first detected in european pig herds in the late s [ , ] and caused epizootic disease outbreaks that resolved shortly within a few weeks [ , ] . however, several recent studies have shown that the dynamics of swiav have changed to a more enzootic form where the virus persists in the herds for months or even years [ ] [ ] [ ] [ ] [ ] [ ] . the altered dynamics of swiav from a short-term epizootic disease to continuous circulation in the herds, is probably a consequence of increased herd sizes and the continuous supply of naïve individuals that maintain the infection [ , ] . in european pig herds, an average prevalence of % has been estimated for swiav infection [ ] . the most prevalent subtypes identified in europe in recent years were the eurasian avian-like swine h n ( . %), the pandemic a/h n (h n pdm ) ( . %), the human-like reassortant swine h n ( %), and the human-like reassortant swine h n ( . %) [ ] . in addition, a number of studies have also shown evidence of reassortants with internal gene segments of h n pdm and the surface gene segments of predominant enzootic swiav [ ] [ ] [ ] [ ] . in denmark, the human-like reassortant h n has never been detected. however, another h n reassortant is widespread among danish pig herds. this subtype is termed "h n dk" and has the ha gene of the eurasian avian-like h n subtype and the na gene of the h n human-like swiav [ ] . in addition to the subtypes mentioned above, introductions of human seasonal iav occurs regularly, increasing the risk of novel swiav reassortants, and making the disease more difficult to control in the herds [ ] . in addition, similar to other rna viruses, swiav has a high mutation rate, which drives the viral evolution and helps the virus evade the immune system by creating novel variants with modified antigenicity [ ] [ ] [ ] . mutations in the antigenic sites (cb, sa, sb, ca and ca ) of the ha protein can lead to the virus being able to escape the binding of neutralizing antibodies [ , ] . moreover, mutations in b-cell epitopes and t-cell epitopes of other iav proteins might also impact the host immunity [ ] [ ] [ ] [ ] . finally, mutations favouring altered n-linked glycosylation (nlg) sites near/within ha and na antigenic sites can also affect the binding of antibodies [ ] . according to the danish agriculture and food council, denmark produces approximately million pigs annually from around three thousand herds of which approximately million are exported as weaners to other countries such as poland and germany [ , ] . in contrast, denmark imports a very limited number of live pigs. in denmark and other countries worldwide swiav is one of the causes of respiratory infection in pigs [ ] . swiav infections lead to destruction of the epithelial cells and impairs the immune system, thereby making the host more susceptible to infection by other viruses and bacteria. co-infection of pigs with distinct variants of swiav and other respiratory pathogens (like pasteurella multocida, mycoplasma hyopneumoniae, haemophilus parasuis, actinobacillus pleuropneumoniae, porcine circovirus type and porcine reproductive and respiratory syndrome virus) are known to cause enhanced disease compared to single pathogen infections, and are all part of the porcine respiratory disease complex (prdc), which can lead to massive economic losses [ , [ ] [ ] [ ] . the aim of the present study was to monitor the molecular epidemiology of swiav circulating in pigs between two weeks of age to weeks of age in a danish pig herd. a newly developed high-throughput real-time pcr (rtpcr) system (fluidigm, south san francisco, usa), which consists of rtpcr assays targeting selected respiratory and enteric viral and bacterial pathogens [ ] was applied for initial detection of swiav in nasal swab samples. moreover, next generation sequencing (ngs) was used to characterize the complete genome of swiav during natural infections, and to examine the genetic variability in the antigenic sites of the virus ha and na proteins. in this study, only nasal swabs were collected and thereby the study did not include the introduction of a needle, which according to the danish law on animal experimentation (lbk number of may ) is the minimum intervention that requires a specific license. a trained veterinarian was involved in the sampling and data collection, and farmer consent was obtained before the sample collection. the study was designed as an observational cohort study to monitor/screen pig health (particularly swiav infection) in a danish farrow-to-finish continuous-flow pig herd. the herd had sows and a farrowing area divided into a number of units. nursery pigs were housed at a separate site with pen places, and finisher pigs were raised at a third site with pen places. according to the danish spf (specific pathogen free) herd health declaration (seges svineproduktion ), the herd was declared positive for mycoplasma hyopneumoniae (m. hyo), actinobacillus pleuropneumoniae (app) type and and porcine reproductive and respiratory syndrome (prrs) type virus, and negative for app type , prrs type virus, brachyspira hyodysenteriae, atrophic rhinitis, demodex mites and lice. the herd had contract with a large abattoir and processing company in denmark, which in initiated the "raised without antibiotics" (rwa) concept [ ] . at present (march ), danish herds including this herd are included in the rwa program, where the producers are payed a premium price to reflect the cost of the increased workload, intensified hygiene measures and other interventions to prevent disease in the effort to reduce the use of antimicrobial agents. the herd produced and recruited gilts internally. after weaning, pigs selected for gilt recruitment were housed in a separate room at the sow site, and vaccinated with a live prrs type virus vaccine (ingelvac ® prrs vet.) at and weeks of age, and additionally with m. hyo, porcine circovirus- (pcv ) (porcilis ® pcv m hyo) and swiav (respiporc flu ) vaccines at , and weeks of age. all adult sows were vaccinated against swiav simultaneously four times a year (respiporc flu ). due to clinical signs like nasal discharge and swiav positive laboratory testing, piglets were vaccinated at four days of age with respiporc flu ( . ml, off label use). during the first week after weaning, pigs were vaccinated against m. hyo and pcv (porcilis ® pcv m hyo). the prevalence of different iav strains in danish pig herds has been reported to be around % [ ] . based on this data, the iav prevalence in this herd was assumed to be between % and %. by using a minimum sample size of , this should, at a very minimum, give at least one iav-positive sample with a high probability of getting iav-positive samples from - pigs. it was assumed from the outset of the study that some piglets may be lost to follow-up or that some may die and hence it was decided to use an initial sample size of . all live-born piglets born within three consecutive farrowing days (n = ) were ear-tagged at birth with consecutive unique id numbers. from every th piglet (id number , , etc.) nasal swabs were obtained (at five to nine day intervals) at week (n = ), week (n = ), week (n = ), week (n = ), week (n = ), week (n = ), week (n = ) and week (n = ). overall, individual piglets/pigs were sampled throughout the study (table s ). due to the variable weaning age (week or week ) of the piglets, the week sampling was done either at the farrowing unit (week f, n = ) or in the nursery unit (week n, n = ) situated at separate buffer stables at the sow site. at week , all weaned piglets were moved into separate nursery units and housed with other pigs from the same herd of the same age until approximately weeks of age. thereafter, the nursery pigs were transferred to a finisher site (~ kilometre away) where they were housed until slaughter at approximately weeks of age. this finisher site received piglets from the nursery units of the same herd. the samples were collected from the april until the september . small-or medium-sized sterile rayon swabs (medical wire, wiltshire, uk) were used to collect the nasal swab samples. the swab was inserted into one nostril of the individual piglet and was then turned a full • . samples were stored in ml phosphate buffered saline (pbs) at - • c until delivered to the laboratory within two days after sampling. at weeks and , piglets were clinically examined before obtaining the nasal swabs. clinical signs of nasal discharge and conjunctivitis were recorded for each individual pig. all samples were processed at the centre for diagnostics (cfd), technical university of denmark (dtu). the pcr analysis was carried out at cfd-dtu, while the ngs was carried out at statens serum institute (ssi), denmark. all the collected samples were vortexed for min, the rayon swab was then removed from the tube and µl of the fluid transferred to a ml eppendorf (ep) tube. the ml ep tubes were centrifuged at × g for min at room temperature. finally, µl supernatant was used for nucleic acid extraction. nucleic acid (both dna and rna) was extracted using the qiacube ht extraction robot (qiagen, hilden, germany) and the cador pathogen qiacube ht kit (qiagen) according to the manufacturer's instructions. the extracted nucleic acids were stored at − • c for further use. the extracted nucleic acids were subjected to reverse transcription using a high capacity cdna rt kit (applied biosystems, foster city, ca usa). a final volume of µl reaction mix was prepared by mixing µl of x rt buffer, . µl of mm dntp mix, µl of x random hexamer, . µl of multiscribe rt enzyme, . µl of nuclease free water and µl of extracted nucleic acids. finally, cdna synthesis was carried out in a t thermocycler (biometra, fredensborg, denmark) with the given cycling conditions: • c for min, • c for min followed by • c for min and finally paused at • c. the cdna sample was pre-amplified using x taqman preamp master mix (applied biosystems). a total volume of µl was prepared by mixing . µl of cdna with µl of x taqman preamp master mix (applied biosystems) and . µl of a nm primer mix (containing the different sets of primers used for the detection of different pathogens) as previously described [ ] . in brief, pre-amplification was carried out in a t thermocycler (biometra) using the program • c for min followed by cycles of • c for s and • c for min. finally, the amplification was paused at • c and the pre-amplified product was stored at − • c for further use. initially, the collected samples were screened for iav by using the high-throughput rtpcr platform biomark (fluidigm, south san francisco, usa) and the . dynamic array (da) integrated fluidic circuit (ifc) chip (fluidigm). a µl sample mix was prepared by mixing . µl of pre-sample mix (prepared by mixing µl of x taqman gene expression mastermix (applied biosystem) and . µl of x sample loading reagent (fluidigm) for each sample) and . µl of pre-amplified sample. similarly, µl primer/probe stock was mixed with µl of x assay loading reagent (fluidigm). three µl of the assay mix and µl of sample mix was loaded into the respective inlets of the . da ifc chip. the . da ifc chip was placed in the ifc controller rx for loading and mixing for approximately min. finally, the chip was inserted into the high-throughput rtpcr platform biomark (fluidigm) for thermal cycling with the following cycling condition: • c for min, • c for min followed by cycles of • c for s and • c for s. all samples were tested in duplicates. positive and non-template (nuclease-free water) controls were included. amplification curves and cycle threshold (ct) values were obtained on the biomark system and finally analysed using fluidigm real time pcr analysis software . . (fluidigm) as previously described [ ] . samples found positive for iav using the high-throughput rtpcr system, were selected for rna extraction. the extraction was carried out for the selected nasal swabs using the qiacube extraction robot (qiagen) and the rneasy mini kit (qiagen) according to the manufacturer's instructions as described previously [ ] . rna was eluted in µl rnase-free water and stored at − • c. detection of iav in the extracted rna was performed in the rotor-gene q (qiagen) pcr platform using a previously published rtpcr assay targeting the matrix gene of iav [ , ] . confirmed iav positive samples with ct values < (using rotor-gene pcr) were used for full genome amplification of iav. a one-tube reaction amplifying each gene segment of iav was performed using a modified version [ ] of a previously published assay [ ] . five µl of the amplified one-tube full genome iav pcr products along with µl of kb dna ladder (invitrogen, carlsbad, ca usa) were run on . % agarose gels (invitrogen) to check if the bands representing all gene segments of iav were visible on a bio-rad gel documentation system (hercules, ca, usa). only fully amplified iav pcr products were selected and were purified using a high pure pcr product purification kit (roche, mannheim, germany) following the manufacturer's protocol. the purified pcr products were sent for ngs on the illumina miseq sequencing platform at the state serum institute (copenhagen, denmark). sequence analysis was performed in clc genomic workbench . . . software (qiagen). all reads obtained from each of the samples were initially trimmed to remove short and low quality reads and primers/adaptors and then consensus sequences of each gene segment was constructed using the function "map reads to reference", using a panel of sequences representing each different lineage of each gene segment known to be present in denmark. the consensus sequences of each gene segment were then aligned using the muscle algorithm [ ] , and then examined for similarities using the function "create pairwise comparison". moreover, the function, blastn, was used to compare the generated consensus sequences with the online ncbi genbank database [ , ] . further analysis was done by selecting some of the closest swiav sequences from the ncbi genbank. phylogenetic analysis was done after aligning all sequences of each gene using clustal-w [ ] and using the maximum likelihood (ml) method with the best fitting substitution model in mega [ ] . the ha subtype numbering was done using the influenza research database (ird) tool at http://www.fludb.org [ , ] . the different amino acids present in the ha antigenic sites (cb, sa, sb, ca and ca ) were calculated by comparing our sequences with reference sequences [ , [ ] [ ] [ ] . b-and t-cell epitopes were analysed by aligning our sequences with reference sequences [ ] [ ] [ ] [ ] using clustal-w [ ] . differences present at seven antigenic sites ( , a, b, c, d, and ) of the na gene segment were calculated by comparing our sequences with reference sequences [ ] [ ] [ ] [ ] . netnglyc . (http://www.cbs.dtu.dk/services/netnglyc/) from dtu bioinformatics (department of bio and health informatics) [ ] was used for the prediction of n-linked glycosylation sites in the ha gene segments only on the n-x-s/t sequons (excluding p at x) with a score threshold > . . fisher's exact test was used to determine any association of data using the analysis tool by .xls at http://itve.dk/ [ ] . the results of the high-throughput rtpcr analysis of nasal swab samples, including an assay for detection of the matrix gene of iav, allowed for estimation of the prevalence of swiav at all sampling times. swiav was detected in pigs throughout the sampling period starting from week until week , indicating continuous circulation of iav in the herd ( table ). the prevalence of swiav increased from the first sampling (week ) ( . %) until week ( . %). among week- -old piglets, . % ( . - . % at % confidence interval) were iav-positive in the farrowing unit while % ( . - . % at % confidence interval) were iav-positive in the nursery unit (weaned at week or week ). after weaning (week ), the prevalence stabilized and then decreased, reaching the lowest prevalence at week ( . %). after the pigs had been transferred to the finisher site, the prevalence increased again, reaching . % at weeks of age. in total, . % of the pigs were iav-positive at least once during the study period (table ). occurrence of iav is defined as the detection of iav in nasal swabs in one or more consecutive weeks in the same pig, whereas recurrence is defined as the detection of iav from the same pig at two or more non-consecutive weeks [ ] . of pigs, ( . %) were found to be positive for iav at least once, whereas ( . %) pigs were found to be negative throughout the study (table s ). all the iav positive and negative pigs were housed together in mixed pens. among the positives, of the pigs ( . %) were found to have recurrent iav (table s ), whereas of pigs ( . %) were found to have only a single occurrence of iav. of these pigs, ( . %) pigs were iav-positive only at a single sampling time, whereas nine pigs ( %) tested positive at two consecutive sampling time points (table s ). among the piglets sampled in week two, ( . %) had nasal secretion and piglets ( . %) had conjunctivitis. however, only two ( . %) piglets had nasal secretion, while four piglets ( . %) had conjunctivitis in week . nasal secretion was recorded in both of the iav-positive piglets at week , while it was only present in two of the positive piglets at week . similarly, conjunctivitis was observed in one out of two positive piglets at week and was only present in one out of positive piglets at week . no association between iav infection in the individual pigs and clinical signs of nasal secretion and conjunctivitis at week and was observed (supplementary tables s , s , s and s ). fifteen samples that had a ct value < in the iav rtpcr assay were selected for one-tube full genome amplification. of these, samples (table s ) displayed clear bands representing all the iav gene segments on the agarose gel and were selected for iav whole genome sequencing (wgs) ( table s ). all the generated sequences for eight full gene segments from all the pig samples have been deposited in ncbi genbank with accession numbers mt -mt . eleven full length ( nt) ha gene segments from the study were used for phylogenetic analysis. twenty three additional ha reference sequences representing avian, human, classical swine and h n pdm subtypes were downloaded from ncbi genbank and included in the analysis. phylogenetic analysis revealed that all the ha gene segments were of eurasian avian-like h nx origin ( c. lineage using the global swine h clade classification system) [ ] . all nine h sequences obtained from pigs sampled between week and were grouped together in one cluster and had pairwise sequence differences of to . % at the nucleotide level and to . % at amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt / (h n ) (accession no.: kr ) in a blastn search with~ % identity at the nucleotide level and~ . % at the amino acid level [ ] . the two h sequences obtained from pigs sampled at week were % identical but clustered separately from the h sequences obtained from the younger pigs ( figure ). these h genes had the highest sequence identity to a/swine/denmark/ - - / (h n ) (accession no.: kr ) in the blastn search with identities of . % at the nucleotide level and . % at the amino acid level [ ] . as mentioned earlier, sequences from two pigs shedding swiav at two non-consecutive sampling times were obtained (pig id sampled at week and week , pig id sampled at week and week ; table s ). in the phylogenetic analysis, the ha sequences of pig id , obtained at weeks and were located in the same cluster and were~ . % ( / ) divergent at the nucleotide level and < % ( / ) divergent at the amino acid level. in contrast, the ha sequences of pig id at weeks and were~ % ( / ) divergent at the nucleotide level and % ( / ) divergent at the amino acid level. from the phylogenetic analysis of pig id it was clearly seen that the ha sequence obtained at the first sampling were located in the cluster defined by the viruses from the younger pigs, whereas the ha sequence obtained at the last sampling (after the pigs had been transferred to the finisher site) were located outside this cluster, thereby representing another h n variant (figure ). the majority of the differences between the two different samplings (week and week ) for pig id and week and week for pig id were located in the antigenic sites of the ha protein between amino acid - . three different amino acid differences were found in the antigenic sites (v a and k n at ca and s h at the sa region) of pig id at week and week . whereas, different amino acids differences were found in the antigenic sites of pig id at week and week (table ) . sequences from different individual piglets sampled at week have one different amino acid (s h) at the sa region and two different amino acids (v a and k n) at the ca region. similarly, sequences from pigs of weeks of age had only one different amino acid (s h) at the sa region. the two ha sequences from week were found to have identical amino acids in the antigenic sites. the cleavage site with one arginine (psiqsr: - ) and fusion peptide sequence (glfgaiagfieggwtgmidgwyg: - ) were found to be conserved in all full-length h ha sequences [ , ] . the ha region of all the ha sequences were found to be more conserved compared to the ha region, as they were only approximately - % divergent at the nucleotide level and - % divergent at the amino acid level. however, the ha region of all the ha sequences were - . % divergent at the nucleotide level and - . % divergent at the amino acid level. eleven full-length ( nt) na gene segments were used for phylogenetic analysis (figure ). twenty seven additional na reference sequences representing danish h n , european h n , h n and asian and american h n na were also used. the phylogenetic tree shown in figure revealed that the n na gene segments from the present study were most identical to the na segment of the danish h n and european h n subtypes. the n sequences obtained from pigs sampled between week and week grouped together in one cluster (danish hxn type) and had pairwise sequence differences of to . % at the nucleotide level and to . % at the amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt / (h n ) (accession no.: kr ) in the blastn search with an identity of . % at the nucleotide level and > % identity at amino acid level, when performing a pairwise comparison [ ] . in contrast, the two n sequences obtained from pigs at week were identical and were positioned apart from the cluster formed by the sequences obtained from the younger pigs. similar to the ha segment, this cluster had the highest sequence identity to a/swine/denmark/ - - / (h n ) (accession no.: kr ) in the blastn search, with an identity of . % at the nucleotide level and > % identity at the amino acid level [ ] (figure ). the n sequences obtained from pig id at weeks and were found to be < . % ( / ) divergent at the nucleotide level and . % ( / ) divergent at the amino acid level. however, the two n na sequences obtained from pig id at weeks and were found to be~ . % ( / ) divergent at the nucleotide level and~ . % ( / ) divergent at the amino acid level. amino acids present in the antigenic sites ( , a, b, c, d, and ) of the n na sequences of pig id at week and week were found to be identical. while the n sequence obtained from pig id at week was similar to the ones from pig id , the sequence from pig id at week were found to be~ . % ( / ) divergent in the antigenic sites of n . similarly, n sequences of pig id at antigenic site d were found to be most divergent, at % ( / ), whereas no changes were found in the antigenic site ( table ). all n na sequences have eight highly conserved amino acids (r , d , r , r , e , r , r and y ) at the inner shell of the na active site, which interact directly with sialic acids. similarly, ten highly conserved amino acids (e , r , w , s , d , i , e , e , n and e ) [ , , ] were also present in an outer shell of the na active site. hence, all the amino acids present at na active sites were found to be conserved. table . comparison of amino acid sequences of neuraminidase (na) antigenic sites of pig id sampled at week and week from the pig herd. amino acid positions were numbered using an n numbering system. amino acid change all the six internal gene segments (pb , pb , pa, np, m -m and nep-ns ) were compared with available online ncbi genbank reference sequences. all the internal gene segment sequences obtained from younger pigs (≤ weeks) were found to be most identical (> %) with the internal gene segments of h n pdm origin. in contrast, only four of the internal gene segments (pb , pb , pa and np) of virus sequences from older pigs (week ) were of h n pdm origin. the remaining two internal gene segments (m -m and nep-ns ) of sequences from older pigs (week ) clustered with the h n dk and european h n swiav subtypes, indicating that these viruses are the result of reassortment events between h n -like viruses and the h n pdm subtypes. in addition, respective internal gene segments obtained from pig id at week and week were compared and were found to be - . % different at the nucleotide level and - . % at the amino acid level. a similar comparison of respective internal gene segments was also done for pig id at weeks and and were found to be . - . % different at the nucleotide level, while at the amino acid level they were . - . % different ( table ). the m -m gene segments of pig id at week and were found to be . % divergent at the nucleotide level as shown in table . similarly, the nep-ns gene segments of pig id at weeks and were found to be most divergent ( . %) ( table ) and also clearly supported by two distinct clusters in the phylogenetic tree ( figure ). nep-ns gene sequences obtained from pigs sampled between week and week made one cluster close to the h n pdm subtypes. in contrast, the nep-ns sequences obtained from pigs sampled at week made a separate cluster and resembled the h n dk and european h n subtypes (figure ). table . pairwise comparison of six internal gene segments of pig id sampled at week and week both at the nucleotide and the amino acid level. week clearly supported by two distinct clusters in the phylogenetic tree ( figure ). nep-ns gene sequences obtained from pigs sampled between week and week made one cluster close to the h n pdm subtypes. in contrast, the nep-ns sequences obtained from pigs sampled at week made a separate cluster and resembled the h n dk and european h n subtypes (figure ). b-and t-cell epitopes present in all the swiav sequences between week and week were identical. one amino acid change (r k) was observed in the b-cell epitope present in the ha part of the ha gene segment after comparing ha sequences from pig id at week and week . similarly, one (e d) and four different amino acids (l f, s n, e v and s t) were found in the t-cell epitope sequence of np and ha gene segments, respectively. t-cell epitopes of other gene segments were found to be identical among the compared isolates. six to seven nlg sites were present in all the ha sequences obtained in this study of which five (at positions , , , and ) were well conserved between all analysed sequences. all the ha sequences obtained from pigs sampled between week and week had one separate nlg site at position , whereas the ha sequences obtained from pigs sampled at week have two other nlg sites at position - and at position . the nlg site at position was located near the ca antigenic site in the globular head domain of the ha protein sequence, whereas the nlg site at position - was located near the sa antigenic site (table ) . similarly, the nlg site at position was located within the sa antigenic site of the ha protein sequence. table . n-linked glycosylation (nlg) sites of h hemagglutinin (ha) gene segments obtained from all the pigs from week to week and week . "+, ++, +++" indicates the nlg potential with score threshold > . . "*" indicates that the nlg site is located in between amino acid - for h numbering system. the study was designed to describe the infection dynamics of swiav in one danish pig herd by following pigs from weeks of age until slaughter (approximately weeks of age). using a high-throughput rtpcr system, we were able to determine the prevalence of iav, and by the use of ngs, we characterized the genetic and antigenic diversity of circulating h n swiav. based on the analysis, it was found that the prevalence of iav in pigs reached a maximum around weaning ( - weeks) and then decreased until weeks and then increased again at weeks of age. at least two different h n variants were circulating in the herd, with one of the h n variants circulating at the sow and nursery sites, and the other circulating in the finisher site. finally, we also demonstrated that individual pigs could have recurrent iav infections either with a very similar h n variant (pig id ) or with two divergent h n variants (pig id ). this confirms previous findings that some pigs can have prolonged swiav infections and be subjected to re-infection, even with closely related swiav [ , ] . the understanding of the epidemiology and the genetic and antigenic diversity of swiav in pigs may help to unravel the layers of swiav infection dynamics and viral evolution from birth to slaughter, thereby helping to design better health interventions for the prevention and control of swiav in the herds. the relative low prevalence of iav before week might be due to the presence of maternally-derived antibodies (mdas), which in this herd were stimulated by the sows being vaccinated with respiporc flu four times annually [ , ] . however, the mdas wane over time, and several studies have shown results indicating that piglets are no longer completely protected from around three to four weeks of age [ , , ] . moreover, the loss of mda occurs at the same time as the piglets are weaned into the nursery, thereby mixing different litters of pigs, creating the optimal environment for iav circulation. this has also been observed in previous studies [ , , ] . similarly, a higher prevalence of iav at week also indicated that the piglet immune system did not respond to the vaccination at day , which is in accordance with a previous study showing no effect of early piglet vaccination against swiav [ ] . one of the explanations for this is that the presence of mda may hinder an active immune response in the piglets, but it could also be due to the reduced vaccine dose used ( . ml) [ ] in the piglets under study. after week , most of the weaned pigs likely developed immunity to the circulating iav subtype and at the same time no new pigs were introduced into the nursery, resulting in a lower prevalence of iav in accordance with other findings [ ] . the increase in prevalence observed at week at a separate finisher site, was most likely due to infections with a second h n dk variant that might have been circulating continuously in the finisher site. this site was managed as a multi-aged, continuous-flow pig herd. this h n dk variant from the finisher site differed significantly in antigenic regions from the h n dk variant circulating at the sow and nursery sites. mutations in the antigenic sites of eurasian avian-like swine h have previously been linked to a lack of cross protection and emphasize that the diversity within the subtypes, especially in the h avian-like viruses, have now reached a level where it makes no sense to consider viruses of the same subtypes as belonging to the same serotype [ ] . however, the lack of a significant cross-reaction should be confirmed by hi-testing, which was not performed in this study. moreover, the na gene and the internal gene cassette were also different between the two h n dk variants, which could also impact the cross-protective immunity between different swiav variants of the same subtype [ , ] . specifically, the internal gene cassette of the h n dk variant that infected the pigs in the sow and nursery sites had a complete internal gene cassette of h n pdm origin, whereas the h n dk variant circulating in pigs in the finisher site had an m -m and nep-ns gene of eurasian avian-like h nx origin. however, the protective role of immunity against the internal gene segments are still controversial [ , ] . pig id was infected twice with the same h n dk variant, which only showed minor genetic differences between samplings. however most of the differences were located in antigenic sites. hence, it can be speculated that even a small number of mutations could facilitate re-infection with the same subtype, thereby confirming the results of a previous study [ ] . in summary, it can be concluded that re-infections can occur with both similar and different variants within the same subtype. the presence of prolonged ( - weeks consecutive) and recurrent (non-consecutive) shedding of iav within week to week pigs also indicated reinfection with the same subtype and this was documented by sequencing. a number of previous studies have shown reinfection with the same strain, leading to prolonged iav shedding [ , , ] . the presence of mda may play role in the prolonged iav shedding, as mda may hinder an active immune response [ , ] . pig id was infected with two different h n dk variants, and most of the mutations were located in antigenic sites as mentioned above. similarly, the acquisition of nlg sites near/within sa and ca antigenic sites of the ha sequence may lead to a shielding effect on the antigenic sites and probably the emergence of new antigenic variants. a range of studies have shown that the shielding effect on ha antigenic sites may lead to an evolution of the ha sequence and be responsible for escaping the pre-existing immunity in the hosts [ ] [ ] [ ] [ ] . the presence of these major differences between variants within the same subtype emphasizes the presence of a massive genetic drift of eurasian avian-like h in danish herds [ ] , which in turn could have consequences for vaccine efficacy as the current swiav vaccine available against the h n subtype has not been updated since - . in our study, the presence of clinical signs of nasal discharge and conjunctivitis in pigs harbouring iav at week was not very evident. reduced levels of clinical signs could be due to the presence of mda. similarly, previous exposure or a low level of exposure to the virus might preclude clinical signs in pigs [ ] [ ] [ ] . in contrast with other studies, our study did not find any association between iav infection and nasal secretion [ , ] . however, clinical signs were only recorded at two stages (at week and week ) of the total samplings in this study. similarly, we did not find any association between iav infection and conjunctivitis, which is also supported by other studies [ , ] . in conclusion, the complexity of swiav infection dynamics in pigs from the farrowing unit to the finisher unit has been demonstrated. a high infection pressure of swiav was identified during the end of the stay in the farrowing unit and the start of the nursery unit. in addition, it has been shown that the prolonged persistence of iav in pigs could be due to re-infection with iavs that are closely related to each other. similarly, re-infections with different strains within the same lineage can also be expected, as the genetic changes affect important antigenic epitopes. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : occurrence and recurrence of iav in pigs from week to week in the pig herd using high-throughput rtpcr. "+" indicates a positive case of iav whereas '-' indicates a negative case of iav. green boxes indicate the non-consecutive detection of iav, yellow boxes indicate the consecutive detection of iav, whereas blue boxes indicate the single detection of iav. 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influenza h n viruses a perspective on the structural and functional constraints for immune evasion: insights from influenza virus a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h n virus hemagglutinin differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity cytokines in the pathogenesis of influenza avian and swine influenza viruses: our current understanding of the zoonotic risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to acknowledge the herd owner for providing access to his property for sampling, and the herd-veterinarian providing assistance during sampling. similarly, we want to acknowledge the laboratory technicians hue thi thanh tran and jonathan rahlff rogersen for their help with laboratory tasks. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. key: cord- -tudl k g authors: opriessnig, tanja; gauger, phillip c.; faaberg, kay s.; shen, huigang; beach, nathan m.; meng, xiang-jin; wang, chong; halbur, patrick g. title: effect of porcine circovirus type a or b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: tudl k g to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-γ) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded, positive-sense rna virus, is characterized by a high mutation rate with the potential of genetically diverse strains evolving over time (forsberg et al., ; hanada et al., ; pirzadeh et al., ; rowland et al., ) . in the past, prrsv isolates have emerged within the swine population with varying degrees of virulence (fang et al., ; han et al., ; nelsen et al., ) possibly due to a high degree of mutation and recombination (yuan et al., (yuan et al., , (yuan et al., , (yuan et al., , . more recently, attention has focused on the occurrence of high mortality in chinese swine herds which veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-g) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. ß elsevier b.v. all rights reserved. was associated with novel prrsv isolates and described as porcine high fever disease in tong et al., ; wu et al., ) . the prrsv isolates involved in porcine high fever disease contained unique nucleotide differences compared to other isolates. specifically, a discontinuous, amino acid deletion was identified within the nsp region which was initially suggested to be correlated with the pathogenicity of the virus wu et al., ). however, more recent reports have concluded that this deletion is unrelated to virulence (zhou et al., ) in spite of the high mortality that was initially associated with this prrsv variant tong et al., ) . interestingly, analysis of samples from affected pigs resulted in the identification of prrsv, porcine circovirus type (pcv ) and classical swine fever virus as the most common co-infection pathogens, suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality (lv et al., ; wu et al., ). pcv is a small, circular, non-enveloped dna virus belonging to the circoviridae family in the genus circovirus. pcv can be further divided into several subtypes of which pcv a and pcv b are prevalent worldwide (patterson and opriessnig, ) . to date, experimental infections comparing pcv a and pcv b in gnotobiotic and conventional pigs have not demonstrated major differences in virulence (beach et al., ; fort et al., ; lager et al., ; opriessnig et al., b) . pcv is the cause of porcine circovirus associated disease (pcvad) with multiple clinical manifestations including respiratory disease . prrsv has become an important component of the porcine respiratory disease complex (prdc) with major economic impact on the swine industry (chae, ) . retrospective studies identified prrsv as the most common cofactor in cases of pcvad (pallaré s et al., ) . experimental coinfection with prrsv and pcv has yielded mixed results. one study completed in reported minimal clinical disease or death loss in conventional pigs coinfected with pcv and prrsv (rovira et al., ) . in contrast, in another study, severe clinical disease and death in of pigs between and days postinfection (dpi) was reported in dually infected, caesarianderived and colostrum-deprived (cdcd) pigs (harms et al., ) . despite differences in severity of clinical presentation, experimental coinfection of pigs with pcv and prrsv has consistently resulted in up-regulation of pcv replication (enhanced viremia and pcv tissue load) and increased severity of prrsv-induced lesions in lung tissues (allan et al., ; harms et al., ; rovira et al., ) . in north america, both prrsv and pcv b have been identified in pcvad outbreaks characterized by excessive mortality suggesting a synergistic relationship between these two viruses gagnon et al., ; horlen et al., ) . the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates in a conventional pig model under the influence of concurrent pcv a or pcv b infection. the severity of clinical disease, macroscopic and microscopic lesions, amount of prrsv and pcv antibodies and nucleic acids in sera, amount of prrsv and pcv antigen associated with lesions, and interferon gamma (ifn-g) concentrations in serum were measured and compared between groups. fifty-three colostrum-fed, crossbred pigs were derived from sows known to be free of pcv , prrsv and mycoplasma hyopneumoniae in two separate batches, pigs in batch (b ) and pigs in batch (b ). in addition, batch (b ) consisted of colostrum-fed crossbred pigs derived from sows free of prrsv and m. hyopneumoniae but seropositive for pcv . b and b pigs were challenged at the same age as b pigs but the experiment was conducted approximately months after b pigs. insufficient numbers of pigs were available from the source herd for singularly prrsv-infected groups to be included with the original experiment. the experimental design and group designations are summarized in table . all pigs were housed under the same conditions and treated in a similar way. all pigs were weaned at three weeks of age and transported to the livestock infectious disease isolation facility at iowa state university, ames, iowa. on the day of arrival, all b pigs were comingled and randomly assigned to one of five rooms each containing or pigs: negative controls (n = ); pigs coinfected with a isolate of prrsv (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with prrsv vr- and pcv b (coi- - b; n = ), pigs coinfected with a isolate of prrsv (nc b) and pcv a (coi- - a; n = ) and pigs coi- - b pcv b nc b prrsv-i- none vr- prrsv-i- none nc b b -prrsv-i- none vr- b -prrsv-i- none nc b a batch and pigs were derived from the same source herd free of prrsv and pcv whereas batch pigs were derived from a different source herd seropositive for pcv . coinfected with prrsv nc b and pcv b (coi- - b; n = ). b pigs were randomly assigned to one of two rooms each containing or pigs which were infected with prrsv vr- (prrsv-i- ) and prrsv nc b (prrsv-i- ), respectively. similarly, b pigs were comingled and randomly assigned to one of two rooms each containing or pigs that were infected with prrsv vr- (b -prrsv-i- ) and prrsv nc b (b -prrsv-i- ), respectively. the pigs from the different batches were kept in different but identical rooms. each room had m of solid concrete floor space, separate ventilation systems and one nipple drinker. inoculation was conducted at approximately days of age. blood samples were collected from all pigs prior to inoculation and at dpi , , and in serum separator tubes ( . ml bd vacutainer, benton dixon, franklin lakes, nj, usa). the blood was centrifuged at  g for min at c and serum was stored at À c until testing. serum samples were analyzed for levels of anti-pcv igg antibody, anti-prrsv-igg antibody, ifn-g, pcv dna, and prrsv rna. in addition, edta tubes ( . ml monoject tm % edta liquid, tyco healthcare group lp, mansfield, ma, usa) were collected at dpi , , , and , stored at room temperature and used within h after collection to determine blood cell counts. all pigs were necropsied on dpi and tissues collected during necropsy were analyzed by immunohistochemistry (ihc) for the presence of pcv and prrsv antigens. the experimental protocol was approved by iowa state university institutional animal care and use committee (iacuc approval # - - -s). prrsv isolate vr- with a rflp pattern - - was recovered in from pig tissues obtained from a sow herd in southwestern iowa affected by severe respiratory disease in - -week-old pigs and high numbers of late term abortions (halbur et al., b; meng et al., ) . the passage virus of the original vr- isolate was used to inoculate pigs in as described previously . serum from the pigs infected with vr- in was used to re-isolate the virus followed by two subsequent passages in marc- cells to produce the virus stock of vr- for this study. prrsv isolate nc b with a rflp pattern - - was isolated in from a clinically affected -week-old pig with systemic pcvad from a group of pigs from north carolina with a history of severe respiratory disease in % of the pigs and approximatley % mortality in the group (gauger et al., ) . the passage virus of the original isolate was used to experimentally infect a set of prrsv-free conventional pigs (data not shown) and the lung tissues from these pigs collected two weeks after infection were used for reisolation of the nc b virus followed by two subsequent passages in marc- cells to produce the nc b virus stock for this study. the two inocula were separated in different aliquots, stored at À c, and virus of the same lot was used for all batches of pigs. on dpi , coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- groups received ml of prrsv isolate vr- at a dose of . median tissue culture infective dose (tcid ) per ml. all pigs in groups coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- received ml of prrsv isolate nc b at a dose of . tcid per ml. inoculation was intranasal by holding the pig in the upright position and slowly dripping ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). two different pcv subtypes were used for the inoculation of pigs. pigs in groups coi- - a and coi- - a were inoculated with the pcv a isolate , which was recovered from an iowa farm in (fenaux et al., ) and has been well characterized genetically (fenaux et al., ) and in the conventional specific pathogen free (spf) pig model (opriessnig et al., (opriessnig et al., , a . both pcv a and pcv b viruses were produced as described previously (opriessnig et al., b) and used for inoculation in this study at a titer of . tcid per ml . pigs in groups coi- - b and coi- - b were inoculated with pcv b isolate nc which was isolated in from a pig farm in north carolina (opriessnig et al., b) . both, pcv b nc and prrsv nc b originated from the same tissues. the pcv groups were inoculated intranasally ( ml) and intramuscularly ( ml) with their respective pcv subtype by injecting ml of the inoculum intramuscularly into the right neck area and ml ( . ml per nostril) intranasally by holding the pig in the upright position and slowly dripping . ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). edta-treated blood samples were analyzed for white blood cells using a multispecies hematology instrument (hemavet hv fs, drew scientific, inc.). the white blood cell (wbc) count was reported as actual numbers of neutrophils, lymphocytes and total wbc per ml of whole blood. in addition to wbc, a ratio was determined between the total neutrophil count and the total lymphocyte count reported as the n/l ratio. values from negative control pigs were considered as baseline for the infected pigs on each dpi. serum samples from all pigs were also tested for the presence of anti-prrsv antibodies by a commercial prrsv elisa (herdchek prrs virus antibody test kit xr, idexx laboratories inc. westbrook, ma, usa), according to the instructions of the manufacturer. samples were considered positive if the calculated s/p ratio was equal to . or greater. all serum samples were tested for the presence of anti-pcv igg antibodies based on an open reading frame (orf ) elisa (nawagitgul et al., ) . samples were considered positive if the calculated sample-to-positive (s/ p) ratio was equal to . or greater. on dpi , three samples were randomly chosen from each group and room and tested for the presence of swine influenza virus (siv) antibodies by an in house nucleoprotein ns elisa (richt et al., ) and for the presence of antibodies to porcine parvovirus (ppv) by hemagglutination inhibition (hi) assay (mengeling et al., ) . a commercial elisa kit (swine ifn-g; invitrogen, camarillo, ca, usa) was used to detect and quantify ifn-g concentrations in serum according to the instructions of the manufacturer. following prrsv/pcv coinfection, the pigs were monitored daily for respiratory disease (dyspnea, sneezing, coughing, nasal discharge). rectal temperatures and behavioral changes such as lethargy and inappetence/ anorexia were also recorded daily. the observers were aware (not blinded) to the treatment status. rna extraction on serum collected at dpi , , , , and was performed using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). the agpath-id prrsv multiplex reagent kit (applied biosystems, foster city, ca, usa) was used for the real-time, reverse transcriptase pcr (rt-pcr) on each extracted rna sample. all samples were run in duplicate. each pcr consisted of ml template rna and ml of pcr master mix. the pcr master mix contained . ml of  rt-pcr buffer, . ml  prrsv primer probe mix, . ml  multiplex rt-pcr enzyme mix, . ml of zenorna- internal control rna and . ml nuclease-free water. each reaction included eight progressive : dilutions of a known copy number of prrsv to generate a standard curve for quantification. each plate was run in the sequence detection system (geneamp sequence detection system, applied biosystems) using the agpath-id company specific conditions ( min at c, min at c, followed by cycles of s at c and s at c). samples were considered negative when no signal was observed within the amplification cycles. dna extraction on serum collected on dpi , , , , and days was performed using the qiaamp dna blood mini kit (qiagen, valencia, ca, usa) and subsequently used for detection of pcv dna by quantitative real-time pcr utilizing primers and a probe designed for pcv orf as described (opriessnig et al., ) . the real-time pcr reaction consisted of a ml pcr mixture containing . ml commercially available master mix (taqman universal pcr master mix, applied biosystems by life technologies), . ml dna, ml ( . mm) of each primer, and . ml ( . mm) probe. the reaction was run in a fast real-time pcr system (abi, foster city, ca, usa) under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. all samples were run in duplicate. serial dilutions of a recombinant pcv dna clone were included on each plate to generate a standard curve. viral concentrations were expressed as the dna copy numbers per ml of sample. samples were considered negative when no signal was observed within the amplification cycles. all dna extracts were also tested for presence of pcv a and pcv b dna by utilizing a forward primer ( -gcagggccagaattcaacc- ), a reverse primer ( -ggcggtggacatgatgaga- ), a probe specific for pcv a ( -cal fluor orange -ggggaccaacaaaatctcta-tacccttt-bhq- ), and a probe specific for pcv b ( -quasar -ctcaaacccccgctctgtgccc-bhq- ), which were designed in the pcv orf as described . the multiplex real-time pcr reaction consisted of a total volume of ml containing . ml of the commercially available master mix (applied biosystems), ml dna, . mm of each primer, and . mm of each probe. all samples were run in duplicate. the reactions were carried out under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. the sensitivity and specificity of the real-time pcr reaction was evaluated using known pcv a and pcv b isolates as well as ppv, prrsv, and pcv type (pcv ) isolates. samples were considered negative when no signal was observed within the amplification cycles. open reading frame (orf) of one prrsv rt-pcr positive pig in each group was sequenced on dpi as previously described (gauger et al., ) . on dpi , all pigs were humanely euthanized by intravenous pentobarbital overdose (fatal-plus vortech pharmaceutical, ltd., dearborn, mi, usa). macroscopic lung lesions were estimated based on the percentage of the lung surface affected by pneumonia ranging from to % (halbur et al., b ). the scoring system was based on the approximate volume that each lung lobe contributes to the entire lung: the right cranial lobe, cranial part of the left cranial lobe, and the caudal part of the left cranial lobe contribute % each to the total lung volume, the accessory lobe contributes %, and the right and left caudal lobes contribute . % each (halbur et al., b) . additionally, lymph node size was scored ranging from (normal) to (four times the normal size) (opriessnig et al., ) . lungs were insufflated with fixative as previously described (halbur et al., b) . sections of lymph nodes (tracheobronchial, mesenteric, mediastinal, superficial inguinal, and external iliac), tonsil, thymus, ileum, kidney, colon, spleen, heart, liver, and brain were collected at necropsy and fixed in % neutral-buffered formalin and routinely processed for histological examination. microscopic lesions were evaluated independently by two veterinary pathologists (to, pcg) blinded to the treatment status. sections of lung were scored for the presence and severity of interstitial pneumonia ranging from (normal) to (severe, diffuse) (halbur et al., b) . sections of heart, liver, kidney, ileum, colon and brain were evaluated for the presence of lymphohistiocytic inflammation and scored from (none) to (severe). lymphoid tissues including lymph nodes (trachea-bronchial, mediastinal, mesenteric, external iliac and superficial inguinal), tonsil, spleen and thymus were evaluated for the presence of lymphoid depletion ranging from (normal) to (severe) and histiocytic inflammation and replacement of follicles ranging from (normal) to (severe) (opriessnig et al., ) . . . immunohistochemistry . . . prrsv detection of prrsv-specific antigen was performed by ihc staining on lung sections as previously described (halbur et al., a) . sections were scored for presence of prrsv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. ihc for detection of pcv -specific antigen was performed on sections of lung, lymph nodes (tracheobronchial, mediastinal, mesenteric, superficial inguinal and external iliac), tonsil, spleen, thymus and small intestine using a rabbit polyclonal antiserum (sorden et al., ) . sections were scored for presence and amount of pcv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. if the results obtained by the two pathologists on a certain tissue differed, the mean of the two scores was used. pcv scores ranged from (no antigen) to (more than % of the lymphoid follicles contain cells with pcv -antigen staining) (opriessnig et al., ) . any tissue or tissue pool with detectable staining was given at least a score of . for the purpose of determining prevalence rates, a score of was considered negative and scores of , and were considered positive. for data analysis, jmp software version . . and sas software version . . (both sas institute, cary, nc, usa) were used. summary statistics were calculated for groups to assess the distributional property and data that were not distributed normally (pcr data) were log transformed prior to analysis. as log transformation can only be applied to numbers above , a constant number ( ) was added to each number in the data set prior to log transformation. a linear mixed model with the random effects ''source'' (source a: b and b and source b: b ) and ''batch'' (b , b , b , nested within ''source'') and the fixed effects ''prrsv strain'' (none, vr- , nc b) and ''pcv subtype'' (none, pcv a, pcv b) was used first on all outcomes. from this, it was determined that the random effect ''source'' contributed to the overall variation whereas ''batch'' did not. to decrease the heterogeneity of the animals in the analysis, all data obtained from the second source, b , were removed from the analysis but were provided as supplemental information throughout the ''results'' and tables. the final model to analyze continuous data collected over time (rectal temperatures, blood cell counts, log transformed pcv and prrsv genomic copies, and elisa s/p ratios) was a repeated measures analysis of variance (anova), where prrsv strain, pcv subtype, dpi and their interactions were the fixed effects and pig was the subject of repeated measures. compound symmetry variancecovariance structure was used to model the within pig correlation. a one-way anova was used to analyze crosssectional data (macroscopic and microscopic lung lesions) where prrsv strain, pcv subtype, and their interaction were the fixed effects. differences among the interacting groups (prrsv strain  pcv subtype) in the repeated measures anova or the one-way anova were assessed using tukey's t-test. a p-value of less than . was considered significant. differences in prevalence of prrsv and pcv antigen between groups (ihc staining) were determined by fisher's exact test. mild, transient lethargy and inappetence were observed in all inoculated groups, although coughing or sneezing was not a feature. pigs in all inoculated groups regardless of coinfection status developed a transient to persistent fever ranging from . c to . c between dpi and dpi . the mean rectal temperature time by group interaction after inoculation was significant (p < . ). all six inoculated groups had rectal temperatures significantly higher than the negative controls at dpi and dpi . by dpi , the mean group rectal temperatures in the prrsv-i- , prrsv-i- , coi- - a, coi- - b and coi- - a groups were significantly (p < . ) higher compared to the negative controls. when the effect of ''prrsv strain'' was evaluated across groups, no differences were found. compared to pigs infected with prrsv alone, coinfected pigs had higher mean rectal temperatures at dpi , and . when the effect ''pcv subtype'' was evaluated among coinfected groups, pcv a pigs had significantly (p < . ) higher rectal temperatures on dpi compared to pcv b pigs (data not shown). b pigs (b -prrsv-i- and b -prrsv-i- ) had similar rectal temperatures as b (prrsv-i- and prrsv-i- ) pigs. hematology results are summarized in table . there was an effect of ''prrsv strain'' on white blood cell counts at dpi with pigs infected with nc b having significantly (p = . ) higher levels of white blood cells compared to pigs infected with vr- ( . ae . versus . ae . ). also, there was a significant effect of ''pcv '' (p < . ): pcv -infected pigs had higher levels of white blood cells at dpi and compared to non-pcv -infected pigs ( . ae . versus . ae . and . ae . versus . ae . ). there was no effect of ''prrsv strain'' on numbers of neutrophils; however, there was a significant effect of ''pcv '' on mean group neutrophil counts at dpi , , and with pcv -infected pigs having elevated levels compared to pigs not infected with pcv ( . ae . versus . ae . , . ae . versus . ae . , and . ae . versus . ae . , respectively). differences in mean group lymphocyte counts were only observed on dpi (table ) and there was an effect of prrsv strain (p = . ): pigs infected with nc b had lower levels of lymphocytes compared to pigs infected with vr- ( . ae . versus . ae . ). additionally, pigs coinfected with pcv had higher levels of lymphocytes (p = . ) compared to pigs infected with prrsv alone ( . ae . versus . ae . ) suggesting an effect of ''pcv ''. all pigs in all groups were negative for prrsv-specific antibodies at dpi and negative control pigs remained negative for anti-prrsv antibody throughout the study. prevalence and group mean s/p ratios are summarized in table . overall, there was a significant effect of ''prrsv strain'' and ''pcv '' on the anti-prrsv antibody response at dpi . specifically, pigs infected with vr- had a significantly (p = . ) higher anti-prrsv antibody response compared to those infected with nc b. similarly, coinfected pigs had significantly (p = . ) higher anti-prrsv s/p ratios compared to pigs singularly infected with prrsv. there was no effect of ''pcv subtype'' on the magnitude of the anti-prrsv-antibody response among coinfected groups. all pigs in b and b were negative for pcv -specific anti-igg antibodies at dpi and the negative controls and b pigs remained pcv seronegative throughout the trial. in the pcv coinfected groups, seroconversion was observed at dpi . the prevalence and mean group anti-pcv -igg s/p ratios table mean group leukocyte values ( /ml of whole blood except for ratios) in the different treatment groups on days post-inoculation (dpi) , , , and . data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. hematology a negative controls (n = ) wbc . ae . . ae . are summarized in table . there was no effect of ''prrsv strain'' or ''pcv subtype'' on the magnitude of the anti-pcv -antibody response. b pigs were seropositive for pcv at the time of arrival (mean pcv elisa s/p ratio: . ae . ) and the s/p ratios remained at a similar level for the duration of the study (data not shown). at termination of the study, pigs randomly selected from each batch tested negative for antibodies against ppv, siv h n and siv h n (data not shown). prevalence of ifn-g positive samples and mean group ifn-g concentrations are summarized in table . there was no effect of ''prrsv strain'' or ''pcv '' on the ifn-g levels and no differences were found among groups; however, analysis of an effect of ''pcv subtype'' among coinfected groups revealed that on dpi , pcv b-inoculated pigs had significantly (p = . ) higher levels of ifng compared to pcv a-inoculated pigs ( . ae . pg/ml versus . ae . pg/ml). all pigs were negative for prrsv-rna in serum at dpi and the negative controls remained negative for prrsv rna throughout the study. the prevalence of prrsv rna positive pigs and group mean genomic copy numbers/ml are summarized in table . sequencing of the orf gene of prrsv and comparison with the original inocula confirmed that the correct prrsv isolates were present in the table prevalence of anti-prrsv antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups on days post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. different superscripts (a, b, c) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of anti-pcv igg antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups except prrsv-i- and prrsv-i- on day post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). grey shaded areas indicate the presence of pcv seropositive pigs (s/p ratio > . ) within a treatment group. different superscripts (a, b) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of ifn-g and mean group concentration (pg/ml) in the different treatment groups on day post-inoculation (dpi) , , , and . data presented as prevalence (mean log group concentration ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. respective groups and rooms. when results were analyzed for a possible effect of ''prrsv strain'', a significantly (p < . ) higher amount of prrsv rna was detected in pigs infected with vr- at dpi and compared to pigs infected with nc b ( fig. ) . when the pigs infected with prrsv alone were removed from the analysis, coinfected pigs with nc b had significantly higher amounts of prrsv rna in serum compared to pigs infected with vr- on dpi ( . ae . versus . ae . ) and dpi ( . ae . versus . ae . ), respectively. an effect of ''pcv '' or ''pcv subtype'' on prrsv replication was not evident. all pigs were negative for pcv -dna in serum at dpi and the negative controls and b and b pigs remained pcv dna negative throughout the study (data not shown). at dpi , / pcv -inoculated pigs were positive for pcv -dna, and all pcv -inoculated pigs were positive for pcv -dna by dpi and remained positive until dpi . the log group mean pcv dna amounts are summarized in fig. . when results were analyzed for a possible effect of ''prrsv strain'' it was found that there was a significantly higher amount of pcv dna in pigs infected with vr- ( . ae . ) compared to pigs infected with nc b ( . ae . ) on dpi . there was a significant effect of ''pcv subtype'' on dpi ; groups infected with pcv b had significantly higher amounts of pcv dna in serum compared to groups infected with pcv a ( . ae . versus . ae . , respectively). an effect of ''pcv subtype'' was not evident in the later stages of infection. all pigs in the pcv a or pcv b groups were correctly infected with their respective subtype as determined by multiplex real-time pcr (data not shown) and crosscontamination between groups and rooms was not detected. table prevalence of prrsv and group mean log prrsv genomic copies per ml in the different treatment groups on days post-inoculation (dpi) , , and . data presented as prevalence (log prrsv rna ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included into the analysis. group negative controls / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a coi- - a / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - a / ( . ae . ) b,c / ( . ae . ) b,c / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) c / ( . ae . ) c / ( . ae . ) b / ( . ae . ) b b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) different superscripts (a,b,c,d) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. fig. . log transformed mean prrsv rna genomic copies (aese) in vr- and nc b infected pigs regardless of coinfection status on day postinoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by asterisks. the lines indicate the linear trend for pigs infected with vr- (gray, dashed) or nc b (black, solid). macroscopic lesions were characterized by mild-tomoderate enlargement of lymph nodes (especially tracheobrochiolar lymph nodes and mediastinal lymph nodes) and mottled-tan lungs with varying degrees of the lung surface affected by visible pneumonia lesions. the group mean lung lesion severity scores are summarized in table and were significantly (p < . ) lower for negative controls compared to all coinfected groups. there were no significant differences in lung lesions severity between the negative controls and the pigs infected with prrsv alone. there was an effect of ''pcv '' on the mean group macroscopic lung lesion scores as evidenced by the coinfected pigs having more severe macroscopic lung lesions compared to pigs infected with prrsv alone. however, there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed macroscopic lung lesions. lung tissues had multifocal-to-diffuse, mild-to-severe, lymphohistiocytic interstitial pneumonia. the mean microscopic lung lesion scores, which are summarized in table , were significantly (p < . ) lower in the negative controls compared to the four coinfected groups; however, the scores in the negative controls were not significantly lower than observed in the groups singularly infected with prrsv. there was a significant effect of ''pcv '' (p < . ) on microscopic lung lesions but there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed microscopic lung lesions. lymphoid lesions were either not observed or were characterized by mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups. significant differences in lymphoid lesion scores were not observed among the groups (data not shown). . . . prrsv all control pigs were negative for prrsv antigen by ihc on sections of lung. the prevalence of prrsv antigen in lung sections was / pigs in the nc b-inoculated group (b : / pigs) compared to / pigs in the vr- -inoculated group (b : / pigs). there were no significant differences in the prevalence rates of prrsv fig. . log transformed group mean pcv dna amounts (aese) in the pcv -prrsv coinfected groups on day post-inoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by different superscripts (a, b). mean group macroscopic (percentage of lung surface affected by lesions) and microscopic (interstitial pneumonia ranging from = normal to = severe, diffuse) lung lesions (mean group amount ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. significant (p < . ) differences between groups are indicated by different superscripts (a, b, c). macroscopic lung lesions ( - %) antigen in lungs between the virus-inoculated groups. the prevalence of prrsv antigen in lungs was independent of ''prrsv strain'' or ''pcv subtype''. all control pigs and all b and b pigs were negative for pcv antigen by ihc. low-to-high amounts of pcv antigen in lung sections were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pcv a-inoculated pigs and / pcv b-inoculated pigs. moreover, pcv antigen was detected in / vr- -inoculated pigs and in / nc b-inoculated pigs. the prevalence of pcv antigen in lung tissues was independent of ''prrsv strain'' or ''pcv subtype''. in lymphoid tissues, low-tohigh amounts of pcv antigen were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pigs inoculated with pcv a and / pigs inoculated with pcv b, as well as / pigs inoculated with nc b and / pigs inoculated with vr- . the prevalence of pcv antigen in lymphoid tissues was independent of ''prrsv strain'' or ''pcv subtype''. the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates recovered from pigs in and in a conventional pig model. to mimic field conditions where coinfections frequently occur, the pigs were concurrently infected with either pcv a or pcv b. the effect of each prrsv isolate was also evaluated in singularly inoculated pigs. however, due to limitations in numbers of available pigs from the source herd, the experiments with singularly prrsv-inoculated groups were conducted separately but under the same study conditions, using the same inocula and assays to analyze the samples. the prrsv isolate vr- used in this experiment has been well-characterized in the cdcd and the conventional pig models and is considered a relatively highly pathogenic prrsv isolate from the s (halbur et al., b meng et al., ) . in contrast, nc b represents a more recent prrsv isolated from an outbreak of respiratory disease on a farm characterized by high morbidity and mortality in (gauger et al., ) . the orf - sequence homology between vr- (genbank accession pru and pru ) and nc b (genbank accession hq ) was approximately . %. the orf region demonstrated the least nucleotide and amino acid homology at . % and . %, respectively (gauger et al., ) . in the past, dual infections with prrsv and porcine respiratory coronavirus (prcv) or prrsv and siv were studied using conventional pigs (van reeth et al., ) and gnotobiotic pigs (van reeth and nauwynck, ) and in general disease was found to be more pronounced in dually inoculated pigs. interestingly, in gnotobiotic pigs the effect of the coinfection appeared additive rather than synergistic (van reeth and nauwynck, ) . more recent studies have shown that prrsv modifies the innate immune response, induces immunosuppression and enhances the inflammatory response to prcv in pigs (jung et al., ; renukaradhya et al., ) . in another study, dual infection of specific pathogen-free pigs with prrsv and pseudorabies virus (prv) resulted in more severe clinical signs and increased pneumonia in pigs inoculated with both viruses compared to pigs infected with prrsv or prv alone (shibata et al., ) . it is also well recognized that pcv replication is enhanced by concurrent prrsv infection in both cd and conventional pigs compared to singularly inoculated pigs (allan et al., ; rovira et al., ) . to the authors' knowledge, the pathogenicity of genetically different prrsv isolates in the presence of concurrent viral infection has not been evaluated in vivo. the combination of prrsv and pcv is one of the most common coinfections associated with swine respiratory disease under field conditions (dorr et al., ; pallaré s et al., ) . both prrsv isolates used in the current study were isolated from field cases of high mortality and experimental infection of pigs with prrsv vr- has resulted in severe lesions and high levels of viremia (halbur et al., b . the two pcv isolates were initially recovered from typical field cases of pcvad in iowa and north carolina and have been characterized in the conventional pig model side by side without identifiable differences between pcv subtypes (opriessnig et al., b; sinha et al., ) . in the current study, clinical disease in the treatment groups was characterized by variable numbers of infected pigs experiencing transient, mild lethargy, mild respiratory disease and inappetence. coinfected groups had significantly higher mean rectal temperatures compared to pigs infected with prrsv alone and the negative controls. interestingly, when organized by coinfection status and analyzed by pcv subtype, pigs inoculated with pcv a had significantly higher mean group rectal temperatures compared to pigs inoculated with pcv b on dpi which was associated with an anti-pcv -antibody response in . % ( / ) of the pcv ainoculated pigs on dpi whereas a delayed antibody response was seen in pcv b-inoculated pigs ( . %; / ). it is well documented that pathogenic differences between type prrsv isolates exist (halbur et al., b . the uniqueness of the current study is the utilization of two temporally and genetically different prrsv isolates both from cases of high morbidity and mortality in the field but isolated years apart. in a separate in vitro study comparing phenotypic traits of the two prrs viruses, nc b demonstrated reduced growth characteristics compared to vr- (gauger et al., ) . nc b plaque sizes were slightly smaller than vr- and the peak viral titer demonstrated by nc b was approximately -fold lower than the vr- peak titer. this is in contrast to the in vivo growth characteristics demonstrated in this report. there were clear differences in initial replication between the two prrsv isolates used in this study. the vr- -inoculated pigs had significantly higher levels of prrsv rna in serum on dpi and . moreover, nc b replicated at higher levels at dpi and dpi compared to vr- which was associated with significantly lower levels of lymphocytes at dpi and a significantly lower n/l ratio at dpi . these results suggest that highly pathogenic prrsvs may replicate more efficiently in vivo in contrast to their decreased growth properties in vitro as previously suggested (johnson et al., ; wang et al., ) . this is further supported by the data obtained from the pigs infected with prrsv alone (b and b ) which clearly show an increase in replication in pigs infected with nc b in the later stages of the in vivo study. similar to other pcv -prrsv coinfection studies (allan et al., ; harms et al., ) , macroscopic and microscopic lesions in coinfected groups were enhanced compared to pigs singularly infected with prrsv. recently, it has been shown that pigs infected with vr- had significantly prolonged (until dpi) pcv viremia and shedding in prrsv-pcv coinfected pigs (sinha et al., ) . a similar approach using prrsv nc b, which replicated differently from vr- in the early stages of infection, could potentially offer new insights on viral interactions in pigs. in the current study, pcv b replication was significantly up-regulated shortly after initiation of the study at dpi compared to pcv a. furthermore, the coi- - b group had significantly higher quantities of pcv b in the serum compared to coi- - a (dpi and ) and coi- - b (dpi ) which was associated with a higher prevalence of pcv antigen in tissues ( . % versus . %) indicating a synergistic relationship between prrsv- (vr- ) and pcv . unlike previous studies where the average trial length ranged from to days (allan et al., ; rovira et al., ) , this trial was terminated at dpi to evaluate prrsv-induced lung lesions which tend to be most severe between dpi and dpi . it remains unknown if the observed trend would have resulted in a difference in clinical disease in the later stages of infection. as expected, and similar to a previous study , the pathological lesions associated with pcv were either not present or they were mild; however, pcv antigen was detected in most tissues in coinfected pigs. in this study, pcv naïve pigs were utilized, thus the relevance of the model to actual field situations is limited considering the majority of young pigs have high levels of passively acquired anti-pcv antibodies (opriessnig et al., b) . therefore, the impact of anti-pcv immunity on the pcv infection could not be ascertained in the experiment; however, this was not a major concern as we know from several experiments that pigs with passively derived antibodies, although protected from clinical pcv associated disease, can still be infected with pcv (mckeown et al., ; opriessnig et al., a) . therefore, we believe that a pcv naïve pig model increases the ability to identify trends and associations between prrsv and pcv . in the current study, prrsv-pcv coinfection was administered intranasally on the same day. this model of simultaneous dual inoculation does not fully mimic the population dynamics due to the variability in timing of exposure to these two pathogens within and between herds in field situations. on many conventional farms, endemic exposure and seroconversion to prrsv often occurs earlier than exposure to pcv . infection of pigs with prrsv prior to pcv may contribute to the manifestation of more severe pcv -induced clinical disease and lesions. prrsv is immunosuppressive, primarily infecting porcine alveolar macrophages (drew, ) , which decreases the pig's ability to clear subsequent infections. in contrast, prior prrsv infection may induce an immunostimulatory effect on the host immune response that serves to enhance pcv replication and lesions (krakowka et al., ) . it is possible that amino acid mutations acquired during serial passaging of prrsv on marc- cells could result in attenuation as reported previously (allende et al., ; an et al., ) . while this is also applicable to the current study, we attempted to minimize this risk, by using a relatively low passage of both viruses with a pig passage followed by only two in vitro passages in marc- cells. inoculation was completed two days after weaning and transport of the pigs to the research facility. it is also possible that the stress from weaning, transport, new socialization, and adjusting to a new environment may have affected the ability of the pigs to respond to concurrent prrsv-pcv infection and influenced the level of prrsv replication in the pigs. however, the data obtained from pigs infected with prrsv alone indicate that this was not the case and that the ability of the pigs to develop a humoral immune response was normal. overall, the data indicate no significant differences between the two prrsv isolates based on clinical signs, gross pathology, histology or hematology even though the prrsv isolates we utilized in this study were isolated from geographically separated herds (vr- from iowa and nc b from north carolina) over a period of years. differences in in vivo replication kinetics were identified. vr- initially replicated more quickly and to higher levels and peaked at dpi and the amount of vr- rna steadily declined thereafter. in contrast, pigs infected with nc b had lower levels of prrsv rna in serum initially and this steadily increased through termination of the study at dpi . concurrent pcv viremia was enhanced by prrsv vr- infection but not by concurrent prrsv nc b infection. a higher prevalence of pcv antigen was demonstrated in the lungs of pigs coinfected with vr- ( . %) compared to pigs coinfected with prrsv nc b ( . %). this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens on prrsv kinetics. additional investigations are necessary to further elucidate the 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contribution of antimicrobial use in human and veterinary medicine to the emergence and spread of resistant bacteria, the use of these substances has to be accurately monitored in each setting. currently, various initiatives collect sales data of veterinary antimicrobials, thereby providing an overview of quantities on the market. however, sales data collected at the level of wholesalers or marketing authorization holders are of limited use to associate with the prevalence of bacterial resistances at species level. we converted sales data to the number of potential treatments of calves and pigs in switzerland for the years to using animal course doses (acd). for each authorized product, the number of potential therapies was derived from the sales at wholesaler's level and the acd in mg per kg. for products registered for use in multiple species, a percentage of the sales was attributed to each authorized species according to their biomass distribution. we estimated a total of , , therapies for pigs and , , for calves in . using the number of slaughtered animals for that year as denominator, we calculated a treatment intensity of . therapies per pig and . per calf. between and , sales of veterinary antimicrobials decreased by %. the calculated number of potential therapies decreased by % for pigs and % for calves. an analysis of treatment intensity at antimicrobial class level showed a decrease of % for colistin used in pigs, and of % for macrolides used in both pigs and calves. whereas the use of rd and th generation cephalosporins in calves decreased by . %, usage of fluoroquinolones increased by . % in the same period. corresponding values for pigs were − . and + . %. this is the first extrapolation of antimicrobial usage at product level for pigs and calves in switzerland. it shows that calves were more frequently treated than pigs with a decreasing trend for both number of therapies and use of colistin, macrolides and cephalosporins rd and th generations. nonetheless, we calculated an increase in the usage of fluoroquinolones. altogether, this study's outcomes allow for trend analysis and can be used to assess the relationship between antimicrobial use and resistance at the national level. to evaluate the contribution of antimicrobial use in human and veterinary medicine to the emergence and spread of resistant bacteria, the use of these substances has to be accurately monitored in each setting. currently, various initiatives collect sales data of veterinary antimicrobials, thereby providing an overview of quantities on the market. however, sales data collected at the level of wholesalers or marketing authorization holders are of limited use to associate with the prevalence of bacterial resistances at species level. we converted sales data to the number of potential treatments of calves and pigs in switzerland for the years to using animal course doses (acd). for each authorized product, the number of potential therapies was derived from the sales at wholesaler's level and the acd in mg per kg. for products registered for use in multiple species, a percentage of the sales was attributed to each authorized species according to their biomass distribution. we estimated a total of , , therapies for pigs and , , for calves in . using the number of slaughtered animals for that year as denominator, we calculated a treatment intensity of . therapies per pig and . per calf. between and , sales of veterinary antimicrobials decreased by %. the calculated number of potential therapies decreased by % for pigs and % for calves. an analysis of treatment intensity at antimicrobial class level showed a decrease of % for colistin used in pigs, and of % for macrolides used in both pigs and calves. whereas the use of rd and th generation cephalosporins in calves decreased by . %, usage of fluoroquinolones increased by . % in the same period. corresponding values for pigs were − . and + . %. this is the first extrapolation of antimicrobial usage at product level for pigs and calves in switzerland. it shows that calves were more frequently treated than pigs with a decreasing trend for both number of therapies and use of colistin, macrolides and cephalosporins rd and th generations. nonetheless, we calculated an increase in the usage of fluoroquinolones. altogether, this study's outcomes allow for trend analysis and can be used to assess the relationship between antimicrobial use and resistance at the national level. keywords: antibiotics, antimicrobial consumption, course dose, pigs, calves introduction use of antimicrobials contributes to the emergence and spread of resistant bacteria in both humans and animals. as early as in the 's concerns arose in relation to therapeutic, preventive and growth-promoting treatments in food-producing animals. the fact that most antibiotic classes are administered to treat infections in both humans and animals was one of the major concerns ( , ) . monitoring antimicrobial usage is therefore a prerequisite to assess the impact of antibiotic treatments on the selection and spread of bacterial resistances. in order to achieve that goal, a number of programs monitoring sales and/or usage of antimicrobials have been established both at national level, for example in switzerland [arch-vet; ( )], denmark ( ), and international level [esvac project of the european medicines agency; ( ) ]. these programs do not only aim at the identification of trends in sales and usage of antimicrobial classes but should also allow establishing a link with changes observed in resistance monitoring programs, thereby providing a basis for risk assessment and evaluation of regulatory interventions ( ) . in order to assess the association between antimicrobial use and resistance, it is of crucial relevance to obtain consumption data at species or, when possible, production type level; there are several species and production type-specific factors that can impact on the relationship between use and resistance. those factors include age at treatment, age and weight at slaughter, products available per species or production type, and especially production structures ( ) ( ) ( ) . antimicrobial sales data are defined as the minimal standard for monitoring programs by the world organization for animal health [office international des epizooties, oie; ( ) ]. they can be collected at either the manufacturer, wholesaler or pharmacy level depending on the national distribution routes of the products. sales data are useful to evaluate long-term trends but do not include information about dose, route of administration, indication or duration of therapy. however, in the context of resistance epidemiology, only data about actual use of antimicrobials collected either at prescription or patient level might deliver the information necessary to establish and evaluate implemented measures. such data can only be currently collected in few countries with advanced collection systems, such as denmark ( ) and the netherlands ( ) among other european countries. the aacting network is maintaining a list of the various collection systems already in place (www.aacting.org). the collection of data at animal level is the ultimate goal of antimicrobial monitoring systems and, until this is available in all participating countries, alternatives using normalization of sales data by the total weight of the food producing animal population as a denominator have been developed. one such denominator is the population correction unit of the esvac project ( ) . other institutions ( ) and countries, including canada ( ) and switzerland [arch-vet; ( )], have implemented similar methods in their surveillance systems. as usage of antimicrobials is strongly dependent on population structure and repartition between high and low-using species, the normalization by weight may provide information on long-term trends but at the same time, a higher usage in one species will be "diluted" by lower usage species/production types (like dairy cows) with a large contribution to the overall livestock biomass ( ) . it is therefore important to measure antibiotic consumption as near as possible to the end users, i.e., to obtain information on species, dosage, duration and whenever possible, indication. the extrapolation of sales data using course doses is an interim measure to data collection at animal level. course dose indicators have been proposed, such as the animal course dose (acd) by the french agency for food, environmental, and occupational health & safety ( ) or defined course dose (dcdvet) from the ema ( ) . an advantage of acd is its product-specific calculation, therefore better representing national specificities than dcdvet units. acds are established for each product using data from the summary of product characteristics (spc) and contain the necessary detail on both dose and therefore potency, and duration of use. the main aim of this study was to provide for the first time an extrapolation of the available national sales data to the number of treated animals in switzerland. we chose to specifically investigate the treatment of pigs and calves because these are mainly reared and treated in groups via oral application. due to the lack of detailed data about repartition of sales, we made assumptions regarding weight at treatment and repartition of sales data between species using a previously published repartition method. we then defined acd for each product containing antimicrobials authorized in switzerland for use in either pigs or calves and combined this information with national antibiotic sales data to extrapolate the number of potentially treated animals during the years to . veterinary antibiotic sales data for the years to were obtained from the federal office of food safety under a confidentiality agreement. since , sales data are collected in switzerland from marketing authorization holders based on article of the ordinance of veterinary medicines ( ) . marketing authorization holders are required to deliver data on every product containing antimicrobials that was sold during a calendar year. products subject to data collection are defined by their atcvet codes ( ) as listed in the esvac project ( ) . additionally, data on antibiotic products not considered by the esvac project, like sprays or products to treat sensory organs, are also collected. data obtained from the federal office of food safety for this study contained the quantity of active antimicrobial ingredient sold in kilogram for each product and year under investigation. the amount of antimicrobials sold of products authorized for a single species was directly assigned to their target species. for each product authorized for more than one species, a repartition had to be determined. we used two distinct methods: the first one was used for premixes, the latter being legally defined in switzerland as being "veterinary medicinal products used to treat groups of animals and incorporated into either water or feed" [ordinance on authorizations for medicinal products, art. ; ( ) ]. for all of these products, periodic safety update reports (psurs) containing data on species repartition submitted to swissmedic, the swiss agency for therapeutic products, during the years to , were used. as premixes represented only products from a total of under investigation but between . % ( ) and . % ( ) of the total sales, another repartition method had to be used for oral solutions, oral powders and injectables. this repartition was done according to biomass repartition as described by carmo et al. ( ) . briefly, for each product authorized for one or more target species, each target species was assigned a percentage of kg of the total sales representing the proportion of its biomass in the total biomass of the list of authorized species for the product. for the present study, food producing animal population numbers were obtained from the federal office of statistics (www.bfs.admin.ch), number of dogs from the anis database (identitas ag, bern, www.anis. ch) and the number of cats from the swiss association of pet food producers (verband für heimtiernahrung, bern, www.vhn. ch). in analogy with calculations of the population correction unit (pcu) of esvac ( ) , the number of slaughtered animals were used for fattening pigs and calves, whereas data for dairy cows, sows, sheep, goats, horses, dogs, and cats represent live animals. throughout the text and in the tables, "pigs" refer to fattening pigs. supplementary table lists the number of animals and the weights used for the biomass repartition. the most likely weight at treatment was sourced from the esvac report ( , ) . as heavy animals with a rather low treatment intensity, like dairy cows, skew the biomass repartition, we chose to only include them in the calculation when they were either explicitly listed as authorized species ("dairy cows") or, when a withdrawal time for milk was given in the spc of the product. for pigs, we did not include the production stage of piglets or weaners. using the number of animals in different production stages presents some challenges, the most prominent one for pigs being the lack number of acds = total quantity of active ingredient sold in one year (mg) daily dose mg kg × duration of tratment days × weight at treatment (kg) therapeutic intensity in species x = number of acds in species x total number of animals for species x of available data for the repartition of use between piglets and e.g., fattening pigs. only few antimicrobials are primarily used in piglets or weaners, colistin being such an example. for almost all other products authorized for pigs, no data are available to stratify antimicrobial consumption per different age classes using sales data. repartition data will only be available once reporting of all treatments with antimicrobials in switzerland is made mandatory at the end of the year . for this reason and because sales data include the use of antimicrobials by all the age categories of the species for the years under investigation, we used slaughtered numbers of pigs as denominator for the therapeutic incidence in this species. finally, for injectable products authorized without indication of the production stage ("bovines" containing dairy cows and "pigs" representing slaughtered pigs and sows) we used raw data provided by experts for the study by carmo et al. ( ) to determine if a use would take place in the particular production stage under consideration. the animal course dose (acd) was calculated for each active pharmaceutical ingredient contained in each product authorized during the years under investigation. data were collected from the authorized summary of product characteristics ( ) and entered into an ms excel sheet containing: name of the product, authorization number, list of authorized species, active ingredient(s), dose and duration. doses given in international units were converted to mg using conversion factors listed in the esvac report ( ) . whenever the recommended dose was a range, the highest recommended dose and longest duration were chosen to reflect the minimal number of animals potentially treated. moreover, when different doses were authorized for different indications, the most likely indication was chosen. this was the case for products presenting both a prophylactic or metaphylactic indication with different doses and duration. acds were defined per kg and the acd per animal obtained by multiplication with the likely weight at treatment. to take swiss specificities into account, the weight at treatment for pigs was taken from a previous study by schnetzer et al. ( ) and the weight for calves based on expert opinion (prof. m. kaske, zurich, personal communication). therapeutic intensity reflects the number of acds per slaughtered animal (pig or calf) in year. for combination products, the number of acds was calculated separately for each active pharmaceutical ingredient. therefore, a single treatment with a combination containing antimicrobials results in acds. acd and intensity were calculated using the following equations: from the year to , sales of antibiotics for use in food producing animals decreased by . % ( table ). in the same time, the percentage represented by premixes decreased from . to . %. therefore, measured in kg, antimicrobials sold in premixes made the largest part of yearly sales of antimicrobials for the veterinary medicine. as a consequence, pigs and calves are the most pertinent species among food producing animals to be investigated for use and trend detection. in tonnage sold for use in these species, the decrease in the years under investigation is comparable: . % in pigs and . % in calves. however, normalizing these numbers to the respective biomass of the produced (slaughtered) population reveals a much higher use per kg of biomass for calves ( . mg/kg biomass in ) than for pigs ( . mg/kg biomass). the difference between both species even increased from . -fold higher for calves in to . in . normalizing sales data to either the overall biomass of food producing animals or to the biomass of a particular species is a crude estimate of antimicrobials use, not taking dose or duration into account. we therefore calculated the number of course doses (acds) per product and species. a summary of the results is presented in table . the total number of acds was approximately . times higher in pigs and decreased by . % over the years under investigation, whereas the decrease for calves was . %. normalization to the number of slaughtered animals showed a much slower decrease of . % for calves between and compared to . % in pigs. as a result, the difference between both species grew from . -fold in the year to . -fold in the final year under investigation. not all antibiotics have the same potential impact on resistance selection and consequences for the treatment of both humans and animals. moreover, different products are authorized for distinct conditions in pigs or calves. the repartition of the number of acds per class of antimicrobials was therefore calculated separately for each species for the year . table presents the repartition by antimicrobial class for ingredients sold in premixes and as parenteral injections. in this year, polymyxins (in form of colistin) were the class with the highest potential numbers of acds per pigs, followed by sulfonamides. in calves, the highest number was represented by penicillins (mainly sold as aminopenicillins) followed by tetracyclines. the total number of acds per animal was . times higher in calves than in pigs. the same calculation was done for injectable products as these may contain antimicrobials of the highest priority [hpcia; ( ) ] not available for oral application. for pigs, the highest number of acds per animal in the year was represented by macrolides, followed by aminoglycosides and fluoroquinolones. for calves (amino)penicillins were the class with the highest number of course doses per animal, followed by macrolides and aminoglycosides. the total number of potential acds per animal for injectable products in the year was . for calves and . for pigs. finally, the evolution of the number of potential acds per animal for hpcias is presented in table . for macrolides used in pigs, a decrease of . % for products sold as premixes was attenuated by a corresponding increase of . % for injectables. this pattern was even more evident in calves where a reduction of . % for premixes was almost completely compensated by an increase of . % in injectables. with respect to the other two classes of hpcias, sales of products containing fluoroquinolones remained stable for pigs (− . %) and an increase of . % was observed for the number of potential acds per animal in calves. courses with cephalosporins of the third and fourth generations showed a comparable decrease in pigs (− . %) and calves (− . %). this is the first study at national level using the acd concept applied to sales of antimicrobials with the objective of extrapolating the number of potentially treated pigs and calves in switzerland. sales of antimicrobials for the veterinary medicine are published at national level since . so far, these data represent the only available source of exhaustive antimicrobial consumption data at national level. sales figures may allow for the recognition of trends, but the lack of information on potency, dose, duration of treatment and repartition per species strongly limits their usefulness. the indicator acd may therefore help to bridge that gap. calculation of acd and repartition of quantities for products authorized for more than one species would not be possible without making assumptions, which might influence the results. the first assumption relates to the weight of the animals. the standard weight has an impact on both the calculation of the species repartition and the acd indicator itself. the impact of using different weights is a topic beyond the scope of this study and the impact on calculations has been studied elsewhere ( ) ( ) ( ) . in this study we used weights at treatment as close as possible to the swiss reality. this should provide the best fitting results, and also guarantees future reproducibility of the method and comparison of results, as these weights are likely to be used when quantifying swiss antimicrobial consumption both at national and international level. this approach is comparable to the one chosen by the esvac project. the method used to stratify antimicrobial consumption by the production types included in the study has some potential bias. as it is based on the total biomass of each animal category, the resulting estimates are highly dependent on the animal demographics and the animal average weight used. this might not always be a representative surrogate of the product repartition by each category. as a reliable repartition is generated by data collected on actual usage, and such data are currently not available in switzerland, we chose an alternative that was applicable at product level that would deliver reproducible results over the years and be as accurate as possible. carmo et al. ( ) have compared three different methods to determine species repartition of antimicrobials. the longitudinal study extrapolation method (based on field data) was not applicable at single product level due to the requirement for minimum, mode, and maximum starting values. the biomass distribution was shown to be the method providing the closest results to the extrapolation based on field data, thereby increasing our confidence on the pertinence of the approach we applied. the two main drawbacks of this method are the dependence on defined average weights and country specific animal demographics. however, the method, limited by the data available in the current swiss context, provides a first insight into antimicrobial consumption patterns in different species/production types. in the future, the data collection system is-abv (description available under http://www.aacting.org/matrix/is-abv/?lid= ) shall provide further insights into these patterns, as well as a basis for comparison with the results from the method and its potential biases. to make our extrapolations as comparable as possible with other projects, we used the same standard weights as in the esvac project ( ) . it must also be noticed that the denominators of the indicators presented were based on the number of slaughtered animals only. the weights used for the calculation of the biomass were likely weights at treatment as defined in the esvac project ( ) . the use of such a calculation might hinder direct comparisons with other studies and should be taken into consideration when benchmarking these results. when using the biomass as a denominator, the result should be interpreted as an indicator for the amount of active ingredient used per kg of animal produced. likewise, the therapeutic intensity indicates the average number of acds per animal produced/slaughtered. both a high proportion of heavier animals like cows or, alternatively, a high treatment intensity in a species of lower biomass are examples of how animal demographics can bias the results of the stratification approach based on the biomass. the repartition across species is mainly influenced by national production structures. in switzerland, dairy production is an important agricultural sector and therefore dairy cows make a high proportion of the food producing animals' sector ( ) . cows represented % of the total biomass in the year and this high proportion leads to an underestimation of the repartition of sales for pigs or calves. this primarily affects the repartition of aminoglycosides and cephalosporins of the third generation, which are antimicrobials frequently used in the treatment of dairy cows. the calculated numbers of acds per animal for these classes presented in table are, therefore, an underestimation. within the same species, biomass repartition could have been used to estimate the use of antimicrobials in different production stages of pigs. however, using piglets, weaners and fattening pigs produced during year introduces the bias of counting a significant but undefined proportion of the animals two or three times. as sales data were only available for one full year, we therefore chose to base our repartition, as well as the denominator for the treatment intensity, on the number of pigs slaughtered during the same year. this indicator is used in this study as a surrogate for all pig production stages. as the numbers of acds represent an extrapolation of usage data based on sales figures, they follow the latter closely. the downward trends in sales is mirrored by the treatment numbers of both calves and pigs. however, differences become evident as soon as additional factors like application route are taken into account. the repartition for pigs in the year shows that % of the active ingredients were used parenterally when based on quantity, whereas they represented % of the treatments when using acds. the main reason for this difference lies in the potency of the active ingredients: antimicrobials are used parenterally with a lower dose as there is no loss of active ingredient compared to the lower bioavailability following oral application. another possible reason is the use of more than one acd for parenterally applied combination products as of injectable products investigated were combinations of two active ingredients. whereas, this approach can be disputed as it shows a higher number of "treatments, " we think that the use of acds is better suited to test for associations between antimicrobial use and resistance. converting sales of antimicrobials to number of treatments per animal allows detection of trends that would not be obvious when only assessing the quantity of active ingredients sold. macrolides used to treat calves provide a good example: our results show a clear shift from the oral application in form of premixes toward an increase in the use of injectables. one possible explanation is the increasing availability of macrolide antibiotics with a long duration of action, e.g., tulathromycin, tildipirosin, and gamithromycin. such active compounds combine the easy use of a single application with a long action. moreover, for parenteral applications, both time to maximal concentration and maintenance of active levels are not influenced by the appetite of the animals, therefore guaranteeing the adequate treatment of sick animals with reduced appetite. on the negative side, studies about macrolides used in human medicine convincingly showed a higher level of resistance selection for longer acting molecules ( ) . our results show a strong difference in the extrapolated usage of antimicrobials in pigs and calves. this cannot be explained by a single factor as the administration of antimicrobials is driven by medical, economic and also psychosocial factors. crowding effect, stress during transport of very young, not yet immunocompetent animals, partially inadequate colostrum feeding and less than ideal stable climate are among the factors favoring respiratory problems in calves ( , ) . in the swine industry, some of the abovementioned factors also exist, but the structure and management of pig production limits the risks. management practices like all-in-all-out including disinfection between the batches or integrated production from piglet to finisher can strongly help to reduce antimicrobial usage. in pigs, there are two main periods at risk for treatment with antimicrobials: the first at weaning with around kg body weight and the second at around to kg body weight ( , ) . in pigs, diarrhea is one of the leading indications for treatment. this is a very unspecific symptom with many different causes, including not only bacterial but also dietary or viral origins. in this context, the availability of vaccines against both circovirus and lawsonia intracellularis infections in the years to contributed to the reduction of diarrheal symptoms, and hence, the rather indiscriminate use of antibiotics to treat such symptoms. for calves, respiratory diseases are much more multifactorial and the introduction of various vaccines (against bovine respiratory syncytial, parainfluenza or corona virus) seems not to have had the same positive effect as in the pig industry. several factors hinder a proper comparison of our results with previously published data. to the best of our knowledge, this is the first time that the acd indicator is used at national level in switzerland. as a matter of fact, its use is not currently widespread in other countries, with the exception of france where it was developed. however, the comparison with french data is difficult. no publication presents the french antimicrobial consumption using acds per animal and year as an indicator. the french indicator for exposure to antimicrobials is alea [animal level of exposure to antimicrobials; ( ) ]. it is obtained by dividing the effectively treated biomass by the total biomass of the same species. the global alea calculated for the year in france was . and represented a decrease of . % compared to . another difficulty is the use of different production categories and standard weights at treatment. for pigs, the french system uses weights up to kg for a specific category of sows and the average for the pig population is set at kg. this is . times higher than the standard weight at treatment of kg identified in previous swiss studies and used here. the differences in the standard weights at treatment also explain the discrepancies in the antimicrobial consumption for france published, for the same year, in the esvac report ( mg/pcu) and in the anses report ( mg/kg). due to the differences in weight and in the categories, and the difficulties in making assumptions and extrapolations, we decided not the compare our figures to the french ones. our data can only be compared with countries where calves are reared for the production of veal meat. besides france and belgium (for which country we could not find adequate data for comparison), this production system also exists in the netherlands. the available report for the year ( ) uses indicators differing from the ones in the present study but still shows a higher treatment intensity in calves compared to pigs. this is in line with the present study, where the antimicrobial use was . -fold higher in calves than in pigs. both examples clearly illustrate the need to harmonize methodologies at international level in order to discuss data collected in different countries. such discussions currently take place within the aactng network (www.aacting.org). this first study of the number of treatments of pigs and calves extrapolated from yearly sales shows both similarities and differences between the two species under consideration. whereas, the sales by species and the number of extrapolated treatments both decreased in a similar way, the difference in the number of treatments per animal between pigs and calves differed over the years under investigation. given that the applied method is based on the extrapolation of sales figures, a similar decrease at species level was to be expected. however, the use of course doses allows to further investigate trends in the patterns of antimicrobial treatments. in our study, this was very clear for the class of macrolides, for which the decreases in oral use were partly (pigs) or completely (calves) compensated by the application of long acting injectables. we, therefore, recommend the use of extrapolated treatment numbers when no exhaustive collection of usage data is in place. the concept of acds can also complement the collection of antimicrobial consumption data at species level allowing for their validation using sales data. all datasets generated for this study are included in the manuscript/supplementary files. rs did all the calculations presented in this work. lc helped with the repartition of sales between species, expert advice and biomass distribution. raw sales 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of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study risk factors for death and unwanted early slaughter in swiss veal calves kept at a specific animal welfare standard antimicrobial drug use and risk factors associated with treatment incidence and mortality in swiss veal calves reared under improved welfare conditions berechnung der therapieintensität bei ferkeln und mastschweinen beim einsatz von antibiotika in fütterungsarzneimitteln monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands in the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fvets. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © stebler, carmo, heim, naegeli, eichler and muentener. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -vu lt w authors: brabb, thea; newsome, denise; burich, andrew; hanes, martha title: infectious diseases date: - - journal: the laboratory rabbit, guinea pig, hamster, and other rodents doi: . /b - - - - . - sha: doc_id: cord_uid: vu lt w although guinea pigs are sensitive and susceptible to the development of lesions from a wide range of viruses, bacteria, protozoa, and parasites, only a small number of organisms cause natural infection and only a portion of that group cause clinical disease. this chapter discusses naturally occurring diseases of guinea pigs, although some data from experimental infections have also been covered as they relate to the pathogenesis of the disease. the material presented includes background, etiology, epizootiology/pathogenesis, clinical manifestations, pathology, diagnosis, prevention, and therapy. the diseases are discussed in an alphabetical order based on the taxonomic groups to which the organisms belong and are independent of the order of perceived importance of the various diseases. the federation of european laboratory animal science associations recommends monitoring for guinea pig adenovirus, guinea pig cytomegalovirus, sendai virus, ectoparasites, endoparasites, e. cunniculi, and a variety of bacteria including bordetella bronchiseptica, chlamydia psittaci, corynebacterium kutscheri, dermatophytes, pasteurellaceae, salmonella, streptobacillus moniliformis, streptococcus, yersinia pseudotuberculosis, and clostridium piliforme. virus-associated necrotizing ronchopneumonia in guinea pigs is a spontaneous multifactorial disease that has low morbidity, high mortality, and a worldwide distribution. although guinea pigs are sensitive and susceptible to the development of lesions from a wide range of viruses, bacteria, protozoa, and parasites, only a small number of organisms cause natural infection and only a portion of that group cause clinical disease. the intent of this chapter is to discuss naturally occurring diseases of guinea pigs, although some data from experimental infections may be covered as they relate to the pathogenesis of the disease. the material is presented under the following headings: background, etiology, epizootiology/pathogenesis, clinical manifestations, pathology, diagnosis, prevention, and therapy. the diseases are discussed in an alphabetical order based on the taxonomic groups to which the organisms belong and are independent of the order of perceived importance of the various diseases. in laboratory colonies, routine surveillance of guinea pigs is conducted monthly, quarterly, or yearly as required. the federation of european laboratory animal science associations (felasa) recommendations include monitoring for guinea pig adenovirus, guinea pig cytomegalovirus, sendai virus, ectoparasites, endoparasites, e. cunniculi, and a variety of bacteria including bordetella bronchiseptica, chlamydia psittaci, corynebacterium kutscheri, dermatophytes, pasteurellaceae, salmonella spp., streptobacillus moniliformis, streptococcus spp., yersinia pseudotuberculosis, and clostridium piliforme. most laboratories also monitor for these additional viruses, simian virus- (sv ), pneumonia virus of mice (pvm), reovirus, lymphocytic choriomeningitis virus (lcmv), and parainfluenza virus- (piv- ). these viruses are routinely monitored both to minimize the risk of transference to mouse and rat colonies (pvm, reovirus, sendai virus, lcmv) and to identify infections that are zoonotic (lcmv, piv- ). the viral taxonomy used follows the recommendations of the viiith international committee on taxonomy of viruses (fauquet, ) , but well-established common names for viruses are used when appropriate. historically, pvm, parainfluenza viruses, cytomegalovirus, and reovirus have been commonly found in guinea pig colonies. from to serologic investigations of laboratory animal colonies originating from ten different european countries found seropositivity for four viral infections in guinea pig stocks; reovirus, pvm, sendai virus, and sv (see paramyxoviridae section for discussion of serological specificity of sendai virus and sv ) (kraft and meyer, ) . in a study guinea pigs reared conventionally were positive serologically for sendai virus, pvm, and reovirus (park et al., ) . adenoviridae: guinea pig adenovirus (gpadv) background virus-associated necrotizing bronchopneumonia in guinea pigs is a spontaneous multifactorial disease that has low morbidity, high mortality, and a worldwide distribution (percy and barthold, ) . after the first description in germany and experimental reproduction of the disease (kunstyr et al., ; naumann et al., ) , sporadic spontaneous cases were reported from the united states, australia, and switzerland (brennecke et al., ) . a -year endemic "in-house" occurrence of adenovirus-associated disease was reported in a british pharmaceutical research facility. in all described spontaneous cases, some additional stress factors were involved (pring-akerblom et al., ) . etiology gpadv is a member of the adenoviridae, genus mastadenovirus that contains canine and bovine adenoviruses as well (fauquet, ) . a portion of the hexon gene was cloned and sequenced and found to be eimeria giardia duodenalis klossiella cobayae leishmania enrietti toxoplasma gondii tritrichomonas caviae trypanosoma cruzi prevention and therapy animals with gpadv are not appropriate for guinea pig pulmonary research projects. rederivation of infected colonies with aseptic hysterectomy or embryo transfer should eliminate the virus. in addition, there could be a concern that natural infection with gpadv would interfere with the use of adenovirus gene vectors. this was examined in a study of gpadv naturally infected guinea pigs that were used as recipients of a human adenoviral vector carrying the gene for green fluorescent protein (hankenson et al., ) . in this model system, no difference in transfection-efficiency was seen in guinea pigs naturally infected with gpadv or gpadv-free. three members of the family herpesviridae that specifically infect guinea pigs are catalogued in the eighth report of the international committee on the taxonomy of viruses (fauquet, ) ; caviid herpes virus (commonly known as guinea pig cytomegalovirus and in some publications as caviid herpesvirus (staczek, ) ), caviid herpes virus (also known as guinea pig herpes-like virus or hsiung-kaplow herpesvirus, or in some publications as caviid herpesvirus (staczek, ) ), and caviid herpes virus (also known as guinea pig x virus) (fauquet, ) . in addition, equid herpes virus , a virus of horses, has been shown to cause severe disease in guinea pigs in one outbreak in a zoo (wohlsein et al., ) . of these viruses, guinea pig cytomegalovirus is the most commonly isolated herpes virus from guinea pigs and serological reactivity to this virus in colonies is not unusual. guinea pig cytomegalovirus is used extensively in research as an animal model for human cytomegalovirus. is consistently recovered from the blood and many other tissues including lung, spleen, and kidney during a -day period beginning at day post-inoculation . viral clearance from the blood and most tissues occurs by days post-infection, but virus remains consistently detectable in the urine and salivary gland during the chronic phase of infection (bia et al., ; hsiung et al., ) . less consistently, virus is found in splenic macrophages, b lymphocytes, kidneys, spleen, pancreas, and cervix of infected animals during this chronic phase. pregnancy can lead to more severe disease and changes in viral load following experimental gpcmv infection griffith and hsiung, ; griffith et al., ) . pregnant guinea pigs may develop interstitial pneumonia, splenomegaly, and necrosis of the liver, kidney, thymus, pancreas, and bone marrow (griffith et al., ) . the mortality rate for pregnant animals during an acute viremic episode following experimental infection is significantly greater than for non-pregnant females. although the fetus is susceptible to gpcmv infection at any time throughout gestation, transplacental transmission occurs most readily during the acute phase of maternal infection. the frequency of stillbirths and viral infection in neonates is highest in animals infected late in gestation. in neonates, brain lesions are observed and virus can be recovered from the salivary glands and, to a lesser extent, from the brain, lungs, pancreas, and liver of the neonates. in some cases, lesions are detected in the salivary glands of offspring up to weeks postpartum. placental infection may be detected after maternal clearance of viremia and maintained despite the presence of significant levels of gpcmv neutralizing antibody. the site of infection is localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobrium of the placenta. whenever infection of the fetus is demonstrated, the placenta is invariably infected; however, infected placentas may be associated with uninfected fetuses. the status of maternal immunity before pregnancy can favorably affect the outcome of maternal or fetal infections with virulent gpcmv strains (staczek, ) . clinical manifestations natural infection with gpcmv causes a latent, persistent infection in the salivary gland generally without serious disease in animals in the vivarium (staczek, ; van hoosier and robinette, ) . however, the death of a sow and fetuses (motzel and wagner, ) and disseminated infection, including pneumonia, in guinea pigs that were not being manipulated experimentally have been reported (van hoosier et al., ) . illness is more likely when associated with pre-existent immune suppression or pregnancy (percy and barthold, ) . transient increase in mononuclear cells is seen as well as histopathological evidence of lymphoid hyperplasia of the spleen in both the t cell and b cell zones, although no changes were noted in the cervical and mesenteric lymph nodes (dowler et al., ) . intranuclear inclusions in tissues have not been noted in vivo, although they are evident in tissue culture cells (dowler et al., ; hsiung et al., a hsiung et al., , b van hoosier and robinette, ) . virus can be recovered from the lymph nodes and spleen of infected animals well after clinicopathological signs of infection end. serologically, this virus is distinct from gpcmv and gpxv and can be differentiated from these viruses both morphologically and serologically . prevention and therapy gphlv is not a clinically important disease of guinea pigs, but could be a complicating factor in research either through interaction with other viruses or cytopathological effects on cultured cells. herpesviridae (fauquet, ) . it was originally isolated from leukocytes of strain guinea pigs that were free of gphlv and gpcmv . in one study of hartley and strain guinea pigs tested serologically for gpxv, % were found to be positive. inoculation of gpxv into hartley guinea pigs caused viremia which persisted for at least weeks . virus could be recovered - months after inoculation suggesting a persistent infection. in this study, % of the animals died between and weeks postinoculation. hepatic necrosis was the only consistent lesion seen. gpxv is both morphologically and serologically distinct from gpcmv and gphlv. prevention and therapy natural disease from gpxv is unknown, but similar to gphlv, this virus may be a complicating factor in research. background and etiology ehv is an equine pathogen that causes a persistent infection in horses with a variety of clinical presentations including respiratory disease, abortion, neonatal death, and neurologic disease (reed and toribio, ) . ehv is a species in the genus varicellovirus of the alphaherpesvirinae subfamily (fauquet, ) . other members of this genus include the type species human herpesvirus (varicella-zoster virus or chickenpox) and bovine herpesvirus (infectious bovine rhinotracheitis virus). one recent report describes infection with ehv resulting in hindlimb paralysis, ataxia, abortion, or stillbirth in of guinea pigs at a european zoo (wohlsein et al., ) . in this outbreak, thomson gazelles (equus thomsoni) kept in the same building as the guinea pigs were first affected and suffered a short course of fatal neurologic disease. the source of the virus was unclear, although a similar strain of virus was isolated months earlier from affected black bears (ursus americanus) and clinically unaffected onagers (equus hemionus kulan). in the second outbreak, a similar strain of virus was also recovered from clinically unaffected zebra (equius quagga boehmi) housed in the same building as the guinea pigs and gazelles. pathology lympho-histiocytic meningoencephalitis was seen predominantly in the olfactory bulb and the frontal cortical regions of the brain with neuronal and glial necrosis, gliosis, and intranuclear inclusion bodies (wohlsein et al., ) . ehv antigen was demonstrated in the neurons, neuronal processes and glial cells by immunohistochemistry and encapsulated herpes viral particles of - nm were detected by electron microscopy. diagnosis differentiation from other members of the herpesviridae is important for diagnosis. immunohistochemistry, virus isolation, pcr, and dna sequencing were used in this report to identify the virus involved (wohlsein et al., ) . prevention and therapy ehv infection of guinea pigs is an example of the severe disease seen when members of the alphaherpesvirinae infect unusual hosts similar to human herpes virus i (herpes simplex virus) which has been shown to infect guinea pigs experimentally (wohlsein et al., ) and has also been reported to cause naturally acquired clinical disease in rabbits (weissenbock et al., ) and chinchillas (wohlsein et al., ) . separation of species, control of fomites, and use of appropriate personal protective equipment can be used to prevent transmission of members of the alphaherpesvirinae subfamily of viruses to aberrant hosts such as guinea pigs. there has been one report of pox-like virus particles obtained from tissue culture samples of fibrovascular proliferations involving the rear limb of half of a colony of guinea pigs (hampton et al., ) . particles resembling pox viruses were observed by electron microscopy. arenaviridae: lymphocytic choriomeningitis virus (lcmv) background the first identified arenavirus, lcmv, was isolated in and shown to cause aseptic meningitis in humans and was named for its ability to cause lymphocytic choriomeningitis in mice and monkeys upon intracerebral injection (amman et al., ; charrel and de lamballerie, ; percy and barthold, ; van hoosier and robinette, ) . lcmv is a rodent-borne zoonotic arenavirus endemic in housemice (mus musculus) worldwide. infection by lcmv in people is known to cause acute central nervous system disease and congenital malformations. etiology the viral genus arenavirus includes viral species, the type species is lcmv (fauquet, ) . like other arenaviruses, lcmv is a non-cytopathic, enveloped, single-stranded rna virus. epizootiology and pathogenesis lcmv is uncommon to rare in guinea pigs (fox, ; percy and barthold, ) . in may , the centers for disease control and prevention reported a cluster of lcmv infections among four solid organ recipients in rhode island and massachusetts who received organs from a single apparently asymptomatic donor (centers for disease control and prevention [cdc], a) . recipients became gravely ill shortly after transplantation; three subsequently died. lcmv was identified as the etiologic agent. viral sequences from the organ recipients were identical to those from a pet hamster acquired by the donor's household days before organ donation. sequence and phylogenetic data provided strong support for the presence of the same lcmv lineage in hamsters and guinea pigs in the rhode island pet store and the ohio distribution center (amman et al., ; cdc, b) . clinical manifestations clinical signs of lcmv are uncommon in the guinea pig, however meningitis with hind limb paralysis has been reported (van hoosier and robinette, ) . pathology lcmv in guinea pigs results in lesions involving the brain including lymphocytic infiltrates in meninges, choroid plexus, and ependyma (percy and barthold, ) . lymphocytic infiltrates are also seen in the liver, adrenal, and lungs. experimentally, neutrophilic destruction of the splenic red pulp, focal bone marrow necrosis, lymphopenia, and death occur following infection with the more virulent (we) strain (djavani et al., ) . diagnosis diagnosis is typically by pcr, ifa, elisa, or mfia (charrel and de lamballerie, ; fox, iii. guinea pigs ; percy and barthold, ) . detection of viral antigen in tissue or section via immunohistochemistry is also feasible. immunity to lcmv is primarily cellular, but virus-specific antibody is produced. prevention and therapy lymphocytic choriomeningitis is a zoonotic disease of concern with rodents kept as pets (pickering et al., ) . human infection occurs most commonly through exposure to secretions or excretions of infected animals. in addition, lcmv can contaminate transplantable tumor or cultured cell lines from mouse, hamster, and guinea pig, and viral stocks (ilar, ; percy and barthold, ) . in mice, lcmv has been shown to limit tumor induction by polyomavirus, mouse mammary tumor virus, and transplantable guinea pig leukemia, delay tumor rejection, and alter sensitivity to endotoxins (ilar, ) . natural infections would be expected to interfere with research studies involving enterohepatic, lymphoid, musculoskeletal, nervous, respiratory, and urinary systems. coronavirus-like particles were found in the feces of young guinea pigs that developed wasting, anorexia, and diarrhea with low morbidity and mortality (jaax et al., ) . at necropsy, there were large amounts of mucoid material throughout the small intestine. histologically, the primary lesion was acute to subacute necrotizing enteritis involving the distal ileum with blunting and fusion of the affected villi, necrosis and loss of enterocytes, and mucosal syncytial cells were evident. by electron microscopy, viral particles consistent with coronavirus were demonstrated in fecal matter from affected animals. coronavirus shedding was also noted in the feces of clinically normal guinea pigs (marshall and doultree, ) . the virus has not been isolated. paramyxoviridae is composed of pleomorphic, enveloped, linear, negative-sense single-stranded rna viruses occurring worldwide in animals and humans, often associated with subclinical infections of the respiratory tract (fauquet, ; simmons et al., ) . the family is split into two subfamilies, paramyxovirinae and pneumovirinae (fauquet, ) . in guinea pigs, three distinct members of the paramyxovirinae have been described; human parainfluenza virus (including human parainfluenza virus , cavian parainfluenza virus , and guinea pig parainfluenza virus ), sendai virus, and simian virus (sv ). in addition, murine pneumonia virus, a member of the pneumovirinae, has been described in guinea pigs. (fauquet, ) . sequence analysis of a guineapig parainfluenza virus isolated in from a colony of guinea pigs in japan indicates that it has . - . % nucleotide identity to human parainfluenza virus and . - . % to bovine parainfluenza virus suggesting this virus was introduced into guinea pigs from a human with human parainfluenza virus (ohsawa et al., ) . a novel paramyxovirus (cavian parainfluenza virus ) was isolated in from a colony of guinea pigs (simmons et al., ) . this virus has % nucleotide identity to guinea-pig parainfluenza virus and human parainfluenza virus and % nucleotide identity to bovine parainfluenza virus . epizootiology and pathogenesis seroconversion in endemic parainfluenza- -positive breeding colony was studied in (blomqvist et al., ) and indicated that pups become infected from weeks to weeks of age. the virus did not persist as sentinels exposed to - -month-old seropositive animals did not seroconvert. experimental infections indicate that seroconversion in naïve animals occurs approximately days following infection (graziano et al., ) . in an infection study guinea pigs inoculated with cavian parainfluenza virus , sendai virus, simian virus (sv- ), murine pneumonia virus, or bovine parainfluenza virus developed no clinical signs of disease or lesions during the eightweek course of the study although they developed robust homologous or heterologous serologic responses to the majority of the antigens (simmons et al., ) . the serologic response, using an elisa, of those inoculated with sv- was modest or equivocal. in addition, sv- elisa resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. in one study, no clinical signs or lesions were seen when guinea pigs were infected with cavian parainfluenza virus (simmons et al., ) . in a separate study, guinea pigs were shown to be susceptible to human parainfluenza virus , developing peribronchiolar and interstitial lesions (clyde, ) . diagnosis identification of the virus via pcr and sequencing is ideal as cross-reactive serological results make interpretation of serology difficult (simmons et al., ) . it is apparent that human parainfluenza virus can be transmitted to guinea pigs and infection-control measures such as the use of face masks, gloves, and laminar flow hoods to prevent transmission in laboratory settings is prudent. it isn't known whether human or cavian parainfluenza virus can be transmitted to humans from guinea pigs. etiology sendai virus, also known as murine parainfluenza virus, is the type species in the genus respirovirus, which also contains the species human parainfluenza virus , bovine parainfluenza virus , and human parainfluenza virus (fauquet, ) . numerous studies have reported seropositivity to sendai virus, including a publication in which describes seropositive reactions by hi or cf in seven out of colonies tested over a -year period in the united states (parker et al., ) . in addition, there is one report of isolation of sendai virus from guinea pigs associated with mice (van hoosier and robinette, ) , although no clinical signs were reported. more recently, five of european guinea pig colonies were seropositive to either sendai virus, human parainfluenza virus , or both (schoondermark- van de ven et al., ) . inoculation of guinea pigs with sendai virus in one report resulted in no clinical signs of disease or lesions (simmons et al., ) . diagnosis as with the other paramyxoviridae, lack of specificity can result in false-positive results with routine serological testing (simmons et al., ) . in one study, sendai elisa-positive samples were seen in guinea pigs infected with pneumonia virus of mice and bovine parainfluenzavirus . guinea pigs were also positive by ifat in the case of bovine parainfluenza virus infection. the importance of sendai virus infections in guinea pigs is unclear. however since it is a significant disease of mice housed in laboratory animal colonies, guinea pigs should be maintained free of sendai virus. in addition, sendai virus is used as a vector in research and natural infection or sero-reactivity may interfere with such uses (baker, ) . sv is a species of the genus rubulavirus which also contains mumps virus and human parainfluenza virus (fauquet, ) . sv was initially isolated from simian kidney cell cultures and has also been shown to infect guinea pig cells in vitro (zakstelskaya et al., ) . serological reactivity to sv has been reported in guinea pig colonies (kraft and meyer, ) . seropositve guinea pigs show no clinical signs or lesions and inoculation with sv results in seroconversion, but not clinical signs of disease (simmons et al., ) . infection of guinea pigs with sendai virus, pneumonia virus of mice, or human parainfluenza virus can result in positive sv elisa results which were negative by indirect fluorescent antibody test, indicating low specificity of the sv elisa (simmons et al., ) . etiology pvm is a member of the genus pneumovirus, species murine pneumonia virus (fauquet, ) . other members of the genus pneumovirus include the human and bovine respiratory syncytial viruses. the complete genomes of two strains of this virus were sequenced in (thorpe and easton, ) . pvm is most often subclinical in immunocompetent mice although in immunodeficient mice, it can cause chronic wasting with cyanosis and dyspnea (percy and barthold, ) . seropositive reactions to pvm have been seen historically in guinea pig colonies in animals without apparent disease (van hoosier and robinette, ) . inoculation of guinea pigs with a strain of pvm that caused mortality in immunocompetent and immunodeficient mice resulted in no clinical signs, although the guinea pigs seroconverted within days after inoculation (griffith et al., ) . prevention and therapy transmission of pvm from guinea pigs to mice, or vice versa, appears to be possible and similar in this regard to sendai virus. in laboratory colonies, it is important to keep guinea pigs free of pvm in order to minimize potential transmission to mice. it is unclear whether laboratory guinea pigs may harbor a poliovirus which, in , was described as the cause of a disease called guinea pig lameness (hansen et al., ; van hoosier and robinette, ) . a similar disease was described by rohrer in (van hoosier and robinette, ) . in these investigations, sera from affected guinea pigs reacted positively to theiler's murine encephalomyelitis virus (tmev) antigens. clinical signs involved a flaccid paralysis and weight loss with an incubation period of - days, and a duration of - weeks. affected animals had meningomyeloencephalitis. more recently, two pet shop guinea pigs were reported to suffer from rear limb paresis and generalized debilitation. the guinea pigs had extremely high titers against "poliovirus", while healthy guinea pigs from the same pet shop were negative (hansen et al., ) . the diseased guinea pigs recovered iii. guinea pigs fully after treatment with vitamin c in the drinking water. further testing of other guinea pig colonies by the same authors demonstrated antibody responses to tmev in out of laboratory guinea pig sera. positive results were found in two out of six breeding centers, and in three out of three experimental units, all of which purchased guinea pigs from one of the seropositive breeding colonies. guinea pig sera from some of the breeding units were also positive serologically for other infections; adenovirus, pvm, reovirus type , sendai virus, sv and encephalitozoon cuniculi. in experimental studies of susceptibility of laboratory animals to six strains of human poliovirus, newborn guinea pigs were the only species of laboratory animals in which multiplication of any of the viruses could not be detected (koroleva et al., ) . naturally occurring antibodies to reovirus have been detected in laboratory guinea pig colonies by ifa, elisa, and mfia (percy and barthold, ; van hoosier and robinette, ) . no clinical signs or lesions have been described. experimentally guinea pigs have been exposed to a reovirus associated with the sars coronavirus (liang et al., ) . background cavian leukemia was first described in by congdon and lorenz as being caused by a retrovirus, however herpes viral inclusions have also been seen in leukemic guinea pigs (jungeblut and opler, ; opler, ; percy and barthold, ; van hoosier and robinette, ) . etiology type c retrovirus particles have been seen in cases of cavian leukemia van hoosier and robinette, ). an endogenous guinea pig retrovirus was induced by bromodeoxyuridine (michalides et al., ; nayak, ) . molecular hybridization techniques were used to show that guinea pig virus nucleotide sequences are endogenous to both domestic (cavia porcellus) and indigenous (cavia aperea) guinea pigs, but cannot be detected in the dna of other mammals tested (dahlberg et al., ) . in , a transplantable leukemia (ksl) of unknown etiology, that had the characteristics of a b cell tumor, was found in a female sewall-wright strain guinea pig. ksl leukemia was also shown to be distinct from another guinea pig lymphatic leukemia (l c) with respect to cell morphology, antigenicity, and in vivo growth rate (key, et al., ) . a c-type virus was reported in the urine of diabetic guinea pigs (lee et al., ) . clinical manifestations clinical signs of cavian leukemia include lethargy, pale mucous membranes, secondary infections, peripheral lymph node enlargement, and death (van hoosier and robinette, ) . leukocyte counts may vary from - , with the preponderance of cells being lymphoblastic. peripheral lymphadenopathy is generally evident. pathology enlarged lymph nodes in the cervical, axillary, mediastinal, retroperitoneal, and inguinal areas are seen in cavian leukemia with splenomegaly and hepatomegaly (van hoosier and robinette, ) . there is a moderate infiltration of leukemic cells in all organs including the spleen, liver, bone marrow, lung, thymus, gastrointestinal associated lymphoid tissue (galt), heart, eyes, and adrenals (opler, ) . treatment and control there is no described treatment. rabies has been reported in a pet guinea pig associated with a bite from a raccoon (eidson et al., ) . most clostridia are large, spore-forming, grampositive, anaerobic rods. the one exception is c. piliforme which is gram negative and is discussed in the section covering gram-negative organisms below. two species of clostridia are discussed here, c. difficile and c. perfringens. background severe profuse diarrhea, associated with clostridium difficile in guinea pigs treated with antibiotics, is an important and preventable disease process. etiology clostridium difficile is most commonly associated with antibiotic-induced typhilitis in guinea pigs, although other organisms including escherichia coli, clostridium perfringens, c. sordelli, c. histolyticum, and bacillus pumilis have been implicated in different studies (boot et al., ; brophy and knoop, ; knoop, ; lowe et al., ; pakes, , ; rehg et al., ; . c. difficile is a common anaerobic, spore-forming, fecal-borne, gram-positive, rod-shaped organism that generally produces both an enterotoxin (toxin a) and a cytotoxin (toxin b) that are crucial in the pathogenesis of disease (greenwood, ) . pathogenesis normal guinea pigs may carry low resident populations of c. difficile in the intestines and toxins can be identified in normal cecal contents (boot et al., ) . in one study, c. difficile was recovered by culture in one out of eight normal guinea pigs and out of guinea pigs with typhilitis. oral or parenteral antibiotic administration, including penicillin, ampicillin, streptomycin, clindamycin, lincomycin, spiramycin, aureomycin, erythromycin, and bacitracin, have all been implicated in causing antibiotic-associated typhilitis by permitting an overgrowth of primarily gram-negative organisms including toxinproducing clostridium such as c. difficile (young et al., ) . the enteric flora of guinea pigs is primarily grampositive and those antibiotics that target gram-positive organisms preferentially are of greatest risk to cause this disease (farrar and kent, ) . one study demonstrated that in the - hours post-administration of penicillin, there was a significant increase in gram-negative anaerobic and aerobic (coliforms predominantly) bacteria bringing the levels of coliform bacteria from less than organisms per gram to - organisms per gram. this shift in the normal gut flora allows bacteria such as c. difficile, which is generally nearly undetectable, to predominate (farrar and kent, ; fox, ; percy and barthold, ) . other gram-negative organisms, e. coli in particular, and other clostridium spp. also can contribute to disease. clostridium difficile (toxin)-associated typhilitis has also been diagnosed in guinea pigs not treated with antibiotics that had been rederived by caesarian section and were exposed to mice as a source of anaerobic flora (boot et al., ) . two years after rederivation, an outbreak of typhilitis was investigated. young guinea pigs - weeks old and a few adults were affected. clostridium difficile was isolated from half of the animals tested and the cytotoxin was identified in cecal contents from % of the affected animals. co-infection with c. perfringens and encephalitozoon cuniculi was present. the authors speculate that the anaerobic flora that the animals were exposed to was not sufficient to protect guinea pigs from c. difficile infection. this same flora has been shown to be unsuitable for rats and rabbits. clinical manifestations generally, affected guinea pigs have weight loss, rough hair coats, anorexia, dehydration, decreased activity, and hypothermia beginning - hours after antibiotic administration (farrar and kent, ; fox, ; knoop, ; . some animals have no stool, while others have diarrhea and acute death has been reported. pathology at necropsy, the cecum is often gasfilled and dilated with hemorrhage evident in the cecal mucosa. histopathologically, there is usually hyperplasia of the ileal mucosa as well as ulceration, edema, and degeneration of the cecal epithelium with leukocytic infiltration. diagnosis c. difficile can be isolated from cecal contents via anaerobic culture (boot et al., ; greenwood, ) . detection of toxin in cecal contents, tissue, or feces via an enzyme immunoassay, cytotoxin activity in cell culture, serum neutralization assay, or pcr are considered the gold standard, however, false positives and false negatives are not uncommon (greenwood, ; houser et al., ; songer, ) . prevention and therapy antibiotic-associated diarrhea is usually treated symptomatically. in humans, administration of metronidazole or vancomycin is standard (greenwood, ; rupnik et al., ; songer, ) . anecdotal information suggests that administration of yogurt or other lactobacillus-containing products in conjunction with antimicrobial agents will prevent or minimize the effects of antibiotic-associated enteritis. a recent study attempted to induce antibiotic-associated enteritis by a single subcutaneous injection of clindamycin . c. difficile and other enteric pathogens were not isolated, however, several guinea pigs that received clindamycin developed enteritis. in affected animals, administration of antibiotics with the oral lactobacillus preparation did not result in any clinical improvement over those animals receiving antibiotics alone. certain pcr ribotypes of c. difficile are found in both human and animal populations, suggesting that this can be a zoonotic disease, although most of these studies are concerned with c. difficile found in food animals rather than guinea pigs (rupnik et al., ) . clostridium perfringens is a ubiquitous gram-positive anaerobic spore-forming bacterium that is the primary cause of enterotoxemia in domestic animals. c. perfringens strains produce a variety of toxins, as many as exotoxins, although four major toxins are associated with typing of the bacteria; alpha, beta, episilon, and iota. infections typically occur via contaminated food, water, bedding, or soil and primarily affect sheep, pigs, and cattle. confirmed c. perfringens infections have been reported in germ-free guinea pigs in and, more recently, in a conventionally housed guinea pig (feldman et al., ; madden et al., ; songer, ) . clinical signs seen in the conventionally housed guinea pig were lethargy and anorexia. at necropsy, there were fibrinous adhesions involving the small intestine and liver (feldman et al., ) . the ileum and cecum had ecchymotic hemorrhages. histopathology revealed acute, multifocal, severe, necrotizing, ulcerative typhlitis and ileitis as well as focal, severe, necrotizing, centrilobular vascular thrombosis of the liver with infarction and coagulative necrosis of the adjacent hepatocyes. pcr was used iii. guinea pigs to identify the toxins of c. perfringens both in the intestine and the liver. corynebacterium etiology corynebacterium spp., members of the family corynebacteriaceae, are gram-positive, non-sporeforming, non-motile, aerobic, pleomorphic rods with coccoid or club-shaped appearance that are catalase-positive and non-acid-fast (boone et al., ; greenwood, ) . pathology corynebacterium spp. can cause disease in guinea pigs, but are also part of the normal flora. specifically, they are commonly isolated from guinea pig conjunctiva. in one study, out of clinically normal pet guinea pigs grew corynebacterium spp. from conjunctival cultures (coster et al., ) . spontaneous disease of guinea pigs associated with corynebacterial infections has rarely been reported, but have involved numerous different species of corynebacterium. in one report, corynebacterium pyogenes was isolated from a guinea pig with septicemia (ganaway, ) . further research with that agent demonstrated it was pathogenic when administered ip to other guinea pigs, but not if it was given orally or intranasally. in another report, c. kutscheri was isolated from the lung of a guinea pig during an epizootic of group c, beta-hemolytic streptococcal disease. further research with this isolate was similar to c. pyogenes, in that intraperitoneal administration resulted in disease while intranasal administration did not. the organism was also given intravenously in this case and was rapidly fatal. in a retrospective study of urinary bladder calculi in guinea pigs, it was reported that corynebacterium renale was the most common isolate from urine and bladder samples (hawkins et al., ) . of the cases submitted for bacterial culture, % were negative. the most common pure culture isolate was c. renale, which was also commonly found in mixed infections with staphylococcus and streptococcus spp. diagnosis diagnosis is made by recovering the organism using aerobic culture conditions, generally on blood agar plates with standard biochemical tests to differentiate corynebacterium from other species (greenwood, ) . pcr and sequencing has been used for further identification (greenwood, ; hawkins et al., ) . prevention and therapy many corynebacterium spp. have significant resistance to a number of antibiotics and this may be true of some organisms in guinea pigs as well (greenwood, ) . in the study isolating c. renale from the urine and bladder wall, many of those animals were already on antibiotics when those isolates were obtained (hawkins et al., ). in addition, the corynebacterium spp. recovered from the normal conjunctiva of guinea pigs, demonstrated some bacterial resistance with six out of nine isolates resistant to oxytetracycline, four out of nine resistant to polymyxin b, two out of nine resistant to bacitracin, and one out of nine resistant to erythromycin, gentamicin, amikacin, and ciprofloxacin (coster et al., ) . guinea pigs are highly susceptible to m. tuberculosis. natural infection has rarely been reported in the literature, but in those cases, the sources of the infections were believed to be from contact with human cases (van hoosier and robinette, ) . background markham and markham ( ) studied the prevalence of staphylococcal organisms in various animal species. a % incidence of coagulasepositive staphylococcus was noted in the nasal passages of guinea pigs. this was the greatest incidence of staphylococcal infection of any species tested, including humans. all of the guinea pigs cultured appeared clinically normal. etiology the staphylococcus genus, members of the staphylococcaceae family, are common inhabitants of the skin, oral cavity, respiratory system, and intestine, and cause suppurative lesions and septicemia in all species (songer and post, ) . these gram-positive cocci are coagulase-positive, catalase-positive, oxidasenegative, non-spore-forming and facultative anaerobic organisms (harkness and wagner, ; songer and post, ) . in guinea pigs, staphylococcus aureus has been isolated from cases of ulcerative pododermatitis and exfoliative dermatitis (percy and barthold, ) . pathogenesis despite the existence of speciesspecific serotypes and biotypes, human staphylococcus aureus phage types have been recovered from animals, and human-animal contact is one means of transmission of s. aureus (harkness and wagner, ) . additionally, direct contact with an infected animal or contaminated surface, as well as spread via aerosol can result in the dissemination of infection. ulcerative pododermatitis, or dermatitis of the footpad, is also known as "bumblefoot". trauma to the footpad from fighting, a sharp cage edge, or abrasive flooring creates a site of entry for bacteria (harkness and wagner, ) . obese guinea pigs, as well as any other natural or experimental cause that results in a sedentary lifestyle, can predispose an animal to bumblefoot, since perfusion is decreased and the pressure on the feet is increased (brown and donnelly, ; harkness and wagner, ) . exfoliative, or acute staphylococcal, dermatitis has also been associated with s. aureus in guinea pigs. strain guinea pigs are reported with this condition more frequently than other strains, and mortality rates due to this condition are higher in young animals, especially if born to an affected dam that is too painful to nurse (ishihara, ; percy and barthold, ) . clinical manifestations swollen, erythemic paws and lameness are noted with bumblefoot (brown and donnelly, ) . paws will often have erosions or ulcerations on the palmar or plantar surfaces. osteoarthritis or osteomyelitis may result, and animals that develop such sequela have a poorer prognosis. alopecia and erythema of the ventral abdomen, leading to cracking and scabbing of the epidermis, are often observed with exfoliative dermatitis (fox, ; ishihara, ; percy and barthold, ) . in addition to its association with ulcerative pododermatitis and exfoliative dermatitis, pneumonia, mastitis, conjunctivitis, cheilitis, and osteoarthritis have been attributed to s. aureus infection in guinea pigs (fox, ) . systemic amyloidosis can develop secondary to chronic s. aureus infection (brown and donnelly, ; fox, ; percy and barthold, ) . pathology hypertrophy of the stratum corneum with a minimal inflammatory response is noted histologically in cases of dermatitis associated with s. aureus (percy and barthold, ) . diffuse cellulitis is the primary lesion associated with ulcerative pododermatitis (brown and donnelly, ) . in cases that progress to amyloidosis, amyloid may be noted in the liver, spleen, adrenal glands, and pancreatic islets (ganaway, ) . diagnosis stained impression smears of lesions can demonstrate clumps of gram-positive cocci (harkness and wagner, ) . s. aureus can often be cultured directly from affected tissues and from the upper respiratory tract and pharynx of affected animals (percy and barthold, ) . blood agar or selective mediums, such as mannitol salt or staph agar, can be used for primary culture (songer and post, ) . s. aureus colonies often present with a golden pigmentation. prevention and therapy prevention of ulcerative pododermatitis and exfoliative dermatitis is easier than treatment. preventing cutaneous wounds by providing sturdy, smooth flooring to guinea pigs, offering non-abrasive feedstuff, and preventing obesity are highly effective in minimizing infection with s. aureus. pododermatitis and exfoliative dermatitis are not usually conditions conducive to lesion drainage and surgical removal (brown and donnelly, ) . lesions can be cleaned, but the possibility of causing greater trauma to the affected area must be considered. also, disinfectants that dry out the skin or agents that are cytotoxic to fibroblasts and reduce white blood cell viability and phagocytic efficiency, such as povidone-iodine or chlorhexidine, should be avoided. long-term antibiotic treatment is necessary, and enrofloxaxin or ciprofloxacin administered twice a day for - months has been reported in the treatment of ulcerative pododermatitis. use of an analgesic agent, especially a non-steroidal antiinflammatory drug such as meloxicam, is also recommended. low-level laser therapy, or phototherapy, has been reported as successful in treating bumblefoot by accelerating angiogenesis and stimulating vasodilatation, although such therapy can only be used after the infection is first controlled. two strains of streptococcus cause disease in guinea pigs. streptococcus pneumoniae can lead to severe pneumonia and streptococcus equi subsp. zooepidemicus is associated with the development of chronic lymphadenitis . infections due to s. equi subsp. zooepidemicus are more prevalent in guinea pigs than are pneumococcal infections. members of the streptococcus genus are part of the streptococcaceae family and consist of gram-positive, catalase-negative, facultative anaerobic bacteria that are non-spore-forming and non-motile (songer and post, ) . background streptococcus pneumoniae was first recognized in guinea pigs in europe in the late s . homberger et al. ( ) are credited with the first report of this organism in guinea pigs in the united states; the animals were from a colony in massachusetts and presented with signs of respiratory infection. etiology streptococcus pneumoniae is an oval-to lancet-shaped, encapsulated coccus that presents in pairs (diplococcus) or short chains (fox, ; percy and barthold, ) . s. pneumoniae is an α-hemolytic streptococcus that ferments trehalose and is optochin-sensitive (harkness and wagner, ; keyhani and naghshineh, ; songer and post, ) . additionally, it is not categorized according to one of the lancefield groupings (songer and post, ) . serovars of pneumococci are differentiated according to their capsular polysaccharide content, and serovars and are the two most often recovered from guinea pigs (fox, ; percy and barthold, ) . asymptomatic guinea pigs can carry pneumococci in the upper respiratory passages; the carrier rate for laboratory colonies can be as high as - % (fox, ; percy and barthold, ) . while iii. guinea pigs pneumonia epidemics due to s. pneumoniae seldom occur in well-managed facilities, stressors which may predispose to infection include poor husbandry, pregnancy, sudden or prolonged changes in temperature, poor ventilation, shipping, inadequate nutrition, concurrent infections, and experimental procedures (nakagawa et al., ; percy and barthold, ) . transmission of s. pneumoniae is by aerosol or via direct contact with abraided skin or oral mucosa (fox, ; percy and barthold, ; wagner et al., ) . infection can also be passed at the time of parturition by an infected dam. abortions and stillbirths can accompany a high mortality rate during an epizootic outbreak in a guinea pig colony (percy and barthold, ) . in , a spontaneous epizootic of respiratory infection involving guinea pigs was caused by s. pneumoniae type (keyhani and naghshineh, ) . during this outbreak, guinea pigs died within days of infection, and a short course of listlessness and failure to thrive were the only noted clinical signs prior to death. in less acute cases of s. pneumoniae, guinea pigs display depression and lethargy, anorexia, and have ruffled fur. they may have a wet nose, nasal or ocular discharge (rhinitis, conjunctivitis), sneezing, coughing, dyspnea, or torticollis (head tilt due to otitis media) (fox, ; wagner et al., ) . wagner, owens, kusewitt, and corley reported that otitis media occurred in of guinea pigs necropsied during a -year period in the s . streptococcus pneumoniae was noted in % of these cases, and was the most common bacteria isolated. gupta et al. ( ) , reported mastitis caused by s. pneumoniae in two guinea pigs. pathology acute bronchopneumonia is most often associated with s. pneumoniae infections. fibrinous exudate, a polymorphonuclear cell infiltrate and thrombosis of the pulmonary vessels may be observed in the lung (fox, ; keyhani and naghshineh, ; percy and barthold, ) . other pyogenic processes noted in infected animals include pericarditis, peritonitis, otitis media, arthritis, endometritis, and suppurative meningitis (fox, ; percy and barthold, ) . additionally, splenitis, fibrinopurulent meningitis, metritis, and lymphadenitis with focal hepatic and ovarian abscessation have been reported (percy and barthold, ) . pneumococci are readily demonstrated on impression smears and in stained tissue sections . diagnosis culture of nasal washings is an antemortem means of diagnosing s. pneumoniae (homburger et al., ) , as is culture of the nasal passages (harkness and wagner, ) . post-mortem, direct smears and culture of inflammatory exudates can be performed. s. pneumoniae is a fastidious bacterium that grows best when incubated under - % co on blood agar or enrichment media (fox, ; percy and barthold, ) . serological diagnosis via elisa has been reported (matsubara et al., ) . prevention and therapy antibiotic treatment of a clinical animal usually results in reversion to a sub-clinical carrier state, so elimination of clinical animals is recommended whenever possible (fox, ) . chloramphenicol, trimethoprim-sulfa combinations, and tetracycline have been used during epizootic outbreaks (harkness and wagner, ; homburger et al., ; keyhani and naghshineh, ) . prevention should focus on good husbandry, adequate diet, early isolation of streptococcal carrier animals, and reduction of environmental stressors. the same s. pneumoniae serovars commonly detected in guinea pigs have been isolated from humans (songer and post, ) . s. pneumoniae can lead to respiratory and neurological disease in humans, especially in older and immune-compromised individuals. to date, interspecies transmission of s. pneumonia has not been reported (harkness and wagner, ; percy and barthold, ) . charles boxmeyer ( ) described his findings of epizootic lymphadentitis in over guinea pigs in . cunningham ( ) reported on guinea pigs from a colony of that had "lumps," and from which "the same unusual type of mucoid hemolytic streptococcus was isolated". cunningham noted a : female to male ratio among the animals with chronic lymphadenitis. etiology streptococcus equi. subsp. zooepidemicus is a lancefield group c streptococcus that is betahemolytic, encapsulated, and ferments sorbitol (percy and barthold, ; songer and post, ) . s. equi subsp. zooepidemicus has a high degree of dna homology with s. equi subsp. equi. s. equi subsp. zooepidemicus has been recovered from human infections, while s. equi subsp. equi has not (songer and post, ) . pathogenesis guinea pigs can carry s. equi subsp. zooepidemicus asymptomatically in the nasopharynx and conjunctiva (percy and barthold, ) , but compared to s. pneumonia, clinical disease is more often noted in guinea pigs infected with s. equi subsp. zooepidemicus. as noted by cunningham, females are indeed more susceptible to disease than males (cunningham, ; fox, ; percy and barthold, ) . strain guinea pigs also appear to be more sensitive than other strains (fox, ; percy and barthold, ; wagner et al., ) . transmission is via aerosol, as well as direct contact with abraded skin or oral mucosa (harkness and wagnerl ; fox, ; percy and barthold, ) . the organism can be transmitted via bite wounds and secondary to abrasion by rough foodstuff (ganaway, ) . while this is likely the route of entry for most natural infections, murphy et al. ( ) showed that the oral mucosa does not need to be abraded in order for entry of the bacterium. the female genital tract can be infected during farrowing (percy and barthold, ) . after initial entrance, the organism travels to the draining lymph node and replicates (harkness and wagner, ) . cervical lymphadenitis is commonly noted in guinea pigs when the point of entry for infection is via the conjunctiva or nasal mucosa (harkness and wagner, ) . clinical manifestations clinical disease associated with s. equi subsp. zooepidemicus is colloquially referred to as "lumps," due to the pyogenic nature of the bacteria and its propensity to travel to and replicate in the lymph nodes (fox, ; percy and barthold, ) . while the cerival lymph nodes are the most common site of s. equi subsp. zooepidemicus replication and abscessation, any lymph node, and practically any organ, can be affected (harkness and wagner, ) . a common presentation for infection with this organism is an animal that appears in good condition except for a swelling in the neck at the position of the cervical lymph node (fox, ) . the swelling is often soft to firm, pus-filled, non-fluctuant and freely moveable (percy and barthold, ) . cunningham's ( ) observations were that "in some cases, one or two nodes were greatly enlarged; in others, as in one animal with cervical node involvement, several plum-sized encapsulated abscesses formed a collar across the front and side of the neck". depending on what additional organs are affected, torticollis (if middle ear involvement), dyspnea and cyanosis or nasal and ocular discharge (if respiratory involvement), or hematuria and hemoglobinuria (if septic) may be noted (fox, ; harkness and wagner, ) . abortions, stillbirths and unexpected deaths are possible (fox, ) . during epizootic outbreaks, septicemia, acute pneumonia, and high mortality can occur (harkness and wagner, ) . pathology the most common finding at necropsy is that of an abscessed, encapsulated cervical lymph node (fox, ) . the node architecture is often destroyed by central necrosis and peripheral fibrosis, and a thick pustular exudate is present (cunningham, ; fox, ; percy and barthold, ) . smears of the exudate reveal numerous gram-positive cocci in short chains (cunningham, ) . a generalized lymphadenitis may be noted, as may retroorbital abcessation, otitis media, pneumonia, pleuritis, pericarditis, or hepatitis (cunningham, ; fox, ; percy and barthold, ) . diagnosis s. equi subsp. zooepidemicus can be cultured from affected tissues or heart blood (harkness and wagner, ; percy and barthold, ; quesenberry and carpenter, ; wagner et al., ) . culture requires blood agar, on which after hours, a clear zone of hemolysis is observed (harkness and wagner, ) . trimming of teeth, elimination of sharp edges to feeders, and feeding nonabrasive feed may assist in prevention of s. equi subsp. zooepidemicus infection (fox, ) . infected animals should be isolated from the colony. surgical removal of abscess contents and capsule can be done with creation of drainage and lavage. antibiotic therapy is required. in epizootic cases, depopulation of affected animals and disinfection of the environment is advised. enzootic disease represents significant potential for research complications. disease-free replacement stock should be obtained and is available from commercial vendors. vaccines are typically type-specific and do not provide reliable protection (fox, ; mayora et al., ) . actinobacillus actinobacillus lignieresii and actinobacillus equuli have been noted in laboratory guinea pigs (songer and post, ) . the genus actinobacillus is a member of the pasteurellaceae family (boone et al., ) . these organisms are pleomorphic gram-negative rods that on gram stain, can have a "morse code" appearance (songer and post, ) . actinobacillus lignieresii and actinobacillus equuli were cultured from guinea pigs, rats and mice during a -month period at the university of missouri research animal diagnostic and investigative laboratory (lentsch and wagner, ) . organisms grew on blood agar inoculated with swabs from the posterior nasopharynx, conjunctiva, and middle ear. conjunctivitis was noted in one animal and otitis media was noted in six. background tartakowsky is credited with one of the first accounts of bordetella infection in guinea pigs in the late s (mccartney and olitsky, ; smith, ) . woolfrey and moody ( ) in their review of this organism stated "ferry, using the name bacillus bronchisepticus; mcgowan, using the term bacillus bronchicanis; and torrey and rahe, using ferry's epithet, independently observed the association of a small gram-negative coccobacillus with outbreaks of "canine distemper" and respiratory tract illness in laboratory animals such as the cat, rabbit, and guinea pig". in the early s, b. bronchiseptica was still considered to be the primary cause of acute pneumonia in guinea pigs and while it is not often noted in modern laboratory facilities, it continues to be a major cause of respiratory disease in pet guinea pigs and this species is thought to be quite susceptible to infection (percy and barthold, ; rigby, ) . etiology bordetella bronchiseptica is a short, gramnegative rod or coccobacillus, that is aerobic, motile, and non-spore-forming (fox, ; percy and barthold, ) . b. bronchiseptica is oxidase, catalase, and urease positive, as well as positive for citrate utilization and nitrate reduction (songer and post, ; woolfrey and moody, ) . b. bronchiseptica is considered a commensal organism in the guinea pig, as well as many other mammalian species, including humans (fox, ; percy and barthold, ; woolfrey and moody, ) . the bordetella genus is a member of the alcaligenaceae family (songer and post, ) . epizootiology and pathogenesis b. bronchiseptica is carried by guinea pigs in the nasal cavity and trachea, and up to % of non-clinical animals in a colony can be carriers (ganaway, ) . stressful events, such as transport or pregnancy, may trigger an acute fatal pneumonia in carriers or animals with previously inapparent infections. transmission of b. bronchiseptica is via aerosol spread, direct contact, direct contact with respiratory secretions of infected animals, or fomites (fox, ; ganaway, ) . b. bronchiseptica adheres to ciliated respiratory epithelium and causes ciliary paralysis (fox, ) . pathogenic b. bronchiseptica produce adhesion and cytolytic toxins that lead to an inflammatory response, antiphagocytic activity, and dermo necrosis. while not explicitly described in the guinea pig, the impairment in mucociliary clearance caused by the binding of bordetella is known to reduce the clearance of additional respiratory pathogens in other animal species. the incubation period is - days (ganaway, ) . the highest levels of morbidity and mortality due to b. bronchiseptica have been noted in young guinea pigs, as well as strain animals (fox, ; ganaway, ) . it is possible for infected animals to develop immunity and clear the organism. yoda et al. ( ) , noted that within - weeks of initial infection, of naturally infected guinea pigs completely recovered from infection. clinical manifestations infection with b. bronchiseptica is often subclinical in nature (fox, ) . it has been suggested that the clinical signs associated with bordetella in the past have actually been due to infections with a secondary invader, such as guinea pig adenovirus, which has not been recognized as an infectious agent of the guinea pig for as long as b. bronchiseptica. during an epizootic outbreak, death may follow respiratory disease and septicemica within a - -hour period (fox, ) . abortion and stillbirths can occur in pregnant dams (percy and barthold, ) . in an enzootically infected colony, sporadic deaths may occur throughout the year, with highest levels during the winter months. inappetance, depression, nasal discharge, dyspnea, and cyanosis may also be observed (fox, ) . pathology at necropsy, cranioventral pulmonary consolidation is seen, and whole lobes or individual lobules will present as dark red to red-gray in color (ganaway, ; percy and barthold, ) . a bloodtinged froth is noted in the trachea. mucopurulent exudates may be noted in affected airways. pleuritis and otitis media may be observed. suppurative bronchopneumonia is noted histologically, with an influx of heterophils and mononuclear cells. uterine infections may result in dead fetuses or pyosalpinx. diagnosis b. bronchiseptica can be cultured on blood or macconkey's agar from nasal or other respiratory secretions (songer and post, ) . in cases of otitis media, b. bronchiseptica can be isolated from the middle ear, and in cases of metritis, from the uterus (fox, ; yoda et al., ) at - °c (songer and post, ) . smith and baskerville medium selects for b. bronchiseptica, and is another option for culture (songer and post, ) . elisa and ifa tests have been developed for the detection of guinea pig bordetella, and have been noted as equally successful in identifying infected animals as cultures of the respiratory tract (boot et al., a; wullenweber and boot, ) . purchasing bordetellafree animals is the best means of control in laboratory colonies of guinea pigs. it is recommended that rabbits and guinea pigs not be co-housed since guinea pigs are highly susceptible to disease and rabbits are frequently sub-clinical carriers of b. bronchiseptica (charles river laboratories, ) . rats and other rodents can be additional sources of infection (rigby, ) . antibiotic treatment with fluroquinolones or trimethoprimsulfonamides may successfully resolve clinical signs, but elimination of infection is rare, with animals often reverting to the sub-clinical state. if infection is suspected in a colony, restocking, test and cull, or rederivation may be more beneficial than treatment (fox, ) . the efficacy of canine, porcine, human, and autogenous bordetella vaccines and bacterins has been evaluated by several individuals; reports suggest that these vaccines do not completely protect guinea pigs from infection, but a decrease in the incidence and severity of clinical disease has been noted in experimentally challenged animals (matherne et al., ; stephenson et al., ) . b. bronchiseptica apparently does not cause epidemic disease in man, but it can cause a whooping cough-like syndrome in immune-competent children or respiratory tract infections in immune-suppressed individuals (songer and post, ) . endocarditis, meningitis, and peritonitis have also been associated with b. bronchiseptica infections in humans. the ease of transmission between guinea pigs and humans in unknown. background and etiology spirochetes of the genus brachyspira (formerly serpulina) have been associated with diarrhea and colitis in a variety of animals including birds and mammals. brachyspira contains seven distinct species. of these, two species have been implicated in guinea pigs, either in natural infections or as an animal model; b. hyodysenteriae, the primary cause of swine dysentery, and b. pilosicoli, a zoonotic agent associated with disease in chickens, pigs, and humans. spirochetes have been associated with disease in guinea pigs in several publications, although purposeful infection to demonstrate causality has not been done and in many cases, identification of the species involved has also not been accomplished (duhamel, ) . guinea pigs have been used as an animal model for b. hyodysenteriae infection. two reports in the s describing tyzzer's disease in guinea pigs also report observing spirochetes on histopathology (mcleod et al., ; zwicker et al., ) . the significance of the spirochete infections in the progression of the disease seen is unclear. a subsequent report describes a series of cases submitted for necropsy between and in belgium (vanrobaeys et al., ) . in of cases when spirochetes were identified, the presenting complaints were either sudden death or a short course of diarrhea followed by sudden death. flagellated parasites were seen in animals and lesions consistent with vitamin c deficiency in six cases. gross necropsy of affected animals most commonly demonstrated liquid, occasionally mucoid or hemorrhagic large intestinal contents, and dilation of the cecum and colon. histologically, the cecum was characterized by hyperemia, mucosal infiltration of neutrophils, a mixed inflammatory infiltrate in the lamina propria, and occasionally, necrosis of crypt epithelium. lesions involving other organs were present in of the animals identified, suggesting multifactorial disease processes. in a report of a single pet guinea pig, clinical signs of tenesmus, tachycardia, pale mucous membranes, and dehydration were associated with acute rectal prolapse (helie et al., ) . histopathology identified superficial mucosal necrosis and ulceration and congestion with hemorrhage in the intussuscepted segment. although spirochetes and organisms consistent with eimeria spp. were seen in the rest of the intestinal tract, no other lesions were noted. b. pilosicoli was identified by pcr. diagnosis diagnosis can be made by identifying typical organisms on histopathology. pcr of intestinal contents is diagnostic (helie et al., ) . prevention and therapy treatment with ranidazole stopped the spread of the disease in some cases as described by vanrobaeys et al. ( ) , although cessation of treatment resulted in resumption of disease. as the majority of reports describe co-pathogens, controlling copathogens may be the most effective prevention. brucella spp. are zoonotic, gram-negative, strictly aerobic, intracellular, non-motile coccobacilli, which infect a variety of species, including guinea pigs (martirosyan et al., ; pappas, ; van hoosier and robinette, ) . natural infections in guinea pigs have been described likely associated with contaminated food, bedding, or other biological materials (van hoosier and robinette, ) . in these outbreaks, guinea pigs developed disease similar to that seen in other species including testicular and joint swelling as well as abscesses in the liver and pancreas. modern husbandry practices should limit transmission of brucella spp. to guinea pigs. campylobacter jejuni is a species of curved, rod-shaped, non-spore-forming, gram-negative, microaerophilic, bacteria that naturally colonize the digestive track of birds and numerous other species, including domestic farm animals, dogs, cats, various wildlife, and rodents (horrocks et al., ). it is a common cause of human gastroenteritis worldwide primarily from contamination of food. it was isolated from laboratory animal guinea pigs in one report where the prevalence of c. jejuni in a multispecies laboratory animal facility was investigated (meanger and marshall, ) . in this report, no clinical signs were seen in any of the animals involved and pathological analysis was not done. guinea pigs have been used as an animal model of c. jejuni infection (burrough et al., ; coid et al., ) . background and etiology guinea pig inclusion conjunctivitis was first described in associated with an experiment to examine the correlation between vitamin deficiency and the development of trachoma virus (murray, ) . the organism was identified as a type of chlamydia psittaci, a member of the psittacosis-lymphogranuloma-trachoma group, first thought to be protozoa, then viruses because they could pass through a -nm filter, and finally in recognized as bacteria (longbottom and coulter, ; moulder, ; songer and post, ) . as members of the family chlamydiaceae, they are iii. guinea pigs gram-negative, obligate intracellular pathogens that infect a variety of animals, including amoeba, birds, sheep, humans, and guinea pigs (greenwood, ) . recently they have been divided into two genera: chlamydia and chlamydophila. chlamydophila species include c. caviae as well as c. pneumoniae (humans, horses, koalas) and c. psittaci (birds) (everett et al., ; murray, ) . unlike most bacteria, the cell walls of chlamydiaceae do not have a peptidoglycan layer, but they do have a major outer-membrane protein, which is the determinant for serologic classification (greenwood, ; songer and post, ) . they also have a unique life cycle which consists of two forms; a small infectious elementary body which is more similar to a spore in that it is environmentally resistant and not metabolically active and a larger metabolically active, non-infectious reticulate body that is responsible for division within the host cell. epizootiology and pathogenesis generally, c. caviae infection is thought to be endemic in guinea pig colonies (percy and barthold, ) , although its detection in colonies has been variable. in a survey done in of five herds of guinea pigs, conjunctival staining revealed the organism in four out of five herds, although not in any guinea pigs less than weeks of age (murray, ) . in , two breeding facilities of conventional guinea pigs in korea were examined by conjunctival staining and no positive organisms were seen (park et al., ) . a survey of pet guinea pigs with normal conjunctiva, also revealed no positive staining (coster et al., ) , which may reflect the sensitivity of the test used or the actual prevalence of c. caviae. in a study using real-time pcr for diagnosis, c. caviae was detected in out of guinea pigs tested, four out of four rabbits, and one dog from samples obtained from a diagnostic laboratory (pantchev et al., ) . c. caviae in a second report was identified via pcr from conjunctival swabs from a rabbit, cat, and human that lived in the same household as infected guinea pigs (lutz-wohlgroth et al., ) . also utilizing pcr and dna sequencing, c. caviae was detected in foals with conjunctivitis, rhinitis, and coughing, indicating the host range of the species of chlamydiaceae may continue to be refined as new diagnostic tests are utilized (gaede et al., ) . one study identified acanthamoebae spp. via pcr from two guinea pigs with conjunctivitis that were also pcr positive for c. caviae (lutz-wohlgroth et al., ) . their role in the disease process is unclear. urogenital disease has been reported in natural infections with c. caviae and has been reproduced in experimental models (lutz-wohlgroth et al., ; mount et al., ) . in experimental infections, inoculation with c. caviae in the lower genital tract of female guinea pigs causes a self-limiting infection lasting - weeks. typically, the infection involves the epithelium of the cervix with very little inflammation. however, in % of the animals, the infection progresses to involve the oviducts, with half of the animals developing pathologic changes in the oviducts including inflammation and fibrosis rank and sanders, ) . infection can be transferred from infected male animals to females during sexual intercourse and vertically from dam to pups (padilla-carlin et al., ; miyairi et al., ) . in experimental infections, both cell-mediated and humoral immunity are important in recovery and resistance to re-infection (miyairi et al., ) . early infection is characterized by influx of polymorphonuclear cells while later in infection, lymphocytes predominate. t-cell depletion results in chronic infection and delayed clearance of the organisms. decreased antibody response also correlates with delayed clearance of the organisms (miyairi et al., ; rank et al., ) . clinical manifestations c. caviae has been primarily associated with conjunctivitis. guinea pigs have reddened, swollen conjunctiva with serous to purulent discharge, follicular hypertrophy, and pannus (deeb et al., ; lutz-wohlgroth et al., ; murray, ; padilla-carlin et al., ; strik et al., ) . generally the conjunctivitis resolves in - weeks. occasionally, conjunctivitis is accompanied by rhinitis. in one study, nasal discharge was present in of of the animals with ocular discharge (lutz-wohlgroth et al., ) . vaginal discharge was seen in % (ten of ) of the animals although the organism could not be recovered by pcr from either the nose or the vagina. abortion was recorded in two animals. an early study also reported rhinitis, bronchopneumonia and abortions, although this study was complicated by infection with streptococcus pneumonia and bordetella bronchiseptica (schmeer et al., ) . pathology histological evaluation of the eyes and surrounding tissue reveals a mixed inflammatory response and congestion beneath the conjunctival epithelium (deeb et al., ; lutz-wohlgroth et al., ) . histological examination of the urogenital tract has primarily been described following experimental infection and consists of inflammation of the endometrium, mesosalpinx, and oviduct with a mixed inflammatory infiltrate and fibrosis rank and sanders, ) . diagnosis diagnosis is by cytology, pcr, culture, tissue assays, and serology (songer and post, ) . giemsa, gimenez, or macchiavello stains on cytologic preparations reveal inclusions in the cytoplasm. inclusions are a dark purple with giemsa and red with gimenez or macchiavello stains. cytology can be insensitive hence pcr can be used in addition (pantchev et al., ; strik et al., ) . in one study, samples were tested via cytology and pcr (lutz-wohlgroth et al., ) . twenty-one were positive by pcr for c. caviae, however, no samples were positive by cytology. as c. caviae is an obligate intracellular pathogen, it cannot be cultured in cell-free media, but only on specific cell lines (songer and post, ) . this is an insensitive and difficult process that is not typically used for diagnosis. direct fluorescent antibody test and enzyme immunoassays can be used to detect antigens in cytology samples or tissues (percy and barthold, ; songer and post, ) . prevention and therapy generally, the disease is self-limiting and treatment is not required (percy and barthold, ; quesenberry and carpenter ) . no current vaccine prevents chlamydial infection, although vaccination studies have demonstrated a decrease in the intensity of the disease following vaccination (padilla-carlin et al., ) . recognition that zoonotic transmission is a possibility should guide appropriate personal protective equipment choices (lutz-wohlgroth et al., ) . citrobacter is a genus of gram-negative coliform bacteria of the family enterobacteriacae (boone et al., ) . in the s and s, a disease eventually named transmissible murine colonic hyperplasia caused by coliform bacteria of the genus citrobacter was recognized (mundy et al., ; percy and barthold, ; petty et al., ) . originally described as unusual biotypes of citrobacter freundii, these organisms were eventually separated into their own species, citrobacter rodentium, on the basis of dna sequencing studies (luperchio et al., ; petty et al., ) . whether the organisms that caused severe septicemia and death in guinea pigs in (ocholi et al., ) and other disease syndromes in hysterectomy-rederived guinea pigs (boot and walvoort, ) are c. freundii as originally indicated or c. rodentium is unclear. pathology there are two reports of disease associated with citrobacter spp. in guinea pigs (boot and walvoort, ; ocholi et al., ) . first, citrobacter spp. were isolated from germ-free guinea pigs that were exposed to defined flora from gnotobiotic mice (boot and walvoort, ) . citrobacter spp. were isolated from the middle ear, spleen, mammary gland, and kidney of some animals as well as the intestinal tract, suggesting an ability to cause varied disease in guinea pigs that are not populated with normal flora. second, an epizootic of citrobacter spp. septicemia in a large colony of conventional guinea pigs was reported (ocholi et al., ) . animals presented with diarrhea, dyspnea, and decreased appetite. mortality in weanlings and breeders was of animals. gross necropsy revealed lung consolidation with pleural adhesions as well as enteritis and thickening of the intestinal wall. fibrinous pneumonia and septic thrombi in the capillaries of the lung, liver, and spleen were seen histopathologically. citrobacter spp. were consistently isolated in pure culture from the lung, liver, spleen, and intestine. diagnosis culture is the traditional method of detecting citrobacter spp. which grow well on normal media, are aerobic or facultative anaerobic, ferment glucose, produce catalase, but not oxidase, and are generally lactose-negative or later lactose-fermenting (greenwood, ) . however, pcr is more sensitive than bacterial culture for detection of c. rodentium in feces from mice (mckeel et al., ) . prevention and therapy c. freundii strains utilize a number of methods to encode resistance to ampicillin and cephalosporins (pfeifer et al., ) . enrofloxacin has been successful in treating mice infected with c. rodentium (maggio-price et al., ) . prevention would include eliminating contact with c. rodentium-infected mice and other rodents. clostridium piliforme (tyzzer's disease) background clostridium piliforme, first described in (tyzzer, ) , causes severe disease in many laboratory animals as well as other mammals (percy and barthold, ) . although rare in modern colonies, outbreaks in mice, rats, rabbits, and guinea pigs have been reported and c. piliforme is found in both wild (wobeser et al., ) and domestic mammals (borchers et al., ; ikegami et al., ) . while tyzzer's disease is not generally considered zoonotic, infection of an hivpositive patient has been reported (smith et al., ) . etiology clostridium piliforme (formerly bacillus piliformis) is a gram-negative, spore-forming, anaerobic rod that can survive for as long as five years in the environment (percy and barthold, ; songer and post, ) . pathogenesis c. piliforme is thought to be transmitted via fecal-oral transmission. in experimental models, oral inoculation was successful when other routes of inoculation did not result in disease (waggie et al., ) . vertical transmission has been reported in one case of hysterectomy-rederived guinea pigs (boot and walvoort, ) . in outbreaks of tyzzer's disease in other species, usually predisposing factors such as stress, overcrowding, poor sanitation, or immunemodulating treatments contribute to the development of disease (percy and barthold, (boot and walvoort, ; mcleod et al., ; zwicker et al., ) typically seen in weanlings. subclinical infections have been noted (boot and walvoort, ) . pathology lesions are usually found only in the gastrointestinal tract in young guinea pigs, although liver lesions have been reported in some animals (sparrow, ; waggie et al., ) . grossly, pinpoint gray foci occur on the ileum, cecum, and colon as well as enlarged, reddened mesenteric and colonic lymph nodes (mcleod et al., ; sparrow, ; waggie et al., ; zwicker et al., ) . white to tan pinpoint foci are also observed on the liver. microscopically, foci of necrosis occur in the mucosa of the ileum, cecum, and colon as well as foci of periportal coagulative hepatocellular necrosis when the liver is involved. clusters of intracellular organisms can be observed via silver stain or giemsa stain in affected enterocytes and hepatocytes. in two reports, affected animals have also had large numbers of spirochetes evident at necropsy (mcleod et al., ; zwicker et al., ) . diagnosis c. piliforme cannot be cultured using standard media, although it can be isolated by inoculation of cell lines, embryonated hen's eggs, and primary mouse or chick embryo cell cultures (niepceron and licoism, ; percy and barthold, ) . therefore, diagnosis typically relies on characteristic lesions and organisms observed on histopathology using a warthin-starry silver stain or giemsa stain (percy and barthold, ) . pcr has been used to detect c. piliforme in feces from rats (furukawa et al., ) . nested pcr was developed to increase the specificity of detection in tissues, better separating c. piliforme from other closely related organisms used conserved regions of s ribosomal rna (niepceron and licois, ). serology to detect antibodies has been used to monitor colonies for this organism (boot and walvoort, ; kraft and meyer, ) . treatment and control treatment of individual animals has not been reported. tyzzer's disease affects a wide variety of animals and immune-compromised people. prevention entails modern husbandry and sanitation practices, preventing contamination of food and bedding, and separation of species. escherichia coli are aerobic, straight gram-negative rods and are part of the normal intestinal flora of most vertebrates, including guinea pigs (crecelius and rettger, ; songer and post, ) . several pathotypes have been reported (e.g. epec-enteropathogenic strains), some which produce cytotoxins. diarrhea, rough hair coats, wasting and death have been reported to be associated with e. coli infection in conjunction with other environmental stressors (ganaway, ) . in addition, there is one report of a compilation of cases of mastitis in guinea pigs from a diagnostic lab over a four year period (kinkler et al., ) . of the cases where cultures were taken, e. coli was found in cases with the next most common isolate being klebsiella pneumonia in six cases. diagnosis may be obtained by culture of affected organs on macconkey agar and differentiation from other enteric gram-negative bacteria by a number of biochemical reactions such as indole production and utilization of certain sugars (greenwood, ) . background perkins ( ) described an epizootic outbreak in laboratory guinea pigs that resulted in the death of all but two animals - hours after the onset of disease. a short bacillus, often paired, was isolated in the blood, spleen, liver, and peritoneal exudates. disease was reproduced in naïve guinea pigs subsequently administered the bacterium by the intraperitoneal route. perkins' findings were similar to those of howard ( ), who inoculated dogs, rabbits, mice, pigeons, and guinea pigs with a bacillus organism isolated from human patients. etiology klebsiella pneumoniae is a gram-negative, rod-shaped bacillus from the genus klebsiella and family enterobacteriaceae (boone et al., ) . k. pneumoniae is facultatively anaerobic, oxidase-negative, and produces acid and gas from lactose. it is an enteric bacterium, noted in the intestinal tract of % of healthy humans (ganaway, ) . it can also reside in the skin and mouth. k. pneumoniae causes pneumonia in guinea pigs of both sexes and all ages, although there are few reports of natural infections of this animal species with this organism. clinical manifestations in the epizoonotic outbreak described by perkins, animals presented as anorexic and ungroomed, and their illness escalated in severity until, in some animals, a comatose condition ensued (perkins, ) . animals usually died - hours after the onset of clinical signs. in other epizootic outbreaks, dyspnea has been reported prior to reaching the comatose state (ganaway, ) . less severely infected animals may be able to recover from disease and develop immunity to re-infection (perkins, ) . otitis media and other signs consistent with a state of septicemia have been associated with clinical disease (fox, ; ganaway, ; kohn, ) . pathology seropurulent or serofibrinous peritonitis is noted at necropsy (fox, ; ganaway, ; perkins, ) . additionally, such exudates may be observed in the pleural cavity and pericardial sac. congestion of the liver, spleen, and kidney has been noted, as has hepatic coagulative necrosis and degeneration of the renal tubule cells. an acute, necrotizing bronchopneumonia may be observed. diagnosis diagnosis of k. pneumonia is usually via culture of the upper respiratory tract. the bacterium has been isolated from blood, spleen, liver, peritoneal exudate, and cerebrospinal fluid (fox, ; ganaway, ; perkins, ) . k. pneumoniae can be differentiated from k. oxytoca, another klebsiella spp. frequently recovered in animals, by the fact that it is indole-negative and does not grow at °c, while k. oxytoca is indolepositive and does grow at °c. antibiotic treatment for klebsiella infection should be based on sensitivity of culture isolates. ceftriaxone and cefodizime were found effective at clearing k. pneumoniae in experimentally infected guinea pigs (drago et al., ) , although generally, chloramphenicol, enrofloxacin, trimethoprimsulfa, and aminoglycoside drugs are considered the safest antibiotic choices for use in guinea pigs. background and etiology proliferative enteritis associated with campylobacter-like organisms has been reported in many species including pigs, hamsters, rats, rabbits, foals, nhps, ferrets, and deer (lawson and gebhart, ) . in the early s, molecular techniques resulted in the identification of lawsonia intracellularis as the causative agent of many of these syndromes. since that time, diagnostics, including pcr, have enabled the diagnosis of l. intracellularis infection (collins et al., ) . pathology there have been two reports of disease in guinea pigs associated with campylobacter-like organisms, both prior to the identification of l. intracellularis. in , one . -month-old guinea pig, treated with steroids for days as part of a study, displayed no clinical signs of disease but on necropsy had marked duodenal mucosal hyperplasia with intracellular warthin-starry stained argyrophilic bacteria in affected epithelial cells (elwell et al., ) . two cagemates of this guinea pig, that had the same steroid treatment, died at days and . one had diarrhea for two days prior to death. histopathology revealed acute enteritis with mucosal erosions without mucosal hyperplasia in both animals. bacteria similar to that seen in the first case were found in the epithelial cells. the authors noted this organism was very similar to the organism associated with transmissible mucosal hyperplasia of hamsters (subsequently identified as l. intracellularis). in , two dams and five young guinea pigs in a laboratory in japan developed diarrhea, anorexia, weight loss, and dehydration. two young animals died - days post-onset of clinical signs. grossly, there was thickened jejunum and ileum. histopathologically, there was hyperplasia and proliferation of the epithelial cells and dilation of the glands primarily in the ileum and the distal part of the jejunum. immature epithelial cells contained various numbers of intracytoplasmic, non-membrane-bound, curved organisms resembling campylobacter spp. bacteria. diagnosis characteristic histopathological lesions, immunohistochemistry, and pcr are diagnostic (collins et al., ; lawson and gebhart, ) . antibiotics have been used to treat a similar proliferative enteritis in swine with success (lawson and gebhart, ) . care should be taken to avoid penicillins and other antibiotics implicated in antibiotic-associated dysbacteriosis (percy and barthold, ) . background and etiology leptospira are tightly coiled, aerobic, gram-negative, flagellated spirochetes belonging to the genera leptospira, family leptospiraceae, phylum, spirochaetes (boone et al., ) . before , the genus, leptospira, was divided into two species, l. interrogans (pathogenic) and l. biflexa (non-pathogenic) which each contained strains separated into numerous serovars (levett, ) . more recently, dna analysis has identified species of leptospira and revisions of these taxonomic groups are ongoing (galloway and levett, ; levett, ) . leptospira are zoonotic organisms with a world-wide distribution that infect a variety of mammals, including mice, rats, carnivores, and ruminants (ko et al., ; levett, ; percy and barthold, ) . wild guinea pigs in south america (cavia porcellus apera), infected with leptospira spp., have been implicated as the natural reservoir responsible for transmitting the disease to domestic cattle (blood et al., ) . in domestic guinea pigs, there have been only a few reports of natural infection, although the guinea pig infection models have been used extensively in leptospira research (ko et al., ; van hoosier and robinette, ) . data from research models and infections in other species indicate that transmission occurs from direct contact with infected urine, soil, or water as leptospira are excreted in the urine (ko et al., ; lourdault et al., ). a report from describes a natural infection where one out of guinea pigs died from leptospirosis (ganaway, ) . pathological findings include jaundice, petechial hemorrhages in the skin, fascia, and muscles, and ecchymosis in the lung. a more recent report of a case of leptospirosis in a -year-old man, described out of guinea pigs that died on his farm in germany. three of the remaining guinea pigs were tested and were serologically positive for leptospira serovar bratislava similar to the man. no necropsies of affected animals were described. diagnosis direct examination of infected tissue, urine, or blood with dark-field microscopy, immunoflourescence, or light microscopy with special stains is useful for diagnosis (greenwood, ; levett, ) . culture and pcr are additional diagnostic tools. growth in culture can be slow. generally, antibody detection is used to demonstrate previous infection. antimicrobial treatment of individual animals is generally successful in other species (pischke et al., ; van de maele et al., ) , although in guinea pigs, care must be taken to avoid antibiotic-associated dysbacteriosis (percy and barthold, ) . prevention includes preventing exposure to wild rodents and leptospira-infected food, bedding, soil, or water. background listeria monocytogenes was first discovered in by murray et al. ( ) , who isolated it from laboratory rabbits and guinea pigs. since that time, few cases have been reported in guinea pigs, although guinea pigs are very susceptible and have been used extensively to model the pathogenesis of listeriosis (cossart, ; disson et al., ; fox, ) . etiology listeria monocytogenes is a facultative intracellular, non-spore-forming, zoonotic, food, soil-, and silage-borne bacterium which causes disease in birds and many mammals. pathology in spontaneous infections of guinea pig colonies, l. monocytogenes infection has been correlated with acute death, reproductive disorders such as abortions and still births, as well as conjunctivitis (chukwu et al., ; colgin et al., ; fox, ) . in an outbreak associated with feeding infected greens to a colony of guinea pigs, the mortality rate was reported to be - % with lung and liver lesions predominating (chukwu et al., ) . conjunctivitis was reported in one outbreak affecting out of newly arrived guinea pigs (colgin et al., ) . l. monocytogenes was cultured from of specimens submitted. the animals presented with unilateral or bilateral serous to purulent discharge with hyperemic conjunctiva. histologically, there was corneal ulceration, stromal edema, and vascular proliferation. the conjunctiva contained an inflammatory infiltrate and the lacrimal gland had multifocal areas of inflammation and necrosis. intracytoplasmic gram-positive rods were seen in the conjunctival epithelium and no chlamydial organisms were identified on conjunctival scrapings or by histopathology. experimentally, guinea pigs have been shown to be susceptible to listeriainduced conjunctivitis. pasteurella multocida background pasteurellosis is rare in presentday guinea pig laboratory colonies. reports of epizootic outbreaks due to gram-negative coccobacilli were reported in europe in the late s (wright, ) . etiology pasteurella multocida, a gram-negative, non-spore-forming and non-motile rod is a member of the pasteurella genus and the pasteurellaceae family (songer and post, ) . spp. are mucosal commensal organisms of the oropharynx and gastrointestinal tract of many species, including man (songer and post, ) . when stressed, the animal's immune system can be overwhelmed and, in guinea pigs, pneumonia may result. p. multocida can be transmitted to man via contact with infected saliva, which is likely to occur as a result of a bite wound or scratch. clinical manifestations wright detailed an outbreak of pasteurellosis that killed guinea pigs over a -year period (wright, ) . clinical signs were not noted prior to death. p. multocida has been isolated from cases of conjunctivitis (percy and barthold, ) . pathology fibrinopurulent serositis is present in cases of guinea pig pasteurellosis (fox, ; ganaway, ; wright, ) . such serositis has been noted in the pericardium, pleura, and peritoneum. lung consolidation may also be observed (fox, ) . diagnosis pasteurella infection is diagnosed via culture. the bacteria do not reliably grow well on macconkey agar, therefore blood agar is routinely used for isolation (ganaway, ; songer and post, ) . plates should be incubated at - % co at °c for - hours (songer and post, ) . in animal tissue or fluids, p. multocida appears as a bipolar rod after aniline dye staining, but in a smear taken from culture, it stains evenly (ganaway, ) . unlike p. pneumotropica, p. multocida is o-nitrophenyl-ß-d-galactoside (onpg) and urease-negative (songer and post, ) . p. pneumotropica is also positive for the fermentation of mannose, while p. multocida is negative. an elisa-based assay has been developed to monitor antibodies to the sp group of pasteurellaceae, which is one of five serologically distinct groups of pasteurellaceae in guinea pigs (boot et al., ) . prevention and therapy antibiotic treatment of p. multocida should be based on culture sensitivities. antimicrobial therapy will eliminate clinical signs, but will not eliminate the organism from the colony (songer and post, ) . background scherago ( ) reported on a fatal epizootic septicemia in a colony of young guinea pigs received from a local breeder. animals died within - hours after showing clinical signs and "gram-negative, non-spore-forming encapsulated rods with rounded ends and straight sides appearing singly and occasionally in pairs end to end" were found on smears from the peritoneal cavities of all examined animals. the route of infection was not determined in this colony, but scherago did complete the required steps to fulfill koch's postulates and demonstrated that the infection was due to pseudomonas caviae. p. caviae has since been reclassified as aeromonas caviae, of the aeromonadaceae family. pseudomonas aeruginosa was noted as the cause of pulmonary botryomycosis in two guinea pigs (bostrom et al., ) . etiology p. aeruginosa, a member of the genus pseudomonas, is part of the pseudomonadaceae family (songer and post, ) . pseudomonas spp. are gramnegative rods that are aerobic, non-spore-forming, oxidase-positive, and non-fermentative. pseudomonas organisms thrive in aqueous environments (songer and post, ) . additionally, they are ubiquitous in soil, decaying organic matter, and vegetation. pseudomonas spp. are opportunistic pathogens of both humans and animals, yet, there are few reports of infections caused by these agents in guinea pigs (fox, ) . bostrum et al. ( ) , did not describe the clinical signs associated with the epizootic outbreak of pulmonary botryomycosis. otitis media, conjunctivitis, and an inflamed prostate gland are signs that have been associated with pseudomonas in other reports of guinea pig infection (fox, ) . pathology a focal, necrotizing bronchopneumonia, along with pulmonary consolidation has been described in cases of guinea pig pseudomonas infection (bostrom et al., ; fox, ; ganaway, ; percy and barthold, ) . bostrum noted atypical "sulfur granules", resembling the spherules of coccidioides immitis, in the lungs of affected animals (bostrom et al., ) . diagnosis p. aeruginosa can be cultured from infected tissues (songer and post, ) . p. aeruginosa grows well on macconkey agar and trypticase soy agar. additionally, cetrimide agar, a commercial formulation, selectively isolates p. aeruginosa. the best means of controlling pseudomonas is to reduce possible sources of infection, such as damp bedding or stagnant water. animal drinking water should be monitored for p. aeruginosa, and in some situations, water may be chlorinated to eliminate contamination (songer and post, ) . reducing stressors, such as overcrowding and concurrent infection, also aids in control. pseudomonas spp. have an inherent resistance to many antimicrobials. salmonella background some of the earliest reports of salmonellosis in guinea pigs date back to the s (nelson and smith, ) . multiple outbreaks of natural infections in guinea pig colonies in the united states were documented throughout the early s (holman, ; nelson and smith, ) . since this period of time, as laboratory standards have grown more stringent and general hygiene and sanitation have improved, the occurrence of salmonellosis in laboratory guinea pigs has declined greatly (fox, ; harkness and wagner, ; percy and barthold, ) . etiology members of the genus salmonella are gram-negative, non-spore-forming bacilli that are facultative anaerobes. salmonella other than s. typhi or s. paratyphi produce hydrogen sulfide, as well as acid and gas from glucose, but not lactose (ganaway, ; songer and post, ) . salmonella is a member of the enterobacteriaceae family. while multiple serovars have been recovered from guinea pigs, the most frequently isolated are s. typhimurium and s. enteritidis (fox, ; ganaway, ; harkness and wagner, ; quesenberry and carpenter, ; percy and barthold, ) . humans are susceptible to disease from the same serovars and phage types as guinea pigs (fish et al., ; percy and barthold, ; rigby, ). pathogenesis guinea pigs are extremely susceptible to salmonella (fox, ) . transmission of salmonella is primarily fecal-oral or via ingestion of contaminated feed (contaminated with feces of infected guinea pig or a wild rodent) (fox, ; harkness and wagner, ; percy and barthold, ; quesenberry and carpenter, ) . entry via the conjunctiva has also been reported in guinea pig epizootics (iijima et al., ; moore, ) . additionally, ingestion of contaminated blood or tissue can transmit salmonella (fox, ) . stressed animals, due to conditions such as weaning, parturition, old age or poor nutrition, are more susceptible to infection (quesenberry and carpenter, ) . recovered animals can develop an asymptomatic carrier state with intermittent shedding in the feces, making elimination of this bacterium from a colony difficult (fox, ; harkness and wagner, ; percy and barthold, ) . the incubation time for salmonella is - days and epizootic or enzootic patterns of infection are possible (fox, ; ganaway ) . salmonella occurs as peracute septicemia or acute, subacute, or chronic enteritis (songer and post, ) . acute disease is characterized by high morbidity and low to moderate mortality. clinical signs include depression, weakness, and fever. dehydration and electrolyte imbalance may be severe. infection spreads rapidly within a colony. conjunctivitis and abortions have also been noted (percy and barthold, ) . although weight loss is present, diarrhea may or may not occur in guinea pigs. in an epizootic outbreak, young guinea pigs may die suddenly without prior signs of illness. pathology in acute and peracute cases, there may be no lesions seen at necropsy (harkness and wagner, ; percy and barthold, ) . splenomegaly may be observed in subacute and chronic infections. minute white foci and/or yellowish-white nodules, up to several millimeters in diameter, may occur in the spleen, liver, or other lymph nodes. similar foci or nodules may also occur in the lung, pleura, peritoneum, and in the wall of the uterus. rupture of the nodules may result in a suppurative pleuritis, peritonitis, and/or pericarditis. gas may be observed in the small and large intestine, along with catarrhal enteritis (fish et al., ) . on histopathology, lesions support bacterial invasion, with subsequent necrosis and abscess formation (ganaway, ) . nodules are noted to be "typhoidlike" granulomas, with areas of central necrosis, surrounded by histiocytes. polymorphonuclear leukocytes and histiocytes are present in the peyer's patches of the ileum and jejunum. diagnosis salmonella can be isolated from blood, feces, and affected organs (most commonly from the spleen) (fox, ) . recovery requires special conditions: enrichment in a broth such as selenite-f or tetracycline and culture at °c on macconkey's or brilliant green agar (fox, ; songer and post, ) . (harkness and wagner, ; rigby, ) . all fresh fruit and vegetables should be thoroughly washed and stored in airtight containers. since so many species of mammals are potential carriers, the exclusion of wild rodents, birds, and other vermin is essential. when increased colony mortality is apparent and a chronic case is recognized at necropsy, salmonella infection is likely widespread and well established in the colony. in situations such as this, it is reasonable to depopulate the colony, sanitize the premises, and restock with salmonella-free guinea pigs. treatment on an individual basis will include the appropriate antibiotics with fluid therapy. animals that become asymptomatic after treatment may become carriers, a problem with zoonotic potential. antimicrobial resistance is expanding; % of salmonella isolates from domestic animals demonstrate multi-drug resistance (songer and post, ) . background streptobacillus moniliformis is one of the causative agents of rat bite fever, a significant worldwide, zoonotic, systemic infection of humans associated with contact with rats (gaastra et al., ; khatchadourian et al., ) . it was described in early recorded history in india and was recognized in the us as early as as a syndrome that developed following contact with rats (gaastra et al., ) . s. moniliformis also causes haverhill fever, a systemic infection of those who eat food contaminated with rat urine or feces (abdulaziz et al., ; mcevoy et al., ; shanson et al., ) . etiology streptobacillus moniliformis, a facultative anaerobic, non-motile, non-capsulate, pleomorphic, gram-negative rod, is the type species of the genera, streptobacillus, family leptotrichiaceae within the phylum fusobacteria (greenwood, ; nolan et al., ) . its genome has been completely sequenced (nolan et al., ) . epizootiology and pathogenesis s. moniliformis is commonly found in the nasopharynx of wild and pet rats (gaastra et al., ; wullenweber, ) , although typically excluded from modern spf laboratory animal facilities (nicklas et al., ; pritchett-corning et al., ) . the organism has also been found in carnivores, calves, pigs, turkeys, and a variety of rodents including guinea pigs presumably following contact with infected rats (gaastra et al., ; wullenweber, ) . most commonly, s. moniliformis in guinea pigs has been associated with cervical lymphadenitis and other localized abscesses (aldred et al., ; fleming, ; percy and barthold, ; van hoosier and robinette, ; wullenweber, ) . severe necrotizing bronchopneumonia with pyogranuloma formation was reported in one guinea pig by kirchner et al. ( ) . no clinical signs of disease were noted in their report. diagnosis gram stain of smears can be suggestive of infection by demonstrating short bacilli in chains or long filaments followed by bacterial culture and isolation (greenwood, ; kirchner et al., ; wullenweber, ) although because of the fastidious nature of s. moniliformis, pcr is more commonly used for detection . if culture is attempted, samples must be cultured on a media containing serum, blood or ascitic fluid, such as loeffler's serum medium (greenwood, ) . cross-reactive sequences between leptotrichia spp. and s. moniliformis have been demonstrated and can result in false positives when pcr is used for diagnosis (boot et al., ; wouters et al., ) . sequence of the amplicon can differentiate these outcomes. as the entire sequence of s. moniliformis has recently been completed (nolan et al., ) , new primers for diagnosis may be forthcoming. antibodies to s. moniliformis can be demonstrated by elisa (boot et al., b (boot et al., , . because the organism is not spread easily, outbreaks can be controlled by culling affected guinea pigs (van hoosier and robinette, ) . treatment of individual animals with antibiotics is effective in other species (gaastra et al., ; wullenweber, ) , although treatment should be approached with caution because of the zoonotic potential of this organism. prevention of contact with infected rats, other animals that contact wild or infected rats, or infected food and/or bedding should eliminate introduction of this organism (van hoosier and robinette, ) . yersinia pseudotuberculosis background ganaway states that "one of the earliest recognized bacterial disease of the guinea pig was caused by yersinia pseudotuberculosis" (ganaway, ) . in present day colonies of laboratory guinea pigs, natural outbreaks of pseudotuberculosis are rare (fox, ; percy and barthold, ) . etiology yersinia pseudotuberculosis, an ubiquitous enteropathogen, is a gram-negative, non-spore-forming, facultative anaerobic rod that is oxidase-negative and catalase-positive (ganaway, ) . it is non-hemolytic and produces both exotoxin and enzyme (fox, ) . growth is optimal at - °c (fox, ; ganaway, ) . the genus yersinia is part of the family enterobacteriaceae and, in addition to guinea pigs, it causes pseudotuberculosis in other rodents, cats, and turkeys (songer and post, ) . epizootiology and pathogenesis y. pseudotuberculosis can result in clinical disease or a sub-clinical carrier state. guinea pigs are very susceptible to infection with this bacterium (fox, ; ganaway, ) . y. pseudotuberculosis is shed in feces, therefore ingestion of food contaminated with feces from a carrier guinea pig, wild rodent, or bird can transmit the organism, as can aerosol spread, or entry via a bite wound or skin laceration (fox, ; ganaway, ; rigby, ) . dams can pass infection to their pups (fox, ) . orally absorbed organisms disseminate from the intestinal tract to the mesenteric lymph nodes. acute and chronic forms of disease have been described (ganaway, ; rigby, ). an acute septicemic form of infection can lead to mortality in - hours. a chronic infection that often lasts several weeks to months and ultimately results in death is the more classic pseudotuberculosis presentation. the classic clinical presentation associated with y. pseudotuberculosis is that of wasting and lymphadenitis, sometimes with accompanying diarrhea, and frequently resulting in death about a month after initial infection (obwolo, ; sebesteny, ) . the acute form of the disease can present with no clinical signs or consist of one to two days of coughing and respiratory distress prior to death (rigby, ) . pathology necropsy of animals chronically or subacutely infected with yersinia pseudotuberculosis reveals nodules throughout the body, including the regional lymph nodes, spleen, liver, lung, and bone marrow (fox, ; percy and barthold, ) . the grayish-white spherical nodules vary from miliary pinpoint to - cm in diameter and may contain creamy to caseous exudate. in acute cases, nodules are noted in the intestinal wall, especially at the location of the terminal ileum and cecum, and often with accompanying mucosal ulceration and acute enteritis (percy and barthold, ) . lung congestion may be seen in acute cases (rigby, ) . yersinia nodules often have a central area of necrosis with neutrophilic infiltration; foamy macrophages are often noted in the periphery (fox, ; ganaway, ). blood vessels within the nodule may contain bacterial emboli. in chronic lesions, fibroblasts and epithelial cells proliferate and nodules may become granulomatous, but do not calcify. diagnosis y. pseudotuberculosis can be cultured from abscesses (rigby, ) . direct smears may also iii. guinea pigs be beneficial. in acute septicemic cases, yersinia may be cultured from the blood (ganaway, ) . yersina spp. can be cultured on standard media and y. pseudotuberculosis can be differentiated from y. pestis, the causative agent of plague, by the fact that it is urease, and rhamnose-positive, while y. pestis is negative for both of these growth conditions (songer and post, ) . treatment and control euthanasia of infected animals is advised due to the fact that antibiotic treatment can result in a carrier state, which, due to its zoonotic potential, puts human handlers and staff at risk. a killed vaccine has been unsuccessful at protecting guinea pigs from infection, but a more recent live vaccine has produced promising results (quintard et al., ) . background dermatophytosis, also known as ringworm or tinea, indicates infection with pathogenic fungi in the phylum ascomycota that cause the bulk of superficial fungal infections in humans and animals (ameen, ; howard et al., ) . the fungi are keratinolytic and keratinophilic and infection is usually limited to cornified layers of the skin, hair, or nails (howard et al., ; weitzman and summerbell, ) . their importance as pathogens is clear in that the majority of dermatophytes infecting animals are zoonotic (chermette et al., ) . originally, the dermatophytes were organized into three anamorphic, or asexual, genera, microsporum, trichophyton, and epidermophyton. the primary structure associated with asexual reproduction is the conidium and classification into these genera was based on the morphology of the macroconidia in culture. subsequent studies revealed sexual reproduction in certain microsporum and trichophyton spp. that occurs by means of an ascospore found within an ascus. the dermatophyte sexual states (teleomorphs) are classified in the genus, arthroderma (weitzman and summerbell, ) . recent efforts at phylogenetic analysis and identification of dermatophytes have been achieved by sequencing s ribosomal dna (rdna) and internal transcribed spacer (its) regions flanking . s rdna (drouot et al., ; frealle et al., ; symoens et al., ) . dermatophyte species can also be divided into three groups determined by their adaptation to a certain habitat. humanassociated dermatophytes are anthropophilic (e.g. trichophyton rubrum), those primarily found on animals and occasionally infecting humans are zoophilic (e.g. trichophyton mentagrophytes), and species persisting in the soil are termed geophilic (e.g. microsporum gypseum) (weitzman and summerbell, ) , however some overlap does occur. zoophilic and geophilic dermatophytes infecting humans cause a more intense inflammatory reaction than anthropophilic species (weitzman and summerbell, ) . etiology dermatophytosis in animals is most often caused by the anamorph species microsporum and trichophyton, however the infecting species can vary with the geographic region (bond, ) . of these genera, trichophyton mentagrophytes is the most common cause (aho, ; balsari et al., ; drouot et al., ; feuerman et al., ; lopez-martinez et al., ; mcaleer, a mcaleer, , b papini et al., ; pombier and kim, ; stenwig, ; vangeel et al., ) . t. mentagrophytes is a species complex with several variants including both anthropophilic and zoophilic pathogens. pollock ( ) considered t. mentagrophytes var mentagrophytes as the most common variant infecting rodents, however the specific variant is not always reported in the literature. the teleomorphs are known for the t. mentagrophytes complex and there are two, arthroderma benhamiae and a. vanbreuseghemii. the a. benhamiae genome has been sequenced (burmester et al., ) . the different variants and sexual states of the t. mentagrophytes complex coupled with more recent phylogenetic analyses presents confusion about proper nomenclature and species assignment (drouot et al., ; fumeaux et al., ) . infection with either m. canis or m. gypseum has also been reported in the guinea pig but less frequently (feuerman et al., ; papini et al., ) . guinea pigs, and other animals, are infected by direct exposure to an infected animal, contaminated environments, or fomites. arthroconidium produced by septation of hyphae are infectious and persist in the environment for months to years (bond, ; weitzman and summerbell, ) . hyphae invade the stratum corneum eventually colonizing the hair follicles. the sequenced genome of a. benhamiae revealed numerous predicted protease-encoding genes and their secretion was confirmed experimentally in a keratin digestion assay indicating an important role in invasion and pathogenesis (burmester et al., ) . young guinea pigs appear to be more susceptible to disease and other predisposing and susceptibility factors include pregnancy, high temperature and humidity, health status, and genetics (pollock, ; pombier and kim, ) . surveys of dermatophytosis in guinea pigs have been reported from around the world, with prevalence ranging from . - % (balsari et al., ; drouot et al., ; feuerman et al., ; lopez-martinez et al., ; papini et al., ; vangeel et al., ) . clinical manifestations infected guinea pigs are most often asymptomatic but can exhibit lesions typical of dermatophytosis. when present, lesions are most common on the head and appear to begin on the nose or muzzle and then can spread over the body to the trunk and limbs (figure . ). circumscribed or irregular lesions with combinations of alopecia, scale, crust, erythema, and ulceration develop, with or without pruritis, and spontaneous remission is possible (drouot et al., ; mcaleer, a; pollock, ; pombier and kim, ) . pustules may be associated with the lesions due to secondary bacterial infections. furthermore, in one outbreak of t. mentagrophytes, affected animals were in poor body condition and there was approximately % mortality in young guinea pigs (pombier and kim, ) . onychomycosis is rare in the guinea pig (drouot et al., ) . pathology microscopic analysis of affected epidermis and adnexa can include acanthosis, vascular congestion, dermal lymphocytic infiltration, folliculitis, perivascular, and interstitial dermatitis, and intra-epidermal pustules (chermette et al., ; pombier and kim, ) . diagnosis microscopy and culture are the primary means of diagnosing dermatophytosis. wood's lamp can be used as a screening tool for m. canis and infected hairs will fluoresce with an apple-green color. however, false negatives can occur (chermette et al., ) . hairs are plucked or the skin scraped from the periphery of an active lesion or from the whole lesion if there is no evidence of inflammation and samples placed in % potassium hydroxide to remove keratin. the samples are then examined for hair or skin invasion with hyphae and/or arthroconidia. to increase sensitivity, fluorescent microscopy with calcofluor white and congo red can be used (bond, ; robert and pihet, ) . geimsa staining will also identify arthroconidia. all the dermatophytes infecting guinea pigs will have ectothrix hair shaft involvement. prior to obtaining a skin or hair sample for culture, the area should be wiped with alcohol to reduce contamination. saprophytic fungi contamination of the fur may hinder isolation of dermatophytes (aho, ) . hair, skin, or crusts are collected using a sterile toothbrush, swab, forceps, scalpel blade, or surgical scrub brush and placed onto culture medium. sabouraud's dextrose agar supplemented with cyclohexamide and chloramphenicol is commonly used. dermatophyte test medium is available, however contamination can cause both false-positive and falsenegative results (robert and pihet, ) . if the animal is simply a mechanical carrier, results of repeated testing will vary (chermette et al., ) . biopsy samples can be stained with periodic acid-schiff or silver stains to detect arthroconidia and hyphae (figure . ) (chermette et al., ) . as mentioned earlier, polymerase chain reaction (pcr) amplification and sequencing of its and s ribosomal dna (drouot et al., ; fumeaux et al., ; symoens et al., ) will identify isolates, and mating experiments can be performed (hironaga et al., ) . reducing exposure to infected animals and contaminated environments is critical in preventing dermatophytosis. assuring a wellmanaged facility with no overcrowding, parasite control, and proper temperature and humidity regulation are important preventive components (pollock, ; iii. guinea pigs pombier and kim, ) . infected animals should be removed, the environment thoroughly decontaminated and any items that cannot be disinfected should be discarded. animal care staff should wear proper personal protective equipment when handling potentially contaminated items or bedding since zoonosis is a concern (mcaleer, a). dilute bleach, benzalkonium chloride, and glutaraldehyde are effective surface disinfectants (pollock, ) . in a laboratory setting, it is doubtful that animals will be treated for ringworm. readers are referred to other summaries of available topical and systemic treatments (pollock, ) . background the microsporidia (members of the phylum microspora) are a group of single-celled intracellular spore-forming eukaryotic parasites that infect a wide range of animals, from insects to mammals. the defining and unique feature of the phylum is the presence of a polar filament (tube) in the mature spore . the microsporidia also have one of the smallest genomes of any eukaryote and lack several key features, such as typical mitochondria. long thought to be protozoa, certain characteristics indicated that these organisms were fungi-like such as presence of a chitinous spore wall, genes for trehalose metabolism, and intranuclear division (brosson et al., ; dunn and smith, ) . recently, lee et al. ( ) concluded that microsporidia are true fungi related to zygomycetes. of the microsporidial species infecting humans, enterocytozoon bieneusi is the most common with lesser, but still significant, numbers of cases due to the encephalitozoon spp. the majority of patients are immunocompromised as a result of hiv infection, aids, or are organ transplant recipients. however, healthy immunecompetent persons are also susceptible to disease (didier and weiss, ; mathis et al., ) . animals are hosts for those common species strongly supporting their zoonotic potential. etiology the primary agent of microsporidiosis in guinea pigs is encephalitozoon cuniculi (syn. nosema). e. cuniculi was first reported in the rabbit as the cause of "infectious motor paralysis" (percy and barthold, ) and subsequently has been identified in a wide range of animal species . katinka et al. ( ) have sequenced the chromosomes of the approximately . -mb genome, highlighting the genome compaction and shortened protein sequences that reflect its intracellular location and host dependence. provide an informative summary of the mammalian microsporidian life cycle. environmentally resistant spores are shed in feces, urine, and mucus and then ingested or inhaled. transplacental transmission has been documented in the rabbit (baneux and pognan, ) and has been claimed in the guinea pig (boot et al., ) . predictably, primary sites of infection commonly include the small intestine, respiratory tract, and placenta. an unknown signal triggers the polar filament (tube) to be extruded from the spore and infectious sporoplasm is injected into the host cell cytoplasm. the sporoplasm then divides into meronts (merogony) that differentiate through intermediary forms before becoming mature spores (sporogony). for e. cuniculi and other encephalitozoon spp., developing spores are packaged in a parasitophorous vacuole. the host cell ruptures releasing spores that commonly disseminate to the kidney, liver, and brain. host protection from infection is primarily achieved by cell-mediated immunity with lesser contributions from the humoral immune system (khan et al., ) . from serologic surveys, prevalence of encephalitozoonosis in guinea pig colonies has ranged from %- %. this variability may be due to differences in housing and husbandry for the particular colony and diagnostic methodology (boot et al., ; gannon, ) . clinical manifestations encephalitozoonosis is subclinical in the guinea pig (boot et al., ; gannon, ; illanes et al., ; moffatt and schiefer, ; wan et al., ) . pathology gross lesions of encephalitozoonosis in the guinea pig are not consistently found and when present have only been described in the kidney. infected animals may have pale kidneys and/or pitting of the renal cortex (gannon, ; moffatt and schiefer, ) . histologic lesions are found primarily in the brain and kidney. focal to multifocal granulomatous encephalitis may be evident in different regions of the brain, with or without associated necrosis, and perivascular and meningeal mononuclear cell infiltrate (moffatt and schiefer, ; wan et al., ) . organisms approximately - . µm in width  . - . µm in diameter (illanes et al., ; can be found within or adjacent to lesions or free in the tissue with no associated inflammation. in the kidney, interstitial nephritis and necrosis, fibrosis, perivascular cuffing and tubular ectasia have all been described (boot et al., ; gannon, ; moffatt and schiefer, ) . here, organisms may occasionally be visualized in renal epithelial cells and collecting tubule lumens. absence of typical lesions in the brain or kidney in a seropositive animal may be due to the multifocal nature of the lesions and insufficient sections being analyzed (gannon, ; wan et al., ) . other lesions reported include necrotic liver foci and interstitial pneumonia with perivascular and peribronchial lymphoid accumulation. diagnosis serology tests including carbon immunoassay, ifa, elisa, multiplex fluorescent immunoassay, and western blot have all been or currently are being used for colony monitoring (percy and barthold, ; . presence of typical histopathologic lesions and special stains confirm the infection. while indistinct with hematoxylin and eosin staining, spores are periodic acid-schiff, gram, and modified trichrome positive (figure . ) . chemofluorescent agents such as calcofluor white can also be used (garcia, ) and spores are birefringent (tiner, ) . pcr primers are available (baneux and pognan, ; ghosh and weiss, ) providing a fast and easily interpretable means of diagnosis and the genome for e. cuniculi has recently been sequenced providing further opportunities for novel molecular diagnostic strategies (katinka et al., ) . urine, brain, and kidney are optimal specimens for testing by pcr. electron microscopy is the gold standard for diagnostic confirmation and offers species identification which is particularly important for human infections (garcia, ) . prevention and therapy e. cuniculi-free guinea pigs are commercially available and therefore, protection from contaminated animals or environments is the primary means of prevention (gannon, ) . faced with contamination, eradication may be successful through serological analysis and euthanasia of seropositive animals (baker, ) coupled with environmental decontamination. e. cuniculi spores are environmentally stable for days to weeks depending on the temperature and humidity, but are susceptible to common disinfectants (jordan et al., ; waller, ) . vertical transmission is thought to occur in guinea pigs (boot et al., ) , so hysterectomy rederivation should be used with caution and offspring extensively tested. it is unlikely that laboratory guinea pigs would be treated for encephalitozoonosis. two of four laboratory-bred guinea pigs were found dead several days to two weeks after adoption (kunstyr et al., ) . diarrhea was noted in one animal prior to death and each had gross evidence of enteritis at necropsy which was confirmed histologically. the yeast candida pintolopesii, a normal inhabitant of the gut, was isolated from the small intestine, lung, and ascitic fluid of one animal. the authors hypothesized that a change in social structure, diet, and environment precipitated disease. cryptococcus neoformans is a basidiomycetous yeast causing serious infections of the lungs and central nervous system in immunocompromised persons (boekhout and guého, ) . betty ( ) reported chronic, subclinical cryptococcal meningitis in laboratory-reared dunkin hartley guinea pigs with no evidence of generalized infection. the source of infection was not identified. van herck et al. ( ) described dermal cryptococcosis of an adult male pet guinea pig. histopathologic analysis of a large ulcerative lesion on the dorsal aspect of the nose showed extensive focal ulcerative dermatitis with infiltrating neutrophils, plasma cells, lymphocytes, and macrophages with edema and ovoid organisms. dermal and subcutaneous tissue also contained large numbers of these thick-walled organisms that were positive by periodic acid-schiff staining. enterocytozoon bieneusi is a significant cause of intestinal microsporidiosis in immunocompromised persons and has been identified in many mammalian and nonmammalian hosts (mathis et al., ) . microsporidian spores were found in the feces of a two-year-old male entered into a prospective study of pediatric enteric parasites in peru. to investigate the source of infection, stool was analyzed from animals in the household, including guinea pigs, chickens, dogs, and cats. seven of eight asymptomatic guinea pigs were positive for spores, whereas all the other animals were negative. polymerase chain reaction and sequence analysis confirmed that both the child and guinea pigs were infected with e. bieneusi and with the same genotype. furthermore, this specific genotype was found in guinea pigs from other unrelated iii. guinea pigs households suggesting that guinea pigs are the natural host and there is zoonotic potential for this organism. there is a single report from brazil of a naturally occurring histoplasmosis outbreak in laboratory guinea pigs (correa and pacheco, ) . clinical signs of disease in adults included progressive emaciation and hindlimb dysfunction before death. young animals exhibited a hunched back, ruffled fur, and conjunctivitis with discharge before dying at two months of age. gross and histologic lesions were numerous; however, for these authors a characteristic lesion of colitis with the wall expanded by numerous lymphocytes, macrophages, epithelioid and giant cells with basophilic organisms free or inside macrophages and culture results established the diagnosis. there may have been a link between this outbreak and histoplasmosis diagnosed in a cow on a farm from which the grass used to feed the guinea pigs was sourced. hortaea werneckii, the causative agent of tinea nigra in people, was diagnosed in a household guinea pig in japan (sharmin et al., ) . lesions included a focal area of ulceration and alopecia on the back and black pigmentation on the palmar aspect of the right forepaw. h. werneckii was detected by mycologic culture of the back lesion and dna sequencing of an amplified region of large subunit ribosomal dna. the authors hypothesized that the animal contracted the infection from the environment, but could not definitively rule out contamination with the fungus rather than a real infection. paecilomyces spp. are occasional opportunists in humans and animals (summerbell, ) . this fungus was identified during routine monitoring from a single conventionally housed laboratory guinea pig with no apparent clinical signs . paecilomyces spp. were most commonly identified in specific pathogenfree rats from one particular animal facility with the trachea and lungs being the most common site of infection in all animals analyzed. mild dermatitis was noted in a group of hairless mice; however, the vast majority of animals had no clinical or histologic evidence of disease. a hairless mutant arose in a closed colony of hartley guinea pigs. in addition to abnormal haircoat development, affected guinea pigs were smaller than their haired siblings and died early due to infections otherwise associated with an abnormal immune system such as systemic cytomegalovirus and balantidiasis, and pneumocystis pneumonia (reed and o'donoghue, ) . abnormal thymic and lymphoid follicle morphology and agammaglobulinemia confirmed the immunodeficiency. protozoa rarely cause disease in guinea pigs and intestinal protozoa are often considered part of the normal flora. in this review, the more frequently reported protozoa of domestic guinea pigs will be discussed. the reader is referred to parasites of laboratory animals (baker, ) and morphology and taxonomy of the intestinal protozoa of the guinea pig (nie, ) for a more detailed description of the protozoa of this species. the nomenclature used in this section is in accordance with the reference. amoebiasis etiology endolimax caviae and entamoeba caviae (syn. entamoeba cobayae) are the causative agents of amoebiasis in guinea pigs (levine, ) . nie ( ) reported the incidence of endolimax caviae to be % and the incidence of entamoeba caviae to be % in a colony of guinea pigs studied at the university of pennsylvania. pathogenesis endolimax caviae and entamoeba caviae are observed in the cecum of the guinea pig (nie, ) . both of these organisms are thought to feed upon the fecal material and microflora of the host intestine, without invasion of tissue or consumption of blood. transmission is via ingestion of cysts passed in the feces. clinical manifestations infection with either organism does not generally result in clinical signs (levine, ) . pathology entamoeba and endolimax are usually non-pathogenic (baker, ; levine, ) . diagnosis trophozoites can be observed in a smear of intestinal contents (baker, ; levine, ) . cysts are detected by zinc sulfate float. endolimax cavieae trophozoites are smaller than those of entamoeba caviae, with an average diameter of . µm and . µm, respectively (nie, ) . the cyst forms of either organism have rarely been observed, and little detail has been recorded on their appearances. prevention and therapy generally, treatment is not necessary for guinea pig amoebiasis. balantidia caviae background upon initial description, controversy developed over whether b. caviae was the same organism as b. coli in swine (vetterling, ) . morphological descriptions and infection studies have since proved that the two species are distinct. etiology balantidium caviae is the causative organism. pathogenesis b. caviae transmission is via ingestion of cysts in the feces (baker, ) . clinical manifestations if the mucosal barrier of the intestine is compromised, b. caviae can become invasive and result in enteritis; otherwise infection is non-pathogenic (baker, ) . pathology b. caviae may be observed in the walls of the cecum and colon, but penetration can occur postmortem, a consideration that should be made when diagnosing infection at the time of necropsy (baker, ; nie, ) . if invasion is ante-mortem, intestinal inflammation should be present; more often, though, b. caviae is considered a commensal protozoan. diagnosis in addition to diagnosing b. caviae in histologic sections of the cecum or colon, ciliated organisms with micro-and macro-nuclei can be observed in fresh fecal smears (baker, ; fox, ) . proper hygiene is most important in controlling b. caviae infection in guinea pigs (baker, ) . tetracyclines have been used to treat balantidiasis in other species. background c. wrairi is named after the walter reed army institute of research where the organism was first identified in a colony of laboratory guinea pigs (vetterling et al., ) . etiology cryptosporidium wrairi is the causative organism. pathogenesis the route of infection for c. wrairi is likely fecal-oral, with ingestion of oocysts in feces (baker, ; fox, ) . experimental infection has shown that the duration of infection for animals older than weeks is usually short, lasting as little as - weeks, after which the organism is cleared (chrisp et al., ) . younger animals have a longer duration of infection. recovered animals appear to be refractory to subsequent infection. clinical manifestations clinical signs are more likely to be seen in young animals, with weight loss noted most commonly (baker, ; fox, ) . if infection is severe, anorexia, a potbellied appearance and a greasy hair coat may be observed with or without accompanying diarrhea (baker, ) . morbidity and mortality, especially in young animals, can reach levels of % during an outbreak (harkness and wagner, ; percy and barthold, ) . echinococcus coli has been associated with clinical cases of c. wrairi, but the significance of this infection is unknown (percy and barthold, ) . subclinical infections with c. wrairi are thought to be possible (baker, ; fox, ) . pathology erosion, hyperemia, and inflammation of the small intestine have been observed with c. wrairi infection (baker, ; chrisp et al., ; harkness and wagner, ) . edema of the lamina propria and hyperplasia of the crypt epithelium occurs. in chronic infections, villous atrophy and bridging, metaplasia of the mucosal epithelium, and lymphocyte infiltration of the lamina propria have been noted. developmental stages of c. wrairi can be seen throughout the intestine, with highest concentrations of the organism noted in the brush border of the ileum. diagnosis oocysts can be seen in fecal floats (gressler et al., ; percy and barthold, ) . kinyoun staining of fecal smears and microscopic examination of mucosal scrapings or histological sections are also diagnostic options. prevention and therapy prevention is the best means of control for c. wrairi. oocysts are resistant to many disinfectants or require high concentrations and/or long contact times before being rendered noninfectious. heating above °c for greater than minutes has been described to be successful for the destruction of cryptosporidium organisms, as has exposure to temperatures below °c (baker, ; harkness and wagner, ) . successful treatment with sulfonamides has been reported, but the efficacy of this treatment has been questioned (fox, ; percy and barthold, ; sebesteny, ) . harkness suggests the "extra-label" use of high doses of the coccidiostat, decoquinate, to treat cryptosporidiosis (harkness and wagner, ) . while c. wrairi is not known to infect humans, the genus has a general lack of specificity and precautions should be taken to prevent zoonotic transmission (baker, ; harkness and wagner, ) . eimeria background reports of coccidia in guinea pigs date back to the late s, although the observed organisms were initially thought to be a variety of rabbit coccidian (vetterling, ) . in the early s, bugge and heinke showed that coccidia noted in the guinea pig were indeed distinct from those of the rabbit and in sheather named the guinea pig coccidia e. caviae. iii. guinea pigs etiology eimeria caviae is the causative organism. pathogenesis transmission of e. caviae is fecaloral, with ingestion of oocysts in feces (baker, ) . clinical manifestations clinical signs due to e. caviae are noted in severe infections and include diarrhea, anorexia and a rough hair coat (baker, ; ellis and wright, ; rigby, ) . the first appearance of diarrhea is often - days after initial infection (baker, ; elsheikha et al., ; percy and barthold, ) . watery, pasty, and hemorrhagic forms of diarrhea have all been reported (baker, ; elsheikha et al., ; fox, ; percy and barthold, ) . severe infections can result in death (baker, ; fox, ) . in mild infections or infections of older animals, diarrhea is likely to resolve in - days (baker, ; fox, ) , after which constipation may follow (fox, ) . stress, such as a diet change or transport, may exacerbate an otherwise nonpathogenic infection of e. caviae in the guinea pig (ellis and wright, ; elsheikha et al., ; rigby, ) . pathology the large intestine is most affected by e. caviae, with organisms most often settling in the proximal colon (baker, ; elsheikha et al., ) . hyperemia, edema, and petechial hemorrhage of the colonic mucosa may be seen (baker, ) . white or yellow plaques may be present in the colon or cecum. watery intestinal contents can contain blood (baker, ; percy and barthold, ) . dilated cystic crypts of liekberkuhn may be observed (baker, ) . developmental stages of e. caviae can be seen in intact epithelial cells or free in the intestinal lumen. enterocytes may slough and an infiltration of polymorphonuclear and mononuclear cells is possible (baker, ; percy and barthold, ) . microgametocytes and macrogametocytes may be present in the cecal and colonic mucosa (percy and barthold, ) . diagnosis oocysts are seen on fecal float (baker, ; fox, ; percy and barthold, ) . fox et al. ( ) recommend using a flotation medium of . specific gravity. the prepatent period of e. caviae is - days, yet diarrhea may occur before day ; therefore, a fecal float performed at the beginning of diarrhea may result in a false-negative finding. multiple floats performed every or days for several weeks is recommended (vetterling, ) . organisms may also be identified on mucosal scrapings or histopathology sections (baker, ; fox, ; percy and barthold, ) . sulfonamides are recommended for treatment. minimizing stress and providing an adequate level of vitamin c help to prevent clinical infection. oocysts in the feces take days to become infective, therefore regular cleaning of pans will help to minimize the spread of infection (rigby, ) . ammonia, followed by a thorough rinsing, has been used to clean cages and pans of infected animals (elsheikha et al., ). steam has also been used to clean enclosures. calhoon notes that eimeria-free colonies can be achieved via cesarean rederivation (calhoon and matthews, ) . etiology the causative agent of guinea pig giardiasis is giardia duodenalis, formally referred to as as g. caviae (baker, ; vetterling, ) . pathogenesis giardia duodenalis is a flagellate of guinea pigs that is transmitted by the fecal-oral route or via ingestion of contaminated food or water (baker, ) . clinical manifestations overt signs of infection, such as diarrhea, are rare, although some animals are severely infected and may present as weak and moribund (baker, ) . pathology the small intestine, primarily the duodenum, is colonized by trophozoites (baker, ) . colonization may result in mild inflammatory lesions, decreased villar height, and cystic enlargement of duodenal crypts. diagnosis diagnosis of g. duodenalis is via direct fecal smear, where trophozoites or cysts may be observed (baker, ) . only trophozoites, and not cysts, are found in diarrheic feces (vetterling, ) . shedding of g. duodenalis is intermittent, so unless multiple fecal smears are performed, there is a high likelihood of obtaining a falsenegative result. felasa recommends including giardia spp. on guinea pig breeding colony health monitoring reports (rehbinder et al., ) . prevention and therapy metronidazole and fenbendazole are treatment options for guinea pig giardiasis (baker, ) . infection can be prevented by thorough environmental sanitation using a quaternary ammonium product or sodium hypocholorite. hot temperatures will destroy cysts. cysts thrive in moist areas, so environments must be kept dry. the zoonotic potential of g. duodenalis of guinea pigs is unknown, but the possibility of transmission between guinea pigs and humans is suspected. klossiella cobayae background k. cobayae is also known as klassia caviae, as it was described and named simultaneously by two individuals, pearce and sangiori (respectively) working independently of each other (vetterling, ) . occurrence in modern laboratory guinea pigs is rare (percy and barthold, ) . etiology klossiella cobayae is the causative organism. pathogenesis k. cobayae is a parasite of the epithelial cells of the renal tubules, as well as the endothelial cells associated with glomerular capillaries and other organs, such as the spleen and lungs (griffiths, ; van andel et al., ; vetterling, ) . transmission is via ingestion of sporocysts in urine (baker, ) . (baker, ; sebesteny, ) . heavy infection can lead to an irregular surface and a gray mottled appearance of the kidney (baker, ; fox, ) . interstitial and perivascular lymphocytic and histiocytic infiltration may occur (baker, ) . a large number of interstitial fibroblasts may also be observed. diagnosis sporocysts are passed in the urine, yet observation of this stage in the urine is difficult (baker, ) . most often, diagnosis is made at the time of necropsy by identifying the different developmental stages of k. cobayae in glomerular capillaries or in the cytoplasm of epithelial cells lining the renal tubules (baker, ; percy and barthold, ) . felasa recommends including klossiella spp. as part of a health monitoring report for guinea pig breeding colonies (rehbinder et al., ) . prevention and therapy control of k. cobayae is best achieved through prevention. contamination of food and bedding with infective urine should be minimized (baker, ) . sulfonamides might be effective forms of treatment. background leishmania enrietti was first noted in laboratory guinea pigs in in brazil (medina, ) . according to the review by machado et al., this flagellate was next reported in in a guinea pig captured from the outskirts of curitiba (machado et al., ) . the sandfly, lutzomyia monticola, which is frequently noted on the trunks of curitibia pine trees, was proposed as a possible vector. etiology leishmania enrietti is the causative organism. pathogenesis little is known about the lifecycle of l. enrietta, but other members of the genus leishmania have indirect lifecycles and sandfly vectors (baker, (baker, , nie, ) . l. enrietta results in cutaneous infection. an infected guinea pig may present with a cutaneous nodule at the site of entry - weeks after infection (baker, ) . additionally, ulcers can develop on the feet, ears, nose, and genitalia of infected animals. ulcers will go through a series of pathologic changes, such as necrosis of surrounding macrophages - weeks after initial infection (especially in naive animals), followed by an infiltrate of giant cells, plasma cells, and lymphocytes at the periphery of the ulcer. by weeks, giant cells are gone and fibroblasts appear, indicating resolution. resolution is usually complete by weeks post-infection. hematogenous spread of the organism can occur and l. enrietti may be observed in the lymph nodes. diagnosis detection is via identification of amastigotes in lesion histology or via culture of the organism (baker, ) . control is achieved by elimination of vectors. treatment with meglumine antimoniate has been attempted, with inconsistent success (thomaz-soccol et al., ) . contact with possible sandfly vectors should be avoided. background this organism was first reported in brazil by carini and migliano (vetterling, ) . in early reports, t. gondii was mistaken for sarcocystis in the guinea pig (kean and grocott, ) . etiology toxoplasma gondii is the causative organism. pathogenesis cats are the definitive hosts of t. gondii (baker, ) . oocysts are released in cat feces and are ingested by guinea pigs which act as intermediate hosts for t. gondii. cysts can remain viable in a guinea pig for up to five years before ingestion by the definitive host. in addition to the guinea pig, a large number of mammalian and avian species are intermediate hosts to t. gondii, including humans. ingestion of contaminated biological material and transplacental transmission are alternative routes of transmission for the guinea pig (baker, ; percy and barthold, ) . clinical manifestations t. gondii infection in guinea pigs is often asymptomatic and, therefore, frequently goes undiagnosed (baker, ; griffiths, ; percy and barthold, ) . infection in pregnant sows can result in vulvar bleeding and abortion (fox, ) . in a case of spontaneous toxoplasma encephalitis in a guinea pig, spastic paralysis, opisthotonos, and loss of urethral and anal sphincter control were reported (markham, ) . pathology hepatitis, pneumonia, encephalitis, and uterine infection (possibly blood-filled), have been noted in infected animals, as have cysts in both the myocardium and central nervous system (baker, ; fox, ; percy and barthold, ) . in spontaneously infected animals that develop toxoplasma encephalitis, congested blood vessels of the meninges were noted and were surrounded by mononuclear leukocytes (markham, ) . diagnosis serology is often used for diagnosis in guinea pigs (baker, ) . cysts can be observed on histology. mice or hamsters can be inoculated with tissue homogenate from suspected positives and act as sentinels of infection. felasa recommends including toxoplasma gondii on guinea pig breeding colony health monitoring reports (rehbinder et al., ) . sulfadiazine and pyrimethamine are reported treatment options for clinical cases of t. gondii, yet the effectiveness of such treatments is questionable (baker, ; fox, ) . temperatures above °c kill oocysts (baker, ) . strict sanitation and periodic colony monitoring are necessary for t. gondii control. background tritrichomonas caviae has also been referred to as trichomonas caviae and t. flagelliphora (tanabe, ) . etiology tritrichomonas caviae is one of the largest intestinal flagellates of the guinea pig, measuring an average of μm long by . μm wide (baker, ) . no cyst stage is known. pathogenesis transmission of t. caviae is fecaloral (baker, ) . clinical manifestations tritrichomonas caviae is a non-pathogenic flagellate of guinea pigs (baker, ) . pathology t. caviae is most often noted in the cecum, although it has been observed in the duodenum, jejunum and lower ileum as well (baker, ) . although rare, tissue invasion and cecal or colonic ulceration are possible. diagnosis diagnosis of t. caviae is via direct fecal smear (tanabe, ) . prevention and therapy t. cavaie does not usually result in clinical signs or pertinent pathology and does not routinely require treatment (baker, ) . guinea pigs, among other mammals, are reservoirs for the flagellate t. cruzi (baker, ; vetterling, ) . reduviid bugs act as vectors, becoming infected after a blood meal from a positive animal. t. cruzi mature and reproduce within the vector. the bugs then go on to deposit t. cruzi-containing feces on humans during another blood meal. the organism enters human skin through a wound (potentially the bug bite) or contact with mucous membranes. chagas disease may result in humans. in the guinea pig, t. cruzi can encyst in multiple organs, including the skin and cardiac muscule. t. cruzi has been noted in domesticated guinea pigs of south america exposed to reduviid vectors. clinical and pathological signs are not described. the reader is once again directed to flynn's parasites of laboratory animals for further discussion on many of the parasites discussed below, including anatomical appearances and detailed life cycles (baker, ) . the nomenclature used in this section is also in accordance with this reference. note: the common names of arthropod organisms have been used in place of the name of the resulting condition (e.g. flies instead of myiasis). ctenocephalides felis has been noted on pet guinea pigs living in a household with cats and/or dogs. infected guinea pigs presented with pruritus, alopecia, dermal crusts, and anemia (white et al., ) . aqueous-based pyrethrin sprays have been recommended as the safest treatment option for such infection. nosopsyllus fasciatus, the northern rat flea, can also inhabit guinea pigs, although similarly to c. felis, infection of laboratory guinea pigs is rare (fox, ) . it has been stated that "fatal myiasis involving at least three species of flies, lucilia sericata, calliphora vicina and calliphora vomitoris, have been reported in guinea pigs" (baker, ) . eggs are deposited on the skin of a guinea pig by an adult fly, where larvae will develop and then feed on living or necrotic animal tissue (baker, ) . (baker, ) . g. porcelli, the most commonly observed of the three, is known as the "slender louse" due to its general appearance, especially when compared to g. ovalis or the "oval louse" (baker, ; harkness and wagner, ; kim et al., ) . several species of sucking lice (order anoplura) have been noted on wild guinea pigs in south america: pterophtirus alata and polyplax spinulosa (dittmar, ) . these later lice are listed in table . , but not discussed here in detail. pathogenesis biting lice attach to hair shafts and abrade the skin to ingest cutaneous fluids (fox, ; kim et al., ) . transmission is via direct contact (harkness and wagner, ; kim et al., ) . the life cycles of guinea pig lice are not specifically described, but in general, biting lice go through the following stages: egg, three nymphal stages, and adult (baker, ) . clinical manifestations g. ovalis, g. porcelli or t. hispidium infection of a guinea pig can be asymptomatic or may result in pruritus and a rough hair coat or alopecia (baker, ; coman et al., ; harkness and wagner, ; kim et al., ; percy and barthold, ; rigby, ) . scabs and crusts are sometimes noted secondary to scratching, especially around the ears (harkness and wagner, ) . pathology signs of louse infection are usually superficial. dermatitis may be noted grossly. diagnosis lice (adults or nits) can be seen on the haircoat of an infected animal, with or without the aid of a magnifying lens (figure . ) (baker, ; griffiths, ). on a dead animal, lice will migrate to the warm tips of the hair as the skin cools (griffiths, ) . tape tests and flea combing can also be used to diagnose louse infestation (coman et al., ) . as stated previously, g. porcelli is slender in appearance ( . - . mm  . - . mm), compared to g. ovalis ( . - . mm  . mm) (figure . ) (huerkamp et al., ) . t. hispidum's dimensions are similar to those of g. ovalis, yet can be differentiated by the fact that t. hispidum has five abdominal segments as compared to g. ovalis' eight. in reports of successful control of guinea pig louse infestations, both infected animals and the environment have been treated (harkness and wagner, ; kim et al., ) . systemic ivermectin (baker, ; white et al., ) , carbamate or pyrethrin-based powders (baker, ; griffiths, ) , romavermectin b (coman et al., ) and a combination of imidacloprid and moxidectin (kim et al., ) have been noted as effective drug choices for treating infected guinea pigs. hypochlorite bleach has been used for environmental decontamination (kim et al., ) . guinea pigs are susceptible to infection with several species of mites, some occurring more commonly than others. chirodiscoides caviae and trixacarus caviae are the most commonly noted infectious mites in guinea pigs, with demodex caviae, myocoptes musculinus, sarcoptes scabies and notoedres muris reported less frequently (baker, ; fox, ; white et al., ) . infection with the astigmatic mite, acarus farris, was described in a case report of two guinea pigs (linek and bourdeau, ) . psocoptes cuniculi has been described on a pet guinea pig in close contact with a pet rabbit (yeatts, ) . white et al. ( ) note cheyletiella parasitivorax as a cause of pruritus and scaling along the dorsum of guinea pigs. the more commonly noted mites, c. caviae and t. cavaie, will be discussed below. the first reports and illustrations of c. caviae were by stanley hirst in the early s (hirst ). hirst noted this mite attached to the hairs of the back of the guinea pig. etiology chirodiscoides caviae is the causative organism (figure . ). pathogenesis c. caviae is a species-specific fur mite that tends to concentrate in the lumbar area and the lateral aspects of the hindquarters (harkness and wagner, ; percy and barthold, ; schonfelder et al., ) . c. caviae feed on the scales of hair shafts (hirsjarvi and phyala, ) and transmission is via direct contact with an infected animal (fox, ; schonfelder et al., ) or with infected cage debris or bedding (fox, ) . the life cycle of c. caviae has not been specifically described, although mites in general commonly go through egg, larval, nymphal, and adult stages (baker, ) . concurrent infection with lice is not uncommon (white et al., ) , and mixed infection with demodex caviae (detected via deep skin scrapping) has also been reported (schonfelder et al., ) . clinical manifestations infection with c. caviae can be asymptomatic or, if infestation is heavy, pruritis, alopecia, hyperemia, and dermal crusts may be seen (coman et al., ; fox, ; hirsjarvi and iii. guinea pigs phyala, ; lumeij and cremers, ; schonfelder et al., ) . anorexia may result secondary to grooming pruritic areas (schonfelder et al., ) . the appearance of c. caviae mites themselves is the major pathologic finding (fox, ) . mites and ova are located on the hair shafts of the guinea pigs, not burrowed into the skin. diagnosis mites may be seen with the unaided eye, hand lens or dissecting microscope. c. caviae can also be found on a superficial scrape (harkness and wagner, ; percy and barthold, ; schonfelder et al., ) . combing, hair plucking, and cellophane tape testing can aid in isolating mites for observation (white et al., ) . prevention and therapy many treatment options have been described in the literature for c. caviae. dilute ivermectin (diluted in aqua/propylene glycol) sprays have been successful, especially for treating large colonies of infected animals (baker, ; hirsjarvi and phyala, ; white et al., ) . selamectin, - mg/ kg, applied twice at -week intervals has also eliminated infection (baker, ; fox, ; schonfelder et al., ) . pyrethrin sprays and powders have been used to rid the environment of c. caviae (harkness and wagner, ; hirsjarvi and phyala, ) . % virkon s ® was used to treat an automatic watering system (hirsjarvi and phyala, ) . older successful treatments include the use of dichlorvos vapors (henderson, ) as well as immersion of shaved animals in . % trichlorfon (lumeij and cremers, ) . background t. caviae was discovered in a colony of albino guinea pigs in the early s by fain and was described as a disease very similar in appearance to sarcoptic mange in other animals (dorrestein and vanbronswijk, ; fain et al., ) . the first report of t. caviae in north america was in at davis, california (mcdonald and lavoipierre, ) . etiology trixacarus caviae is the causative organism (figure . ). trixacarus caviae is a species-specific sarcoptic mite that most commonly affects guinea pigs that are - years of age (dorrestein and vanbronswijk, ) . if left untreated, the parasite load peaks month post-infection, after which it slowly regresses (fuentealba and hanna, ) . the life cycle of t. caviae includes an egg, larval, two nymphal, and a final adult stage, all of which inhabit the host guinea pig (baker, ) . transmission is by direct contact, with nymphal or larval stages indicated in establishing new infestations (baker, ; rothwell et al., ) . within hours, pups born to an infected guinea pig dam will show clinical signs consistent with t. caviae infestation (beck et al., ; kummel et al., ) . humans have been reported to develop transient pruritic, papulovesicular dermatitis after contact with infected animals, although mites appear to be incapable of persisting on human skin (fuentealba and hanna, ; kummel et al., ; mederle and indre, ) . t. caviae may be asymptomatic and result in non-clinical carrier animals, t. caviae has been reported as the most common and important cause of dermatitis in guinea pigs (dorrestein and vanbronswijk, ; fuentealba and hanna, ; percy and barthold, ; white et al., ) . severe pruritis and excoriation along with erythema and alopecia can accompany dermatitis in some animals (ackerman, ; beck et al., ; dorrestein and vanbronswijk, ) . in those guinea pigs that are clinical, grayish to yellow or white crusts may be present on the skin and can be dry or slightly greasy (ackerman, ; dorrestein and vanbronswijk, ) . secondary bacterial infections of infected skin areas are possible (fuentealba and hanna, ) . infection usually starts on the face and ears, spreading to the lumbar regions and lateral aspects of the legs (dorrestein and vanbronswijk, ; percy and barthold, ) . the intense pruritic response in some guinea pigs is reported to be due to an initial allergic reaction to mite antigen (fox, ) . extreme restlessness and emaciation have been noted in affected animals, especially in those that seemingly scratch uncontrollably (beck et al., ) . this constant scratching can lead to "fits" of muscular spasms or sporadic epileptiform seizures (ackerman, ; beck et al., ; dorrestein and vanbronswijk, ; kummel et al., ) caused by generalized pruritus-induced hyperesthesia (baker, ) . pathology orthokeratotic hyperkeratosis and acanthosis have been noted in animals showing clinical signs of t. caviae infection (ackerman, ; dorrestein and vanbronswijk, ; fuentealba and hanna, ; zenoble and greve, ) . a polymorphonuclear leucocytic infiltrate is present in the dermis (dorrestein and vanbronswijk, ; percy and barthold, ; rothwell et al., ; zenoble and greve, ) . adult mites may be noted in "tunnels" or burrows located in the hyperkeratotic areas (dorrestein and vanbronswijk, ; zenoble and greve, ) . such tunnels approach the epidermis perpendicularly, with some entering the mouth of hair follicles (zenoble and greve, ) . eggs may also be present in tunnels (dorrestein and vanbronswijk, ) . t. caviae mites have sharp spines on the cuticle of their dorsums (zenoble and greve, ) , which differentiates them from notoedres (ackerman, ) . the anus of the female t. caviae mite is located dorsally, while this structure is located terminally on s. scabiei (ackerman, ; baker, ; fuentealba and hanna, ) . diagnosis deep skin scrapes of crusted areas with % koh reveal t. caviae (dorrestein and vanbronswijk, ; fain et al., ; fuentealba and hanna, ; mederle and indre, ). zajac et al. ( ) report that live mites could be demonstrated for days postmortem from deep skin scrapings taken from a guinea pig held at °c after death. prevention and therapy treatment of both affected animals and the environment has been employed to rid guinea pig colonies of t. cavaie. multiple treatment regimens have been reported as successful over the years, including lindane baths (dorrestein and vanbronswijk, ; zajac et al., ) , weekly lime sulfur dips (ackerman, ; mcdonald and lavoipierre, ; zenoble and greve, ) , ivermectin injections (white et al., ) , and monthly spot-on treatment with % imidacloprid and % moxidectin (beck et al., ) . dilute lime sulfur can also be used to treat the environment (white et al., ) . valium has been used to control seizure-like activity related to t. cavaie infection by some, whereas others suggest that treating the mite infection alone eliminates this sequela to infection (beck et al., ; white et al., ) . according to harkness and wagner ( ) , tapeworms rarely infect guinea pigs. anoplocephala spp. and monoecocestus parcitesticulatus (table . ) have been reported in the intestines of south american guinea pigs (baker, ; sardella and fugassa, ). etiology baylisascaris procyonis is the causative organism. pathogenesis guinea pigs are paratenic hosts of b. procyonis and contract infection by ingesting eggs in raccoon feces (baker, ) . once ingested, larvae will hatch and penetrate the small intestine of the guinea pig, migrate through the liver to the lungs and disseminate throughout the body via the circulatory system. larvae then encapsulate where they remain until ingested by a raccoon. clinical manifestations no signs are present in the guinea pig due to b. procyonis unless organisms migrate to the brain. migration can result in lethargy, head tilt and ataxia, which may progress to cachexia, stupor, hyperexcitability, lateral recumbency, torticollis, or opisthotonos (van andel et al., ) . pathology in cases were b. procyonis migrates to the brain, multifocal eosinophilic granulomatous inflammation and neutrophilic infiltration is seen, along with perivascular lymphoid cuffing and malacia (van andel et al., ) . eosinophilic granulomata have been noted in the lungs of some infected animals. diagnosis histology is diagnostic for b. procyonis (baker, ) . the baermann extraction technique can be used to extract organisms from the cerebral tissue of clinical animals (van andel et al., ) . prevention and therapy prevention, rather than treatment, is the most logical approach for b. procyonis control, as clinical signs are often not noted until the end stage of infection and diagnosis relies on histopathology. contamination of bedding or food with raccoon feces must be avoided. ova can be viable for years in soil and weeks to months in straw and are resistant to most disinfectants (fox, ) . removal of contaminated bedding and the autoclaving of caging may be required (baker, ) . humans, similarly to guinea pigs, can contract infection by ingesting raccoon feces, but transmission from guinea pigs to humans is not feasible (fox, ) . etiology paraspidodera uncinata is the causative organism. epizootiology and pathogenesis p. uncinata is the cecal worm of guinea pigs and the most common nematode infecting this species (griffiths, ) . the prevalence of p. uncinata in wild guinea pigs of south america was found to be %, while the prevalence was noted as . % in a laboratory colony of guinea pigs (coman et al., ; dittmar, ) . transmission is via ingestion of an embyronated egg in feces (baker, ; fox, ) . eggs become infectious - days after they are initially shed (fox, ) . the life cycle is not well described (baker, ) . clinical manifestations infections are normally asymptomatic, but with heavy parasitic loads weight loss, diarrhea, and disability are possible (fox, ) . pathology infection can, and often does, occur without pathologic changes (baker, ; percy and iii. guinea pigs barthold, ; rigby, ) . if cecal worms migrate through the intestinal mucosa, a hemorrhagic typhlitis may be seen along with capillary ectasis in the submucosa (coman et al., ). diagnosis adult worms may be seen in the cecum at necropsy and eggs may be observed in feces ( figure . ) (baker, ; rigby ) . prevention and therapy levimasole administered at mg/kg, either orally or subcutaneously, has most often been reported as an effective treatment for p. uncinata in the guinea pig (baker, ; eliazian et al., ) . other reported treatment regimens include oral mebendazole at mg/kg (baker, ) and romavermectin b given subcutaneously at a dose of . mg/kg twice at a three-week interval (coman et al., ) . etiology pelodera strongyloides is the causative organism. pathogenesis p. strongyloides organisms are usually found in damp soil or vegetation (white et al., ) . bedding can be a possible source of infection. clinical manifestations while typically nonpathogenic, if p. strongyloides invade hair follicles dermatitis can result (baker, ; white et al., ) . pathology organisms may be found in hair follicles of clinically infected animals. the observation of larvae in skin scrapings or biopsies signifies infection (white et al., ) . adults may be observed in the bedding. prevention and therapy cages should be kept dry, as organisms thrive in moist environments (white et al., ) . the bedding of any positive animal should be replaced. a - % chlorhexadine bath has been recommended for infected animals as a means of preventing secondary infections. trichinellosis due to trichinella spiralis is possible in guinea pigs, but rare, especially in laboratory animals that seldom come in contact with infected material (raw or undercooked meat) (sebesteny, ) . enteritis could develop in infected animals if t. spiralis organisms migrate through the intestinal wall. fasciola background infection in present laboratoryhoused guinea pigs is rare, but when fasciola organisms are noted it is often in conjunction with recent diet supplementation of a leafy vegetable carrying encysted trematodes (baker, ; fox, ) . etiology two fasciola species are noted in guinea pigs, fasciola hepatica and fasciola gigantica (baker, ) . f. gigantica is slightly larger in size than f. hepatica, although f. gigantica rarely reach patency in guinea pigs. pathogenesis guinea pigs ingest encysted fasicola metacercariae on vegetation (baker, ; fox, ) . once ingested, metacercariae hatch in the small intestine and migrate through the wall to the peritoneal cavity and the liver. juvenile flukes develop in the liver and, once mature, burrow in the bile ducts. eggs are shed and enter the gut, where they pass in the feces. a snail intermediate host is necessary for maturation from cercariae to infectious metacercariae. metacercariae encyst on leafy vegetation. clinical manifestations emaciation and anemia have been associated with fasciola infection (sebesteny, ) . posterior paresis was noted in animals that developed cysts in the lumbar musculature after aberrant fluke migration (baker, ) . pathology hepatic congestion and hemorrhage, especially around portal vessels, central veins and sinusoids is possible (baker, ) . fibronecrotic tracks and granulomas may also be observed in the liver. other pathology due to fluke infection is often the result of aberrant migration, resulting in cysts in areas such as the kidney, the peritoneal cavity and the pelvic cavity. cysts often contain coffee-brown-colored material. diagnosis fasciola spp. can be observed on fecal flotation (baker, ) . prevention and therapy prevention is best achieved by limiting the amount of leafy vegetation supplemented in a guinea pig's diet; if supplemented, do not feed vegetation from fluke-endemic locations. fasciola in ruminants is treated with albendazole and clorsulon (baker, ) . haverhill fever with spine involvement trixacarus caviae infestation in a guinea pig studies on fungal flora in hair from domestic and laboratory animals 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protozoa of the guinea-pig, cavia-porcella development of a high-sensitivity nested pcr assay for the detection of clostridium piliforme in clinical samples complete genome sequence of streptobacillus moniliformis type strain ( ) the pathology of experimental yersiniosis in guinea pigs an epizootic infection of citrobacter freundii in a guineapig colony: short communication genetic characterization of parainfluenza virus derived from guinea pigs morphology of cavian leukemia the guinea pig as a model of infectious diseases detection of all chlamydophila and chlamydia spp. of veterinary interest using species-specific real-time pcr assays survey of dermatophytes isolated from the coats of laboratory animals in italy the changing brucella ecology: novel reservoirs, new threats microbiological monitoring of guinea pigs reared conventionally at two breeding facilities in korea susceptibility of inbred and outbred mouse strains to sendai virus and prevalence of infection in laboratory rodents pathology of laboratory rodents and rabbits report of a laboratory epizootic among guineapigs, associated with gaseous emphysema of the liver, spleen, and kidneys, due to bacillus mucosus oapsulatus the citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic escherichia coli resistance to cephalosporins and carbapenems in gram-negative bacterial pathogens exposure to nontraditional pets at home and to animals in public settings: risks to children of guinea pigs and men--an unusual case of jaundice fungal diseases of laboratory rodents an epizootic outbreak of ringworm in a guinea-pig colony caused by trichophyton mentagrophytes polymerase chain reaction for detection of guinea pig adenovirus contemporary prevalence of infectious agents in laboratory mice and rats ferrets, rabbits, and rodents: clinical medicine and surgery efficacy of an oral live vaccine for veterinary use against pseudotuberculosis pathogenesis of endometritis and salpingitis in a guinea pig model of chlamydial genital infection humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis a new guinea pig mutant with abnormal hair production and immunodeficiency equine herpesvirus and report of the federation of european laboratory animal science associations (felasa) working group on animal health accepted by the felasa board of management clostridium difficile antitoxin neutralization of cecal toxin(s) from guinea pigs with penicillin-associated colitis implication of clostridium difficile and clostridium perfringens iota toxins in experimental lincomycinassociated colitis of rabbits toxicity of cecal filtrates from guinea pigs with penicillin-associated colitis natural infections of guinea-pigs conventional methods for the diagnosis of dermatophytosis haematological and pathological responses to experimental trixacarus caviae infection in guinea pigs clostridium difficile 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and mating behaviour of the trichophyton mentagrophytes species complex a study of trichomonas from the guinea-pig new isolation of leishmania enriettii muniz and medina, in paranastate, brazil, years after the first description, and isoenzymatic polymorphism of the l. enriettii taxon genome sequence of the non-pathogenic strain of pneumonia virus of mice and comparison with the genome of the pathogenic strain j birefringent spores differentiate encephalitozoon and other microsporidia from coccidia a fatal disease of the japanese waltzing mouse caused by a spore-bearing bacillus (bacillus piliformis cerebrospinal larva migrans due to baylisascaris procyonis in a guinea pig colony leptospirosis in dogs: a review with emphasis on clinical aspects dermal cryptococcosis in a guinea pig disseminated cytomegalovirus disease in the guinea pig viral and chlamydial diseases prevalence of dermatophytes in asymptomatic guinea pigs and rabbits typhlitis caused by intestinal serpulina-like bacteria in domestic guinea pigs (cavia porcellus) the biology of the guinea pig cryptosporidium wrairi sp. n. from the guinea pig cavia porcellus, with an emendation of the genus lesions of experimentally induced tyzzer's disease in syrian hamsters, guineapigs, mice and rats otitis media of guinea pigs sensitivity of encephalitozoon cuniculi to various temperatures, disinfectants and drugs diagnostic exercise: granulomatous encephalitis in guinea pigs therapeutic efficacy of oral lactobacillus preparation for antibiotic-associated enteritis in guinea pigs mammalian microsporidiosis naturally occurring herpes simplex encephalitis in a domestic rabbit (oryctolagus cuniculus) the dermatophytes dermatologic problems in guinea pigs tularemia, plague, yersiniosis, and tyzzer's disease in wild rodents and lagomorphs in canada: a review fatal epizootic equine herpesvirus infections in new and unnatural hosts spontaneous human herpes virus type infection in a chinchilla (chinchilla lanigera f. dom.) human infections associated with bordetella bronchiseptica dogs as vectors of streptobacillus moniliformis infection an epidemic of pasteurella infection in a guinea-pig stock streptobacillus moniliformis -a zoonotic pathogen. taxonomic considerations, host species, diagnosis, therapy, geographical distribution interlaboratory comparison of enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence (iif) for detection of bordetella bronchiseptica antibodies in guinea pigs rabbit mite infestation development of resistance to reinfection of bordetella bronchiseptica in guinea pigs recovered from natural infection an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs mange caused by trixacarus caviae in guinea pigs persistent sv virus infection in continuous cell cultures sarcoptid mite infestation in a colony of guinea pigs naturally occurring tyzzer's disease and intestinal spirochetosis in guinea pigs key: cord- -smlq y authors: dhakal, santosh; renukaradhya, gourapura j. title: nanoparticle-based vaccine development and evaluation against viral infections in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: smlq y virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. the economic burden caused by virus infections such as porcine reproductive and respiratory syndrome virus, swine influenza virus, porcine epidemic diarrhea virus, porcine circovirus , foot and mouth disease virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. pigs can also have a role in zoonotic transmission of some viral infections to humans. inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. thus, improvements over existing vaccines are necessary to: ( ) increase the breadth of protection against evolving viral strains and subtypes; ( ) control of emerging and re-emerging viruses; ( ) eradicate viruses localized in different geographic areas; and ( ) differentiate infected from vaccinated animals to improve disease control programs. nanoparticles (nps) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. nps help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. some of the nps-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (m) cells and dendritic cells (dcs). in conclusion, nps-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. this review highlights the recent developments in nps-based vaccines against porcine viral pathogens and how the nps-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy. viruses are the obligate intracellular nano-sized particles, which depend on host cell machinery for propagation and survival. they carry deoxyribonucleic acid (dna) or ribonucleic acid (rna) as their genomic material. there are several viruses from both dna and rna virus families that infect and produce disease in pigs [ ] . there are many economically important swine viral infections which cause considerable morbidity and mortality, and responsible for significant economic losses to the pork industry (table ). depending on their cellular and tissue tropisms, viruses cause pathological changes and clinical signs associated with respiratory system, reproductive and gastrointestinal tracts, nervous system, skin and extremities, alone or in combinations [ , ] . porcine reproductive and respiratory syndrome virus (prrsv), an enveloped and positive-stranded rna virus of arteriviridae family, causes porcine reproductive and respiratory syndrome (prrs) [ ] . prrs is responsible for over one billion dollar loss per year through direct and indirect costs in the us swine industry [ ] . two entirely distinct genotypes of prrsv circulate in european (genotype /prrsv ) and north american countries (genotype /prrsv ) and cause tremendous economic loss. prrsv is transmitted through oral-nasal secretions and semen. the clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. it is the most common pathogen associated with porcine respiratory disease complex (prdc) [ ] . swine influenza (flu) constitutes another persistent health challenge to the global pig industry. flu infection is caused by influenza a virus of orthomyxoviridae family which has negative-sense, single-stranded, segmented rna genome. influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets [ ] . the clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses [ ] . influenza of swine origin occasionally infect humans and can even lead to pandemics as of [ ] . porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) are enteric pathogens of young pigs [ ] . these viruses belong to coronaviridae family and have positive-sense, single-stranded rna genome. tgev did serious economic damage to the swine industry in s but with the advent of vaccines it has been largely controlled [ ] . pedv still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. in / , pedv outbreak in the us led to over a billion-dollar loss [ ] . rotaviruses are double-stranded rna viruses of reoviridae family, cause enteric infections in pigs. rotavirus of groups a, b, c, e and h are involved in porcine enteric infections. some of these porcine rotaviruses also have zoonotic potential [ ] . foot and mouth disease (fmd) is another highly contagious, acute viral disease in pigs. the etiologic agent, fmd virus (fmdv), is a positive-sense, single-stranded rna virus of picornaviridae family [ ] . fmdv is transmitted through direct contact with infected animals or contaminated sources. clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. fmdv causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions [ ] . classical swine fever (csf) or hog cholera can result in high morbidity and mortality in pigs. it is caused by csf virus (csfv), an enveloped, positive-sense, singlestranded virus of flaviviridae family. transmission of csfv occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets [ ] . clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. csf is a notifiable disease to world organization for animal health (oie). the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . for example, the / outbreak of csfv in the netherland resulted in death of million pigs and economic losses of . billion dollars [ ] . united states is free of csfv; however, this virus is endemic in many parts of the world including central and south america, africa and asia. porcine circovirus (pcv ), a single-stranded dna virus of circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (pcvad). pcv is transmitted horizontally as well as vertically. direct contact is the most efficient way of horizontal transmission of this virus. the clinical signs of pcv infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures [ ] . five genotypes of pcv (pcv a to pcv e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide [ ] . porcine parvovirus (ppv) is the common cause of reproductive failure in swine herds. this single-stranded dna virus of parvoviridae family is transmitted through oral-nasal routes. stillbirths, mummification, embryonic death, and infertility (smedi syndrome) are linked to ppv infection. conventionally, ppv was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate [ ] . aujeszky's disease or pseudorabies in pigs is caused by suid herpesvirus , a double stranded dna virus belonging to herpesviridae family. the causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [ , ] . african swine fever (asf) causes hemorrhagic infection with high morbidity and mortality. the etiologic agent, asf virus (asfv), is a double stranded dna virus of asfarviridae family [ ] . virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus ornithodoros. clinical disease may range from asymptomatic infection to death with no signs. acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death [ ] . asf is oie notifiable disease. united states is free of asfv, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the us and other nonendemic regions through animal trades [ ] . the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . besides the rna and dna viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis e virus, porcine endogenous retrovirus, porcine sapovirus, japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [ , , ]. different types of vaccines that are available against economically important swine viruses are listed in table . vaccines against prrsv are being used in the us since s [ ] . both inactivated and modified-live virus vaccines are available and used globally. these vaccines are effective in reducing clinical disease and viremia mainly against homologous but not against heterologous infections [ ] . therefore, different strategies are ongoing to develop live, inactivated, subunit and mucosal prrsv vaccines to induce better immunity and broader protection [ , [ ] [ ] [ ] . swine influenza vaccines are also most effective when the vaccine strains closely match with the circulating strains [ , ] . to increase the immunity and protection, vaccines containing multiple strains of influenza a virus (iav) and autogenous vaccines are widely used [ , ] . cocirculation of multiple lineages of iav and frequent antigenic drift are responsible for reduced field efficacy of current swine influenza vaccines. moreover, the most commonly used whole inactivated iav vaccines administered via intramuscular route do not induce adequate mucosal antibody and cellular immune responses, suffer maternal antibody interference in young piglets and even can cause enhanced respiratory disease [ , ] . the emergence of highly virulent strains of pedv in recent years has highlighted the need of safe and effective vaccines against porcine enteric coronaviruses that prevents clinical disease, mortality and virus shedding in neonates [ ] . modified live vaccines against rotavirus are available for use in pigs against rotavirus a but their efficacy under field conditions is questionable indicating the need of alternatives for porcine rotavirus management [ ] . the available inactivated vaccines provided great help in prevention and control of fmd outbreaks in many countries. however, the development of these vaccines needs high level biocontainment facilities. further, the fmdv serotypes undergo continuous antigenic drift and escape the vaccine-induced immunity [ ] . thus, fmd vaccines with less stringent regulatory procedures and multi-serotype protective efficacy are needed in the future. safe and highly efficacious live-attenuated vaccines are available against csfv but differentiation of infected from vaccinated animals (diva) is not possible with these vaccines, which limit their use during outbreak control or disease eradication programs [ ] . inactivated whole virus or subunit vaccines based on pcv a are highly adopted in pig farms and are efficacious in reducing clinical signs and improving the production parameters. however, infections are still widespread in vaccinated farms [ , ] . further, the replacement of pcv a to pcv b and recently to pcv d is in part contributed by the selection pressure exhibited by pcv a-based vaccines [ ] which highlights the need of vaccines that protect against multiple genotypes. the currently used inactivated vaccines of porcine parvo virus protect against old ppv strains but not against the newly emerging strains demanding for more efficacious vaccines [ , ] . fortunately, pseudorabies has been eradicated in many countries including the us by using inactivated and attenuated vaccines together with stringent biosecurity measures. however, it is still a problematic disease in many countries including china and is also maintained in feral swine populations in other countries [ , ] . the frequent emergence of virulent strains even in the vaccinated herds demands updated vaccine technology to achieve efficient control and ultimate global eradication of pseudorabies [ , ] . vaccine is not available so far against asfv, and the control measures depend entirely on early identification and culling of infected herds and adoption of strict sanitary measures [ ] . vaccine development is hindered by the antigenic diversity and multitude of immune-evasion strategies used by the virus. an effective vaccine will definitely help in control and eradication of asfv from endemic countries and prevent its geographical expansion [ ] . [ , ] porcine epidemic diarrhea (ped) rna particle, inactivated and live-attenuated virus (in asia) protective immune response in sows better mucosal immunity [ , ] foot and mouth disease (fmd) inactivated virus less stringent requirements in vaccine production protection against multiple serotypes [ ] classical swine fever (csf) live-attenuated virus diva potential [ ] porcine circovirus associated disease (pcvad) inactivated, recombinant subunit multi-genotype protection [ , ] porcine parvovirus infection inactivated virus protection against novel strains [ , ] pseudorabies inactivated, live-attenuated virus protection against novel emerging strains [ , ] african swine fever (asf) none novel cross-protective vaccine [ ] importance of nanoparticle-based vaccine delivery platforms development of vaccines has made significant impact on reducing the viral infectious disease burden in both humans and animals. however, there are still many diseases for which either we do not have vaccines or need substantial improvements over existing ones [ , ] . in the past few decades, nanoparticles (nps)-based technologies have elicited significant interests in the development of novel vaccine candidates as they offer multiple benefits over inactivated virus or subunit soluble antigens. nps-based vaccines (nanovaccines) are prepared either by encapsulating vaccine components within the nps or by decorating the particle surface with viral antigens. nps protect antigens from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release. all of these properties help in induction of better immune responses compared to soluble antigen vaccines [ ] . the different mechanisms used by various nps to facilitate immune modulation of antigen presenting cells (apcs) are depicted graphically in figure . briefly, nps can enhance antigen adsorption and uptake by apcs; they can also facilitate antigen processing mechanisms; nps can induce maturation of dcs and promote antigen cross-presentation through major histocompatibility complex (mhc) class i to cd + t cells; and induce production of different innate cytokines that regulate humoral and cellular immune responses. nps-loaded antigens are readily phagocytosed by apcs; soluble antigens are not [ ] . moreover, dendritic cells (dcs), the key player involved in bridging innate and adaptive immunity, preferentially internalize nps compared to microparticles (> nm). for example, when poly(lactic-co-glycolic acid) (plga) particles of size nm to µm encapsulating ovalbumin were tested on mouse bone-marrow derived dendritic cells, nm sized particles were taken up efficiently compared to larger ones [ ] . the nm sized plga nps resulted in greater activation of dcs and stronger antigen-specific t cells responses in immunized mice compared to soluble antigens and larger particles [ ] . besides controlled delivery of antigens, nps also provide adjuvant-like functions. vaccine adjuvants either work as antigen delivery systems facilitating antigen uptake and presentation by apcs or they activate innate immune receptors for cytokine production and maturation/migration of dcs [ ] . adjuvant-induced innate immune responses determine the type of adaptive immune responses generated such as t helper (th ) versus t helper (th )-biased immunity [ ] . alum, the most widely used adjuvant in humans, is safe and inexpensive. its compatibility has been proved favorable with different vaccine antigens. however, despite inducing potent antibody responses, alum is a weak-inducer of cell-mediated immunity. adverse reactions are observed at injection site with alum-based adjuvants [ , ] . in veterinary vaccines, oil-in-water emulsions or saponins are the most common adjuvants. these can also cause adverse reactions at the injection sites [ , ] . while number of adjuvants are available for parenteral vaccinations, very limited options are available for intranasal (in) or other alternative routes of immunization [ , , ] . nps can serve as an alternative adjuvant for human and animal use as they act both as antigen delivery system and activate the innate immune responses [ ] [ ] [ ] . further, the modern vaccination approach has shifted from traditional whole pathogen-based antigens to small fraction (subunit) of the pathogen. however, purified whole inactivated pathogen and subunit or recombinant antigens by themselves are poorly immunogenic and require a potent immunostimulatory platform to augment the immune response. this can be achieved through nps-based technologies [ , ] . nps-based platforms can be used to deliver multiple antigens or antigen/adjuvant combinations, which improves antigen uptake and concurrent activation of apcs leading to innate immune programming [ , ] . co-delivery of cpg oligodeoxynucleotide and tetanus toxoid in nanospheres induced significantly greater t cell proliferative response and to times greater igg antibody isotypes in mice after subcutaneous immunization compared with the group that received tetanus toxoid and cpg oligodeoxynucleotide in soluble form [ ] . likewise, co-delivery of melanoma antigen and toll-like receptor (tlr) agonist in plga nps induced therapeutic anti-tumor effects that are mediated through potent cd + t cell activation [ ] . nps can be surface modified to target microfold (m) cells, macrophages or dcs, and could be used for mucosal vaccination through oral, nasal or other mucosal routes of immunization. in mice, surface coating of plga nps encapsulating hepatitis b virus vaccine antigens with lectin resulted in efficient targeting of oral delivered nps to mucosal m cells and induced secretary iga antibody response in mucosal surfaces [ ] . likewise, dcs targeted chitosan nps loading plasmid dna encoding nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) induced better nucleocapsid protein-specific mucosal iga antibody response compared to soluble unentrapped antigens after nasal immunization in mice [ ] . a targeted t-cell mediated immune response is critical in protection against intracellular pathogens such as viruses. beneficially, nps-delivered antigens are useful in antigen cross-presentation to cytotoxic t lymphocytes (ctls) and development of robust cell-mediated immune response [ , ] . plga-based particulate vaccines are shown to induce efficient t-cell immunity in mice and pigs [ ] [ ] [ ] [ ] . similarly, rodent and pig studies have shown that polyanhydride nps-based vaccines also enhance cellular immunity [ , ] . thus, immunogenic properties of different polymer-based nps could be exploited to improve the efficacy of vaccines for use against porcine viral infections. in this review, only studies conducted in pigs related to the development and evaluation of nps-based vaccine candidates by using virus-like particles (vlps), biodegradable polymers, polysaccharides and liposomes against porcine viral infections are included (table ) . vlps are constructed using viral structural proteins, which can self-assemble but are non-infectious as they lack the viral genomic material. vlps mimic the virion and can effectively induce innate and adaptive immune responses [ ] . vlps are produced using different bacterial, insect, yeast or mammalian expression systems [ ] . due to their smaller size and particulate nature, vlps-based vaccines are processed and presented not only through mhc class ii but also through mhc class i pathway leading to the generation of antibodies as well as ctl responses [ , ] . the potential use of vlps in porcine viral vaccine development is evident through the success in commercialization of human papilloma virus (hpv), hepatitis b virus and malaria vaccines by adapting this technology [ ] . in one study, prrsv vlps containing five (gp , gp , gp , gp a and m) and two (gp and m) viral surface proteins were generated using the baculovirus expression system. prrsv vlps vaccine was mixed at : ratio with mycobacterium tuberculosis whole cell lysate (m. tuberculosis wcl) adjuvant and administered into pigs. vlps-vaccinated pigs were partially protected with -log reduction of virus titers in lungs. vlps-vaccinated pigs also had enhanced ifn-γ response compared to mock challenge pigs [ ] . however, in another study, when pigs were vaccinated in with prrsv vlps expressing n, m, gp and e proteins, enhanced viremia accompanied with higher level of ifn-α cytokine response was observed [ ] . the contrasting results in prrsv vlps study suggest the need for further research to fully evaluate the potential of vlps-based prrsv vaccines for swine. influenza-associated vlps expressing ha, na and m proteins of pandemic (h n ) virus were inoculated twice intramuscularly with or without emulsigen (mvp lab, usa) adjuvant to pigs. this vaccine induced robust serum igg, mucosal iga and virus neutralizing antibody responses in pigs. after homologous virus challenge, vlps-vaccinated pigs had significantly reduced pneumonic lesions and virus titers were substantially lowered in upper and lower respiratory tracts compared to mock vaccinated animals [ ] . many studies have been conducted with the goal to develop vlps-based fmdv vaccine using various expression systems encoding different viral antigens. rabbit hemorrhagic disease virus (rhdv) vlps expressing t-cell epitope of a protein of fmdv (rhdv- a-vlps) was generated. this vlps vaccine induced maturation of bone marrow derived dendritic cells in vitro [ ] . pigs immunized im with rhdv- a-vlps together with montanide isa adjuvant (seppic, france) induced higher serum igg and iga antibody responses. this vaccine also increased number of ifn-γ secreting cells and lymphoproliferative responses in pbmcs compared to vaccine delivered without adjuvant and in rhdv- a-vlps inoculated pigs; however, challenge experiments were not performed [ ] . guo et al. constructed fmdv vlps expressing capsid proteins vp , vp and vp and immunized pigs by im route [ ] . vlps-vaccinated pigs produced virus-specific neutralizing antibodies and ifn-γ response in peripheral blood mononuclear cells (pbmcs) as good as the inactivated fmdv vaccine control. after challenge with homologous virus, vlps-vaccinated pigs did not show specific clinical signs [ ] . in another study, vp epitope peptides (ep - ) of fmdv were inserted into the coat protein genes of male-specific coliphage (ms ) (cp-ep - vlps) and injected im to pigs. this formulation resulted in induction of virus neutralizing antibodies and protected % of the immunized pigs compared to only % protection in peptide alone vaccinated animals. however, the protection was lower than inactivated vaccine ( %) indicating the need : of further improvement in this vlps either by using longer sequence of epitope or addition of other adjuvants [ ] . vlps generated by insertion of vp epitopes of fmdv into porcine parvovirus vp were administered im to pigs. this vlps-vaccine induced higher virus neutralizing antibodies compared to synthetic peptide vaccine and resulted in better protection to challenge fmdv infection [ ] . vlps have also been developed and tested against porcine neurotropic viruses [ , ] . porcine encephalomyocarditis virus (emcv) vlps containing structural protein p , nonstructural protein a and protease c were generated. after im administration together with montanide ims n vg adjuvant (seppic), vlps-vaccine induced sustained production of virus neutralizing antibodies comparable to commercial vaccine control. there was absence of any severe injection site reactions in vlps-vaccinated pigs [ ] . this suggests the potential of developing vlps-based vaccine against emcv disease in pigs. likewise, in a recent study, japanese encephalitis virus genotype i (gi) vlps encoding premembrane (prm) and envelope (e) proteins were constructed. after subcutaneous immunization, this vaccine formulation induced robust neutralizing antibody response and protection against both homologous gi and heterologous giii jevs viruses. this finding indicates the cross-protection potential of vlps-based jev vaccine in pigs [ ] . early study on pcv vlps used full length cap protein in escherichia coli expression system [ ] . pigs vaccinated against pcv using cap vlps and isa adjuvant (seppic) by im route induced cap-specific igg antibodies. vaccinated animals were apparently healthy with normal body weight gain and absence of any clinical signs of disease [ ] . li et al. [ ] showed induction of cap-specific igg antibodies in pigs vaccinated by subcutaneous (sc) injection of cap vlps. vaccinated pigs demonstrated reduced fever, viremia and mild pathological changes in lungs and lymph nodes compared to unvaccinated challenge animals. in another study, vlps co-expressed with cap protein and porcine gm-csf were administered im to pigs. this vaccine formulation induced significantly higher virus neutralizing antibodies in pigs. after virus challenge, vlps-vaccinated pigs had normal body weight gain compared to cap protein alone and commercial pcv vaccine groups. virus clearance, however, was observed in equally in vlps as well as other control vaccine groups [ ] . only a single vlps-based vaccine study for porcine parvovirus was found [ ] . ppv-vlps expressing major structural protein vp were administered im with double oil emulsion (doe) mineral oil adjuvant to weaned pigs. there was an induction of significantly higher neutralizing antibodies in vlps-vaccinated animals compared to inactivated vaccine group. further, when gilts immunized with this formulation were challenged with virulent ppv, virus was not detected in any of the fetuses. thus, ppv-vlps can be a potential vaccine candidate to prevent ppv-induced reproductive failure [ ] . in summary, vlps of various origin can be used to develop more efficient vaccines against porcine viral infections. further studies are needed to evaluate their immunogenicity and protective efficacy under field conditions. plga is a co-polymer of lactic acid and glycolic acid. it is the most widely explored synthetic polymer in vaccine studies. it is a safe and non-toxic compound, and its hydrolysis products are readily assimilated into existing metabolic pathways [ ] . plga nanoparticles are prepared either by oil in water emulsification or nanoprecipitation methods [ , ] . plga nps bear a net negative charge. they enter apcs through pinocytosis and endocytosis, undergo reversal of charge and endolysosomal escape of entrapped vaccine cargo leading to antigen processing in cytoplasm, resulting in cross-presentation of antigen to cd + t cells through mhc class i pathway [ , ] . plga nps are involved in maturation of dcs of mice and human origin, and controlled release of entrapped antigens leading to efficient expansion and differentiation of memory t-cells [ , ] . in rodent studies, induction of robust t-cell immunity is observed with plga nps-based vaccines containing various vaccine antigens [ , ] . further, plga is approved for drug deliveries in humans by the us food and drug administration (fda) and european medicine agency (ema) [ ] . plga nps enhance antigen uptake and induce maturation of porcine apcs [ , , ] . single dose of in immunization with plga nps-encapsulated inactivated/ killed prrsv antigen (nps-kag) induced activation of innate natural killer (nk) cells, γδ t-cells and secretion of innate cytokine ifnα [ ] . nps-kag vaccine also induced greater frequency of cd + t cells; increased secretion of ifn-γ; lowered frequency of t-regulatory cells; and reduced secretion of inflammatory cytokines compared to control kag-vaccinated animals [ , ] . in a subsequent study, when nps-kag was co-administered in with m. tuberculosis wcl adjuvant, a balanced th / th immune response and augmentation of mucosal iga antibody response was observed. after heterologous prrsv challenge, pigs that received nps-based vaccine showed no clinical signs and also had significant reduction in lung virus load [ , ] . plga nps were also used to encapsulate highly conserved influenza peptides and evaluated for efficacy in pigs after in administration. plga nps-based subunit vaccine resulted in induction of epitope-specific t-cell response but not the antibody response [ ] . the t-cell biased immune response was also observed in pigs after in immunization with plga nps-encapsulated inactivated/killed influenza virus (plga-kag) vaccine in pigs [ ] . in plga-kag vaccine administered animals observed reduced fever; lowered pneumonic lesions; and increased virus clearance from lungs after heterologous virus challenge compared to kag vaccine controls [ ] . in another study, pedv kag was encapsulated in plga nps and used to immunize pregnant sows by in route. this nanovaccine induced higher virus-specific igg and neutralizing antibodies in serum and greater igg, iga and neutralizing antibody responses in colostrum. it also induced greater cell proliferation and ifn-γ responses in restimulated pbmcs compared to kag vaccine controls. importantly, piglets born to nps-vaccinated sows had higher virus neutralizing antibodies and were better protected against homologous virus challenge than kag controls [ ] . these studies suggest that plga nps can be used as an efficient means of enhancing virus-specific cell-mediated immune responses in pigs. polyanhydrides are another type of synthetic polymer widely studied for vaccine deliveries [ ] . polyanhydride nps are synthesized by polycondensation or emulsification processes and are biodegradable, biocompatible and safe for vaccine delivery [ , ] . they activate innate immune responses in a manner similar to lipopolysaccharides (lps) [ ] . the surface-eroding nature of polyanhydride nps provides safe microenvironment for the encapsulated antigens and facilitates slow and sustained antigen release [ , ] . induction of better antibody and cell-mediated immune responses by polyanhydride npsbased vaccines has been reported against viral, bacterial and parasitic infections [ , ] . inoculation of polyanhydride nps-based siv kag vaccine (kag-nanovaccine) by in route enhanced cell-mediated but not the antibody responses in pigs [ ] . after heterologous virus challenge, kag-nanovaccine group had six to eightfold reduction of nasal virus shedding compared to kag vaccine controls [ ] . in a subsequent study, when kag-nanovaccine formulation was supplemented with cpg-odn adjuvant, both cell-mediated as well as mucosal iga antibody responses were improved [ ] . after heterologous virus challenge, cpg-odn-adjuvanted kag-nanovaccine provided better protection through a significant reduction in influenza-induced fever, -fold reduction of nasal virus shedding and -fold reduction in lung virus titers compared to pigs immunized with five-times greater quantity of soluble killed antigen (kag) vaccine [ ] . this study also indicates the dose-sparing ability of polyanhydride nps. thus, polyanhydride nps can also be used to induce better cellular as well as humoral immune responses in pigs. chitosan, alginate and other polysaccharides have also attracted attention as materials for nps formulation and drug delivery studies. chitosan is a natural polymer derived from deacetylation of chitin and is composed of glucosamine and n-acetylglucosamine residues [ ] . due to the availability of amino and carboxyl groups in an acidic microenvironment, chitosan nps have net positive surface charge which makes them highly mucoadhesive and increases their half-time of antigen retention on mucosal surfaces [ , ] . further, chitosan nps can reversibly open the epithelial cell tight junctions thereby improving paracellular and intracellular antigen transport across mucosal epithelial surfaces [ , ] . chitosan nps also enhance antigen uptake by apcs, induce apc maturation and active secretion of innate cytokines [ , ] . thus, chitosan nps form an attractive mucosal vaccine delivery vehicle. chitosan-based nps are used in pigs to deliver adjuvants such as bee venom and plasmid encoding porcine il- and il- /il- genes, which improved induction of better virus-specific immune responses of respective vaccines against prrsv and pcv [ , ] . chitosan nps enhance antigen uptake by porcine apcs and activate them to produce innate cytokines including ifn-alpha, tnf-alpha and il- β [ ] . chitosan nps encapsulated siv kag (cnps-kag) vaccine administered twice through in route without any additional adjuvant in pigs induced the cross-reactive mucosal iga antibodies. chitosan nps-based vaccine also induced ifn-γ response in pbmcs and tracheobronchial lymph nodes (tbln) better than kag vaccine controls. this vaccine formulation substantially reduced the challenge heterologous virus titers by up to -fold in both the upper and lower respiratory tracts compared to soluble kag vaccine. this finding emphasizes the potential benefits of using chitosan nps in future development of mucosal swine influenza vaccine for pigs [ ] . recently, dendrimer-like-alpha-d-glucan (nano- ) nps derived from sweet corn variety sugary- was examined as an alternative, safe, cost-effective and potent adjuvant [ , ] . nano- are positively charged nps which efficiently adsorb negatively charged antigens through electrostatic interactions. rodent studies have shown that nano- nps enhance antigen uptake by dcs, induce their maturation, activate them to produce pro-inflammatory cytokines and help in induction of antigen-specific antibodies [ , ] . in a recent study, we observed that nano- nps with or without addition of siv killed antigen (kag) can stimulate porcine apcs and produce cytokines such as ifn-α, tnf-α and il- β [ ] . pigs immunized via in route with nano- nps adsorbed siv kag at two-to-one ratio (nano- + kag) resulted in cross-reactive mucosal iga responses better than kag controls. moreover, pigs immunized im with nano- adsorbed ovalbumin (nano- + ova) had significantly greater igg and igg antibodies in serum compared with pigs vaccinated with ova alone [ ] . these findings highlight the possibility of using cornderived nano- nps as a potential adjuvant in porcine viral vaccine development. liposomes can encapsulate both hydrophilic and hydrophobic molecules in aqueous and non-aqueous phases of their vesicles [ ] . liposome vesicles protect antigens from enzymatic degradation, enhance antigen internalization by apcs and maintain controlled release of antigens [ ] . liposome-encapsulated antigens can enhance both cellular and humoral immune responses [ , ] . in a pig study, liposome nps were used as an im adjuvant for a pcv dna vaccine [ ] . liposome nps-adjuvant induced higher neutralizing antibodies and ifnγ response in pigs and reduced viremia of a challenge virus compared to alum-adjuvanted vaccine, providing the evidence that liposome nps can be a potent adjuvant in pigs [ ] . in our recent study, we used liposome nps to encapsulate ten highly conserved peptides of different influenza viruses of human and pig origin and immunized pigs through in route co-administered with monosodium urate (msu) crystal adjuvant [ ] . the liposome-adjuvant based vaccine enhanced the mucosal iga antibody response and induced peptide and virusspecific t-helper/memory cells and ifnγ responses resulting in reduced fever and modest reduction in virus titers in the respiratory tract of pigs [ ] . these studies highlight the fact that liposome-based nps can be used as an attractive vaccine delivery platform against porcine viral infections. virus infections have significant impact on pig industry worldwide. use of available vaccines have definitely helped in achieving strong control over some of the porcine viral infections such as food and mouth disease, transmissible gastroenteritis, classical swine fever and pseudorabies. vaccination also helped in reducing the clinical signs and increasing the production parameters in pcv -associated disease. however, for many other porcine viruses, further improvements in existing vaccine platforms and development of novel vaccine delivery systems are necessary to: ( ) induce better mucosal and cell-mediated immunity; ( ) protect against emerging and re-emerging strains; ( ) enhance the breadth (heterologous, cross-genotype and heterosubtypic) of immunity; and ( ) differentiate between infected and vaccinated animals. nps-based vaccine delivery platforms such as vlps, biodegradable polymers and liposomes have great potential as they-( ) protect vaccine antigens from degradation; ( ) facilitate antigen uptake and processing by apcs; ( ) impart adjuvant potential; ( ) can be used in mucosal and other alternate routes of immunizations; and ( ) induce effective mucosal and cellular cross-protective (broader) immunity. research efforts are ongoing to develop porcine viral vaccines using nps-based technologies. however, more collaboration(s) and in-depth studies are warranted to make this innovative vaccine antigen delivery technology successful and practical for application in food animal industry. to date, almost all of the immunomodulatory mechanisms of nps-based vaccine delivery platforms have been studied in rodent disease models, which may or may not reflect the situation in pigs or other domestic animal species [ ] . likewise, proper understanding of effect of size, charge and other physicochemical properties of nps after delivery through different routes of immunization in pigs is 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manuscript. authors' contributions sd wrote the article; gjr edited and revised the article. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -pij jaa authors: li, jun-yu; yong, yan-hong; gong, dong-liang; shi, lin; wang, xiao-min; gooneratne, ravi; yadnyavalkya, patil; ju, xiang-hong title: proteomic analysis of the response of porcine adrenal gland to heat stress date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: pij jaa abstract heat stress (hs) and its associated pathologies are major challenges facing the pig industry in southern china, and are responsible for large economic losses. however, the molecular mechanisms governing the abnormal secretion of hs-responsive hormones, such as glucocorticoids, are not fully understood. the goal of this study was to investigate differentially expressed proteins (deps) in the adrenal glands of pigs, and to elucidate changes in the immune neuroendocrine system in pigs following hs. through a functional proteomics approach, we identified peptides, corresponding to proteins. of these, we found deps between heat-stressed and control porcine adrenal gland tissue; of these were up-regulated and were down-regulated in response to hs. these deps included proteins involved in substrate transport, cytoskeletal changes, and stress responses. ingenuity pathway analysis was used to identify the subcellular characterization, functional pathway involvement, regulatory networks, and upstream regulators of the identified proteins. functional network and pathway analyses may provide insights into the complexity and dynamics of hs-host interactions, and may accelerate our understanding of the mechanisms of hs. rising global temperatures have been accompanied by increased interest in researching the detrimental effects of heat stress (hs) on the swine industry. pigs enter a state of hs when the ambient temperature exceeds their thermal neutral zone ( - °c for adult pigs) (coffey et al., ) . due to their high production of metabolic heat, accelerated fat deposition, and lack of sweat glands, pigs there are more sensitive to hs than many other mammals (d'allaire et al., ) . heat stress in pigs not only decreases food intake and body weight gain, but also has immunosuppressive effects, all of which may result in large economic losses for the swine industry (cruzen et al., ; pearce et al., ) . for instance, hs is estimated to cost the us swine industry losses of over $ million each year (st-pierre et al., ) . understanding the stress-associated mechanisms involved in immune system function and the increased susceptibility of livestock to heat-related illness is more important now than ever, as the majority of emerging animal diseases are zoonotic and can potentially threaten public health. when exposed to a high-temperature environment, the central nervous system of mammals, including livestock, engages in physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (hpa) axis and the sympatho-adrenal axis. the predominant hormone regulating the synthesis and secretion of adrenal glucocorticoids is adrencocorticotropic hormone (acth) (minton, ) . in pigs, cattle, and sheep, both corticotropin-releasing hormone and vasopressin regulate the secretion of acth, suggesting that these two proteins interact to enhance acth secretion. acth acts on the adrenal gland with the circulation, it to induces the expression and secretion of glucocorticoids, which suppress the production of cytokines and other pro-inflammatory mediators, including tnf-α, interferon-γ, il- β, il- , il- , il- , and prostaglandins (wilckens and rijk, ) . glucocorticoids also facilitate the release of anti-inflammatory mediators, such as transforming growth factor-α, il- , and il- , and have apoptotic effects and strong anti-proliferative properties in immune cells (visser and nagelkerken, ) . ultimately, cytokines activate the release of glucocorticoids, which in turn suppress cytokine synthesis fin a negative feedback loop (barrat et al., ; haddad et al., ) . thus, it is of interest to understand how higher core temperatures alter adrenal function. isobaric tag for relative and absolute quantification (itraq) is a powerful quantitative proteomics technique. in recent years, several proteomic studies have explored protein expression in a number of porcine cells and tissues, including pulmonary alveolar macrophages (lu et al., ) , mesenchymal stem cells (huang et al., ) , liver (liu et al., ) , heart (cabrera et al., ) , and intestine (colladoromero et al., ) . nevertheless, no large-scale proteomic analysis to date has examined the molecular complexes or pathways involved in the pathogenesis of the hpa axis. the purpose of the present study was to investigate protein expression in the adrenal gland of pigs in response to hs, and to elucidate potential changes in that occur in the endocrine system under hs. pigs were maintained and studied in accordance with the national institutes of health (nih) guidelines for the care and use of laboratory animals, and all protocols were approved by the guangdong ocean university animal care and use committee, china. six castrated bama miniature pigs (sus scrofadomestica) months, weighing - kg were obtained from the bama miniature pigs breeding farm in the guangxi zhuang autonomous region of china. the six pigs were randomly divided into a heat stress group ( barrows, ha) and a control group ( barrows, ca). control pigs were housed with an ambient temperature of ± °c, and the relative humidity was kept at approximately %. pigs assigned to the heat stress treatment were kept at ± °c(maintained using an artificial climate chamber) in a manmade climate room, with a relative humidity of approximately %. all pigs were given access to water ad libitum. diet (see diet composition table) was formulated according to the recommended nutrient allowances for this breed of pig and the feeding was done twice a day, in the morning as well as in the evening. pigs were euthanized by a head-only electric stun tong apparatus on the th day under heat stress, followed by manual exsanguination. immediately after slaughter, adrenal tissue was removed and weighed. subsequently, tissues were washed with pbs to remove any blood and contaminants on the tissue surface. adrenal tissue was placed into sterile tubes and snap frozen in liquid nitrogen three pigs were used in the control group and three pigs were used in the heat-stressed group. the adrenal tissues of the pigs in each of the groups were pooled together to form one pooled sample and utilized as one sample for further analyses. once in the laboratory, frozen specimens were stored at − °c until biochemical and molecular analyses were performed. frozen samples of adrenal tissue from all pigs in the two groups were crushed in a mortar containing liquid nitrogen. the powder (approximately mg per sample) was transferred to a sterile tube containing ml lysis buffer (lb; containing m urea, m thiourea, % chaps, mm tris-hcl, ph . , mm dithiothreitol, dtt). tissue homogenate was further disrupted using an ultrasonic cell disruptor (vcx , usa) at % power output for min, cycling between s on and s off. afterwards, the lysate was centrifuged at , ×g for min at °c, and the supernatant was collected for protein quantification. protein concentration was measured using the pierce bca protein assay kit (thermo scientific, usa). protein digestion was performed as per the fasp procedure described by wisniewski, zougman et al. (wiśniewski et al., ) ; and the resulting peptide mixture was labeled using the itraq reagent- plex multiplex kit (ab sciex, framingham, usa), according to the manufacturer's instructions. after h of incubation at room temperature, labeled samples were mixed at equal ratios. subsequently, labeled peptides were combined and fractionated by strong cation exchange (scx) chromatography (han et al., ) and desalted on c cartridges ( -u; sigma, st. louis, mo, usa). the dried peptide mixture was reconstituted and acidified with ml buffer a ( mm kh po in % of acn, ph . ) and loaded onto a column ( . × mm). peptides were eluted at a flow rate of ml/ min with a gradient of %- % buffer b ( m kcl, mm kh po in % of acn, ph . ) for min, - % buffer b for - min, %- % buffer b for - min, and %- % buffer b for - min. the elution was monitored by absorbance at nm, and fractions were collected every min. the tryptic peptides were extracted, and the peptide mixtures were concentrated by speed vac centrifuge to dryness, and were again dissolved with % acetonitrile (acn) in . % formic acid before lc-ms/ms analysis. the fractions from . were subjected to liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis. initially, samples were loaded onto pre-columns ( μm × mm; μm-c ; waters, usa). peptide mixtures were separated on analytical columns ( μm × mm; . μm-c ; waters, usa) at a flow rate of nl/ min over min. thermo easy-nlc is a binary buffer system used for high performance liquid chromatography (hplc), consisting of . % formic acid (buffer a) and % acetonitrile (acn) in . % formic acid (buffer b). the related liquid phase gradient was as follows: - min with % to % buffer b; - min with %- % buffer b; and - min with % buffer b. peptides eluted by hplc were directly injected into a q-exactive mass spectrometer (thermo fisher scientific). data were acquired in the positive ion mode with a selected mass range of - mass/charge (m/z). q-exactive survey scans were obtained at , (m/z ) and , (m/z ), with the resolution for higher-energy collisional dissociation (hcd) spectra and maximum ion injection times fixed at ms and ms, respectively. dynamic exclusion ( . s duration) was used. ms/ms data were collected using the top most abundant precursor ions. the normalized collision energy was ev, and the underfill ratio defined as . %. the instrument was operated with peptide recognition mode enabled. protein identification was performed using the mascot . . search engine (matrix science, london, uk). according to the relative abundance of different itraq tags, the peptides derived from different groups were quantified by the scaffold software, and represent the ratio of one group to another. the relative quantification of the protein is calculated by using the relative quantification of the peptide, which is expressed as the average ratio. to determine the deps in the adrenal gland between the hs and the control groups, the average ratio of identified proteins was calculated by proteinpilot based on the weighted average log ratios of the peptides. the deps were further analyzed for significant down-or upregulation, which was not determined by the size of the ratio but was calculated by software perseus. a cutoff level of significance of % (or p < . ) was chosen as a criterion. gi numbers of all significantly regulated proteins and some unaltered proteins were imported into the ingenuity pathway analysis software (ipa, www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as gene ontology, uniport and trembl. the canonical pathways and proteins interaction network of the deps were analyzed using the ipa. statistical analysis was performed using spss statistics . . differences analysis in physiology indices between the ha group and the ca group were performed using a t-test, and p < . was taken to indicate statistical signifcance. we measured the body temperature, heart rate and respiratory rate of pigs at days , and . it was found that body temperature and heart rate were increased significantly in heat-stressed pigs at days and . (p < . ), however, the difference between body temperature and heart rate was not significant at days. compared to controls, the respiratory rate, too increased substantially in pigs under heat stress at days , and (p < . ) (see table ). the sample size used for this was pigs each in control and hs groups respectively. we used the itraq method to identify proteins differentially expressed in the adrenal gland between the heat stress and control groups. we identified differentially expressed proteins (deps), of which were up-regulated and were down-regulated in the ha group vs. the ca group. table provides information about key deps, upregulated and downregulated (see table ). all the deps have been provided in the supplementary table . all protein and peptide identifications were obtained by database searching and stringent data filtering. the lc-ms/ms analysis produced , spectra, corresponding to unique peptides; the effect of heat stress on the body temperature, heart rate and respiratory rate at days , , and . we found that heat stress increased the body temperature (bt), heart rate (hr) and respiratory rate (rr) of pigs significantly. the sample size used for this was pigs each in control and hs groups respectively. the asterisk '*' and double-asterisk '**' denote a significant difference between stressed pigs and control pigs on the same day (p < . and . , respectively). the units of the above indices are centigrade (°c), beats per minute and breaths per minute, respectively. proteins were identified at a false discovery rate (fdr) of ≤ . (fig. a) . the molecular weights of the identified deps ranged from to kd (n = ), - kd (n = ), - kd (n = ), - kd (n = ), - kd (n = ), or > kd (n = ) (fig. b) . in addition, the identified deps had high peptide coverage; % of deps had > % sequence coverage, and % of deps had > % sequence coverage (fig. c) . . % of the identified deps were represented by three or more peptides (fig. d) . to gain functional insights into the cellular proteome, gene ontology annotation was used to determine the subcellular localization of the identified deps. the deps identified from adrenal gland tissue altered by heat stress localized to various subcellular regions: high density lipoprotein particles ( . %), extracellular area ( . %), endoplasmic reticulum ( . %), protein-lipid complexes ( . %), cytoplasmic lipoprotein particles ( . %), triglyceride-rich lipoprotein particles ( . %), serosa ( . %), platelet alpha particles ( . %), endoplasmic reticulum inherent ( . %), extracellular matrix ( . %), chromatin ( . %), pigment granules ( . %), extracellular space ( . %), serosa integrity ( . %), neuronal processes ( . %), secretory granules ( . %), cytoplasmic vesicles ( . %), and cytoskeleton ( . %). (fig. ) . gene identifications of the identified deps (supplementary table ) were converted to human geninfo identifier (gi) numbers, since the pig genome database has poor annotations compared to the human genome, and because many proteins were unassigned or uncharacterized. to better understand these deps, we used ingenuity pathways analysis (ipa) tool to examine canonical pathways; the top enriched pathways are shown (fig. ) , including pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il- signaling and production in macrophages'. the deps identified in adrenal gland tissue during heat stress by itraq were clustered according to different functions. four functional groups were found: diseases and disorders, molecular and cellular functions, physiological system development, and toxicity functions were significantly enriched (p ≤ . ; fig. ). the deps from adrenal gland under heat stress, which correspond to diseases and disorders (fig. a) , included proteins that are related to neurological disease, psychological disease, metabolic disease, skeletal and muscular disorders, hereditary disorders, hematological disease, immunological disease, inflammatory disease, inflammatory response, respiratory disease, dermatological disease and conditions, connective tissue disorders, infectious disease, cardiovascular disease, cancer, and endocrine system disorders. these deps were assigned to molecular and cellular function groups (fig. b) , including cell death and survival, molecular transport, cellular growth and proliferation, cellular assembly and organization, cellular function and maintenance, cellular movement, lipid metabolism, small molecule biochemistry, free radical scavenging, protein degradation, protein synthesis, and cell morphology. these deps were also enriched in physiological system developmental functions groups (fig. c) , including tissue development, nervous system development and function, organ morphology, organismal development, embryonic development, hematological system development and function, immune cell trafficking, and tissue morphology. finally, these deps were enriched in toxicity function groups (fig. d) , including renal necrosis/cell death, liver hyperplasia/ hyperproliferation, kidney failure, cardiac inflammation, and heart failure. proteins that changed significantly in the adrenal gland under heat stress were mapped to functional networks (fig. ) . the main networks of interest correspond to ( ) cell assembly and tissue, cellular function and maintenance (fig. a); ( ) cell migration, intercellular signal interactions (fig. b) small molecule biochemistry (fig. d ). proteins that are present in these pathways and identified as up-regulated deps in our analysis are depicted in shades of red; those that were identified as down-regulated deps are shown in green. proteins in the network but not identified as deps in our study are depicted in white. we also predicted the upstream regulators of adrenal deps by ipa analysis and found that cytokines, kinases, chemical agents, chemical kinase activators, mature micrornas, and growth factor were activators of these deps, as an example, chemokine(c-c motif)ligand , curcumin and brain derived neurotrophic factor while cytokines, mature microrna, auxins, transcription regulators, and chemicals were inhibitors of these deps, such as, il- , ccaat enhancer-binding protein β and dexamethasone etc. these predicted upstream regulators of deps responsive to heat stress may have an important role in regulating hormone secretion and signal transduction in pigs. . the previous research of our laboratory found that: the analysis of plasma cortisol levels in bama miniature pigs revealed that the levels increased with the duration of heat stress. although there was no significant difference in the cortisol levels of control and heat-stressed pigs on the first day. however, at subsequent time points, cortisol levels were significantly higher in heat-stressed pigs compared with those in the control animals on the th day (ju et al., ) . in the present study, we used a method combining proteomics and bioinformatics to identify proteins that are differentially expressed in the adrenal gland of pigs under heat stress, and to elucidate molecular pathways and cellular functions that might mediate the heat stress response through these deps. we identified a total of deps in the pig adrenal gland under heat stress conditions. ipa analysis software was used to analyze the cell localization, molecular function, signal pathway, regulatory network, and upstream regulators of these deps, which laid the foundation to elucidate mechanism of heat stress and stress-induced immunosuppression. the function of tubulin is mainly to interact with microtubule-associated proteins and related proteins that activate microtubule structures and maintain microtubule polymerization and depolymerization, modulate cell morphology, and are involved in cell division, cell movement, and transport of intracellular substances. cytoskeletal changes are associated with trans-cellular membrane trafficking. together, β-tubulin and α-tubulin (table ) participate in the formation of microtubules, the integrity of which is essential for the segregation of chromosomes during cell division, the maintenance of cell shape, and the intracellular trafficking of macromolecules and organelles. changes in β-tubulin and vimentin levels have been detected in sars-cov (jiang et al., ) and infectious bursa disease virus (ibdv) (zheng et al., a) . β-tubulin was one of the deps identified in this study, and its expression was approximately . -fold up-regulated in adrenal tissue under heat stress, suggesting that differentially expressed cytoskeletal proteins could promote stress response in the adrenal gland. further large-scale studies are required to understand the roles and interrelation of β-tubulin and vimentin in porcine heat stress response. the heat shock protein (hsp) response is a highly conserved cellular response to external stress in all species. hsps take part in antigen presentation, intracellular trafficking, and apoptosis, and acting as molecular chaperones by assisting nascent polypeptides in assuming their proper conformations (khar et al., ) . several hsps were identified as down-regulated deps in this study, including hsp and hsp . several members of the hsp family are expressed on the surfaces of cells; these can stimulate immune effector cells directly or can play crucial roles in antigen cross-priming. in this situation, hsps act as shuttle molecules for exogenous antigens and can directly stimulate t cells by prompting apc cytokine secretion (kotsiopriftis et al., ) . hsps appear to play a part of the innate immune response since the emergence of phagocytes in early multicellular organisms, and were commandeered for adaptive immune responses with the appearance of immune specificity (srivastava, ) . hsp is an endogenous ligand for the toll-like receptors (tlrs) that bind to microorganism-and tumor-specific antigens, then combine with major histocompatibility complexes (mhc) i and ii, which activate tumor-specific pathogens and t cells (han et al., ). mortaz found that high temperature induced mouse bone marrow mast cells to release hsp , and the secretion of hsp in turn activated the toll-like receptor (tlr ) pathway (mortaz et al., ) . the expression of hsp is enhanced under stress conditions, and is generally considered to act through its role as a molecular chaperone, but recent reports indicate that hsp also modulates inflammatory responses by inhibiting activation of the inflammatory transcription factor, nuclear factor-kappa b (zheng et al., b) . in addition, hsp may directly interfere with cell death pathways, such as those involved in apoptosis and necrosis (yenari et al., ) . in this study, hsp was up regulated( . fold reduction) in porcine adrenal gland tissue under heat stress, further indicating the role of hsp in adrenal gland injury and highlighting its relevance to inflammatory responses. hsp has direct modulatory effects on inflammatory cells, and can activate monocytes and granulocytes to produce inflammatory cytokines, tumor necrosis factor-α (tnf-α), il- and il- (wells and malkovsky, ) . hsp can also specifically bind tlrs, especially tlr , which is involved in human and rat atherosclerotic lesions (grundtman et al., ) . hsp belongs to a family of small heat shock proteins, can affect protein assembly, and may also participate in protein degradation. this conclusion follows directly from data suggesting that heat stress reduces the expression of heat shock proteins, handicapping their ability to induce a protective immune response when immunocytes are confronted with foreign entities. antigen presentation by hsps activates the innate and adaptive immune systems to initiate an acute response to stress factors, and suppression of hsps obstructs this response. these properties allow hsps to be used in immunotherapy in novel ways, which could lead to a greater understanding of how heat stress modulate the immune response, and why heat stress induces immunosuppression in pigs afflicted by post weaning multi-system wasting syndrome (pmws). histone h a was significantly down-regulated in porcine adrenal gland under heat stress. ipa analysis shows that histone h a plays a fig. . ingenuity pathway analysis of proteins significantly altered in heat stressed pigs. red, up-regulated proteins; green, down-regulated proteins significantly; white, proteins known to be in the network but were not identified in our study. the colour depth shows the magnitude of the change in protein expression level. the shapes are suggestive of the molecular class(i.e., protein family). lines connecting the molecules indicate the relationship between molecules. dashed lines demonstrate indirect interactions, and solid lines demonstrate direct interactions. the arrow styles demonstrate specific molecular relationships and the directionality of the interaction. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) role in cancer, infectious diseases, reproductive system diseases, liver diseases, and immune diseases. however, in a bovine proteome study, histone h a was considered a new natural immune molecule, and was down-regulated in neutrophils of immunosuppressed dairy cows. the immunological function of neutrophils releases a series of dna, histones, and antimicrobial peptides, forming a microbial kill 'trap' (lippolis and reinhardt, ; john and lippolis, ; kimura et al., ) . histone h a belongs to a group of conserved eukaryotic cationic proteins that are involved in antibacterial activity, and function in dna folding. further studies are needed to determine if down-regulation of h a expression in the adrenal gland under heat stress conditions is related to immunosuppression. annexin (anxa) is a calcium-dependent phospholipid-binding protein widely found in eukaryotes. phosphatidylserine (ps) is transferred from the inside of cell membrane to the outside after cell apoptosis. in the presence of calcium ions, anxa has a high affinity for ps, and can specifically bind to ps exposed on cell membrane. thus, anxa specifically recognizes apoptotic cells, and acts as a novel molecular probe to detect apoptosis. anxa is one of the most widely distributed and abundant members of the anxa family, and is involved in the anti-coagulation activity, calcium channel activity, protein kinase inhibitory activity, and many other important physiological processes. anxa is also closely related to inflammatory responses and cell stress (gauer et al., ; lokman et al., ) . anxa , , and , were down-regulated deps identified in our study. these proteins participate in cell function and maintenance, intercellular communication and interaction, cell survival and death, cell migration, neurological diseases, and other processes. whether these down-regulated proteins are involved in the regulation of heat stress-induced apoptosis is still unclear (gu et al., ) , and must be explored in further studies. cui performed similar studies on heat stressed pig livers and found that chronic heat stress caused a reduction in the weight of the liver. they also found proteins in the liver that were differentially abundant between heatstressed pigs and control pigs. proteins found were responsible for heat shock and oxidative stress response, cellular apoptosis, metabolism, signal transduction, cytoskeleton and immune system responses. their research found that heat caused an innate immune response whereas the voluntary diet restriction resulted in stress and cellular apoptosis (cui et al., ) . in summary, in this study we identified deps in the porcine adrenal gland under heat stress were characterized, and we constructed a comprehensive network of protein regulation in the porcine adrenal gland in response to heat stress. although many significantly differentially regulated proteins and pathways are closely related to the symptoms or pathological responses of heat stress, further functional investigations ought to be carried out to facilitate our understanding of the pathogenic mechanisms and molecular responses of pigs to heat stress. further work may identify novel therapeutic targets or lead to the development of new methods of preventing heat stress. in conclusion, we identified deps, of which were up-regulated and were down-regulated, from pig adrenal gland tissue under heat stress. among these deps, tubulin and heat shock protein (hsp) such as hsp , hsp , hsp , and histone h a might be involved in the regulation of heat stress. ingenuity pathways analysis (ipa) tool examined canonical pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il- signaling and production in macrophages'. our expression profiles provide new insights into the key proteins involved in heat stress in the pig. supplementary data to this article can be found online at https:// doi.org/ . /j.rvsc. . . . in vitro generation of interleukin -producing regulatory cd + t cells is induced by immunosuppressive drugs and inhibited by t helper type (th )-and th -inducing cytokines altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning feeding growing-finishing pigs to maximize lean growth rate quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to salmonella typhimurium effects of long term heat stress in utero or during finishing on pork carcass composition chronic heat stress induces immune response, oxidative stress response, and apoptosis of finishing pig liver: a proteomic approach sow mortality associated with high ambient temperatures membrane modulates affinity for calcium ion to create an apparent cooperative binding response by annexin a 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protection proteomics analysis of host cells infected with infectious bursal disease virus anti-inflammatory effects of the kda heat shock protein in experimental stroke not applicable. the authors of this study have no conflicts of interest. jl conceived the study designed the experiments, prepared the samples for mass spectrometry, analyzed the mass spectrometry data, conducted the experimental work created the figures and wrote the manuscript. yy participated in the enrichment analysis, created the pathway figure, aided in revising the manuscript. dg, ls and pt analyzed the mass spectrometry data, participated in the enrichment analysis, participated in writing and revision of the manuscript. rg obtained and analyzed flow cytometry data and aided revising the manuscript. xwang was responsible for animal care xj provided the financial support, and designed the experiments and revised the manuscript. all authors have confirmed the final version of the manuscript. key: cord- - lgup yj authors: robbins, r.c.; almond, g.; byers, e. title: swine diseases and disorders date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . - sha: doc_id: cord_uid: lgup yj swine diseases and disorders that are significant in modern, commercial swine production systems are organized by body system; the reader will need to know basic anatomy and physiology. the industry significance, etiology, epidemiology, pathogenesis, clinical signs, postmortem and histpathologic lesions, diagnostic testing, and generic treatment, control, and prevention are described. diseases of a particular system are summarized in a differential diagnosis table. r elsevier inc. all rights reserved. autogenous vaccines vaccine made from microorganisms isolated from the animal it will be used on; in the united states these are killed and, by permit, are extended for use in a farm, production system, or region. farrow process of parturition; location where a pig is born and stays till weaning, usually - weeks of age. grow-finish phase of production that follows the nursery period where pigs reach slaughter weight; used to describe pigs from to weeks of age. histopathology; -ic the science and microscopic examination of formalin-fixed and paraffin-embedded sections of diseased tissues. lesion visible (microscopic or macroscopic) deviation from normal. national animal health monitoring service a program administered by united states department of agriculture-animal and plant health inspection service. nursery phase of production that begins after weaning, used to transition pigs from the farrowing house to finishing; used to describe pigs from to weeks of age. oie world organization for animal health created on january . pathognomonic characteristic of a specific disease or disorder that, when present, is sufficient to make a diagnosis. veterinary diagnostic laboratory location where samples are submitted and tests are run to determine the cause of disease. in an approach to investigating any suspected disease or disorder in swine production, a history should be gathered first. important history to understand from caretakers includes: age of pigs affected, duration of clinical signs, morbidity rate, mortality rate, treatments administered, response to treatments, and any other important information regarding previous diagnoses or disease in the affected group of animals. this is also the time to examine any production records that have been kept on the affected group of swine as well as previous groups for comparison. records include but are not limited to: where the animals originated from; number in the herd; age; daily mortality; number treated; name of treatment, route of delivery and dose; feed and water usage; high-low temperatures; and vaccinations received or administered. after examining the production records and obtaining a history, proceed with a visual examination of the herd. typically, it is a biosecurity custom to observe youngest groups first; however, in cases of suspected infectious diseases, it may be best to begin with the healthiest group advancing in order of increasing severity or prevalence. often, a definitive diagnosis is not achieved without an extensive clinical and pathological investigation. a postmortem examination, or necropsy, of affected pigs should occur last. any pigs recently deceased of natural causes should be examined to establish trends, with the understanding that submission of tissues from these animals may not yield valuable diagnostic results. tissues for diagnostic evaluation should be collected from clinically affected pigs that are euthanized immediately before necropsy. sampling of five or more pigs may be required to obtain a valuable diagnosis. when investigating signs referable to the central nervous system (cns), it is important to preserve brain and spinal cord tissue for microscopic evaluation in cases of neurological disease; therefore, blunt force trauma and brain penetration by captive bolt are not preferred methods of euthanasia. at minimum, fresh and formalin-fixed tissue samples should include: brain, tonsil, heart, lung, lymph nodes, spleen, kidney, liver, and intestine. additional samples that may be beneficial for diagnosis include: premortem whole blood and ethylenediaminetetraacetic acid-chelated blood (for serum chemistry and complete blood count), spinal cord, intact stifle and hock joints (remove the leg at the hip), intact eyeball with optic nerve attachment, urine, feed, and water. consult a diagnostic lab regarding any additional samples that may be required in determining an etiologic diagnosis. the etiologic diagnosis should be based on consistent history, signs, and pathology derived from a list of differential diagnoses that are most common or most likely to occur in that herd or production system. a treatment, control, or prevention program should be formulated simultaneously. before using any chemical, pharmaceutical, or biologic in swine intended for food, know the domestic use guidelines, importer requirements or producer-packer agreements regarding withdrawal times, residue and tolerance limits, prescribing guidelines, and prohibited substances. this section will focus on a practical approach to investigating signs of neurological disease in swine summarized in table . it is important to determine if clinical signs are consistent with cns or peripheral nervous system lesions (pns). common cns signs in pigs include behavioral abnormalities (most commonly stupor), ataxia, loss of righting, seizures or seizure-like activity (paddling), nystagmus, and blindness. musculoskeletal disorders may clinically confuse or complicate perceived pns signs and must be differentiated from each other. streptococcus suis is a gram-positive cocci with reported serotypes. observational studies implicate sows as carriers and piglets are colonized as they pass through the birth canal (amass et al., ) . disease occurs most frequently during the suckling and postweaning period. commingling pigs from different herds, concurrent infection with porcine reproductive and respiratory syndrome (prrs), and other stress factors may increase the risk of developing s. suis meningitis (villani, ; thanawongnuwech et al., ) . variable morbidity and mortality: mortality depends on early recognition and treatment. clinical signs of s. suis meningitis include paddling, recumbency, nystagmus, and seizure. isolation of s. suis from the lung, nasal secretions, or tonsil from normal pigs is clinically insignificant. in contrast, s. suis isolation from cerebrospinal fluid (csf), meninges, joints, endocardium, or serosal surfaces with or without lesions is relevant (pijoan, ) . few to no gross lesions may be observed during necropsy. early recognition of clinical signs followed by injection with an antimicrobial that s. suis is susceptible is the most effective means of treatment. administering an antimicrobial that s. suis is susceptible to in the drinking water has been proposed to control morbidity (villani, ) . antimicrobial susceptibility patterns for s. suis isolates from regional diagnostic laboratories can be used to assist in selection of an appropriate antimicrobial while diagnostic tests are pending; ceftiofur is effective . commercial and autogenous vaccines are available but due to s. suis serologic diversity may not be effective . haemophilus parasuis (hps), also called glässer's disease, causes bacterial meningitis, arthritis, and polyserositis similar to s. suis. infections are not clinically or grossly distinguishable from s. suis. definitive diagnosis is by bacterial isolation. however, hps is a fastidious gram-negative rod and culture media must be supplemented with v factor for successful isolation. owing to the difficulty in isolating hps, polymerase chain reaction (pcr) tests are a suitable alternative (oliveira et al., ) . like s. suis, isolation from the airways has little significance unless lesions are present (hoefling, ) . antimicrobial susceptibility testing identifies ceftiofur or florfenicol that are typically effective first choice therapeutics (oliveira, b) . prevention may be achieved with medicated early weaning. edema disease results when a fimbrial (f or f ) and shiga-like toxin (stx- e) positive strain of escherichia coli successfully attaches to brush border receptors releasing toxin that damages blood vessels including those of the blood-brain barrier causing edema and encephalomalacia. edema disease most commonly affects rapidly growing pigs, weeks postweaning. morbidity is moderate to high and mortality is high. acute death of robust pigs, ataxia, eyelid swelling, and diarrhea are typical clinical signs (rademacher, ) . at necropsy, edema may be observed in the mesentery between the loops of the spiral colon and in the cardiac region of the gastric mucosa. stomachs are usually full of feed. bacteriologic isolation of a β-hemolytic strain of e. coli from affected pigs with meningoencephalitis is not sufficient for a diagnosis. genotyping is necessary to confirm that the e. coli isolated was f or f and stx- e positive and thus capable to induce such lesions. there is no effective treatment. vaccination using an avirulent live culture of e. coli postweaning, thorough cleaning and disinfection between groups, and use of genetically resistance breeds that lack the fimbrial receptor are preventatives (fairbrother and gyles, ) . pseudorabies (prv), also known as aujezsky's disease, is caused by a herpesvirus. prv was eradicated from the us commercial swine herd in (usda aphis, ) . feral swine are potential reservoirs. cattle, sheep, dogs, and cats can also be infected with prv. high morbidity is due to large quantities of virus shed in saliva and nasal secretions for several weeks following infection. mortality is inversely related to age approaching % in neonates. clinical signs are also age dependent. neonates may die without signs. suckling and recently weaned pigs are those that commonly exhibit ataxia, tremors, excess salivation, and seizures. at necropsy, the brain appears congested and hemorrhagic. necrotic foci occur in the spleen, liver, lung, lymph node, and specifically tonsils. histopathologic lesions are characterized by nonsuppurative meningitis and intranuclear inclusion bodies. pcr, virus isolation (vi), immunohistochemistry (ihc), or fluorescent antibody can be used to confirm the diagnosis. no specific treatment is available. vaccination and eradication are effective for control (usda aphis, ) . in areas free of prv, suspicion of the disease should be reported to state and federal agencies as required. congenital tremors result when hypomyelination or demyelination of the brain and spinal cord. clinical signs are clonic muscle contractions that cause a general tremor of the entire body. pigs are affected at birth but severity subsides with note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). age (dewey, ) . mortality is variable and is the sequela of malnutrition because piglets are unable to nurse. there is no known treatment or prevention. hypoglycemia occurs when piglets fail to nurse the sow. this condition is observed within h afterbirth. there is a low morbidity but high mortality. pigs may appear disoriented, ataxic, recumbent, or dead. on necropsy, affected piglets will have empty stomachs. assigning an employee to attend farrowing to ensure piglets nurse will reduce incidence. water deprivation, also referred to as salt poisoning, is an idiopathic disease resulting from a period of inadequate water intake (carson, ) . high morbidity with variable mortality occurs. the disease is suspected when there is a history of power outage or poor management (thacker, ) . fighting over water access is the first clinical sign and occurs within hours. dog-sitting, opisthotonous, convulsions, and fighting over water follow and develop after h without water. removal of the brain from an affected animal reveals edema and eosinophilic meningoencephalitis with perivascular cuffing and this is pathognomonic (gudmundson and meagher, ) . serum or csf with a sodium level above meq l À may also be used for supporting evidence (osweiler and hurd, ) . treatment of swine showing signs with an anti-inflammatory is variably effective. when water is restored, limit intake to short, - min intervals until all animals have had a chance to drink and fighting has ceased after which water can be provided ad libitum. prevention is daily observation to ensure each animal can access water, adequate water delivery system, and equipping the facility with a generator or alternative method to deliver water during power outages. gastrointestinal diseases and disorders can occur in all ages of swine as summarized in table . most digestive diseases are referable to the gastrointestinal tract and result in diarrhea and occasional vomiting. diarrhea is the result of an intestinal dysfunction caused by malabsorption, excessive secretion, or effusion. unfortunately, this is not an exclusive characterization of diarrhea and overlap occurs (moeser and blikslager, ) . rather, differentials for diarrhea should be referable to age at onset and site of infection. gastric ulcers are noninfectious and result when glandular mucosa specifically the pars esophagea is traumatized by gastric acid. gastric ulcers have a wide variety of causes but are most commonly associated with small feed particle size (ayles et al., ) and interruption of feed intake whether caused by disease or poor management. it is common to see signs consistent with gastric ulceration increase following an acute prrs or influenza outbreak. morbidity and mortality vary with the scope of the underlying cause. clinical signs include regurgitation, vomiting, pallor or jaundice, and acute death. an acutely dead pig with blood in its stomach is indicative of an active ulcer and is sufficient evidence for a diagnosis. in chronic cases, ulceration causes hyperplasia resulting in stricture of the pars esophagea and regurgitation. feeding a coarse ground diet for weeks significantly decreases severity (ayles et al., ) but is impractical in modern production facilities. rotavirus is a nonenveloped rna virus with a doublelayered capsid allowing it to remain stable and infective in the environment for months and intrinsically resistant to some disinfectants. four serogroups infect swine: a, b, c, and e (the latter only reported from the united kingdom). in addition, infections with particular serogroups vary by age: type c mostly in suckling piglets and type a predominately in nursery pigs (stephenson et al., ) . type a is the most prevalent serogroup. severity of disease decreases with age and is self-limiting. the virus infects and destroys villous enterocytes resulting in villous atrophy. in response, crypt cells fill in the gaps but, because they are incapable of absorption, suckling piglets quickly loose body condition and have a gaunt or wasted appearance. neither clinical signs nor gross lesions are pathognomonic although loops of small intestine appear thinwalled with moderate to large amounts of watery contents. a histopathologic report of blunted villi and crypt hyperplasia is suggestive of rotavirus infection. infection with rotavirus can be confirmed by pcr or electron microscopy (em). enzymelinked immunosorbent assay (elisa) is also available but limited to detection of serogroup a. ihc and em detect rotavirus and confirm its role in pathology, but both tests lack table common gastrointestinal diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). sensitivity. treatment is supportive by administration of oral rehydration solutions. acidifiers and antibiotics are sometimes administered to control secondary bacterial infections. treatment success is variable and depends on the degree of malnourishment. prevention among neonatal piglets is through ingestion of lactogenic virus neutralizing antibody from the sow, which is stimulated by administering feedback of rotavirus positive piglet feces or intestines (arruda et al., ) or a modified-live commercial type a vaccine no less than weeks before farrowing. a modified-live commercial type a vaccine is also available for pigs. it does not induce cross-protection for other serogroups and may be cost-prohibitive. tge is caused by tgev, a coronavirus that is heat labile at temperatures above c, prone to dessication and photosensitization (bay et al., ) . the epidemic form causes acute disease in all age groups within as little as h of infection. morbidity and mortality is high, approaching %, in an epizootic outbreak. the severity of disease is age dependent but all ages will develop diarrhea (moeser and blikslager, ) . postweaning infections result in high morbidity but low mortality; the most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. necropsy reveals that the small intestine and colon are fluid filled, the small intestinal wall is thin almost translucent, and lacteals are empty. necrosis and atrophy are observed throughout the length of the villus. the colon and cecum are spared. the endemic form occurs when susceptible animals are introduced to the herd or after maternal antibody wanes. prior exposure to porcine respiratory coronavirus (prcv) may cause false-positive antibody test results. a tgev/prcv differential elisa is available. in an outbreak, sows are fed tissue of diarrheic pigs to stimulate herd immunity and new introductions of animals are stopped. after the exposure and a subsequent cool-down period of - months or after clinical signs cease, sentinels can be introduced and monitored for seroconversion (saif and sestak, ) . absence of seroconversion indicates successful elimination of tgev. commercial vaccines are available but should be used with caution and only when elimination is not an option. ped is caused by pedv, a coronavirus that causes signs and histolopathologic lesions indistinguishable from tgev. unlike tgev, pedv is more environmentally resistant making elimination more difficult. the disease has been described in europe, asia, and, as of , the united states. prevalence of the enzootic form is approximately % (chae et al., ) . morbidity approaches % and mortality is % or more in a naïve sow herd resulting in - weeks of production losses. clinical signs appear within h; piglets develop a watery, fetid diarrhea leading to dehydration, metabolic acidosis, and death before caretakers are able to humanely euthanize them. vomiting also occurs. the severity of disease is age dependent but all ages will develop diarrhea. postweaning infections result in a high morbidity but low mortality; most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. viral shedding occurs up to days postinfection. reproductive failure and inefficiency is a sequela of an outbreak (olanratmanee et al., ) . tgev/pedv differential pcr is available to confirm a presumptive diagnosis of ped. serum can be submitted for elisa or immunofluorescent antibody but collected no sooner than weeks after diarrhea was o bserved. immunoprophylaxis using egg antibody or hyperimmune serum and supportive care including electrolyte administration have been used for treatment. in an outbreak, sows are fed tissue and feces from diarrheic pigs to stimulate herd immunity (olanratmanee et al., ) . hygiene is the key to reducing environmental contamination. preventing introduction of virus into a herd with biosecurity alone may not be sufficient because the virus has been found in aerosol up to miles from a positive farm (goede et al., ) . porcine coccidiosis is most often caused by isospora suis. farm hygiene, specifically farrowing rooms, and sow infestation influence the persistence of disease; however, age at infection rather than infectious dose has the greatest impact on severity (worliczek et al., ) . the prepatent period is approximately days. morbidity is variable and mortality is low. pasty diarrhea, unthrifty to potbellied appearance of - day old pigs, and below average wean weight is suspicious for coccidiosis. on necropsy, the small intestine often is thickened and the mucosal surface is necrotic and has an adherent pseudomembrane. histopathologic examination of the affected portion of the intestine reveals larvae in the lamina propria. sensitivity of fecal flotation is moderate. there is no effective treatment. prevention is by oral administration of an anticoccidial (maes et al., ) . heat treatment (flaming) of flooring may reduce environmental contamination. concrete, rubber coated and plastic flooring in the farrowing crates are difficult to clean and disinfect so removal may be the only option. swine dysentery (sd) is a spirochete of the genus brachyspira that is an oxygen-tolerant anaerobe giving it the ability to survive for long periods of time in manure, pits, and lagoons (schwartz et al., ) . rodents, particularly mice, are known vectors and can serve as reservoirs. brachyspira hyodysenteriae is the species known for causing sd. other species of brachyspira have been recently described in dysentery-like disease (burrough, ) . incubation is - days but disease occurs in a - week cycle. administration of tiamulin or lincomycin in the feed or water may alter the time to onset of signs after exposure. morbidity is high and mortality is low to moderate characteristically causing disease in only the finisher and mature groups. economic significance is mostly lost performance due to reduced daily gain and feed conversion. the specific mechanism of pathogenesis is not well understood but the spirochete does not invade the lamina propria. clinical signs are the presence of mucohemorrhagic diarrhea containing flakes of frank blood or appearing as a generalized brick red to rust color. lesions are mostly observed in the spiral colon where epithelial sloughing and mucosal invasion cause necrosis resulting in the formation of a pseudomembrane. the colonic walls may be thickened due to vascular congestion and mucosal hyperplasia. bacterial culture produces strong β-hemolysis. pcr test for confirmation and speciation is recommended for any isolate with characteristic growth. introduction of infected pigs and contaminated equipment or facilities are the source of infection. pleuromutilins, like tiamulin, and the lincosamide, lincomycin, are effective for treatment. however, if the environment remains contaminated, clinical signs will recur. depopulation has resulted in successful eradication (harms, ) . medicated elimination that combines thorough pulse medication with tiamulin, cleaning and disinfection, and employment of an aggressive rodent control program is also effective (burrough and sexton, ) . escherichia coli is a gram-negative rod that infects all ages of swine but must express virulence factors to cause diarrhea. escherichia coli colonize the small intestine by fimbria that binds receptors on the villous surface of enterocytes. enterotoxigenic e. coli (etec) then produce toxin(s) that increase osmolality leading to diarrhea (moeser and blikslager, ) . etec is subdivided by fimbria, toxin, and age of pig affected. neonatal diarrhea (nd) is most common in pigs - days of age. the onset of postweaning diarrhea (pwd) caused by f is delayed, occurring - days postweaning, compared to that caused by f and its severity is indirectly related to wean age. clinical signs are profuse diarrhea, rapid dehydration leading to emaciation, or death due to metabolic acidosis. fluid filled and hyperemic sections of jejunum and ileum may be present at necropsy but few consistent gross lesions occur. intestinal contents have a distinctly alkaline ph. isolation of large numbers of e. coli and with dense layers of rod-shaped bacteria covering villi seen on histopathology in samples from pigs with diarrhea is sufficient for diagnosis of e. coli but not etec. genotyping is necessary to determine fimbria and toxin types, which are essential to confirm diagnosis of etec. treatment of affected pigs/litters/groups includes administration of antibiotics and oral rehydration solution or electrolytes to correct hyperkalemia (kiers et al., ) . control and prevention of nd is by passively derived lactogenic immunoglobins from vaccinated females (kohler, ) . prevention of pwd include selection of genetically resistant breeds lacking k and f receptors, administration of an oral avirulent live culture to stimulate active immunity or competitively exclude field strains (genovese et al., ) , feeding ppm zinc oxide postweaning and probiotics. immunity and exclusion is unique to each fimbria; vaccines should include the prevalent genotype(s) causing the diarrhea. clostridium perfringens type a (cpa) is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of neonatal infection. frequency of cpa diarrhea is on the rise in the usa. in uncomplicated cases, mortality is low whereas morbidity is high and below average weaning weights result. cases of cpa diarrhea are associated with the expression of α and β toxin. cpa is cultured from the stomach and upper third of small intestine but does not bind intestinal epithelium causing few to no histologic lesions. because of its ubiquitous nature and prevalence among health pigs, cpa may be an opportunist and its role as a primary cause of neonatal enteritis is not definitive. large numbers ( þ or þ ) of gram-positive bacilli cultured from feces or intestinal contents of diarrheic pigs is suggestive of cpa. genotyping by pcr to confirm presence of cpb gene in cpa isolates and rule out of other causes of nd are supportive to the diagnosis (bueschel et al., ). treatment has variable success rates and is limited to administration of empirically selected antibiotics and oral rehydration solutions to affected piglets. control of cpa enteritis is best accomplished by preventing other causes of nd. following a thorough cleaning, sporicidal disinfectant should be applied to farrowing crates and equipment between litters and be allowed to dry before reloading. feeding of bacitracin to sows has resulted in significant increases in weaning weights (schultz, ) . a commercial cpa toxoid vaccine is available (hammer et al., ) . autogenous whole cell vaccines are also in use. if vaccine is unavailable, feedback might be considered but should be pursued with caution (robbins and byers, a) . cpc is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of infection. pathogenesis of type c is due to expression of β toxin leading to necrosis of intestinal epithelium resulting in hemorrhagic diarrhea or acute death of piglets less than days of age. gross necropsy reveals hemorrhagic and blood-filled loops of small intestine. a pseudomembrane may form on the luminal surface, and intestinal mucosa is edematous. gross and histopathologic lesions in the presence of large numbers ( þ or þ ) of grampositive bacilli cultured from feces or intestinal contents warrant a presumptive diagnosis. genotyping by pcr to confirm presence of the cpb gene is confirmatory (songer and uzal, ) . treatment of affected piglets is unrewarding due to the rapid and debilitating course of this disease. prevention is accomplished by vaccination of gestating females with a commercial toxoid and ensuring piglets consume sufficient colostral antibodies to result in protection. clostridium difficile is a gram-positive bacillus that easily sporulates making it environmentally resistant to many disinfectants. clostridium difficile associated diarrhea leads to a - % reduction in wean weights (songer and uzal, ) . although more than a third of piglet diarrhea involves c. difficile, it is the better known to cause healthcare-associated infections among humans. the pathogenesis of c. difficile infections is in response to the expression of toxins a and b. a watery diarrhea occurs in - day old piglets. mesocolonic edema may be observed at necropsy. clostridium difficile is difficult to culture and can be isolated from healthy piglets. therefore, volcano lesions on histologic exam and confirmation of toxins in fecal contents by antigen elisa are diagnostic. treatment is ill-defined but is likely similar to that for cpa enteritis, because it is likely to be initiated based on clinical signs, which are similar. autogenous vaccines are used to aid in prevention but efficacy is unclear. ppe, commonly referred to as ileitis, is the general categorization of infections caused by lawsonia intracellularis, an obligate intracellular bacterium. because the bacteria cause lesions in the ileum, ppe is also referred to as ileitis. seroprevalence in grow-finish herds can reach %. ppe can further be divided into four clinical forms (kroll et al., ) . porcine intestinal adenomatosis (pia) is most common in - week pigs and causes little mortality. porcine hemorrhagic enteritis (phe) affecting pigs weeks of age and older including breeding swine and can be associated with increased mortality and dark, bloody stools. necrotic enteritis (ne) and subclinical ileitis, the most common form, occur among postweaning pigs. in all forms, transmission is by the fecaloral route. crypt enterocytes infected with l. intracellularis become hyperplastic. the altered ratio of villous and crypt enterocytes leads to malabsorption and subsequent increases in feed conversion and time to reach market weights. pia results in variable degrees of thickened ileum that can be found at necropsy. the ileal lumen may contain a blood clot in phe or pseudomembrane in ne. when diarrhea ranging in color from normal (pia, ne, and subclinical) to dark-red or black (phe) is observed, ppe should be considered as a possible cause. subclinical ileitis usually causes no clinical signs (gebhart, ) . histopathologic lesions containing intracellular s-shaped organisms are suggestive of lawsonia infection but ihc should be used to confirm diagnosis. pcr is helpful to detect infection and is highly specific but moderately sensitive. cross-sectional or longitudinal serologic profiling using a widely available elisa is the best tool for determining timing of exposure. treatment is with effective antibiotics, such as tylosin, administered by injection or in the feed or water. control is by administration of a commercially available modified-live oral vaccine before infection or feeding antibiotics when infection is known to occur. vaccination should take place at least weeks before seroconversion (walter et al., ) . salmonellosis causing gastrointestinal disease in swine is most commonly associated with the species typhimurium. salmonella typhimurium is commonly isolated from swine. isolation of multidrug resistance strains of s. typhimurium from swine at slaughter have garnered attention from public health and food safety professionals and it is this that make this infection significant to the pork industry (foley et al., ) . some european union member states have implemented meat-juice serologic monitoring at slaughter to assess on-farm salmonella control programs. pathogenesis of s. typhimurium is similar to salmonella cholerasuis by invading enterocytes and subsequently macrophages leading to an infectious carrier state. initial infection causes inflammation and cytokine release that result in watery, yellow diarrhea containing feed particles. button ulcers may be visible on the mucosal surfaces of the colon and cecum on gross necropsy examination and, on histopathology, can be found to extend into the lamina propria. bacterial isolation without using enrichment media and the presence of histopathologic lesions is consistent with a diagnosis of salmonella enteritis. treatment is with antibiotics administered symptomatically to diarrheic pigs. antibiotic susceptibility of the isolate should be considered before initiating treatment. rearing pigs on slatted floors, decreasing stocking density, and acidification of digesta are effective in reducing the prevalence of salmonella infections in swine (funk and gebreyes, ; boyen et al., ) . cross-protection with s. cholerasuis vaccine has been reported and reduces carcass colonization (husa et al., ) . whipworm infestations of swine are the result of trichuris suis infection. pigs kept on pasture, in outdoor lots, or facilities with a history of t. suis diagnosis are at greatest risk for disease (pittman et al., a) . the prepatent period is - weeks. the egg is not immediately infective, which requires - weeks in the environment. the infectious larva hatches from the egg and invades enterocytes in the small intestine and cecum. the entire life cycle of t. suis is completed in the intestine. ulcerations in the mucosa and damage to capillary blood supply of intestinal epithelium lead to hemorrhage, anemia, and hypoalbuminemia. clinical signs are depressed weight gain, increased feed conversion, bloody diarrhea, ill thrift, and death. adult worms imbedded in the ileum, cecum, or proximal colon are sufficient for diagnosis of whipworms. eggs are intermittently shed and thus not a reliable method of diagnosis (pittman et al., a) . treatment and control are synonymous and require administration of an effective anthelmintic like fenbendazole. prevention is by steam sanitation and drying; however, eggs are resistant to common disinfectants and remain infective for years. the porcine integument or skin, like that of other domestic species, serves as a protective barrier between fragile internal tissues and harsh external hazards. skin is comprised of layers (from external to internal): epidermis, dermis (superficial and deep), and subcutis. blood vessels, hair follicles, sebaceous glands, and muscles are found in the dermis. notably, the pig's skin does not contain sweat glands; therefore, modern swine facilities are outfitted with evaporative cooling systems for thermal regulation in hot climates. skin diseases and disorders can be the result of viral or bacterial infections, parasite infestations, immunologic reactions, and idiopathic or iatrogenic causes that are summarized in table by their various macroscopic and microscopic lesions. greasy pig is a skin disease of swine caused by a toxin produced by staphylococcus hyicus. a break in the skin is the typical sequela. gilt litters reportedly have a higher incidence of this disease, presumably due to deficient maternal immunity. all ages of pigs may be affected but suckling and nursery pigs are most likely to develop disease. affected pigs develop focal crusts on the face, neck, and axillary region, and the crusts may coalesce as the disease progresses. affected areas are greasy to touch and may appear black due to dirt adhering to it. if pigs are untreated or fail to respond to treatment, the trunk and extremities may become involved. pyrexia and lethargy can be observed in severe cases and are followed by growth reduction. gross appearance of affected skin is rarely confused with other skin conditions of swine. submission of formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis for histopathologic examination is needed for a diagnosis. the pathognomonic histologic lesion is exudative epidermitis. table common integument diseases and disorders of pigs greasy pig x x x erysipelas x x porcine dermatopathy and nephropathy syndrome (pdns) x x x sarcoptic mange x x the first column provides the diseases. the remaining columns represent the type of lesion that occurs. the s. hyicus can be cultured from the surface of clinically normal skin sections. treatment includes topical application of antimicrobials or disinfectants. unaffected pigs with direct contact with affected pigs should also be treated to control spread. in cases where pigs exhibit systemic signs, administration of an injectable antimicrobial and anti-inflammatory is warranted. in the united states, no antimicrobials are labeled for the treatment, control or prevention of s. hyicus so all antimicrobial therapy is extra-label. prevention should focus on facility hygiene and include a soap degreaser and disinfectant regimen to reduce contamination. in addition, scarification of the skin of breeding age females with the farmspecific s. hyicus strain can reduce disease incidence in suckling pigs (murray and rademacher, ) . erysipelas or diamond skin disease is caused by a soilborne gram-positive bacterium, erysipelothrix rhusiopathiae. this zoonotic pathogen is transmitted by migratory fowl, turkeys, and pigs. humans may become sickened when direct contact with blood from affected animals contaminates an open wound (brooke and riley, ) . the finding of lesions at slaughter results in partial or complete carcass condemnation (bender et al., ) . the disease is most common in growing, finishing, and breeding age swine. bacterial emboli lodge in blood vessels causing vasculitis, thrombosis, and ischemia leading to lameness, abortions in gestating females, and raised, red to purple rhomboid skin lesions for which erysipelas is best known. skin biopsies from the affected area should include epidermis and dermis, but histologic lesions are only supportive. bacteriologic isolation or pcr identifying e. rhusiopathiae confirms the diagnosis. treatment with β-lactam antibiotics including penicillin is effective. commercial bacterins and avirulent live cultures are available for prevention (wood, ) or in the face of outbreaks to prevent the chronic form. pdns has been associated with porcine circovirus type (pcv ) infection, but any disease process resulting in ischemia could cause result in pdns. the condition is characterized by red to purple discoloration of skin that begins on the caudal surface of the hind limbs and the ventral surface of the abdomen resulting from ischemia. on necropsy, gross examination of the kidney cortex may be speckled with pinpoint, white foci caused by infarcted blood vessels. pig of any age can be affected with pdns, but it is more commonly observed during growing and finishing stages. submission of fresh and formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis is required. there is no specific treatment or prevention; rather, diagnose the underlying cause to determine appropriate therapy ( figure ) . sarcoptic mange is the result of an allergic reaction to the saliva of ectoparasites, sarcoptes scabiei. mange may also be caused by demodex phylloides. mortality is low and morbidity is moderate. economic losses are the result of reproductive inefficiency, growth reduction, and carcass condemnation. infestation and subsequent clinical signs in the breeding herd, most notably an incessant scratching, develop following the purchase of infested genetic replacements. in addition, growing pigs placed in facilities that previously housed infected swine or facilities that reuse straw bedding or have solid wood partitions may also become infested. the mite is rare in modern, high-health swine operations. the burrowing mite causes red pustules and flaking skin. individual pigs may develop signs in as few as weeks but a herd may not show signs for several months. in the chronic stage, thick crusts develop at the corners of and inside the ears. examination of a scraping from the crusts will reveal the mite (averbeck and stromberg, ). an elisa test is used to determine prior exposure and determine success of eradication programs. treatment can be applied topically using an antiparasitic, such as amitraz, to temporarily alleviate clinical signs. control and eradication programs utilize feeding or injection of ivermectins (mohr, ) . the musculoskeletal system is comprised of tendons, ligaments, muscles, and bones. disorders and disease of this system are typically characterized by lameness. lameness is any deviation in normal locomotion including favoring a limb or failure to bear weight on the limb. neurologic conditions, which also cause changes in locomotion, may be ruled out by postmortem examination of articular surfaces and diagnostic testing. investigation of musculoskeletal diseases and disorders should always start with the claws that are easily traumatized causing pain resulting in lameness. flooring and genetics also influence the incidence of lameness. common musculoskeletal diseases and disorders of swine can be divided into osteopathies and myopathies and summarized in table . mycoplasma hyosynoviae colonizes upper airways and tonsils resulting in a carrier state. transmission is vertical from sow to pigs and lateral between pigs (ross and spear, ) . m. hyosynoviae is most often diagnosed during the grow-finish phase. morbidity is variable but mortality is low. clinical signs are a stiff gait and difficulty in standing, most often the stifle or elbow and less frequently the hock, hip, and shoulder. signs often occur - weeks after a stressful event; lesions begin to resolve weeks postinfection. the affected joint contains yellow or blood-tinged effusion with moderate villous proliferation but is not always observed despite lameness and does not necessarily correlate with presence of histopathologic lesions. aseptic collection of synovial fluid by needle aspiration or sterile swab or submission of the affected joint intact is recommended for diagnosis. pcr is the most sensitive test; culture requires special media and lacks sensitivity (gomes neto et al., ) . histopathologic examination of formalin-fixed synovium reveals nonsuppurative fibrinous polyarthritis and lymphoplasmacytic perivascular synovitis. elisa is also available. lincomycin has historically been an effective therapeutic choice (burch and godwin, ) . treatment should be initiated when lameness is first observed; however, spontaneous resolution is common. no commercial vaccines are available. mycoplasma hyorhinis is a ubiquitous bacterium that is an early colonizer of upper airways. transmission is vertical from sow to pigs and then between pigs postweaning (rovira, ) . infection can progress to polyarthritis, polyserositis, and otitis in the pre-or postweaning phases; arthritis develops postweaning. clinical signs include lameness, arthritis, and fever that develop - days after septicemia occurs and persists for - days (gomes neto et al., ) . disease may become chronic resulting in ill thrift, reduced growth, and death. articular surfaces may be eroded. in cases of lameness, synovial fluid and formalin-fixed synovium can be submitted. alternatively, the entire affected leg can be submitted; disarticulate above the infected joint keeping the affected joint intact. submission of fibrin or fibrin covered tissue(s) should be included for pcr testing to differentiate m. hyorhinis from other bacteria that form fibrin on serosal surfaces like hps and s. suis (rovira, ). histopathology reveals fibrinosuppurative inflammation in affected tissues. treatment is empirical. erysipelas is the result of a chronic e. rhusiopathiae infection causing arthritis and endocarditis that follows the initial septicemia. lameness and joint swelling is mostly noticeable in hock and carpal joints. lameness may also occur in stifle and elbow but swelling cannot be appreciated. synovial fluid appears serosanguinous and can be submitted for testing by bacterial culture or pcr. alternatively, the entire affected leg can be removed to prevent contamination; disarticulate the leg above the infected joint. histopathologic examination of formalin-fixed synovium reveals a proliferative synovitis. other lesions that occur are nonsuppurative fibrinous polyarthritis and erosion of cartilage that can progress to pannus and ankylosis. treatment with β-lactamase antibiotics including penicillin is effective. an anti-inflammatory is added to a treatment program for pain management. commercial bacterins or avirulent live cultures are available for control and prevention. oc is the result of a delay in ossification of articular cartilage, and represents the most common lesion among culled sows. morbidity is most often reported in adult and breeding age pigs (dewey et al., ) . mortality is variable and is the result of humane euthanasia because the animal becomes nonambulatory. oc causes lameness, pain, and joint swelling. a noninfectious lameness most often affects the distal part of the humerus or femur. lesions are typically bilateral and symmetrical. diagnosis is made by ruling out other causes of lameness. ricketts occurs as a result of phosphorus deficiency, vitamin d deficiency, or secondary to iron toxicity but is not caused by dietary calcium deficiency. the condition should be suspected when there is an increase in nonambulatory pigs and broken bones during the finishing stage particularly at and immediately before marketing. occasionally, joint swelling in the nursery stage is observed. rachitic rosary (enlargement of costochondral junctions) and soft bones are observed on necropsy. if rickets are present, a bone ash analysis of the second rib will be below normal. feed analysis can identify low levels of vitamin d or phosphorus. low levels of vitamin d or phosphorous serum chemistry also will occur (madson et al., ) . supplementation of vitamin d is the only reported treatment and response that may be considered diagnostic. prevention includes proper diet formulation for the stage of production. mhd is a noninfectious disease of muscle caused by deficiency of vitamin e or selenium. it can occur if pigs are fed grain grown in selenium deficient soils (dewey, ) . clinical signs are limited to acute death of large, robust pigs. on necropsy, the heart muscle has a mottled appearance. feed analysis, response to vitamin e supplementation, and ruling out other causes support diagnosis of vitamin e/selenium deficiencies like mhd (hooser, ) . supplementation with selenium is impractical in the united states because of environmental regulations, and overzealous supplementation may cause toxicosis. splayleg is a noninfectious, congenital condition resulting from delayed myofibril development with no known cause. splayleg has low morbidity and mortality as long as it is identified and corrected before it leads to starvation or being crushed by the sow. treatment includes the use of nonslip note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). flooring in farrowing crates and application of harness or tape that holds the rear legs under the pig until it is strong enough to walk on its own. reproductive failure occurs when insemination fails to result in pregnancy or pregnancy fails to produce viable pigs due to infectious and noninfectious causes summarized in table . reproductive failure should be considered when a low conception or farrowing rate, irregular returns to estrus, abortions, stillbirths, or mummies persist at an abnormal rate. infertility occurs when fewer than four embryos are present at the time of maternal recognition of pregnancy resulting in a regular return to estrus and reduced conception rate for that breeding group. irregular returns to estrus result from embryonic death or early term abortion after implantation but before calcification of the fetuses. embryonic death of some or all of the embryos will result in low total born or irregular return to estrus, respectively. early term abortion also will reduce farrowing rates. mummies and stillborns can occur any time after calcification of the fetuses. the normal rates for mummies and stillborns are o . and o pig per litter, respectively. late-term abortions are classified as those occurring after days of gestation. total abortion rate should remain o % of a breeding group. these are general guidelines; thus, familiarity with the herd's normal reproductive performance is the most sensitive means to identify a reproductive problem. prrs is, at this time, known only to occur among swine. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrs usually results when susceptible swine are infected with either the leylystad or north american strains of prrs virus (prrsv), a member of the arteriviridae family. viremia lasts up to days, but shedding of infectious virus can last much longer (murtaugh and genzow, ) . prrsv is most commonly transmitted by introduction of infected swine or contaminated fomites, use of contaminated semen, and aerosol. the pathogenesis of the reproductive form is believed to be arteritis of fetal umbilical cords during gestation (lager and halbur, ) . swine may show no signs when reinfected with a homologous strain. conversely, infection with a heterologous strain will reproduce lesions and disease but is usually less severe than that of naïve swine (murtaugh and genzow, ) . clinical signs of prrs in a breeding herd start with an epidemic of abortions followed by an increase in low viable piglets, stillbirths, and mummies. abortions result due to fetal death or pyrexia of the gestating female. sows and gilts may be anorexic, pyrexic, or lethargic. periparturient females may become agalactic. in severe outbreaks of prrs, sow mortality also increases. in utero infection of feti can result in persistently infected piglets (rossow, ) . prewean mortality commonly increases and may remain above the herd average for weeks. diagnosis can be made by submitting lung, spleen, and lymph node from fetuses or low viable piglets. whole fetuses can also be submitted but should be refrigerated to prevent autolysis. lesions are not pathognomonic so confirmatory testing such as pcr, ihc, or vi should be conducted. tissues and thoracic fluid from stillbirths, aborted, or mummified feti can be submitted but may result in false negatives. serum collected from aborted sows or low viable piglets and tested for prrsv by pcr is another option for diagnosis. prrsv elisa indicates previous exposure but is not useful in a previously exposed herd. treatment of prrs is supportive. anti-inflammatories to reduce fever and antibiotics for control and treatment of secondary bacterial pneumonia may be necessary. the most common methods for control include depopulationrepopulation and herd closure and rollover, also called loadclose-homogenize, using commercial vaccine or herd-specific live virus exposure (corzo et al., ) . periods of closure vary based on facility capacity but a minimum of days is recommended. commercial modified-live and killed vaccines are available but do not prevent infection and should be used in accordance with label and domestic guidelines. ppv is sometimes described by the acronym smedi (stillborns, mummies, embryonic death, and infertility). ppv is an enzootic infection of swine breeding herds in the united states. the virus is ubiquitous and is transmitted through ingestion of infected feces, afterbirth, or fetal tissue. the disease most commonly affects gilts and younger parity sows (christianson, ) . the pathogenesis is through damage to table common reproductive diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the clinical signs. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). the placental epithelium resulting in fetal death. clinical signs of ppv range from low total born, mummies of various sizes, irregular returns to estrus, and females diagnosed pregnant but fail to farrow. diagnosis is based on vaccination history, clinical signs, and pcr testing of mummified fetuses. ppv elisa may provide diagnostic value if acute and convalescent serum samples are used. there is no effective treatment for ppv; however, commercial killed vaccines are available and very effective. exposure of unbred females to tissue or cull sows from a seropositive herd has been used for immunization when vaccine is unavailable. leptospirosis is caused by infection by spirochete bacteria. leptospira species may be zoonotic (leptospira canicola, l. icterohemorrhagiae), swine-adapted (l. pomona and l. bratislava), or incidentally infect swine (l. grippotyphosa and l. hardjo). infection has been associated with exposure of swine to contaminated soil or untreated surface water, and exposure to urine from infected vectors, such as rodents. infected swine can become carriers resulting in chronic disease. the pathogenesis is due to bacteremia resulting in transplacental infection followed by fetal death. clinical signs include pyrexia, low conception rate, abortion, stillbirths, and low viability pigs resulting in increased prewean mortality. diagnosis is made using dark field microscopy or ihc performed on tissues, particularly kidney, of aborted feti or stillbirths. paired or matched serology for hemagglutination inhibition (hi) testing may be useful if suspected. treatment with antibiotics, such as chlortetracycline, may be pursued (henry et al., ) . commercial killed bacterins are available to aid in prevention and should be given at least semiannually to breeding stock (christianson, ) but may not be available in all countries. for example, federal regulations prohibit the use of these bacterins in france and the netherlands (figure ) . pcv is a ubiquitous virus in swine facilities. pigs become infected with pcv through ingestion (oral nasal contact). in addition, breeding females can become infected via insemination with contaminated semen (madson et al., a) . gilts and low parity sows are affected most often, whereas boars show no clinical signs. pcv -associated reproductive failure may occur in conjunction with ppv. infection results in variable lengths of viremia. pcv -reproductive failure is due to transplacental infection of fetuses. clinical signs depend on the stage of gestation when the infection occurs. embryonic death, early term abortions, stillbirths, mummies, low total born, or low viable pigs can result from infection. mummies may vary in size, like ppv, and measuring crown to rump length is useful to determine the time when that fetus was infected. pcv -reproductive failure is diagnosed by the presence of viral antigen confirmed by ihc or deoxyribonucleic acid confirmed by pcr along with the presence of lesions in fetal tissue notably myocardial mineralization. pcr testing of fetal thoracic fluid is sufficient to diagnose in utero infection of piglets (madson and opriessnig, ) . commerical killed baculovirus vectored vaccines are available and effective for prevention of disease but not infection or viremia (madson et al., b) . prv or aujezsky's disease virus was eradicated from the us commercial swine herd in ; a comprehensive review is available (usda aphis, ). prv is a member of the herpesviridae family and, like other herpesviruses, infection can result in a carrier state or latency within nervous tissue with the potential for recrudescence. the pathogenesis of prv results from viremia, and then replication and necrosis of epithelial tissue including the placenta (christianson, ) . the period of viremia gives prv time to cross the placenta and cause fetal death. clinical signs following acute infection include embryonic death, abortion, mummies, and stillborns. necrotic foci can be found in fetal spleen, liver, lung, and lymph node. histopathology is not definitive; ihc is required to confirm presence of antigen. diagnosis may also be made through serology; a commercial elisa test is available and can differentiate between exposure to the gene-deleted vaccine and wild-type virus used extensively in the us eradication. commercial prv vaccines are available but only should be used in accordance with federal guidelines. brucellosis is a zoonotic infection caused by the bacteria, brucella suis biovars and . brucella suis is transmitted through direct contact with susceptible swine, ingestion of infected tissue, or fluids including milk and contaminated semen. pathogenesis of b. suis is initiated when the mucosal epithelium is penetrated, thereby resulting in bacteremia that commonly persists for weeks and results in placentitis among other lesions. clinical signs of infection in gilts and sows include abortion with or without vaginal discharge, whereas boars have reduced libido and fertility. bacteriologic isolation of b. suis from vaginal discharge or tissue confirms diagnosis. serology reflects prior exposure (or vaccination) to b. suis but not for diagnosis of acute disease. the us commercial swine herd is brucellosis free. swine erysipelas (se) is a zoonotic, gram-positive bacterium, which is ubiquitous among swine. erysipelothrix rhusiopathiae is the sole causative species. carrier swine shed the bacteria in saliva, nasal discharge, and feces. infection may result from direct contact with carriers, exposure to infected facilities or soil (wood, ) . a bacteremia lasting several days precedes lesions. reproductive failure is most often due to abortion but infertility and low total born following high fevers or endometritis at the time of breeding is also possible. clinical signs including rhomboid skin lesions, high fevers, lethargy, inappetence, withdrawal, and response to treatment with penicillin of affected sows and gilts are suggestive of acute and subacute se. serology is available; availability is by veterinary diagnostic laboratory (vdl) and value is limited when vaccine is in use. bacterial culture and histopathologic examination of fetal tissue is unrewarding for diagnosis of se but is helpful to rule out other causes of abortion. in chronic se, culture of e. rhusiopathiae from vulvar discharges was successful (gertenbach and bilkei, ) . treatment involves injections of antibiotics and anti-inflammatories. commercial vaccines are available and effective. co poisoning induces hypoxia resulting in an increased number of stillborns (hooser, ) . concentrations of ppm are toxic. malfunctioning heating units or poorly ventilated farrowing rooms are the cause. diagnosis is done by ruling out infectious causes of stillbirths. fetal blood or thoracic fluid can also be measured for co concentrations. zearalenone is a luteotropic mycotoxin produced by fusarium rosea. it binds estrogen receptors resulting in irregular returns to estrus, signs of estrus in prepubertal gilts, and reduced litter size (hooser, ) . diagnosis is by detection of elevated levels in feed samples. however, definitive diagnosis is rarely possible because the contaminated feed has long been consumed by the time reproductive failure occurs. aas and seasonal infertility is a noninfectious cause of reproductive failure. the declining photoperiod and temperature fluctuations during the fall months result in declining progesterone levels. high-ambient temperature experienced during lactation and the postweaning period are suspected but not confirmed as a cause. diagnosis is done by ruling out infectious causes and careful assessment of management, facilities, and reproduction records (rueff, ) . modern facilities that utilize gestation crates and evaporative cooling systems may improve but not prevent infertility during the fall months (leman, ) . the respiratory system can be simply divided into upper and lower portions. the upper portion includes the nasal cavity and sinuses, throat, trachea, and bronchi for air conduction. the lower portion is the lung comprised of bronchioles and alveoli responsible for air exchange. the respiratory system is commonly involved in numerous infectious diseases of swine summarized in table . the most notable infectious agents are the viral pathogens, prrs and pcv , which cause primary pathologic lesions to both the respiratory and the immune system. this damage to the immune system often leads to respiratory or systemic disease incited by secondary infectious agents. such mixed respiratory infections can occur at any age, and, when they occur in growing and finishing pigs, are termed porcine respiratory disease complex (prdc). multifactorial respiratory disease can obscure histopathologic lesions complicating the diagnostic process. app is a host-adapted, fastidious, and gram-negative encapsulated rod that is transmitted vertically from sow to piglet. morbidity and mortality are strain-specific; virulence varies with expression of apxi and apxii toxins. inhalation of strains of app expressing apx toxins results in lung lesions within - h. the disease is economically significant because mortality occurs during the later part of the finishing phase, usually just before slaughter. clinical signs of fever, lethargy, dyspnea, and acute death are common. pigs found dead may have a frothy, blood-tinged discharge from the nose and mouth. focal hemorrhage occurs in the diaphragmatic lung lobe, which is firm, and its appearance is likened to that of a bull's eye. fibrinous, necrotizing bronchopneumonia containing streaming leukocytes is a key histopathologic feature. bacterial culture is difficult and requires nicotinamide adenine dinucleotide (nad)-supplemented media so diagnosis is traditionally made on finding characteristic postmortem and histopathologic lesions. a pcr test is also available. in an outbreak, the entire population should receive antimicrobial therapy parentally. unlike many other gram-negative bacteria, app is sensitive to a variety of antimicrobials; at iowa state university vdl % of isolates were sensitive to ceftiofur, enrofloxacin, florfenicol, tiamulin, tilmicosin, and tulathromycin. prevention is aimed at eliminating carrier swine through depopulation or by pulse medication (marsteller and fenwick, ) . actinobacillus suis causes a hemorrhagic, necrotizing pneumonia during nursery and grow-finish phases. actinobacillus suis infection has similar clinical signs and pathologic appearance to app. affected pigs are frequently observed in a dog-sitting position with elbows abducted. unlike app, lung lesions are random in their distribution and petechial table common respiratory diseases and disorders of pigs postweaning nursery postweaning grow-finish adult note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). hemorrhages may be seen in other organs due to the septicemia that follows a. suis infection (yaeger, ) . a pcr test is available to help differentiate disease from app and hps (oliveira, a) . like app, outbreaks should be treated by parental delivery of an antimicrobial; however, treatment of only those individual pigs with clinical signs is usually sufficient. autogenous vaccines can be used for control but response is variable because most pigs are already seropositive at the time of vaccination. ascaris suum, the swine roundworm, is the most common parasitic infection of swine. the reduced growth performance and liver condemnations are responsible for economic losses (stewart and hoyt, ) . the prepatent period is - days. adult roundworms are present in the manure but it is the migration of larvae through the lungs, occurring - days after ingestion of an infective egg that causes respiratory signs. a persistent cough and dyspnea result due to verminous pneumonia. the liver develops whitish spots, called 'milk spots' that are the cause of condemnations but resolve within days (stewart and hoyt, ) . the presence of eosinophils is suggestive of a parasite infestation. treatment and control is accomplished using anthelmintics: dichlorvos, fenbendazole, levamisole eliminate adults and larvae; piperazine kills only adults. proper cleaning and disinfection particularly removing fecal material between groups reduces potential for exposure but it is virtually impossible to get rid of a. suum once a premise is infested (pittman et al., b) . it is necessary to prevent access to contaminated soil. ar is described in two forms: progressive (par) and nonprogressive (npar). par is caused by toxigenic strains of pasteurella multocida, whereas npar is the result of toxigenic strains of bordetella bronchiseptica. in both forms, the bacteria attach to cilia in the nasal passages and the cytotoxin production causes hypoplasia of nasal turbinates. clinical signs include sneezing, deviated snouts, and, in cases of par, bloody nasal discharge occurring in a large number of grow-finish pigs. mortality is low but the reduced growth that results due to ar makes it economically important. beacause the cytotoxins are responsible for ar, isolation of either bacterium from nasal passages is not sufficient for diagnosis. in addition, b. bronchiseptica and p. multocida colonize the lung leading to bronchopneumonia causing cough and dyspnea in pigs postweaning, often part of prdc (hansen et al., ) . therefore, examination of nasal turbinates at slaughter is the recommended method for diagnosis of ar (gatlin et al., ) . transmission is vertical; therefore, prefarrow vaccination of sows can protect piglets up to weeks of age. if vaccination does not prevent ar, depopulation of the herd may be necessary. hps is also called glässer's disease. there are serovars identified and prefer to colonize the nose (macinnes et al., ) . hps may not actually result in pneumonia but does cause signs of respiratory disease including nasal discharge and dyspnea. in addition, fever, lethargy, and acute death are observed. on necropsy, one or all of the pleural, pericardial, epicardial, and peritoneal serosal surfaces become covered in fibrin. effusion commonly occurs. histopathologic lesions are described as fibrinopurulent. definitive diagnosis is by bacterial isolation on culture media supplemented with v factor. owing to the difficulty in isolating hps, pcr testing is now available (oliveira et al., ) . isolation from the airways in the absence of lesions has little significance (hoefling, ) . ceftiofur, enrofloxacin, or tulathromycin delivered parenterally to affected animals are effective therapeutic drugs. use of water-soluble antimicrobials is for control. maternal immunity, medicated early weaning, and controlling infections with prrs, pcv , and influenza postpone or prevent disease onset (rapp-gabrielson et al., ) . commercial and autogenous vaccines are available but may experience limited efficacy due to serologic diversity; controlled exposure to low dose, live virulent culture is another option (oliveira et al., ) (figure ) . mh is known to infect pigs in production systems worldwide causing reduced growth performance and mortality. the disease is classified as enzootic pneumonia or a component of prdc. both manifestations of mh cause paralysis of the mucociliary escalator resulting in a severe cough and dyspnea known as thumping. vertical and lateral transmission can occur; but, owing to its slow rate of transmission between pigs, the disease primarily occurs in grow-finish pigs (meyns et al., ) . in addition, time of colonization with mh and disease severity are directly related (fano et al., ) . on necropsy, well-demarcated (red to purple lobular consolidation occurs in the apical) diaphragmatic, and accessory lung lobes is visible. histopathologic lesions characteristic of mh is bronchopneumonia with lymphocytic perivascular, peribronchial, and peribronchiolar cuffing. because mh is difficult to isolate, pcr is the most sensitive method of detection. elisa is available and is helpful in establishing herd status but must be interpreted in the context of vaccination as tests do not distinguish between antibodies produced subsequent to vaccination or field infection. treatment of affected pigs with parental antimicrobials like enrofloxacin, tulathromycin, or lincomycin or administration of water-soluble lincomycin, tiamulin, or tetracyclines to affected groups is effective in outbreaks. control can be achieved through pulse-medication in feed of chlortetracycline (thacker et al., ) beginning figure epicarditis, heart, nursery pig. fibrin gives surface a granular appearance, caused by hps infection. note the enlarged (draining) mediastinal lymph nodes located cranial to the base of the heart and the excess thoracic fluid (reddish-brown) indicative of septicemia. courtesy dr. glen almond. week before the historical onset of disease . commercial vaccines are whole cell bacterins marketed to reduce lesions but do not prevent disease or slow transmission rate. simultaneous infection with prrsv reduces efficacy of mh vaccination thacker, ) . eradication from the herd is preventative but practically difficult to accomplish. pcvad is any disease process where pcv infection results in lesions and includes pmws (ellis et al., ) and pdns. infection with pcv is widespread. morbidity and mortality is variable, often dependent on the occurrence of secondary infections and their virulence. survivors of pcvad remain stunted, owing to the economic significance of this collection of diseases. clinical signs include wasting, dyspnea, depression, ill thrift, and diarrhea. lungs are wet, heavy, and fail to collapse; pulmonary edema and lymphadenopathy also can be found at necropsy. histopathology results include presence of interstitial pneumonia, lymphoid depletion, enteritis, nephritis, and dermatitis. for a diagnosis of pcvad the following must occur: pcv antigen within characteristic lesions and lymph nodes are depleted (sorden, ) . ihc is used to confirm presence of pcv antigen within the histopathologic lesion. pcr has little value in diagnosing pcvad unless the herd is considered free. commercial vaccines are very effective and available with flex labels for administration to sows and pigs and as or doses (chae, ) . nonvaccinated, subclinically infected pigs have poorer weight gain compared to their vaccinated counterparts (kristensen et al., ) ; therefore, it is part of most vaccination protocols by us pork producers ( figure ) . prrs is the result of infection with the leylystad or north american strain of prrsv. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrsv is the most commonly diagnosed viral respiratory pathogen at vdls (gauger, ) . infection is observed to increase susceptibility to other infections, particularly opportunistic bacteria. this apparent increased susceptibility to secondary and opportunistic infections is the result of the pathologic process in which prrsv recruits and replicates in pulmonary alveolar macrophages, and then disseminates systemically (rossow, ) . clinical signs are nonspecific including fever, lethargy, and dypsnea but not cough. signs also depend on the type of secondary infection(s) present. lungs fail to collapse and appear heavy, wet, and gray on postmortem examinations. lymphadenopathy is caused by hyperplasia of germinal centers. interstitial pneumonia, alveoli are lined with hyperplastic type ii pneumocytes and contain necrotic debris, whereas the lining of bronchi and bronchioles is normal (rossow, ) . vasculitis also occurs. pcr is the most sensitive method for confirming infection. owing to the genetic diversity of prrsv, sequencing of the orf region is a common adjunct to pcr testing. sequences are then used to create dendrograms for use by production systems pursuing prrs control and epidemiologic investigations (murtaugh, ) . prrsv elisa is helpful for establishing herd status; national animal health monitoring service reports that a large percentage of us herds are seropositive. treatment is limited to maintaining pig comfort, minimizing stress, and controlling secondary infections. commercial modified-live vaccines (mlv) are available and administration during the nursery phase significantly reduces mortality and improves growth performance during the grow-finish phase of production (robbins et al., b) . mlv vaccines do replicate and should not be used in negative populations. salmonella cholerasuis is the swine-adapted salmonella from the c serogroup and, unlike s. typhimurium, is not a foodborne pathogen. ingestion or inhalation of the bacteria causes a septicemia resulting in low to moderate morbidity with high mortality within - days of infection that occurs postweaning, predominately during the grow-finish phase (baskerville and dow, ) . signs include high fevers ( c), lethargy, dyspnea, acute death, and cyanotic extremities and abdomen. the latter makes it impossible to differentiate clinically from classical swine fever virus (csfv). pleuropneumonia, interlobular edema, mediastinal, and tracheobronchial lymphoadenopathy, and occasionally white foci in the liver are apparent postmortem (turk et al., ) . acute histopathologic lesions that form in the lung are purulent bronchitis, lobular necrosis, and abscessation, whereas paratyphoid nodules are observed in the liver. isolation is best achieved from the draining lymph nodes, lung, or liver using selective culture media. serogrouping and typing is necessary for speciation and diagnostic confirmation. owing to the rapid onset of disease, parental treatment is recommended. salmonella cholerasuis isolates are commonly susceptible to ceftiofur. increased hygiene particularly eliminating access to waste and vaccination is preventive (husa et al., ) . siv is more accurately described as influenza, to encompass the infections occurring in swine, avian, and human species. influenza virus is classified by its hemagglutinin and neuraminidase proteins; the three predominant strains in pigs are h n , h n , and h n . rapid transmission and onset are characteristic; in the experimental inoculation of one nonvaccinated nursery age pig resulted in . more becoming infected (romagosa et al., ) . virus is shed for - days and uncomplicated lesions resolve days postinfection (gramer, ) . nasal discharge, fever, and lethargy occur but resolve quickly. cough and dyspnea can last up to figure pulmonary edema, lung, grow-finish pig. interlobular edema associated with pcvad, ventral portion of apical and diaphragmatic lung lobes is consolidated (purple) due to secondary bacterial infection. courtesy dr. glen almond. weeks postinfection. pcr and vi detect virus for diagnosis of clinical cases. elisa and hi detect antibodies; elisa is helpful in establishing herd status, whereas hi is best for vaccination timing and measuring postvaccination titers (allerson et al., ) . necrotizing bronchitis, bronchiolitis, and alveolitis as lesion resolves affected areas appear vacuolated. pigs recover quickly so treatment should focus on maintaining pig comfort, minimizing stress, and controlling secondary infections. all licensed vaccines are killed; commercial and autogenous products are in use in the united states. vaccination reduces lung lesions and rate of transmission, but does not prevent infection and is complicated by antigenic shift and drift. in the united states, it is typical to vaccinate the sows rather than pigs to control disease and infection (allerson et al., ) . trade diseases are those listed by the oie. when one of these diseases is suspected or confirmed, it results in closure of international market access, which would be economically devastating to import-export businesses. the primary method for managing diseases that affect trade is to prevent their introduction. foot-and-mouth disease (fmd) is caused by a picornavirus, fmdv, which causes mucosal lesions exclusively in cloven hoofed species. clinical signs are excessive salivation, anorexia, and lameness causing high morbidity but low mortality. gross lesions are vesicles at cutaneous junctions, on the snout, or in the oral cavity. similar lesions can be caused by seneca valley virus, vesicular stomatitis, swine vesicular disease, and vesicular exanthema of swine; therefore, any blister in swine warrants diagnostic investigation. fmdv is highly transmissible within and between species. african swine fever (asf) is caused by asfv, currently classified as an iridovirus. soft ticks can act as reservoirs or vectors. current outbreaks are reported throughout eastern europe and russia that have been associated with improper garbage feeding. the virus damages blood vessels resulting in clinical signs and gross lesions consistent with septicemia; including red to purple skin discoloration and enlarged spleen, liver, and lymph nodes. excess blood and fluid in body cavities may occur. classical swine fever, historically referred to as hog cholera, is caused by csfv, a pestivirus, eradicated from the united states in . transmission is associated with infected feeding, uncooked or undercooked garbage containing pork or pork by-products to swine. the virus remains infectious for months when refrigerated and years when frozen. clinical signs are nonspecific and are easily confused with s. cholerasuis. the virus replicates rapidly in tonsils, which makes it the ideal tissue to collect for diagnosis of csfv. see also: animal health: ectoparasites. animal health: foot-and-mouth disease. animal health: global antibiotic issues. slum livestock agriculture. vaccines and vaccination practices: key to sustainable animal production. zoonotic helminths of livestock the impact of maternally derived immunity on influenza a virus transmission in neonatal pig populations maternally derived antibody transfer to piglets following siv vaccination streptococcus suis colonization of piglets during parturition model for characterizing oral controlled exposure in a field setting sarcoptic mange in swine effect of dietary particle size on gastric ulcers, assessed by endoscopic examination, and relationship between ulcer severity and growth performance of individually fed pigs pathology of experimental pneumonia in pigs produced by salmonella cholera-suis some properties of the causative agent of transmissible gastroenteritis in swine erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations non-typhoidal salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen prevalence of cpb , encoding beta toxin, in clostridium perfringens field 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pleuropneumonia in missouri swine pseudorabies (aujeszky's disease) and its eradication: a review of the u.s. experience a retrospective evaluation of actions taken to control streptococcus suis infection serologic profiling and vaccination timing for lawsonia intracellularis swine erysipelas − a review of prevalence and research age, not infection dose, determines the outcome of isospora suis infections in suckling pigs actinobacillus suis septicemia: an emerging disease in highhealth herds key: cord- - fctxk authors: proudfoot, chris; lillico, simon; tait-burkard, christine title: genome editing for disease resistance in pigs and chickens date: - - journal: anim front doi: . /af/vfz sha: doc_id: cord_uid: fctxk nan for thousands of years, humans have used selective breeding to improve desirable traits in both livestock and companion animals. in livestock, targeted breeding has been common practice since the british agricultural revolution of the th century, with measurable production traits such as feed conversion in cattle or wool production in sheep actively selected for. in the late th century, genomic selection was added to the livestock breeding tool box; by reading specific locations in the genome and assigning them to measurable production traits, faster improvement in livestock production efficiency has been achieved. one of the inherently difficult production traits to measure is resistance to a specific disease, as animals with less severe symptoms or pathology may simply have been exposed to less pathogen. experimental infections guaranteeing equal pathogen exposures are expensive and require large numbers of animals for genetic association studies, making them ethically questionable. genome editing offers new opportunities to livestock breeding for disease resistance, allowing the direct translation of laboratory research into disease-resistant or resilient animals. made? genome editors are custom enzymes that allow scientists to cut the dna strands in the nucleus of a cell at a specific position. the researcher can then influence how the dna is repaired, introducing very precise genetic changes at a target locus in their species of interest. this technology has been revolutionary and provides exciting possibilities for the production of livestock resistant to viral diseases. such opportunities are particularly pertinent given state efforts to improve global food security and reduce food waste throughout the production chain. the most prominent editor technology today, crispr/cas, uses a nucleotide rna guide to target its enzyme component to a designated locus in the genome. the probability of off-target cutting with a high fidelity cas enzyme is very low, because with four potential base combinations at each of the nucleotides there are over one trillion unique guide combinations. once the enzyme has cut the dna strands, the predominant repair pathway in most cells is nonhomologous end joining, an error-prone process which often introduces small insertions or deletions into the genetic code at the break site. if the target is within a gene, such perturbations can result in a disruption to the function of that gene, potentially leading to a loss of protein function. this can be very useful to basic science as it allows researchers to discover functions associated with novel genes. for many applications, a more precise change to the genome is required. to that end, scientists regularly make an alternative dna repair process called homology-directed repair. to do this, researchers provide a novel dna sequence alongside the crispr/cas reagents, whereby the cellular repair machinery uses the new dna as a template when fixing the break. this approach facilitates the introduction of defined implications • genome editing technology enlarges the tool box of trait-selective breeding. • methods for genome editing have developed over the past decades, making the technology more efficient and specific. • technology to generate edited pigs and chickens is developing alongside genome editors to generate animals faster and more affordable. • for two major pig diseases, it has been shown that resistant animals can be generated that are refractory to infection. in chickens there are promising laboratory results but no genome-edited, resistant chickens yet. • genome editing allows us to overcome bottlenecks in trait-selective breeding and allows the incorporation of genetic traits from other breeds, related species, or laboratory results. • two major hurdles still to be faced prior to the implementation of this promising technology are consumer acceptance and the regulatory framework. changes at the genomic target locus and has sufficient refinement to alter a single nucleotide, allowing precise modification of gene function. finally, by introducing a pair of editors, it is possible to generate two concurrent dna breaks on the same chromosome. the cellular repair machinery then joins the ends of the cut sites, promoting the deletion of the intervening sequence. all the editor reagents introduced to the cell are rapidly degraded, with only the alteration to the genomic sequence remaining to be propagated following cell division. genome editing has been applied to a wide variety of agricultural species including salmonids, poultry, and ruminants. however, due to its global economic value, relatively short generation time, and multiparous nature, the most edited livestock species to date is the pig. there are two main methods widely used for the generation of edited pigs: cloning of edited fibroblasts or direct injection of the zygotes with editor reagents. both work well, and each has specific advantages. in cloning, fibroblast cells can be maintained in the lab for prolonged periods. this allows researchers to introduce editor reagents into the cultured cells typically by lipofection, electroporation, or microinjection. editing events in each cell of a population can be characterized and individual cells with the desired alteration to their genome selected for the cloning process, whereby the fibroblast cell is fused with an enucleated oocyte shell in a process called somatic cell nuclear transfer ( figure a ). the reconstituted "zygote" is then transferred to a recipient gilt or sow (carlson et al., ) . despite the benefit of being able to prescreen the donor cells, cloning is generally inefficient with hundreds of reconstituted zygotes being transferred to a single recipient. cloning also yields reduced litter sizes when compared with standard breeding and offspring often demonstrate reduced viability. as an alternative to cloning, newly fertilized zygotes can be directly microinjected with genome-editing reagents and transferred immediately back to the oviduct of a recipient animal ( figure b ). in contrast to cloning, this approach (lillico et al., ) results in the efficient establishment of pregnancies and robust litters. however, without the prescreening of cells that is routine in cloning, offspring from direct zygote manipulation inevitably encompasses a range of editing outcomes, since selection of a specific edit is not possible. porcine zygotes can also be generated by maturation of oocytes extracted from slaughterhouse-derived ovaries and in vitro fertilization. unfortunately, in vitro fertilization in pigs often results in polyspermy, rendering the resulting embryo inviable. however, in this controlled environment, editing rates can be increased and costs and animal use reduced. an emerging alternative to these proven methods could be the use of surrogate sires. as a first step towards this goal, pigs have been edited to remove a gene required for male fertility, generating an empty spermatagonial stem cell niche in the testis . spermatogonial stem cells can be isolated and cultured in vitro, opening the possibility to edit and characterize these cells before transfer to a recipient (park et al., ) ( figure c ). genetic modification of poultry poses unique challenges due to the very different physiology of the avian egg compared with a mammalian oocyte. as a result, isolation and transfer of a chicken yolk is not practical. one approach that has been taken is in ovo electroporation of editing reagents, which allowed the analysis of gene function in the neural crest (gandhi et al., ) . however, others reported that electroporation resulted in mosaicism with editing limited to a subset of cells as the chicken embryo is already much further developed when an egg is laid compared with a zygote (veron et al., ) ( figure d ). as a result, it is unlikely that this approach could be efficiently utilized to generate edited birds. an alternative approach involves sperm transfection-assisted gene editing, whereby sperm are lipofected with editing reagents before use in artificial insemination (cooper et al., ) ( figure e ). however, advances in chicken stem cell technology show the greatest promise for genome editing in chicken. primordial germ cells (stem cells that eventually develop into germ cells) can be isolated from the blood of developing chicks in ovo and cultured in vitro. as with mammalian fibroblasts, these cells can be edited and selected in vitro before transfer into the bloodstream of a stage-matched recipient where they migrate to and populate the developing gonad. the chicken embryo is accessed through an opening in the egg shell, which is sealed again until the chicken hatches. genome editing in primordial germ cells has been successfully demonstrated by a number of groups (park et al., ; taylor et al., ; idoko-akoh et al., ) and one group has generated modified birds (park et al., ) . the founder birds generated from this editing method are chimeric due to the presence of preexisting germ cells. the resulting offspring generated from breeding with the founders will be a mixture of edited or nonedited. recipient chicken embryos devoid of germ cells are currently being developed that will significantly increase the efficiency of this process (m. mcgrew, unpublished results) ( figure f ). genome editors will undoubtedly have a significant role on the generation of disease-resistant animals as exemplified below. it is important to note that currently the technology is limited to modifying a single gene or a snp with large effects; however, disease resistance in many cases is likely to be a polygenic trait. multiplexing technology is under development such that in the future polygenic traits could be altered in a single step. progress so far? porcine reproductive and respiratory syndrome virus. porcine reproductive and respiratory syndrome (prrs) is arguably the most economically important pig disease worldwide. the causative agent of prrs is an arterivirus, named prrs virus (prrsv), that affects pigs of all ages but most importantly causes late-term abortions and stillbirth in sows and severe respiratory disease in piglets with severe morbidity and high mortality. prrsv also incapacitates the pig's immune response, providing an ideal breeding ground for severe secondary infections, mostly by bacteria, which leads to increased use of antibiotics. prrsv exclusively infects cells of the monocyte/macrophage lineage and two macrophagespecific proteins, cd and cd , were identified as receptors for the virus: cd acting on the surface of the cells and cd inside the internalizing transport vesicles (calvert et al., ; van gorp et al., ) . the virus was thought to attach to cd to be taken up into the cells; however, genome-edited pigs lacking cd were not resistant to prrsv infection (prather et al., ) . cd on the other hand is thought to act through a key-lock interaction with the virus to allow it to escape from the internalizing transport vehicles into the cytosol where it replicates. cd consists of nine globular domains, organized like beads on a string, with domain determined to mediate the key-lock interaction allowing viral entry into pig cells (van gorp et al., ) . using genome editing to generate pigs lacking cd whitworth et al. showed for the first time that this approach could be used to produce livestock resistant to important viral diseases, in this case prrs (whitworth et al., ) . cd is known to have a range of important biological functions in homeostasis, inflammation, and immune responses. as a refinement on functional knock out of the entire cd protein, editing reagents were designed to remove only domain leaving the remainder of the protein intact. the resulting animals were completely resistant to prrsv infection and maintained the biological functions associated with the remaining domains of cd (burkard et al., ; burkard et al., ) . porcine epidemic diarrhea virus/transmissible gastroenteritis virus. the two coronaviruses porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) both cause severe diarrhea in preweaned piglets and are associated with high morbidity and mortality. in vitro host-pathogen studies identified aminopeptidase n as the receptor for tgev and a potential receptor for pedv (delmas et al., ; li et al., ) . the use of genome editing to generate pigs lacking aminopeptidase n successfully showed that pigs resistant to tgev infection could be generated. however, the edited animals remained susceptible to pedv infection (whitworth et al., ) . aminopeptidase n is important for peptide digestion in the small intestine and knockout mice were shown to have delayed mammary gland development. in humans, aminopeptidase n defects are associated with different types of leukemia and lymphoma. therefore, further investigation into the potential consequences of the absence aminopeptidase n in pigs is warranted as it may affect the overall health and/or productivity of the animals. african swine fever virus. african swine fever virus (asfv) causes a severe hemorrhagic disease in domestic pigs (sus scrofa domesticus) and wild boars (sus scrofa ferus) with high mortality in pigs of all ages. asfv is highly contagious and can be transmitted by soft ticks of the ornithodoros genus. it was identified in and contained to africa with occasional transmission around the strait of gibraltar into portugal and spain. in an introduction of the virus into the caucasus region showed that the virus does not solely rely on ticks for transmission in the wild, as transport of contaminated material and direct contact between animals have been shown to be major routes of disease dissemination. since then, the virus has spread across eastern europe and russia and was recently found in western europe and china. asfv poses a huge risk to the pig industry worldwide and is a limiting factor to a sustainable pig industry in many parts of africa. interestingly, asfv also infects wild suids, such as warthogs (phacocherus africanus) and bushpigs (potamocherus porcus), without causing overt disease. such infected wild suids are thought to act as a reservoir of the virus in africa. in the late stages of asfv infection, a cytokine storm, i.e., an overreaction of the immune system, is observed, which is thought to strongly contribute to the lethal outcome of disease. a comparison of the warthog and domestic pig genomes identified differences in the rel-like domain-containing protein a (rela, also known as p ) protein, which is involved in nf-κb cytokine signaling, was thought to underlie the different responses of the related species to asfv infection (palgrave et al., ) . researchers used genome editing to convert a key region of the encoded domestic pig protein sequence to the warthog equivalent (lillico et al., ) . data on susceptibility of the edited animals to asfv infection have yet to be reported. in this instance, it is important to differentiate between disease resistance, the ability of an animal to suppress the establishment and/or development of an infection, and disease resilience, where an infected host manages to maintain an acceptable level of productivity despite challenge pressure. should these pigs prove to be resilient to asfv infection it is likely that their use may not be permitted in many jurisdictions, since they could act as reservoirs of infection. however, in environments where the disease is endemic use of such animals could be beneficial. avian leucosis virus. avian leukosis virus infection results in inappetence, diarrhea, weight loss, a reduction in eggs laid, and often causes tumor formation in the chicken. the virus is divided into six subgroups, with the avian leucosis virus subgroup j (alv-j) shown to be responsible for major disease outbreaks in china. the cellular receptor of alv-j was identified to be the chicken sodium/hydrogen exchanger protein on the cell surface. chicken somatic cell lines have been edited to introduce changes to this gene-conferring resistance to avian leucosis virus in vitro (lee et al., ) . despite cells showing resistance to alv-j infection, no edited chickens have been produced to date. in both mice and humans, a lack of the sodium/hydrogen exchanger protein is associated with severe neurological disease; however, targeted changes to single amino acids may retain biological functions of the protein in chicken while resulting in disease resistance. avian influenza virus. in chickens, disease resistance to avian influenza is at the top of the wish list due to the serious impact on chicken health but also the risk of transmission to humans. similarly, influenza a is also one of the diseases on the resistance wish list for pigs, as they can act as an intermediate host-aiding virus adaptation to humans. the acidic leucine-rich nuclear phosphoprotein- a (anp a) was found to play a key role in avian influenza virus replication in both chicken and water fowl. although the virus polymerase protein readily interacts with the avian anp a, the human version of the same protein supports only limited replication of the viral genome. it has been demonstrated in vitro that deletion of a small region of chicken anp a can prevent replication of avian influenza virus (long et al., ; long et al., ) . although the functional consequence of edited anp a has yet to be demonstrated in vivo, such approaches offer exciting opportunities that have the potential to benefit both industry and animal welfare. as exemplified above, currently many gene editing approaches focus on targeting host genes involved in mediating entry of the virus, with a special focus on receptors. however, as the example for avian influenza shows, host genes play an important role in other steps of the pathogen replication cycle and also provide editing targets for disease resilience or resistance. more in-depth host-pathogen interaction studies, including genome-wide editing studies in vitro, will no doubt produce a variety of further candidate genes for genetic disease resistance. an alternative antipathogen approach pursued for decades is the generation of transgenic livestock, expressing antiviral or antibacterial agents, such as enzymes or small interfering rnas. genome editing can be used to improve the integration efficiency of these transgenes at specific locations in the genome; however, the discussion of transgenic disease-resistant animals is beyond the scope of this review. how does genome editing fit within existing selective breeding structures and how will it be regulated? selective breeding has generated highly productive, robust animals that are adapted to a modern production environment. livestock production is dynamic, with evolving challenges such as climate change and disease outbreaks coupled with societal pressure to reduce antimicrobial use. selective breeding for disease resistance has proven difficult, as outbreaks are often sporadic and resistant/resilient animals often difficult to identify. in circumstances where a genetic trait for disease resistance can be identified in the breeding population, then selection through the selective breeding can be achieved. a good example of this is pigs with resistance to f type enterotoxigenic e. coli. association studies revealed that a polymorphism in the fucosyl transferase gene conferred resistance to these bacteria. there was initial concern that selection for the locus figure . genetic resistance to disease and how genome editing can help integrate traits into highly productive lines. (a) genetic resistance to disease may be present in a small percentage of production animals and genetic selection for these animals may be associated with the risk of inbreeding, productivity loss, or the risk of losing other desirable traits. genome editing allows integration of the disease-resistance trait into a wider selection of pigs, ensuring genetic variability and maintenance of desirable traits. (b) genetic resistance to disease may be present in an indigenous or less productive breed. crossbreeding would result in productivity loss and the risk of losing other desirable traits, such as fur color. genome editing allows for incorporation of genetic disease resistance into highly bred lines without losing productivity. (c) genetic resistance may be observed in a closely related species, e.g., wild boar or wild suids in the case of the domestic pig. integration into highly bred domestic pig lines would only be possible by genome editing. (d) resistance genes may be identified in laboratory research but not in highly bred lines, making integration into those productive animals only possible using genome editing. harboring this gene may counterselect for another gene associated with stress resistance. however, this proved not to be the case and genetic selection for the favorable fucosyl transferase allele has been integrated into many pig-breeding programs (coddens et al., ) . this was possible, in part, because the favorable allele was present at sufficient prevalence (in most studies between % and %) in the breeding population to allow for selection while avoiding inbreeding. in circumstances where an allelic variant associated with a resistant phenotype is present at a much lower frequency, it may prove difficult to incorporate effective selection into a standard breeding regime without the risk of inbreeding and related longer-term productivity loss (figure a) . genome editing has the potential to contribute in such circumstances, allowing the direct introgression of a beneficial allele into the offspring of diverse, highly productive animals. similarly, disease-resistance traits associated with less productive indigenous breeds are unlikely to be introduced to highly productive populations by standard crossbreeding as this would result in a significant set-back in productivity, abrogating decades or even centuries of advances made through genetic selection ( figure b ). in circumstances where resistance or resilience is observed in a related species, crossbreeding is simply not possible. genome editing could bridge these gaps. one example of this is resilience of wild suids to african swine fever virus while domestic pigs can suffer from severe disease. it is not possible to crossbreed these species, so introduction of the genetics underlying resilience is not possible by this route. genetic comparison can be used to identify the functional differences underlying such traits, and genome editing employed to introduce appropriate variants into domestic pigs ( figure c ). finally, with a good understanding of host-pathogen interactions, novel genetics that has not been observed in live animals can be created and tested for efficacy in a laboratory environment. this was the case for both the cd /prrsv and apn/tgev examples in pigs and would be the case for the anp a/ influenza and the alv-j resistance in chicken, described above. in such circumstances, integration through genome editing presents a practical route to benefit from the findings ( figure d ). it is imperative that in such circumstances thorough phenotypic characterization of the edited animals be carried out as deletion of all or part of a functional protein could result in a loss of (systemic) biological function. a second measure worthy of consideration before embarking on an editing project is whether the gene is located within a locus that has been actively selected in breeding programs. this could indicate whether a potential target is associated with known production traits. this approach has been taken for prrsv-resistant pigs, with evaluation as to whether the cd gene locus has been selected for in pig breeding programs (johnsson et al., ) . overall, genome editing holds vast promise for the future production of animals resistant or resilient to disease, improving productivity and animal welfare while reducing food waste throughout the production chain. through reduction of primary and secondary infections, it should also be possible to reduce antimicrobial use in livestock production. technical improvements in the generation of genome editing animals will undoubtedly reduce the cost implications of this technology. the two major hurdles still to be faced prior to implementation of this promising technology are consumer acceptance and the regulatory framework. approval of edited animals for human consumption relies on national and multinational legislation, which is currently at early stages. encouragingly, some international jurisdictions such as argentina and brazil have about the authors dr. chris proudfoot is research fellow at the roslin institute/university of edinburgh since . his work centers on generation of genome-modified livestock, with particular emphasis on genome editors, to improve disease resistance or to accurately model human disease. dr. proudfoot has worked extensively with zfns, talens, and crispr/cas to produce a variety of edited animals. he was a member of the team that produced the first edited livestock using this method. dr. simon lillico is a research associate at the roslin institute/university of edinburgh. he joined the institute on an industrial collaboration to produce highvalue therapeutic proteins in hens eggs and then applied his expertise in lentiviral transgenesis to generate livestock models of human diseases. the rapid expansion of the field of genome editors over the last yr has made practicable genome modifications which had previously been unattainable. dr. lillico has been at the forefront of application of these editors to livestock, creating either disease-resistant/resilient strains, or accurate models of human disease. dr. christine tait-burkard is an assistant professor at the roslin institute/university of edinburgh since in the departments of genetics and genomics and infection and immunity. her research focuses on understanding host-pathogen interactions on a cellular and genetic level, developing new in vitro tools for virus research, improving and developing easy-to-use diagnostics, and devising strategies to combat viral disease in livestock in general and pigs in particular. she employs genome editing and genetic selection to generate animals genetically resistant to viral disease. corresponding author: christine.burkard@roslin.ed.ac.uk already ruled that modifications, such as the prrsv-resistant pig, that do not have any new genetic information integrated into the animal, will be exempt from regulation. precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses efficient talen-mediated gene knockout in livestock the possibility of positive selection for both f (+)escherichia coli and stress resistant pigs opens new perspectives for pig breeding generation of gene edited birds in one generation using sperm transfection assisted gene editing (stage) aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev optimization of crispr/cas genome editing for loss-of-function in the early chick embryo high fidelity crispr/cas increases precise monoallelic and biallelic editing events in primordial germ cells precise gene editing of chicken na+/h+ exchange type (chnhe ) confers resistance to avian leukosis virus subgroup j (alv-j) porcine aminopeptidase n is a functional receptor for the pedv coronavirus live pigs produced from genome edited zygotes mammalian interspecies substitution of immune modulatory alleles by genome editing species difference in anp a underlies influenza a virus polymerase host restriction avian anp b does not support influenza a virus polymerase and influenza a virus relies exclusively on anp a in chicken cells species-specific variation in rela underlies differences in nf-κb activity: a potential role in african swine fever pathogenesis successful genetic modification of porcine spermatogonial stem cells via an electrically responsive au nanowire injector generation of germline ablated male pigs by crispr/cas editing of the nanos gene targeted gene knockout in chickens mediated by talens an intact sialoadhesin (sn/siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus efficient talen-mediated gene targeting of chicken primordial germ cells sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus crispr mediated somatic cell genome engineering in the chicken gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs we acknowledge financial support from the biotechnology and biological science research council (bbsrc) (bb/ r / , bb/n / ) and the bbsrc institute strategic programme grant funding to the roslin institute (bbs/ e/d/ and bbs/e/d/ ). key: cord- - uop z authors: montoya, maría; foni, emanuela; solórzano, alicia; razzuoli, elisabetta; baratelli, massimiliano; bilato, dania; córdoba, lorena; del burgo, maria angeles martín; martinez, jorge; martinez-orellana, pamela; chiapponi, chiara; perlin, david s.; del real, gustavo; amadori, massimo title: expression dynamics of innate immunity in influenza virus-infected swine date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: uop z the current circulating swine influenza virus (iv) subtypes in europe (h n , h n , and h n ) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h n swine iv and four different h n iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at , , and days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h n strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin- responses in bronchoalveolar fluids (balf) at day post infection, as opposed to the other non-swine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. the current circulating swine influenza virus (iv) subtypes in europe (h n , h n , and h n ) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h n swine iv and four different h n iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at , , and days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h n strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin- responses in bronchoalveolar fluids (balf) at day post infection, as opposed to the other nonswine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. keywords introduction the first line of defense after viral infections is based on innate immunity, which has the capacity to respond to pathogens by sensing pathogen-associated molecular patterns (pamps) through germline-encoded pattern recognition receptors. this process leads to the production of several cytokines such as type i (α and β) interferons (ifns). these bind to the type i ifn receptor to signal the induction of hundreds of interferonstimulated genes in the local uninfected and virus-infected cells, thus creating an antiviral state that suppresses virus infection and serves to promote the onset of adaptive immunity ( , ) . innate immune responses are crucial not only for blunting viral replication in the first instance but also for orchestrating effective adaptive immune responses. therefore, a transient induction of cytokines and chemokines is required for efficient antiviral responses. however, overreacting and prolonged immune responses may lead to undesired secondary effects, contributing to immunopathology. as for influenza virus (iv), the innate immune response may vary between mild and severe infections, and include widely different soluble innate immune inhibitors: sialic acid-based inhibitors, ca-dependent lectin inhibitors, anti-microbial peptides, complement, natural igm, ifns, to name a few ( ). toll-like receptors and (tlr- and tlr- ) have been found to play an important role in influenza a virus recognition and initiation of the immune response in human respiratory epithelial cells and plasmacytoid dendritic cells (pdcs) ( ). activation of both tlrs and cytoplasmic receptors leads to a potent type i ifn release and simultaneous pro-inflammatory cytokine expression. type i ifns, ifn-γ, and pro-inflammatory cytokines, such as interleukin (il)- , il- , il- , il- , and tumor necrosis factor-alpha (tnf-α), have been shown to be upregulated in lung tissue and lung lavage after experimental infection of pigs with swine iv ( - ). three iv subtypes (h n , h n , and h n ) are currently circulating in swine herds in europe ( ) ( ) ( ) and have been associated with disease occurrence and gross lesions in swine ( ) . also, pigs are susceptible to infection with low pathogenic and highly pathogenic avian ivs (lpaiv and hpaiv, respectively) ( ) . most lpaiv subtypes diagnosed in field samples possess a h or h hemagglutinin usually restricted to birds. hpaiv are able to infect pigs under natural and experimental conditions ( ) . besides, it is well known that some ivs are able to infect humans and pigs, as it was the case in the last h n pandemic infection ( , ) . in vitro, different iv strains can interact with porcine dendritic cells (dcs) and induce sequential waves of cytokine production that are dependent on time and virus strain ( ) . this effect could account for the different immune responses generated by iv strains. on the other hand, we have previously examined the potential of h n iv from canine, equine, avian, and seal origin to productively infect pigs. our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation ( ) . however, no studies in vivo have addressed whether innate immune responses of pigs to swine-adapted and non-adapted iv strains might be different. the above findings outline the main working hypothesis of our study, which implies that swine iv strains might give rise to peculiar innate immune responses and time courses thereof in pigs, clearly different from those triggered by other iv strains. in order to answer these questions, samples from pigs infected with a swine (h n ) and four different non-swine-adapted h n iv strains circulating in different animal species (dogs, horses, wild aquatic birds, and seals) from our previous study ( ) were analyzed and innate immune responses in the respiratory tract were thoroughly investigated. in line with the above operational framework, six groups of pigs ( - weeks old, landrace × pietrain, free from common pathogens) were housed in separate isolation rooms and adapted to the new environment and stockmen over a -week period under veterinary supervision. the animals used in our study were seronegative at that time to influenza a viruses by competition elisa (id screen ® influenza a antibody competition elisa, id-vet, france). also, they had been always healthy and thrifty before the present study. the experimental infection of pigs with different iv strains was described in our previous paper ( ) . pigs were infected with the following iv strains: at day , each pig in groups , , , , and was intratracheally infected with ml of virus suspension in pbs, containing × chicken embryo infectious doses % (eid ) of the corresponding virus strain. animals were clinically inspected on a daily basis. rectal temperature and weight were measured at the same times. four pigs of each virus-infected group were euthanized at day , , and post infection (p.i.), respectively. in the mock-infected group, two pigs were euthanized at each of the same times. blood serum samples in vacuum tubes were collected from jugular veins at day and just before sacrifice at the aforementioned days. after sacrifice, bronchoalveolar fluids (balf) were collected by lung lavage with pbs, as described by busquets et al. ( ) . briefly, the right lung of sacrificed pigs was used to perform a bronchoalveolar lavage (bal) using around ml of pbs, and the left one was sampled for histopathological and virological studies. a complete necropsy was performed. lung lesions were classified depending on the extent of pneumonia. mild lesions were recorded when affecting small areas (< cm ) of cranial or medial lung lobes, moderate lesions when affecting extended areas ( - cm ) of cranial or medial lobes, and severe lesions when affecting large areas (> cm ) of cranial, medial, diaphragmatic, and accessory lobes. histopathology samples from nasal turbinates, trachea, and lungs were collected, fixed in % buffered formalin and processed for histopathology, i.e., they were dehydrated through graded alcohols and embedded in paraffin. the -µm thick sections were cut, stained with hematoxylin-eosin and examined in a "blind-fashion" manner. in particular, we aimed to evaluate bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles, as well as inflammatory changes in alveoli. in the lung, bronchointerstitial pneumonia intensity was assessed by means of a semi-quantitative lesion score. the pathological scores for tracheal and pulmonary tissues were as follows: : no lesions, : mild lesions, : moderate lesions, : severe lesions. in order to check iv replication in the lower respiratory tract of pigs, balf samples collected from pigs killed at day , , and were tested for gene m of the influenza a virus using a quantitative real-time pcr procedure ( ) . the sensitivity of the test was equal to tissue culture infectious doses % (tcid )/ microliters. anti-influenza a virus nucleoprotein (np) antibody levels were investigated in serum using the id screen ® influenza a antibody competition elisa (id-vet, france) following the manufacturer's instructions at days , , and p.i. as previously described ( ) . the threshold level was set at < % of the od level observed in the control wells without any swine serum. the kit detects antibodies against all influenza a subtypes and antigenic variants thanks to the use of a monoclonal antibody against a highly conserved epitope of the influenza a virus np and it was used with pig serum as previously described ( , ) to avoid background. acute phase proteins (app) of pigs (haptoglobin and serum amyloid a, saa) were investigated in balf samples by commercial colorimetric kits (haptoglobin kit, tridelta development, code tp . multispecies saa elisa kit, tridelta development, code tp ), according to the manufacturers' directions. interferon-alpha was measured in balf samples by two different assays. first, a sandwich elisa with monoclonal antibodies (mab) f and k to porcine ifn-alpha was used as previously described ( ) . ifn-alpha was also measured by a cytopathic effect inhibition assay on mdbk cells with vesicular stomatitis virus ( ) . the test was calibrated with a preparation of porcine recombinant ifn-α (pbl biomedical laboratories, cat. - ). the units of this preparation are determined with respect to the international reference standard for human leukocyte ifn (ga- - ) provided by national institutes of health (bethesda, md, usa). this assay for porcine ifn-α in serum had been also validated in a previous study ( ) . identification of the cytokine was performed by a neutralization assay on mdbk cells of ifn α-positive balf samples ( ) , using monoclonal antibody (mab) g to porcine ifn-α (serotec, cat. mca z). the assay was calibrated with porcine recombinant ifn-α . interleukin- and tnf-α were determined in balf samples by bioassays on td and wehi cells, respectively, as previously described ( , ) . cytokine concentrations were determined from a standard curve created with reference preparations of human recombinant il- and tnf-α (pierce endogen, rockford, il, usa). immunophenotyping of balf cells was carried out by flow cytometry. this was performed using indirect labeling of cells by hybridoma supernatants for cd (clone ppt ), cd a (swc ) (clone - - a), and swine mhc (sla) ii (clone f ) ( ) . the antibody against γδ tcr (clone pgbl a) was purchased from euroveterinaria s.a. and the one against cd (clone - - ) was purchased from serotec s.a., both conjugated with r-phycoerythrin (pe). as for the hybridoma supernatants, the concentration of antibody was empirically calculated after titration of each preparation and the secondary antibodies were either pe or apc-conjugated goat anti-mouse igg (jackson immunoresearch, suffolk, uk) used following the manufacturer's instructions. each primary antibody was compared with its relevant isotype control in each sample using the same concentrations. briefly, . × cells/ μl/well were labeled for h at °c with µl of each monoclonal antibody (relevant and irrelevant) at a pre-established, optimal dilution. after -h incubation at °c, cells were washed with cold pbs with % fcs by centrifugation at × g, °c, min. then, the secondary antibody conjugated to r-phycoerythrin diluted : was added. cells were incubated for a further h at °c, then washed as before and resuspended in pbs with % fcs. stained cells were acquired using facsaria i (becton dickinson ® ) and analyzed by software facsdiva v. . . . a gating strategy was applied to living cells using their forward and side scatter (fs/ss) characteristics. staining with isotype controls did not exceed % in all the samples. total rna was extracted from balf cells using rneasy mini kit (qiagen, milan, italy) by the qiacube system (qiagen, milan, italy) in accordance with the manufacturer's instructions. the protocol included a dnase treatment for eliminating genomic dna. rna concentration was evaluated by uv absorbance (biophotometer, eppendorf, milan). three µl of rna ( ng/µl) were added to the reaction mix for cdna synthesis. this was performed in the presence of random hexamers as previously described ( ) . then, the expression of porcine ifn-α, il- , il- , il- β, and tnf-α genes was determined using the primer sets shown in table . the choice of the porcine ifn-α subtypes shown in table was dictated by their important role in constitutive expression of type i ifns ( ) and, therefore, by their possible expression before iv infection. porcine beta -microglobulin was used as housekeeping control gene ( table ) to normalize the test results at different sampling times. evagreen real-time pcr amplification was performed over cycles in a cfx real-time system (bio-rad, milan, italy) as described by razzuoli et al. ( ) . in each sample, the relative expression of the selected genes was calculated using the formula Δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct (cycle of threshold) values were the mean of three test replicates ± sd, as previously described ( ) . negative samples were given a ct fictitious value for further statistical examination. gene expression data (Δct values) obtained on balf cells were checked for normality by the kolmogorov-smirnov test. the data sets passing the normality test were checked for significant differences between time points (days , , p.i.) by one-way anova for unpaired data, whereas the others were evaluated by the non-parametric kruskal-wallis procedure (graphpad prism . , graphpadsoftware, san diego, ca, usa). the threshold for significance was set at p < . . statistical analyses on flow cytometry data were carried out using the sas system v. . . (sas institute inc., cary, nc, usa). for all analyses, the individual pig was used as the experimental unit. shapiro wilk's and levene tests were used to evaluate the normality of the distribution of the continuous variable and the homogeneity of variances, respectively. a statistical analysis was performed to study the association between the different variables with the different experimental groups at the different time points ( , , and days post-infection). to analyze the association between continuous, normally distributed variables and the different experimental groups, an anova test was used at , , and days post-infection. finally, a wilcoxon rank-sum test was used for the continuous, non-normally distributed variables at the different time points. no clinical signs were shown during the experimental procedure, such as fever, weight loss, anorexia, depression, nasal discharge, or altered breathing. temperature was not higher than . °c in any animal at any time point. there were no traumatic lesions as a result of intratracheal virus inoculation. gross lesions were mostly shown by animals infected with the swine iv, but some lesions were also detected in lungs from pigs infected with other iv strains. pulmonary lesions ( table ) were consistent with bronchointerstitial pneumonia at different grades of severity ( - ). the pigs infected with swine iv presented the highest level of severity followed by those infected with seal iv, avian iv, and either equine or canine iv in descending order. histopathology results are shown in figure . nasal turbinates sections did not differ between iv and mock-infected animals. tracheal lesions of iv-infected pigs consisted of variable grades ( - ) of lymphoplasmacytic infiltration in the lamina propria. in the most severe cases, focal ulceration of the mucosa with fibrin deposition and neutrophilic infiltration was observed. a lymphoplasmacytic infiltration was observed in the bronchioli with extension to surrounding alveolar septa. in the most severe cases, at day p.i., necrosis of the bronchiolar epithelium was observed with accumulation of necrotic cells in the bronchiolar lumen and alveolar spaces. in agreement with the gross lesion scores shown in table , histopathological lesions were much more severe in pigs of the swine group at day p.i. followed by the animals infected with seal or avian iv. the pigs infected with either equine or canine iv showed the least severe lesions ( table ) . to confirm the virological data (iv titration in embryonated chicken eggs) shown in our previous paper ( ) , viral replication was evaluated by quantitative real-time pcr on balf samples of animals sacrificed at days , , and p.i. (figure ) . the swine-adapted iv actively replicated in the pig respiratory tract at both day and p.i., as shown by ct values ranging from to . instead, we could detect only one iv-positive balf sample in the equine group at day p.i. with a ct value of and none in the canine group, indicating very little replication of those viruses. on the contrary, the avian and seal virus strains showed a moderate replication at day in most pigs, with ct values ranging from to . one animal from the seal group was still iv-positive at day p.i. with a ct value of . the above pcr data are in agreement with iv titrations in embryonated chicken eggs, reported in our previous paper ( ) . total anti-np antibody levels were measured by a competition elisa kit in serum samples from all animals at the beginning of the assay (day ), days , and p.i. all animals were seronegative at the beginning of the experiment (day , figure ) . also, no antibody response was observed in mock-infected animals. on the other hand, all iv-infected animals seroconverted (od < the threshold) by day p.i. most of them seroconverted by day p.i. in groups infected with swine, avian, and seal viruses, whereas the response was delayed in groups infected with the equine and canine viruses (figure ). the above findings confirmed a different invasiveness in pigs of swine-adapted and non-adapted iv strains in terms of virus replication and pathological lesions. on this basis, we investigated innate immune responses during iv infection in the respiratory tract that might differ between pathogenic and non-pathogenic iv strains in pigs. protein cytokine levels were measured in all animals from all groups by different assays. firstly, levels of ifn-α in balf were analyzed by elisa. all samples were negative except those collected from animals infected with swine iv at day p.i. (figure a) . these findings were confirmed by a bioassay for type i ifn on mdbk cells, and the specificity of the response was confirmed by an ifn-neutralization assay based on a mab to porcine ifn-alpha (data not shown). there was only one pig (sw- ) with a slight tnf-α response in balf at day p.i. in the swine iv-infected group. balf samples were always tnf α-negative in the other groups (data not shown). on the other hand, il- was clearly detected at day p.i. in the balf samples of swine iv-infected pigs. in the other groups, there was no significant difference with respect to mock animals, with the exception of pig se- in the seal group (day p.i.) ( figure b) . the high il- response in balf of the swine group at day p.i. led us to investigate haptoglobin and saa in balf samples of the mock, swine, and equine groups at day p.i. all the samples were haptoglobin-negative (< . mg/ml). on the contrary, all the equine iv-infected pigs were saa-positive (range: . - . mg/ml), as also was one sample of the swine group ( . mg/ml). all the other samples were saa-negative (< . mg/ml). the expression of some cytokine genes was investigated by quantitative real-time pcr on balf cells. results are shown in figure and detailed hereunder. mock group: there was no significant difference between the different sampling times for each cytokine gene under study. swine group: there was no significant difference of gene expression at the three time points. only tendencies (p < . ) were shown for il- , il- , and ifn-gamma genes. equine group: significant differences were shown for ifna , ifn-gamma, and il- genes. in particular, at day p.i., an increase was observed of ifn-gamma and il- and a decrease of ifna gene expression. canine group: significant differences were shown for ifna / , ifna / , and ifn-gamma genes. at day p.i., there was a downregulation of ifna / and / , as opposed to a significant increase of ifngamma gene expression at day p.i. no other significant change of expression was detected. avian group: there were significant differences for ifna / , ifna , ifna , and ifn-gamma gene expression and tendencies for ifna / and ifna genes. in particular, a downregulation of these genes occurred at day p.i. seal group: ifna / was downregulated at day p.i., whereas ifn-gamma and il- genes were upregulated at the same time. based on the fact that local responses in balf from swine iv-infected animals were different from those of the other iv-infected pigs, we investigated whether there was any difference in cells infiltrating the lungs of infected pigs (figure ) . therefore, cells from balf were analyzed by flow cytometry to detect changes among animals of each group. swine iv-infected animals had significantly more t cells (cd +) at day and p.i. than the rest of the infected groups (p < . ), and the highest concentrations also corresponded to the appearance of a low concentration of the γδ t cell receptor phenotype. at day p.i., γδ t cell percentages were significantly different from those of the other infected pigs (p < . ). also, only swine iv-infected animals showed a clear increment of pdcs (defined as cd a + cd + cells) at day p.i., with a statistical tendency (p = . ). no major changes were detected concerning the surface expression of sla-ii or cd in balf cells (data not shown). in this study, the pathogenicity of iv strains with different host specificity was characterized in swine in terms of clinical symptoms and tissue lesions. also, we aimed to define a possible association between innate immune responses to iv strains, and clinical and postmortem findings. the main findings of our study are outlined in table . despite wide variations among individual pigs, the iv strains under study could be discriminated in terms of pathogenicity. our results confirmed a widely different replication of iv strains in the lower respiratory tract of swine: the swine-adapted virus replicated to a large extent, as opposed to the other iv strains under study. these pcr results were fully in agreement with virus titration results in our previous study ( ) . interestingly, the avian and seal h n strains replicated more than the equine and canine strains. replication of the canine and equine strains could have transiently taken place at days - post infection, before the first sampling. this is consistent with low-grade postmortem lesions and histopathological analyses on samples at day p. i., as reported in tables and . these findings highlight different invasiveness of swine, seal and avian, equine, and canine viruses, in descending order (figure ) . the replication to a higher level of swine, avian, and seal iv was associated with an earlier antibody response compared with groups equine and canine (figure ) , where a possible low-titered replication could account for the late antibody response to np. also, we cannot rule out that defective or partially assembled particles in the viral inoculum could also have induced antibodies to np, and that different iv strains could elicit antibodies with different reactivity in the assay. please notice, however, that amino acid identity of nps with respect to swine-adapted iv was very high, ranging between % (canine strain) and % (avian and seal strains). therefore, the combination of an ab response to iv np with macro and microscopic lesions and the clear discrimination from mock-infected animals indicate that pigs can be productively infected by h n viruses, which is also in agreement with the replication of both seal and avian h n strain in tracheal explants ( ) . most importantly, the pathogenic role of swine-adapted iv strain was confirmed by both gross and histological lesions in lungs, not induced by the other viruses under study. in turn, the presence of lesions could be temporarily associated with local innate immune responses in the lower respiratory tract (balf samples): ifn-α and il- ( figure ) . these findings are in agreement with those of other experimental infections of pigs with ivs ( , ) . moreover, previous results in our group showed that ifnα secretion was only detected in vitro when swine iv-infected myeloid porcine dcs but not when other ivs from human or avian origin were used ( ) . interestingly, the ifn-α titers in bal fluids of swine iv-infected pigs were not associated with the expression of ifn-α genes, in agreement with our previous data in vitro ( ) and another study in vivo ( ) . but for one animal, the il- local response in balf samples of the swine group at day p.i. was not in agreement with the presence of app. unexpectedly, saa was clearly expressed in balf samples of the equine group at day p.i. this indicates that saa responses can be induced despite poor virus replication in the lower respiratory tract. also, the equine strain widely affected the expression of cytokine genes (see figure ). this result suggests that innate immune responses can be triggered by some iv strains regardless of their replication efficiency. also, our findings confirm the possible extrahepatic expression of app observed in other disease models ( ) . the swine iv strain also caused detectable changes of the balf-infiltrating leukocyte populations. in particular, some cd +cd a+ cells were observed in balf from swine iv-infected animals but not in balf from the other iv groups. these are considered pdcs in swine ( ) , and their main function is the release of high levels of type i ifns. this is in agreement with the fact that ifn-α was only observed in balf samples of the swine iv-infected group (figure a) . ifn-α was detected at day p.i., whereas the increased prevalence of pdcs was detected at day p.i. this apparent discrepancy could be explained by our detection limit for pdcs in balf, as a certain minimum concentration would probably be required for detection by flow cytometry. also, we do not rule out a distinct or overlapping release of ifn-α by alveolar macrophages or other cells (e.g., epithelial cells) at an early time point after infection. despite the lesions caused by the swine iv strain, no disease signs were observed. this result is in contrast with an experimental infection of pigs with a danish h n iv strain, which led to the development of typical clinical signs of influenza ( ) . the absence of clinical signs was probably associated in our study with the reduced infectious challenge ( × eid ), which was necessary to standardize the challenge infection with iv strains showing high or poor replication in embryonated chicken eggs ( ) . yet, our data are in agreement with the fact that many pigs become infected with one or more iv subtypes without showing clinical signs ( ) . the gene expression data point at a crucial neglected issue: iv strains seem to cause a long-term regulation of the innate immune response, by far beyond the actual period of virus replication and persistence in organs and tissues. instead, the modulation of cytokine genes was more related to tissue damages, as indicated by the gross and hystopathological lesions persisting in some pigs until day p.i. retrospectively, earlier sampling of lung tissues and/or balf samples could have provided a more clear picture of cytokine gene expression levels, which were probably in a descending phase at day p.i. after a possible major peak at day ( ) . this is particularly true of ifn-β, which is highly up-regulated for a short time after iv infection ( ) . indeed, our previous results actually showed that ifn-β gene upregulation was only detected h after swine iv infection of myeloid porcine dcs in vitro ( ) . this is also in agreement with its role as immediate-early type i ifn gene in non-lymphoid cells ( ) . some innate immune responses were triggered by the swineadapted iv strain, only. diverse iv strain-specific factors could account for these responses, such as hemagglutinin receptor specificity and affinity, polymerase efficiency and activity of iv anti-ifn proteins, which may show additive or synergistic interactions with figure | rt real-time pcr for some cytokine genes was carried out on bronchoalveolar fluids cells collected at the indicated times after infection. for each sample, the relative expression of the selected genes was calculated using the formula Δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct values were the mean of three test replicates. results are shown as n-fold change in gene expression ( −Δct ). the same superscripts (a, b, c) on the bar indicate significant differences (p < . ) in one-way anova or kruskal-wallis test. samples from day p.i. are shown as gray bars, from day p.i. as striped bars, and from day p.i. as black bars. the host's immune system. on the basis of our previous results on tracheal explants of pigs ( ) , components other than affinity for sialic acid receptors of swine iv strains are likely to account for high innate immune responses. in particular, gross and histological lesions are probably induced following recognition of pamps of the swine-adapted iv strains and activation of the nlrp inflammasome (cryopyrin) ( ) . such a response is probably less intense after infection with other iv strains, which can be accounted for by different causes: (a) amino acid changes of some viral pamps. (b) suppressive regulations by odns of non-swine iv strains [as shown, e.g., for porcine circovirus ( ) , and/or by ifn-α subtypes with anti-inflammatory control activity ( ) ]. (c) third, the innate immune response to the swine-adapted iv could be simply due to the much higher and/or quicker replication of this strain. in this respect, viral replication measured by quantitative real-time pcr correlated with the degree of lesions. beyond the above hypotheses, a biological explanation of the observed results could be obtained by reverse genetics studies: changes or disruptions should be engineered in the viral genome, and effects of such alterations should be checked in vitro or in vivo. on the whole, the emerging picture outlines a host/virus relationship characterized by a strict control of the innate immune responses of pigs to non-adapted iv strains. the swine-adapted ones can circumvent these controls, in the framework of a rapid, effective, non ab-dependent containment of the primary virus infection in the lower respiratory tract. interestingly, the uncomplicated exposure to the swine iv strain did not induce clinical signs in our study. this finding points at a crucial role of the infectious pressure caused by common pathogens on farm for disease onset. this feature probably exerts additive and/or synergistic effects with common environmental stressors (animal density, seasonal changes, hierarchy fights after commingling) and genetic traits. as author contributions mm supervised the entire study. er performed real-time pcr assays for cytokine genes and data analysis thereof. mb and pm-o performed flow cytometry experiments and data analysis thereof. db, lc, and jm supervised sample collection, storage and distribution, as well as cytokine, clinical immunology, histopathology, and antibody assays. ef and cc performed realtime pcr assays for ivs and data analysis thereof. as, dp, mab, and gr provided scientific support. ma supervised clinical immunology assays, cytokine protein assays, and the manuscript writing. acknowledgments regulation of adaptive immunity by the innate immune system type i interferons in host defense the 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infection of pigs ) for the invaluable help in measuring some clinical immunology parameters, dr. l. fraile (udl, spain) for assistance in statistical analysis dendritic cells in innate and adaptive immune responses against influenza virus porcine innate and adaptative immune responses to influenza and coronavirus infections differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- caspase- : the inflammasome and beyond identification of a sequence from the genome of porcine circovirus type with an inhibitory effect on ifn-alpha production by porcine pbmcs differential biological activities of swine interferon-alpha subtypes we would like to thank dr. jaime maldonado the research leading to these results has received funding from: the european community's seventh framework programme (fp , (fp , - , research infrastructures action, under the grant agreement no. fp - (nadir project), and from the project agl - -c - of spanish ministry of science and innovation. key: cord- -fudeixy authors: xu, kui; zhou, yanrong; mu, yulian; liu, zhiguo; hou, shaohua; xiong, yujian; fang, liurong; ge, changli; wei, yinghui; zhang, xiuling; xu, changjiang; che, jingjing; fan, ziyao; xiang, guangming; guo, jiankang; shang, haitao; li, hua; xiao, shaobo; li, julang; li, kui title: cd and papn double-knockout pigs are resistant to prrsv and tgev and exhibit decreased susceptibility to pdcov while maintaining normal production performance date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: fudeixy porcine reproductive and respiratory syndrome virus (prrsv) and transmissible gastroenteritis virus (tgev) are two highly infectious and lethal viruses causing major economic losses to pig production. here, we report generation of double-gene-knockout (dko) pigs harboring edited knockout alleles for known receptor proteins cd and papn and show that dko pigs are completely resistant to genotype prrsv and tgev. we found no differences in meat-production or reproductive-performance traits between wild-type and dko pigs, but detected increased iron in dko muscle. additional infection challenge experiments showed that dko pigs exhibited decreased susceptibility to porcine deltacoronavirus (pdcov), thus offering unprecedented in vivo evidence of papn as one of pdcov receptors. beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit papn or cd for entry. porcine reproductive and respiratory syndrome (prrs) is a highly infectious viral disease characterized by reproductive disorders including premature birth, late abortion, stillbirth, weak and mummy fetuses, and respiratory dysfunction in piglets and in growing pigs (wensvoort et al., ) . since its discovery in the united states in , prrs has rapidly spread worldwide, with frequent outbreaks causing large economic losses (holtkamp et al., ) . three surface receptors on porcine alveolar macrophages (pams) have been shown to function in prrsv invasion in vivo: heparin sulphate (hs), sialoadhesin (sn), and cd (calvert et al., ; crocker and gordon, ; jusa et al., ) . multiple studies have reported that cd is an essential receptor for prrsv infection, with scavenger receptor cysteine-rich domain (srcr ) serving as the core domain for virus recognition (calvert et al., ; van gorp et al., ; patton et al., ) . gene editing technology has been emerging as an important approach of livestock animal and plant germplasm improvement. the technology makes possible for precise modification of more than one gene simultaneously, which is particularly desirable for obtaining important economic traits that are controlled by multiple genes. in , prather's group was the first to use crispr/cas technology to generate srcr domain-targeted cd knockout pigs. they demonstrated that a cd knockout line was completely resistant to genotype prrsv infection (whitworth et al., ) . subsequently, several laboratories have generated anti-prrsv pigs targeting cd . for example, the cd srcr domain was replaced with human cd -like srcr domain to generate prrsv genotype resistance (wells et al., ) . wei et al., reported homozygous geneedited large white pigs with a bp deletion in exon of the cd gene (wei et al., ) that are fully resistant to genotype prrsv. there are also examples of deletion of the srcr domain seeking resistance to both prrsv genotypes (burkard et al., ) , or introducing a premature termination in the cd srcr domain to generate hp-prrsv (highly pathogenic prrsv)-resistant duroc pigs . deleting the srcr lbp region has also been reported to generate a prrsv genotype resistant pigs . all these studies demonstrate that prrsvresistant pig breeds can be generated by editing the cd gene, enabling alleviation of the severity of prrsv. in addition to prrsv, transmissible gastroenteritis virus (tgev), an acute high-contact infectious virus, is known to frequently occur to co-infect with other porcine diarrhea-associated viruses such as porcine epidemic diarrhea virus (pedv), porcine rotavirus (porv) (zhang et al., ) . tgev is globally distributed and causes tremendous economic losses in pork production (gerdts and zakhartchouk, ) . characterized by vomiting, severe diarrhea, and dehydration, the mortality rate of tgev-infected piglets under the age of days approaches %. tgev is a single-stranded, positive-sense rna coronavirus which targets pig intestinal epithelium for infection (brierley et al., ; wesley and lager, ) . studies have shown that the papn protein acts as a receptor in mediating tgev infection. the viral glycoproteins bind to papn receptors on the surface of small intestinal epithelial cells and mediate membrane fusion, thus resulting in the virus entering into elife digest pig epidemics are the biggest threat to the pork industry. in alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. the porcine reproductive and respiratory virus (prrs virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. two coronaviruses -the transmissible gastroenteritis virus (or tgev) and the porcine delta coronavirus -cause deadly diarrhea and could potentially cross over into humans. unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. traditionally, breeding pigs to have a particular trait is a slow process that can take many years. but with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. when viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the prrs virus relies a protein called cd , and tgev uses papn. xu, zhou, mu et al. used gene editing technology to delete the genes that encode the cd and papn proteins in pigs. when the animals were infected with prrs virus or tgev, the nonedited pigs got sick but the gene-edited animals remained healthy. unexpectedly, pigs without cd and papn also coped better with porcine delta coronavirus infections, suggesting that cd and papn may also help this coronavirus infect cells. finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. these experiments show that gene editing can be a powerful technology for producing animals with desirable traits. the gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale. epithelial cells (delmas et al., ; hansen et al., ) . inhibition or direct knockout of papn in small intestinal epithelial cells can mitigate tgev infection zhu et al., ) . papn knockout pigs are resistant to tgev (luo et al., ; whitworth et al., ) . pdcov is a highly virulent porcine coronavirus discovered in that causes watery diarrhea and vomiting in sows and piglets, with piglet mortality rates of % to % woo et al., ) . there is controversy about whether or not papn is a functional receptor for pdcov. wang et al., showed that papn functions as a receptor to promote pdcov entry into cells , while zhu et al., confirmed its involvement but showed that papn was an unnecessary important functional receptor for pdcov infection (zhu et al., ) . li et al., suggested that pdcov infection may require a co-receptor, in addition to papn . using cells isolated from papn knockout pigs, however, stoian et al., showed that these pig cells were still susceptible to pdcov infection in vitro. it was suggested that papn may be one of the receptors for pdcov, and an unknown receptor or factor may compensate for papn function in the absence of papn (stoian et al., ) . however, whether papn knockout pigs may be resistant to pdcov infection in vivo remains unknown. although gene-edited cd knockout (prrv resistant) pigs and papn knockout (tgev resistant) pigs have been previously generated, respectively, pigs that are resistant to the infection of both viruses are lacking. our objectives in the present study were ( ) to knockout cd and papn simultaneously using a gene editing approach; ( ) to verify if the resultant dko pigs are simultaneously resistant to infection by prrsv and tgev; ( ) to use the dko pigs as an in vivo experimental model to test for potential papn-mediated resistance to pdcov infection. we report successfully generated gene-edited large white pigs with both cd and papn gene knockouts using crispr/cas and somatic cell nuclear transfer (scnt). through viral challenge experiments, we found that these dko pigs exhibit complete resistance to genotype prrsv and tgev, and exhibit decreased susceptibility to pdcov infection. in addition, with the exception of meat color score and iron content, no differences in the production performance, reproductive performance, or pork nutrient content were observed between dko pigs and wt pigs. thus, in addition to demonstrating that our dko pigs are robustly resistant to both prrsv and tgev without suffering deleterious effects for production performance, our study also provides insights into ongoing controversy about the papn protein as a potential receptor for pdcov infection of pigs. in order to generate cd and papn dko cloned pigs, we constructed sgrna delivery plasmids targeting these genes, and selected successful dko pig fetal fibroblasts (pefs) as nuclear transfer donors ( figure a ). for cd , the srcr domain-binding site for prrsv in exon (van gorp et al., ; ma et al., ) was selected as the sgrna recognition site. to inactivate the papn protein, a sgrna target site in exon two immediately downstream of the atg start codon was selected ( figure b) . successful dko colonies were cultured as donor cells for scnt (supplementary file ). the cloned pigs generated in this experiment were obtained via both primary and secondary clonings. for primary cloning, the selected dko cells are used as donors for nuclear transplantation. for secondary cloning, the ear-derived fibroblasts of the primary cloned pigs are re-cloned, which rapidly provided a large number of high-quality dko donor cells, thus improving cloning efficiency and resulting in many genotypically identical pigs. in our primary cloning, a total of reconstructed embryos were transplanted into surrogate sows, of which two were pregnant and gave birth to eight live piglets. of these piglets, four survived after weaning ( figure c and supplementary file ). we determined the cd and papn genotypes of the four surviving piglets using pcr and sanger sequencing. the genotypes of the three piglets (# , # , and # ) matched that of cell colony # , which had an bp deletion on both copies of cd near the target site, and a copy of papn carrying a bp deletion on one copy and a bp deletion on the other, both resulting in frameshift mutations or premature termination after the target site ( figure d ). papn protospacer pam in order to generate more dko pigs for viral challenge experiments, we collected ear tissue samples from three piglets (# , # , and # ) and isolated ear-derived fibroblasts. a total of reconstructed embryos generated from ear-derived fibroblasts of # were transplanted into nine surrogates. four sows successful gave birth to a total of live piglets, among which survived post-weaning (supplementary file ). the genotypes of these piglets matched that of # , and the three dko primary clones used for subsequent experiments. we used flow cytometry and western blotting for cd , immunohistochemistry (ihc) and western blotting for papn, and confirmed that expression of both proteins was undetectable in dko pigs but detectable in wt pigs of the same age and breed ( figure e -g). we designed multiple pairs of amplification primers for the px vector backbone to confirm that no random integration of px vector fragments were in cloned pigs (figure -figure supplement ). we also tested for off-target modifications in dko pigs using potential off-target sites for each of the two sgrnas and found no alteration in any of these predicted sites in the cloned pigs (supplementary file ). this data demonstrates that clones of sus scrofa line with multiple gene-edited can be generated through primary and secondary cloning with high efficiency and no off-target detected. for testing of prrsv resistance in pams derived from dko pigs, we selected the highly pathogenic genotype prrsv strain wuh to challenge dko and wt pams at a multiplicity of infection (moi) of . . qrt-pcr and western blot analyses were used to assess prrsv proliferation in pams. at hr post-infection (hpi), dko pams carried a significantly lower prrsv load compared with wt pams, and no viral rna or prrsv-n protein was detected thereafter in dko pams (figure a and b). the low level of prrsv rna that was initially detectable in the dko line at hpi is likely attributable to the adsorbed prrsv independent of the existence of cd , as cd is thought to be primarily responsible for the uncoating and viral rna release processes of prrsv infection van gorp et al., ) . we next sought to examine if dko pigs are resistant to prrsv in vivo. four -day-old dko pigs and six wt control pigs of the same age were challenged with the prrsv strain wuh . nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml) were used to infect both experimental groups. the phenotypic data of body temperature, feed intake, respiration, defecation, and mental condition were recorded daily after infection. as shown in figure c , while fever (over ˚c) began at day post-infection (dpi) and persisted throughout the remainder of the experimental period in the wt group, the body temperature of the dko pigs stayed normal throughout the days of the post-viral challenge observation period. scoring for other clinical symptoms of prrsv at dpi showed that wt pigs exhibited decreased appetite, shortness of breath, cough, malaise, drowsiness, and difficulty walking, whereas the dko group displayed no abnormalities except for a brief cough and diarrhea in two pigs at dpi and dpi, respectively ( figure d ). the body weight of the dko pigs increased, throughout the day post viral challenge observation period: the detected body weights of the wt pigs were all lower than dko pigs after dpi ( figure e ). of the six challenged wt pigs, one was slaughtered at dpi to harvest pams, and the five remaining wt pigs died within dpi. in sharp contrast, all four pigs in the dko group remained healthy, and survived for the entire duration of the -day experiment ( figure f ). among the dead and slaughtered wt pigs, the lungs were swollen, with severe bleeding, and obvious lesions, while the lung tissues of dissected dko pigs did not exhibit lesions or any other distinct symptoms associated with prrsv ( figure g ). hematoxylin and eosin (h and e) staining showed thickening of the alveolar walls and infiltration of a large number of inflammatory examination of prrsv antigens in lung tissue via ihc, it was revealed that the viral antigens were present in the lungs of the wt group, but not that of the dko pigs ( figure h , lower panel). moreover, we measured the prrsv viral load in the serum of both groups at , , , , and dpi and found that in the wt group, the prrsv load increased rapidly and significantly by dpi, reaching its maximum at dpi. in agreement with other experiments showing viral resistance, the prrsv viremia in the dko group remained negative throughout the challenge ( figure i ). we also tested the prrsv viral load in pams, lung tissues, and tonsil tissues of the two groups of pigs after viral challenge. whereas a high titer of prrsv was detected in all tissues examined in the wt group, prrsv was almost undetectable in dko pigs ( figure j ). from dpi, the amount of prrsv-specific elisa antibodies in the serum of wt pigs increased significantly, and antibody levels were positive (s/ p! . ) at and dpi, while such antibodies in dko pigs remained consistently negative (s/p< . ) ( figure k ). taken together, these results provide compelling in vitro and in vivo evidence that the dko pigs are resistant to prrsv infection. following characterization of prrsv resistance, we next sought to determine if double knockout of cd and papn also conferred resistance against tgev. four -day-old dko pigs and six wt control pigs of the same age and breed were fed under the same conditions and infected with tgev. a total of ml of tgev (  tcid /ml) were orally administered to each pig in two doses (day and day , ml/day). at dpi, one dko pig and one wt pig were slaughtered to collect intestinal tissues for pathological examination, and the remaining pigs were housed under regular husbandry conditions until slaughter, and tissues were sampled at dpi. body temperature was recorded daily beginning at day , prior to inoculation, and piglet weighing and blood sampling for serum separation were conducted at , , and dpi. during the viral challenge period, no abnormalities were observed among the pigs, with the exception of two wt pigs that had diarrhea. there was no significant difference in weight gain between the two groups (data not shown). detection of tgev-specific neutralizing antibodies in serum showed no neutralizing antibodies in the dko pigs throughout the experiment, while two of the wt pigs were positive for neutralizing antibodies at dpi, and all wt pigs were positive by dpi ( figure a) . all slaughtered pigs from both wt and dko groups (sampled at dpi and dpi) were dissected to examine potential lesions in small intestine tissues. for the dko group, no lesions were found in the small intestine samples collected at either dpi or dpi ( figure b ). in marked contrast, wt group tissues collected at dpi demonstrated a thin and yellowing small intestine wall, with hemorrhages typical of tgev clinical symptoms, and by dpi there were notable duodenum, jejunum, and ileum hemorrhages, accompanied by intestinal wall thinning and enlarged mesenteric lymph nodes ( figure b ). pathological examination of small intestine tissue sections revealed pathological changes, including necrosis and shedding of intestinal mucosal epithelial cells, intestinal villi fusion, plasma cells accumulating in the lamina propria, and infiltration of eosinophils in the duodenum, figure continued in serum. wt: to dpi, n = ; dpi, n = ; dpi, n = . dko: to dpi, n = . data are expressed as means ± sd. statistical significance was determined by student's t test; ns, p> . ; *p< . ; **p< . ; ***p< . . the online version of this article includes the following source data for figure : source data . the qrt-pcr detection of prrsv load in pams. source data . rectal temperatures of pigs. source data . clinical symptoms scores of pigs. source data . body weights of pigs. source data . the survival rate of pigs. source data . the qrt-pcr detection of prrsv load in serum. source data . the qrt-pcr detection of prrsv load in pams, lung tissues and tonsil tissues. source data . prrsv-specific antibodies in serum (s/p ration). jejunum, and ileum of wt pigs at dpi and dpi, while the same small intestine tissues in dko pigs appeared healthy ( figure c ). we also analyzed the ratio of intestinal villus height (vh) to the crypt depth (cd). the smaller the ratio, the more severe the intestinal villi atrophy. we found that compared with the mock group, the three intestinal segments of the wt group had significant intestinal villous atrophy, and the intestinal villi of these intestinal segments in the dko group did not show atrophy; that is, the degree of intestinal villous atrophy in the three intestine segments in the wt group was significantly higher than that in the dko group ( figure d) . these results consistently demonstrate that our cd /papn dko pigs exhibit strong resistance to tgev infection. pdcov is a highly pathogenic virus that has recently been shown to cause diarrhea in newborn piglets, although the functional receptors for pdcov have not yet been confirmed stoian et al., ; zhu et al., ) . whether papn functions as a receptor or co-receptor in pdcov infection of pigs remains controversial. to test the hypothesis that papn may functionally mediate pdcov infection, we tested the susceptibility of our dko pigs to this virus. two -day-old dko pigs and four wt pigs of the same age and breed were challenged with pdcov. a total of ml of pdcov ( .  tcid /ml) was orally administered to each pig in two doses (day and day , ml/day). during the days of pdcov challenge study, both the dko and wt pigs appeared normal, with no distinct differences in body temperature or weight (data not shown). blood was collected at , , and dpi to assay for levels of virus-specific antibodies. at and dpi, wt pigs were all antibody-positive, while the dko pigs were all antibody-negative at dpi, but carried antibody levels comparable to that of the wt group by dpi ( figure a ). this suggests that the double-gene knockout led to a delayed onset of humoral immunity in pigs, possibly due to delayed-immune system exposure to the virus. all pigs were slaughtered at dpi, and the small intestine tissues were collected to evaluate disease severity. it was found that the intestinal wall of the wt had become thinner, with watery fluid in the small intestine, and mesenteric hyperemia, none of which was observed in the small intestine of the dko pigs ( figure b ). pathological examination of small intestine tissue sections revealed significant lesions in the small intestine tissues of both of the wt and dko groups, which included intestinal villi fusion, infiltration of lymphocytes in the intestinal mucosa, with many lesions in the intrinsic membrane in the duodenum and jejunum tissues. in the ileum, there were signs of necrosis and shedding of intestinal mucosal intraepithelial cells and naked lamina propria. the extent of lesions in the wt pigs was more severe than that of the dko pigs ( figure c ). we also detected the ratio of intestinal villus height to the crypt depth, and found that compared with the mock group, the three intestinal segments of both of the wt group and the dko group had intestinal villous atrophy, but the degree of villous atrophy in the ileal tissue in the dko group was lower than that of the wt group ( figure d) . in addition, we tested the resistance of pams derived from dko pigs to pdcov. dko and wt pams were infected with pdcov, and indirect immunofluorescence assays (ifa), tissue culture infectious dose (tcid ) assays, qrt-pcr, and western blot analyses to assess pdcov proliferation in pams all indicated that dko pams exhibit significantly decreased susceptibility of pdcov infection compared to wt pams (figure -figure supplement ) . these data suggest that although the dko line is still susceptible to pdcov infection, the viral invasion and damage to the small intestines was partially inhibited compared to that of the wt line. we next evaluated the growth and performance indices of dko pigs. three -month-old dko large white boars and three wt large white boars of the same age were selected for slaughter testing. the live weight at slaughter, carcass weight and length, dressing percentage, ham percentage, lean rate, loin eye area, average backfat thickness, muscle ph, marbling, and drip loss were determined. as shown in table , with the exception of meat coloring score, dko pigs showed no difference in comparison with wt pigs for these indices. in addition, there was no significant difference in birth weight or in the average daily gain between wt and dko pigs (supplementary file ). most notably, the meat color score in the dko pigs ( . ± . n= ) was significantly higher than that of wt pigs ( . ± . n= ), although both were within the normal range of to according to the guideline of 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) ( table and figure a -b). since the cd protein is known to play a role in the degradation of haemoglobinhaptoglobin (hb-hp), and considering that fe is an important component of haemoglobin, we reasoned that the increased meat color score (redness) may be due to the decreased hb metabolism as a consequence of cd knockout, and subsequently mild accumulation of fe containing hb in the meat. to test this hypothesis, the meat fe level was analyzed, it was found that the concentration of fe was significantly higher in dko pigs compared to wt pigs ( figure c ). we also tested the serum haptoglobin (hp) content and found that the hp content in dko pigs was significantly higher than that of wt control pigs ( figure d ). evaluation of the nutritional components of pork such as total protein, total fat, ash, moisture, specific minerals, and amino acid content was also performed. as shown in table and supplementary file , no differences in these indices were observed between the two groups. in order to test the reproductive performance of the dko boars, semen from dko male pigs (n = ) and that of wt pigs (n = ) of the same age and breed were analyzed. it was revealed that the concentration, motility, and velocity distribution of the sperm from dko boars did not differ from wt boars (table ) . furthermore, there was no difference in the litter size between the two genotypes: dko litters were . ± . (n = , litter size from to ) and the wt litters were . ± . (n = , litter size from to ). in addition, these three dko pigs did not show any growth abnormalities or disease phenomena during the -month rearing process, and no abnormalities were observed in the main tissues and organs after slaughter (data not shown). taken together, with the exception of slight meat coloring score increase, these results show that the simultaneous, editing-based disruption of the cd and papn loci, does not affect the normal growth and reproductive performance of the resultant dko pigs. conventional breeding for complex traits using molecular marker-assisted selection is a lengthy process, requiring multiple rounds of crosses and backcrosses to introgress each individual gene. crispr/cas gene editing not only allows bypassing of this long process, but also provides a possible means to obtain multiple beneficial genotypes in a single generation while also avoiding gene penetration from donor species, thus maintaining the desirable qualities of the original species. zhou et al., first used crispr/cas in combination with scnt to generate knockout of park and pink genes, whose dysfunction are known to contribute to the early onset of parkinson's disease in humans . huang et al., got the pig model with metabolic disorder successfully by editing apolipoprotein e and low density lipoprotein receptor genes simultaneously . our study is the first report on how multiple gene edits can be combined in livestock animal to offer simultaneous resistance to two major viral infection. similar to the previous reports above, double knockout efficiency using crispr/cas -mediated dual gene editing method without any drug or flow cytometry screening was high in our study, reaching . % ( dko cell colonies out of cell colonies). in this experiment, we quickly generated a large number of dko pigs by re-cloning. we found that the re-cloning efficiency ( . %, / ) was much higher than the primary cloning efficiency ( . %, / ). a possible reason for this elevated efficiency could be that the monoclonal cells used for the primary cloning must be cultured in vitro for a long time, which has been reported to inhibit cloning efficiency (li et al., ; magnani et al., ; mastromonaco et al., ) . the donor cells used in re-cloning were ear-derived fibroblasts isolated directly from dko pigs, eliminating the requirement for a long-term, in vitro screening process. our findings support the notion that the efficiency of this approach is not gene specific, and may be applicable to the knockout of other genes that allow improving disease resistance or animal production. in , calvert et al. first discovered that cd functions as a prrsv receptor protein during pams infection, which has since been confirmed by several studies (calvert et al., ; van gorp et al., ; guo et al., ; patton et al., ) . structural studies of cd revealed that the srcr domain corresponding to cd exon seven is necessary to mediate prrsv infection (van gorp et al., ) . in recent years, several groups have successfully generated prrsv-resistant gene-edited pigs by targeting exon of the pig cd gene (burkard et al., ; the online version of this article includes the following source data for the online version of this article includes the following source data for table : source data . comparison of the concentration, motility, and velocity distribution of the sperm between dko and wt large white pigs. ; whitworth et al., ; yang et al., ) . in the present study, we used a single sgrna targeting exon of cd , generated an bp double-stranded deletion that terminated protein translation near the target site. our finding on the complete resistance to prrsv genotype in our knockout line is consistent with those previous reports. cd is known to play a role in promoting the clearance of plasma free haemoglobin (kristiansen et al., ) . our finding that the dko pigs have higher meat fe content and have elevated serum hp levels is consistent with this idea, and may explain the observed darker red color in our dko meat. interestingly, and consistent to our finding, wells et al., also reported that the serum hp levels are elevated in cd knockout pigs (wells et al., ) . despite the slight color score increase, no abnormal growth or reproductive performance was observed in our dko pigs, and the meat color of both dko pigs and wt pigs were within the normal range. production performance evaluations and identification of pork nutritional components showed that our dko pigs were indistinguishable from that of the wt pigs in growth rate and reproductive performances, except for the meat color score and iron content. however, the number of dko pigs tested by us is still small, and the production performance of dko pigs still needs to be verified in large populations in the future. apn is known to be a receptor for many coronaviruses, and studies have shown that separate domains function in virus recognition vs. hydrolase catalytic activity (reguera et al., ) . two research groups have recently demonstrated that papn knockout pigs block tgev but not pedv infection (luo et al., ; whitworth et al., ) . our data showing that papn knockout can completely prevent tgev virus infection are consistent with these recently published findings. in addition to tgev and prrsv, we also determined if papn deletion conferred protection against pdcov. apn is a receptor for multiple coronaviruses and is abundantly expressed on small intestinal epithelial cells, which has led to the speculation that papn may also be a receptor for pdcov. wang et al., and li et al., proposed that papn functions as a receptor in mediating pdcov infection wang et al., ) . however, another study found that knockout of papn in ipi- i cells inhibited but did not completely block pdcov infection, suggesting that papn was not essential for viral recognition (zhu et al., ) . taken together, these studies suggest that papn may be involved in pdcov infection, but pdcov may also be able to enter cell through other pathway(s). our results on the delayed pdcov-specific neutralizing antibodies production, and a reduced extent of gross and histopathological lesions on small intestine in dko pigs compared to wt pigs are consistent with this previous suggestion that papn may play a role but is not the only path for pdcov cell entry. interestingly, a recent study showed that pams, but not lung fibroblast-like cells, from papn knockout pigs showed resistance to pdcov infection (stoian et al., ) , a finding consistent with our in vitro experiments showing that dko pams exhibit decreased susceptibility to pdcov infection. in addition, papn knockout pigs are susceptible to pdcov when virus levels were detected using qrt-pcr, and virus neutralization activity was measured, although the extent of tissue lesions between the ko and wt groups was not compared (stoian et al., ) . our findings are in line with this study reporting that papn knockout pigs are still susceptible to pdcov. however, as reflected by the delay in neutralizing antibody response, and much lighter intestine damage in the dko pigs, the susceptibility of the papn knockout group to the virus is reduced compared that of the wt pigs, indicating the potential role of papn in mediating pdcov infection. additionally, the effect of cd knockout in the delayed adaptive immune response cannot be ignored. despite the important role of cd in innate immunity, an inhibiting effect of soluble cd on the adaptive immune system has also been reported (frings et al., ; o'connell et al., ) . it is thus possible that the delayed adaptive immune response we observed in pdcov-infected dko pigs may be associated with cd knockout-induced immunosuppression. in summary, the dko pigs generated in this study are simultaneously resistant to prrsv and tegv, and exhibit decreased susceptibility to pdcov, while maintaining the same growth and reproductive production traits when compared to wt animals. these pigs may offer breeding starting points for disease-resistant pig colony generation and will be a valuable model to help deepen our understanding of the role and mechanisms of these receptor proteins in the infection mechanisms of multiple viruses. the cd and papn genotypes of colonies and piglets born after nuclear transfer were detected by pcr and sanger sequencing. one third of the cells in the -well plate and the ear tissue were used to extract the genomic dna. the primer pairs cd -f/cd -r and papn-f/papn-r were used to amplify the sequences near the sgrna target sites in the cd and papn genes, respectively. the primer sequences are shown in supplementary file . the pcr products were genotyped by sanger sequencing. potential off-target sites were predicted using an online software: crispor (http://crispor.tefor.net/ ). we identified the potential off-target sites for each of the two sgrnas. twenty pairs of primers were designed to amplify the potential off-target sites from the genomic dna isolated from the dko pigs ( #, #, #). sanger sequencing was performed to determine whether any mutations occurred. the primer sequences are shown in supplementary file . the total protein extracted from lung tissue, liver tissue, and spleen tissue of non-challenged wt pigs and dko pigs was used to detect cd , and protein extracted from duodenal, jejunal, and ileal tissues were used to detect papn expression. whole cell lysates of prrsv-infected pams and pdcov-infected pams were used to quantify the expression levels of prrsv nucleocapsid (n) protein and pdcov nucleocapsid (n) protein, respectively. the protein samples were separated by % or % sds-page and transferred to a polyvinylidene fluoride membrane (millipore). the membrane was blocked with % skim milk for hr, and then incubated with primary antibody at ˚c overnight and secondary antibody at room temperature for hr. chemiluminescent signals were developed with supersignal west pico plus chemiluninescent substrate (thermos scientific) and captured with a tanon- (tanon). cd rabbit polyclonal antibody ( - -ap; proteintech) was used to detect porcine cd . apn polyclonal antibody (a ; abclonal) was used to detect papn, anti-prrsv-n antibody (made in our laboratory) was used to detect prrsv-n protein, anti-pdcov-n antibody (made in our laboratory) was used to detect pdcov-n protein, gapdh rabbit antibody ( ; cell signaling) or b-actin rabbit antibody (ac ; abclonal technology) was used to stain gapdh or b-actin as a loading control. hrp-conjugated affinipure goat anti-rabbit igg(h+l) (sa - ; proteintech) and hrp-conjugated affinipure goat anti-mouse igg(h+l) (sa - ; proteintech) were used as the secondary antibody. pams were isolated from dko piglets and wt piglets. the lungs were obtained from the euthanized piglets. the lung surfaces were rinsed with pbs, and pams were subsequently obtained by bronchoalveolar lavage with prmi- medium (gibco, usa). the collected lavage solution was dispensed into a ml centrifuge tube, centrifuged at g for min, and the supernatant was discarded. pams were washed again with prmi- medium and then frozen in cryopreservation solution containing % fbs and % dmso. for further in vitro infection experiments, pams were cultured in rpmi- medium with % fbs and  antibiotic antimycotic ( ; invitrogen) at ˚c/ % co , and then infected with a highly pathogenic prrsv (hp-rrsv) strain wuh (gen-bank accession number hm ) (li et al., ) at a dose of moi = . and pdcov strain chn-hn- (genbank accession number kt ) at a dose of moi = . the production of progeny prrsv was evaluated through western blot, and qrt-pcr assays, and the production of progeny pdcov was evaluted through ifa, tcid , qrt-pcr and western blot assays. pams were fixed in % formaldehyde for min at room temperature. the cells were then blocked with % bsa overnight at ˚c and incubated with mouse anti-pig cd mabs (mca pe; bio-rad) at ˚c for hr in the dark. after washing with pbs three times, pams were resuspended in pbs and immediately analyzed using a bd facsverse flow cytometer (bd biosciences, ca) and flowjo software (treestar, ca). all wt pigs used in the infection experiment were born from natural breeding, and they were matched by age and breed with the dko pigs. the four dko and six wt pigs used for prrsv wuh viral challenge were both about days old. viral inoculation was conducted by nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml). during the days of prrsv challenge, piglet rectal temperature and clinical symptoms data (feeding, breathing, defecation, mental state) were collected every morning. at the same time, piglet survival rate was recorded, blood was collected, and the piglets were weighed regularly. if any pigs died during the course of the prrsv challenge, pictures were immediately taken and samples were collected. all surviving pigs were slaughtered at days post-infection (dpi) and lung tissue was examined for disease symptoms. four dko pigs and six wt pigs were used for tgev challenge. pigs were inoculated with a total of ml of tgev strain wh- (genbank accession number hq ) (  tcid /ml) that were orally administered to each pig in two doses (day and day , ml/day). for the pdcov challenge, two dko pigs and four wt pigs were orally administered a total of ml of pdcov strain chn-hn- ( .  tcid /ml) divided into two doses delivered on day and day ( ml/day). for both the tgev and pdcov groups, the rectal temperature of the pigs was measured daily for the full day experiment and the diarrhea of the piglets was observed. blood was collected and the piglets were weighed regularly. in the tgev group, a dko pig and a wt pig were slaughtered on day , and the remaining pigs in the tgev group and all pigs in the pdcov group were slaughtered at dpi. after slaughter, the pigs were dissected to observe the gross lesions in small intestine tissue, to collect small intestine tissue samples, and to detect any pathological changes by h and e staining. meanwhile, during these days, wt pigs and dko pigs were reared under the same conditions without any virus infection, and these pigs were used as the mock group. lung tissues of pigs in the prrsv challenge group, and duodenum, jejunum, and ileum tissues in the tgev and pdcov groups were collected. the tissues were fixed in % paraformaldehyde fixative, dehydrated, embedded, and cut into ~ mm-thick sections. for histopathology, the sections were stained by h and e. for ihc, tissue sections were stained with antibodies specific to the corresponding protein antigens. tissue sections were then observed and photographed with a fluorescence microscope. the antibodies used to detect papn protein were purchased from abcam (ab ); the antibody used to detect prrsv-n protein was made by our laboratory. the blood tissues of three experimental groups of pigs were collected at different times after viral challenge and the sera were separated. for the prrsv group, the sera from all samples were subjected to prrs antibody detection by commercially available enzyme-linked immunosorbent assay (elisa) kit (idexx, me). the antibody level was determined to be negative or positive according to the s/p value. if s/p< . , the antibody is negative, and if s/p! . , the antibody is positive. in order to detect tgev-specific and pdcov-specific antibody levels in serum, we used a serum neutralization test (snt). briefly, sera were heat inactivated by min of incubation in a ˚c water bath. then serial -fold dilutions of serum samples in four replicates were mixed with tcid of tgev strain wh- in a : ration. after incubation, ml of the mixture was added into st cells (a swine testicular cell line permissive of tgev infection; atcc crl- ) at a confluence of~ %, seeded in well cell culture plates. appropriate serum, virus ( tcid , tcid , tcid , and . tcid ), and cell controls were included in this test. for about hr after incubation, the cells were monitored for tgev-specific cytopathic effects. neutralization titers were calculated as the reciprocal of the highest dilution resulting in complete neutralization. similarly, sera were diluted mixed with tcid of pdcov strain chn-hn- . in contrast, pdcov titers were assessed using llc-pk cells (a porcine kidney cell line permissive of pdcov infection; atcc cl- ) that were washed twice with dulbecco's modified eagle's medium (dmem) (invitrogen, ca), and supplemented with . mg/ ml trypsin (gibco, usa) prior to and after hr incubation with these mixtures. cells were then cultured in dmem supplemented with . mg/ml trypsin for approximately hr, and the neutralization titers of sera from pdcov group were calculated. to quantify the copies of prrsv and pdcov in the infected experimental group, we extracted prrsv rna from pams, serum, lung tissue, and tonsil tissue from both the challenge and the mockinoculated group, and extracted pdcov rna from dko pams and wt pams after infected with pdcov. rna extraction was performed using trizol reagent (omega bio-tek). the rna was reverse transcribed into cdna according to the instructions for a transcriptor first strand cdna synthesis kit (roche). the cdna was then amplified with sybr green real-time pcr master mix (applied biosystems) in an abi real-time pcr system (applied biosystems). rna copy numbers were calculated from a standard curve drawn from positive standards at different dilutions. the primers used for qrt-pcr are listed as follows: '-gcaattgtgtctgtcgtc- ' and '-cttatcctccctgaatc tgac- ' for prrsv; '-gccctcggtggttctatctt- ' and '-tccttagcttgccccaaata- ' for pdcov. dko pams and wt pams in -well cell culture plates were infected or mock-infected with pdcov at a multiplicity of infection (moi) of . at hpi, cells were fixed with % paraformaldehyde for min and permeabilized with methanol for min at room temperature. the cells were then blocked with bovine serum albumin ( %) diluted in phosphate-buffered saline (pbs) for hr, and incubated with a pdcov-n-protein-specific monoclonal antibody for hr and an alexa fluor -conjugated donkey anti-mouse igg for hr. the cell nuclei were counterstained with ', -diamidino- -phenylindole (dapi) for min at room temperature. after three washes with pbs, the stained cells were observed with an inverted fluorescence microscope (olympus ix , japan). pdcov-infected pams were frozen and thawed repeatedly to completely release viruses. next, llc-pk cells (a pig kidney cell line known to be highly permissive to pdcov infection) were seeded in -well plates and were infected with -fold serial dilutions of virus samples in eight replicates. at hpi, pdcov titers were calculated based on cytopathic effects and expressed as the tcid value per milliliter, using the reed-muench method. the amount of hp in serum was measured using an enzyme-linked immunosorbent assay (elisa) kit ( - , alpha diagnostic) specific to pig hp, as previously described . assays were performed in triplicate for each sample. the quality and performance of pigs related to slaughter were determined by a third-party testing center (the national breeding swine quality supervision and testing center (chongqing), ministry of agriculture and rural affairs of china). all testing followed the guidelines stipulated in the 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) . briefly, dko pigs and control wt pigs were weighed before slaughter, euthanized after fasting for hr, and hairs, heads, hoofs, and internal organs were removed after carcass dissection. the weight of carcass, length of carcass, loin eye areas, thickness of skin, and backfat thickness of carcass were all measured. ham, skin, bone, lean, and fat were dissected from the left side of the carcass and their individual weights were determined. to evaluate meat quality, we measured muscle ph, meat color score, intramuscular fat, marbling, and drip loss of longissimus dorsi. for analysis of pork nutrition, total protein, total fat, ash, moisture, amino acid, and individual minerals, amino acids were analyzed for the longissimus dorsi. the nutritional content of the pork was tested by the beijing institute of nutritional sources. semen from dko pigs and wt control pigs were collected and returned to the laboratory in a ˚c incubator for testing their quality. the detection system was hamilton-thorne research ivos ii computer-assisted sperm analyzer to measure the concentration, motility, and velocity distribution of the sperm. all data are presented as the mean ± standard error of mean (sem). data from each of the two groups of pigs were compared with an unpaired t-test when a normal distribution was not obtained. the significance levels were set at . , . , and . , as indicated by *, **, ***, respectively. the data was analyzed with graphpad prism . . for windows (graphpad software, la jolla, california). animal experimentation: all experimental protocols related to animal work described in this study were reviewed and approved by the institutional animal care and use committee (iacuc) at the institute of animal sciences, chinese academy of agricultural sciences. all experiments were performed in accordance with the approved guidelines for animal care and management of research projects. (ias - ). decision letter and author response decision letter https://doi.org/ . /elife. .sa author response https://doi.org/ . /elife. .sa supplementary files . source data . amino acid content of dko lean meat and wt lean meat. . source data . birth weights and average daily gains of wt pigs and dko pigs from birth weight to slaughtering weight. table , table and table . quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses generation of pigs resistant to highly pathogenic-porcine reproductive and respiratory syndrome virus through gene editing of cd properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev isolation, genomic characterization, and pathogenicity of a chinese porcine deltacoronavirus strain chn-hn- only the soluble form of the scavenger receptor cd acts inhibitory on phorbol ester-activated t-lymphocytes, whereas membrane-bound protein has no effect vaccines for porcine epidemic diarrhea virus and other swine coronaviruses modulation of cd expression by metalloprotease adam regulates porcine reproductive and respiratory syndrome virus entry highly efficient generation of pigs harboring a partial deletion of the cd srcr domain, which are fully resistant to porcine reproductive and respiratory syndrome virus infection the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers crispr/cas -mediated apoe-/-and ldlr-/-double gene knockout in pigs elevates serum ldl-c and tc levels aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells n-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus identification of the haemoglobin scavenger receptor effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses broad receptor engagement of an emerging global coronavirus may potentiate its diverse crossspecies transmissibility porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-b production aminopeptidase n-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus the crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine cd reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer monocytelymphocyte cross-communication via soluble cd directly links innate immune system activation and adaptive immune system suppression following ischemic stroke modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies highly efficient crispr/cas -mediated transgene knockin at the h locus in pigs the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus porcine coronavirus hku detected in us states porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry generation and propagation of cluster of differentiation biallelic gene edit ing pigs replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cd knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus occurrence and investigation of enteric viral infections in pigs with diarrhea in china generation of crispr/cas -mediated gene-targeted pigs via somatic cell nuclear transfer contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. for the cd gene, the sgrna was designed to target exon , and for the papn gene, the sgrna was designed to target exon . the sequences of the two sgrnas are as follows: ggaaacc-caggctggttggaggg (cd -sgrna) and gcatcctcctcggcgtggcgg (papn-sgrna). the pam is indicated in bold font. the two sgrna sequences were cloned into the px vector (addgene plasmid # ) and named px -cd and px -papn, respectively. two plasmids were extracted (tiangen, dp ) in large quantities and used to transfect the fetal fibroblasts of large white pigs. the fetuses of large white pigs at -day-old were used to isolate pefs, which were then cultured in dmem medium containing % fbs. when the cells grew to % confluence, approximately cells were transfected with px -cd ( . ug) and px -papn ( . ug) plasmids. a lonza b nuclear transfection system was used for transfection with nucleofector program t- . the entire transfection process was performed according to the kit instructions (lonza, vpi- ). cells were cultured for hr after transfection and then seeded into cm dishes at a density of cells/dish. the culture medium was changed every days, and cells were cultured for days to form singlecell colonies. single-cell colonies were transferred to -well plates for expansion culture. when cells in the -well plates reached confluence, / of the cells were taken for genotype identification, and the remaining cells continued to expand. cells with genotypes identified as double-gene mutations were cultured and frozen for scnt. the oocytes for scnt were derived from a nearby slaughterhouse, and the nuclear donor cells were the dko fibroblasts. the nuclear transfer donor cells were transferred into enucleated oocytes, and the reconstructed embryos were activated and cultured to develop into blastocysts. we then selected well-developed recombinant embryo clones to be surgically transferred into the oviduct of recipient gilts on the day after estrus was observed. after the embryo transfer, the technicians observed the estrus of the sow, and regularly checked the pregnancy by b-ultrasound. changli ge: is affiliated with shandong landsee genetics co., ltd. the author has no financial interests to declare. key: cord- -a fynm authors: riggs, shannon m. title: chapter guinea pigs date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: a fynm publisher summary this chapter deals with the health and medical care issues of guinea pigs. guinea pigs have wide bodies with short limbs. they have a short, flat nose, laterally placed eyes, and hairless external pinnae. the dentition of the guinea pig is described as aradicular hypsodont. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. heavy food containers are recommended to make dumping of the receptacle more difficult. all food containers should be easy to disinfect and should be cleaned regularly, because guinea pigs have a habit of soiling their food bowls. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than °f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. c h a p t e r guinea pigs shannon m. riggs guinea pigs, cavia porcellus, are members of the caviidae family of the order rodentia. guinea pigs are native to mountainous regions of south america where they were domesticated as long as years ago. wild species of guinea pigs, or cavies, still inhabit columbia, peru, venezuela, argentina, brazil, and paraguay. domesticated cavies in these countries are used for food. in the wild, guinea pigs live in small groups and, therefore, are often more comfortable in the presence of other guinea pigs when maintained as companion animals. extensive breeding has resulted in numerous varieties of coat color and characteristics. the most common breeds are the american (or english) which has a short, smooth coat and the multicolored teddy (figure - ); abyssinian ( figure - ), which has a medium length coat in a whorled pattern; and the peruvian, which has a very long, smooth coat. guinea pigs have wide bodies with short limbs. a distinctive anatomic characteristic of species in the family caviidae is the number of digits on the front and rear feet ( digits front feet and digits rear). tails are usually very short or absent. the guinea pig has a short, fl at nose, laterally placed eyes, and hairless external pinnae. adult guinea pigs usually weigh between and g, with the males being slightly larger than females. the average life span of the companion guinea pig is approximately to years. the dentition of the guinea pig is described as aradicular hypsodont (e.g., all teeth have a relatively long crown and are "open rooted"). the maxilla is slightly wider than the mandi-ble, and the occlusal angle of the premolars and molars is marked compared to other rodent species. the dental formula of the guinea pig is (i / , c / , pm / , m / ) = . the maxillary incisors are much shorter than those set in the mandible. the molars and premolars are not easily visualized without special instrumentation because of the small size of the oral cavity and tendency for the involution of the buccal surface. females are sexually mature at weeks of age, whereas males on average reach puberty approximately weeks later. gestation is long, when compared to other rodents, at days. as a result of this long gestation period, young are precocial when born. juvenile pigs usually eat solid foods by or days of age. litter sizes range from to , with an average of to young. a female guinea pig should deliver her fi rst young before she is months of age. if birth has not occurred before months of age, the pubic symphysis becomes mineralized, with future pregnancies resulting in an inability of the sow to naturally deliver the babies. female guinea pigs that become pregnant after months of age invariably require cesarean section deliveries. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. wire-bottom cages may also be used; however, care must be taken to ensure that the mesh is small enough that a limb cannot become entrapped. an area of solid fl ooring should be provided, as uninterrupted time on wire mesh may predispose the guinea pig to pododermatitis. adequate space is needed in the enclosure for the guinea pig to move about unencumbered with enough space for a hide box. hide boxes or a secluded space is required for prey species (e.g., rodents) to reduce stress that may lead to disease problems. substrate products that contain aromatic oils (e.g., cedar and pine shavings) should not be used, as they can act as contact and respiratory irritants. appropriate bedding materials include recycled newspaper products, shredded paper, and aspen shavings. the enclosure should be cleaned thoroughly on a regular basis (e.g., times per week) because unsanitary conditions predispose the guinea pig to pododermatitis, respiratory, and other health problems. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. however, the sides of the enclosure should be high enough to prevent escape (approximately cm). heavy food containers are recommended to make dumping of the receptacle more diffi cult. all food containers should be easy to disinfect and cleaned regularly, as guinea pigs have a habit of soiling their food bowls. most guinea pigs readily accept drinking water from a sipper bottle, which will decrease spillage and will keep feces, urine, and bedding from contaminating the water. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than ° f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. all animals are very sensitive to environmental and/or nutritional changes. therefore, if changes have to be made, gradual exposure of the animal to the changes is recommended. an appropriate guinea pig diet includes a formulated, pelleted diet for that species, high-quality hay (e.g., timothy, orchard grass, oat) ad libitum, and ample fresh vegetables. as the animal's food intake is more dependent on volume consumed rather than calories consumed, a pet fed a predominantly pelleted diet (higher nutritional concentration) has a tendency to become obese. fruits and grains, if they are offered at all, should comprise a very small portion (< %) of the total diet and offered only as treats. because guinea pigs lack the enzyme l-gulonolactone oxidase, they are unable to synthesize ascorbic acid from glucose. therefore, guinea pigs require supplemental vitamin c in their diets. although commercial guinea pig pellets are manufactured with vitamin c, the supplement often degrades rapidly, especially if the pellets are subjected to high heat and humidity. vitamin c placed in drinking water also degrades rapidly and should be changed daily. to ensure that a guinea pig is receiving a proper amount of vitamin c, it is necessary to supplement a diet of pellets and hay with plenty of fresh foods or often a specifi cally manufactured vitamin c supplement tablet (oxbow, inc., murdock, ne). many green, leafy vegetables, such as kale, mustard greens, dandelion greens, parsley, and many others, are excellent sources of ascorbic acid (box - ). the vitamin c requirement of an adult, nonbreeding guinea pig is mg/kg/day. , in the united states, guinea pigs are not routinely vaccinated for infectious diseases. however, owners should be encouraged to have annual examinations that include an oral examination and a complete blood count. being prey species in the wild, guinea pigs are adept at hiding illness, and routine evaluations by a qualifi ed veterinarian may help in detecting abnormalities early. many health problems of guinea pigs are related to improper husbandry. during a routine veterinary visit, the owners should be asked to provide a detailed description of the animal's housing environment, including substrate, frequency of cleaning, ambient temperature, and exercise time. the diet history is also important. owners should be asked not only what the guinea pig is offered, but also in what proportions and of what foods the animal actually eats. most guinea pigs are quite docile and do not require aggressive restraint. often a hand on the animal's dorsum is adequate to restrain a guinea pig patient on the examination table ( figure - ). when transporting a guinea pig, support the body with one hand under the thorax and abdomen while placing the other hand on the back to prevent the patient from falling or jumping ( figure - ) . if chemical restraint is required, gas anesthesia with isofl urane or sevofl urane is generally well tolerated. anesthetic gases can be delivered via mask or induction chamber. mild sedation can be achieved with an intramuscular injection of a combination of midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg) intramuscularly. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. before beginning a physical examination, it is important to observe the animal before it has been stressed by restraint. a healthy guinea pig should be alert and aware of its surroundings. as guinea pigs are often shy animals, they may attempt to hide or escape. the examiner should use a thorough, systematic approach to focus on the respiratory character and rate, posture, and attitude of the animal. it is also important to have any instruments (e.g., transilluminator, stethoscope, thermometer, blood-collecting supplies) that may be necessary to decrease the amount of handling time for the patient. obtaining an accurate body temperature, heart rate, and respiratory rate is best accomplished at the beginning of the examination, as these parameters will invariably change with handling. an accurate weight should be obtained using an electronic gram scale. a "hands-on" physical examination should begin with the head; the veterinarian should assess the eyes for symmetry or discharge and check that the external pinnae of the guinea pig are hairless, as normal. the external ear canals often contain a small to moderate amount of dark, ceruminous debris. the nasal planum should be dry and fl at, whereas palpation of the ventral mandible may reveal deformities secondary to overgrowth of molar and premolar apices. the guinea pig coat varies somewhat with breed but, in general, should be smooth and shiny. guinea pigs often have a mild to moderate amount of dark sebaceous debris on the skin of the dorsum. older male guinea pigs may develop a focal accumulation of this debris at the base of the vertebral column, which may be referred to as the "grease gland." thoracic auscultation and abdominal palpation can be performed as in other patients. heart and respiratory rates will vary depending on the degree of stress a patient experiences. as pulse and respiratory rates can be very rapid, careful auscultation is necessary to detect subtle abnormalities (e.g., murmurs, crackles, wheezes). auscultation of gut sounds is also an important part of the guinea pig physical exam. a healthy guinea pig should have to borborygmi per minute. the practitioner should keep in mind that stress will decrease gastrointestinal (gi) motility; therefore, a stressed animal will often have a decrease in borborygmi. structures normally found on abdominal palpation include kidneys, urinary bladder, cecum, and intestines. fecal pellets are often palpable in the colon. careful examination may reveal the presence of abnormalities such as gi distention, masses, or, in females, ovarian cysts. limbs and joints should be carefully evaluated because thickened or painful joints may be indicative of a vitamin c defi ciency. a complete oral examination is an important part of the guinea pig exam. because of the potential stress associated with the oral exam, this evaluation should be reserved for the end. the oral cavity of the guinea pig is very narrow with a small opening, making visualization diffi cult ( figure - ) . instruments such as an otoscope with cone or a human nasal speculum will increase visualization of the caudal oral cavity ( figure - ). however, many dental lesions may be overlooked using these methods, and anesthesia is often required to adequately determine oral health. obtaining blood samples from guinea pigs for routine diagnostics is a challenging procedure. peripheral venipuncture sites available for most mammals (e.g., lateral saphenous vein and cephalic vein) are diffi cult to visualize in guinea pigs and only allow for the collection of small sample volumes. the jugular vein may be used; however, the short neck of the guinea pig and poor tolerance of the aggressive restraint by the patient restrict the availability of this site for blood collection. often to obtain an adequate blood sample, it is necessary to anesthetize the patient. once anesthetized, the large central vessels may be accessed with less stress on the patient and phlebotomist. acceptable venipuncture sites while the patient is anesthetized include the cranial vena cava ( figure baseline blood work is an important tool in the routine monitoring of health as well as in the diagnosis of disease. a complete blood count is essential for assessing red blood cell and white blood cell parameters (box - ). guinea pigs have heterophils rather than neutrophils as the predominant circulating granulocyte. heterophils lack myeloperoxidase, the enzyme that causes purulent, liquid exudates. therefore, the debris contained in guinea pig abscesses is often found to be very thick and caseous, a fact that must be understood when treating these conditions. a normal guinea pig white blood cell differential will consist of primarily heterophils and lymphocytes, usually with a greater proportion of lymphocytes. the remaining leukocyte types (e.g., monocytes, eosinophils, basophils) are normally present in very low numbers. early stages of a guinea pig's infl ammatory response are often characterized by a shift in the differential white cell ratio (e.g., increased heterophils, decreased lymphocytes) rather than an increase in total leukocyte count. this ratio shift makes evaluating the entire leukogram essential for health evaluation (box - ). the platelet count is also an important marker of infl ammation in guinea pigs and other small mammal species. large increases in the platelet count (> , , /μl) can be seen without an increase in the total white blood cell count. a cell type that is unique to guinea pigs is the kurloff cell. these large lymphocytes contain a cytoplasmic inclusion (e.g., a kurloff body). kurloff cells are noted most often in the peripheral blood of reproductive age females, although they are also identifi ed in male guinea pigs. the number of circulating kurloff cells will increase in response to exogenous estradiol and testosterone administration, although the effects were more dramatic with estradiol administration. , other studies have shown the disappearance of kurloff cells in spayed female and castrated male guinea pigs. the function of the kurloff cell is not completely understood, and these cells appear to lack lysosomes. at this time, there is no evidence that the kurloff cell has phagocytic activity. the activity of kurloff cells appears to most closely correlate with that of natural killer cells found in other species. plasma biochemical analysis is necessary for evaluation of organ function as well as plasma glucose and proteins. it is important to interpret the results of a chemistry panel in conjunction with history, physical exam fi ndings, and the results of other diagnostic tests. reference intervals for biochemical parameters are shown in box - . urinalysis is a useful diagnostic test in guinea pigs with signs of upper or lower urinary tract disease. urine samples may be obtained by free catch, fl oor catch, or cystocentesis. if urine is to be cultured, it is ideal to use the cystocentesis method of collection. ultrasound guidance and/or mild sedation of the patient will aid the veterinarian and/or veterinary technician in obtaining the urine sample. the urine of guinea pigs is typically yellow to amber in color, but it may be darker and more orange depending on the patient's diet. pigments in the urine can sometimes be mistaken for hematuria, so differentiation is important. because guinea pigs are herbivores, the ph of guinea pig urine is alkaline, usually . to . . crystalluria may be seen but is not a normal fi nding. if crystals are found in a urine sample, the guinea pig patient should be examined for urinary calculi. to minimize rotation, care should be taken to extend the limbs symmetrically when positioning the patient. because guinea pigs have stocky builds with short limbs, and because they resent aggressive restraint, sedation or anesthesia is helpful in obtaining diagnostic radiographs as well as in reducing the patient's stress ( figure - ). table - is a guideline for radiographic techniques used in common small mammal radiographic studies. dental malocclusion is a common disease problem in guinea pigs. radiographs of the skull are helpful in assessing the degree of malocclusion as well as potential bone involvement. in addition to lateral and dorsoventral views of the skull, right and lateral oblique views can help to localize lesions. magnifi ed views of the skull can be obtained by placing the patient on an elevated platform under the x-ray beam without changing the distance between the x-ray cassette and beam. ultrasound is another imaging modality that is very useful in the diagnosis of common guinea pig disease processes, such as ovarian cysts (figure - ) and urinary tract calculi. as with radiographs, sedation or anesthesia can assist in reducing patient stress and improve the quality of images. guinea pigs often have a large amount of gas accumulation in the gi tract, which obscures the ultrasound image, sometimes making this imaging technique of limited value. advanced imaging techniques, such as computed tomography (ct) and magnetic resonance imaging (mri), are useful tools in the diagnosis of disease in guinea pig patients. limitations of these modalities include limited availability, expense, and decreased image quality compared with that of larger patients. mri has the added limitation of requiring a significant amount of time (often > min) under anesthesia with limited monitoring ability. regardless of the present limitations, veterinarians should be aware that these techniques are available and that reference material exists that depicts normal anatomy. as guinea pig owners continue to demand high-quality care for their pets, these imaging techniques will likely become more commonplace in small mammal practice for these patients. microbiologic samples can be obtained for diagnosis of various guinea pig infections. exudates from nasal or ocular secretions can be examined for abnormal fl ora. some bacterial organisms (e.g., bordetella bronchiseptica) are diffi cult to culture. laboratories need to be advised as to the organisms in question so they can perform more specifi c diagnostic tests to obtain organism identifi cation. abscesses are another disease condition that may require culture for proper treatment. because guinea pigs form caseous abscesses, the purulent debris itself is typically not useful for bacterial isolation. for the best chance of identifying organisms within an abscess, a portion of the abscess capsule should be submitted to the laboratory. both aerobic and anaerobic bacterial cultures should be requested, especially if it is suspected that the abscess may have originated from a dental problem. roundworms and coccidia are seen in guinea pigs and can be identifi ed by fecal fl otation or fecal direct smear, as in other species. cryptosporidiosis has also been reported. identifi cation of cryptosporidium organisms usually requires acid-fast staining or immunofl uorescent antibody testing in addition to fecal fl otation. ectoparasites (e.g., mites, lice, fl eas) commonly infest guinea pigs. visualization of the parasites and/or their waste, as well as skin scrapings, microscopic examination of hair follicles, and tape preparations can be useful in the identifi cation of these parasites. the term enterotoxemia refers to the overgrowth of toxinproducing bacteria in the gi tract, particularly clostridium diffi cile. this can occur with stress, an abrupt change in diet, gi stasis, or inappropriate antibiotic administration. the gi fl ora of guinea pigs is predominantly gram positive, and administration of antibiotics with a primarily gram-positive spectrum (e.g., beta-lactam antibiotics, macrolides, lincosamides) can lead to the depletion of normal gut fl ora, allowing colonization by opportunistic bacteria (e.g., gram-negative organisms, clostridium spp.). the dentition of the guinea pig is described as aradicular hypsodont, meaning all teeth grow throughout the animal's life and are open-rooted. the dental formula of the guinea pig is i / , c / , p / , m / . the mandible of these animals is wider than the maxilla. the occlusal angle of the molars and premolars in the guinea pig is quite severe when compared with that of rabbits and other rodents (figure - ). guinea pigs lack the layer of yellow enamel present on the rostral surface of the incisors in most other rodents. dental malocclusion is a common disease process in guinea pigs. the development of malocclusion can be the result of multiple etiologies or a combination of factors (e.g., improper diet, genetics, trauma). incisor malocclusion alone is rare in guinea pigs, so a thorough oral examination is necessary to determine the presence of cheek teeth malocclusion. when the molars and premolars do not occlude properly, overgrowth of the crowns and reserve crowns takes place. with improper wear, sharp points can form on the buccal aspects of the maxillary cheek teeth and the lingual aspects of the mandibular cheek teeth. the mandibular cheek teeth can overgrow to such an extent that they entrap the tongue, making eating and drinking diffi cult (figure - ) . clinical signs can include inappetence or dysphagia, decreased fecal output, ptyalism, poor coat quality, and leth-argy. abscesses commonly occur at the apical aspects of overgrown molars and premolars. because the premolars and molars are elodont (open-rooted), it is possible that these teeth can overgrow and impinge on the nasolacrimal duct, causing ocular and nasal discharge. the reserve crowns can also overgrow into the nasal cavity, where oral bacteria may be seeded, causing rhinitis and sinusitis. diagnosis of dental disease is based on physical and oral examination fi ndings. careful palpation of the ventral mandible and maxilla may reveal bony protuberances corresponding to overgrowth of the apical surfaces of the cheek teeth. proper instrumentation is very important for adequate visualization of the oral cavity. dental speculums and pouch dilators are invaluable aids in obtaining a good view of the molars and premolars (figure - ) . however, many abnormalities can be overlooked in a conscious animal, so anesthesia is often required to obtain a thorough oral examination. visualization of the oral cavity and occlusal surfaces can be facilitated by the use of an endoscope (figure - ) . most endoscopes available in veterinary practice have a degree of angulation of the fi eld of view, allowing greater focal area. abnormal oral examination fi ndings may include an uneven occlusal surface or angle of the cheek teeth, formation of sharp points with or without associated ulceration of the oral mucosa, food impaction, and abnormal spaces (diastema) between teeth. imaging, including routine radiographs, magnifi ed skull radiographs, and computed tomography can be incorporated to better evaluate the extent and seriousness of the process (figure - ) . radiographic studies should include dorsoventral, lateral, and right and left oblique views. treatment is centered on restoring a normal occlusal plane to the teeth, a procedure that should be performed while the animal is under general anesthesia. unstable animals should be provided with supportive care before the anesthetic event. a high-speed surgical dental handpiece can be used to restore a normal occlusal plane and to reduce any sharp points. handheld trimmers are not acceptable, as they tend to crush teeth and can cause fractures and pulp exposure. dremel tools are also not considered acceptable because the small size of the guinea pig oral cavity will not allow for appropriate trimming. with any dental instruments, care should be observed to minimize oral soft tissue trauma. when a guinea pig is diagnosed with dental malocclusion, it is important to convey to the owners that this will be a lifelong problem for their pets. with many cases, routine occlusal adjustments will be necessary for the remainder of the animal's life. tyzzer's disease is the common term for bacterial enteritis caused by clostridium piliforme. infection with this organism is more commonly described in small rodents such as mice and hamsters, although it has also been described in guinea pigs as well as other mammalian groups. tyzzer's disease often presents acutely, with signs such as diarrhea, depression, and poor coat quality, often progressing rapidly to death. transmission is via the fecal-oral route. guinea pigs can be infected with c. piliforme without showing clinical signs. animals that are carrying the organism but showing no signs of illness have still been found to shed organisms. animals that carry the organism and then become immunocompromised (e.g., stress, immunosuppressive drug therapy) often develop clinical disease. clostridium piliforme is an intracellular bacterium that will not grow on routine culture media, so antemortem diagnosis is diffi cult. tyzzer's disease begins as an intestinal infection, but later spreads to the liver hematogenously, causing areas of necrosis. , positive identifi cation of the organism on necropsy requires examination of hematoxylin and eosin or silver staining of affected tissues. treatment should consist of fl uid and nutritional support as well as appropriate antibiotic therapy. unfortunately, the progression of the disease is rapid, making treatment unrewarding. prevention is key, focusing on owners' reducing housing stress, providing a proper diet, and maintaining a clean environment. cryptosporidiosis has been described as causing disease in guinea pigs in a laboratory setting. diagnosis was made histologically from affected animals that lost weight, had diarrhea, and suffered an acute death. cryptosporidium organisms were found in the brush border of the intestinal tract from the duodenum to cecum, with associated infl ammation. suspected coronavirus infection has been described in young guinea pigs. clinical signs in affected animals included anorexia, weight loss, and diarrhea. approximately half of the affected animals in the reported outbreak recovered. necropsies performed on animals that died or were euthanized showed lesions consistent with coronavirus infection resulting from an acute to subacute necrotizing enteritis involving primarily the distal ileum. coronavirus-like virions were identifi ed on transmission electron microscopy of feces collected from the lower gi tract at necropsy. outbreaks of salmonellosis have occurred in guinea pig colonies. some of the organisms that have been isolated include s. enteritidis, s. dublin, s. fl orida, s. poona, and s. bredeney. , salmonellosis has been described to affect guinea pigs in acute and chronic disease processes. with acute salmonellosis, guinea pigs often die after a brief illness characterized by nonspecifi c signs of illness and diarrhea. usually only a few pathogenic abnormalities are found at necropsy, and confi rmation of a diagnosis is dependent on culture of affected tissues. chronic salmonellosis is a wasting disease, lasting several weeks. splenomegaly, hepatomegaly, and mesenteric lymphadenopathy are common postmortem fi ndings in affected animals. guinea pigs that recover can remain chronic, intermittent shedders of salmonella organisms. yersinia pseudotuberculosis causes several disease syndromes in guinea pigs. , the most common presentation affects the mesenteric and colonic lymph nodes, infi ltrating these lymph nodes with caseous nodules. clinically, affected guinea pigs will lose weight, have diarrhea, and develop a generalized lymphadenopathy. transmission of the disease can be either vertical or horizontal. as stated previously, guinea pigs require dietary supplementation of ascorbic acid because they lack the enzyme l-gulonolactone oxidase necessary for synthesis of this compound. although diets formulated for guinea pigs are supplemented with ascorbic acid, it is important to remember that it breaks down very rapidly, usually within the fi rst days after production. if a guinea pig is not receiving vitamin c supplementation in its diet, the veterinarian can assume that the diet is defi cient in this important nutrient. ascorbic acid is a necessary component of collagen, and defi ciencies are often noted as manifestations of abnormal collagen synthesis. clinical signs of hypovitaminosis c can include lameness, hemorrhage, lethargy, anorexia, poor coat quality, and bruxism. diagnosis of hypovitaminosis c is based largely on clinical signs and history. radiographs will reveal changes to the costochondral junctions and widening of the epiphyses of long bones. recommended treatment includes fl uids and nutritional support, pain control, and parenteral vitamin c supplementation. attention should also be paid to improvement of the guinea pig's diet at home. in addition to causing skeletal and cartilage abnormalities, vitamin c defi ciency has been known to reduce immune function. in vitro, vitamin c defi ciency was demonstrated to cause a reduction in migration of macrophages in guinea pigs. urogenital dystocia dystocia most frequently occurs in primiparous sows that are bred after approximately months of age. at this time, the symphysis between the pubic bones becomes fused and will not expand to allow the passage of fetuses. cesarean section must be performed in these cases to save the sow and the young. factors other than age that can predispose a sow to dystocia include large fetuses in relation to sow size, uterine inertia, and obesity. urolithiasis occurs commonly in pet guinea pigs, and the common clinical signs associated with the disease include stranguria and pollakiuria, vocalizing when urinating, and hematuria. the underlying cause(s) of this condition is not completely understood but is likely associated with a genetic predisposition and/or the presence of a high-calcium diet. other less common underlying etiologies associated with urinary calculi formation include ureteral neoplasms (e.g., papilloma). calculi are primarily composed of calcium carbonate, although magnesium ammonium phosphate hexahydrate and calcium phosphate calculi will also occur. radiographic or ultrasonic imaging can be used to confi rm the location of the urinary calculi, which may be present in the renal pelvis, ureters, urinary bladder, or urethra (figure - ) . urinary tract calculi often require surgical removal. ideally, once removed, the calculus and/or a portion of the bladder wall should be cultured. it may also be helpful to analyze the composition of the stone to help determine ways of preventing recurrence. ovarian cysts are a common fi nding in middle-aged to older female guinea pigs. this disorder has a reported prevalence of % in female guinea pigs aged . to years. , multiple cysts can be present on one or both ovaries. clinical signs related to cystic ovaries can include abdominal distention, lethargy, and anorexia related to the space-occupying nature of the cysts. ovarian cysts that are actively producing hormones can produce bilaterally symmetrical alopecia of the fl anks. ovarian cysts can be quite large (up to several centimeters) and can often be identifi ed on physical examination. abdominal ultrasound is the most useful diagnostic tool for a defi nitive diagnosis. the most defi nitive treatment for cystic ovaries is ovariectomy or ovariohysterectomy, curing the current problem and preventing recurrence. in animals where surgery is not a viable option, ultrasound-guided percutaneous aspiration of ovarian cysts can be performed as a palliative treatment. however, without removal of the ovaries, the cysts will recur, requiring repeated procedures. pregnancy toxemia typically occurs in pregnant sows during the last weeks of gestation. as with other species, pregnancy toxemia is the result of a negative energy balance and the metabolism of fat. sows that experience pregnancy toxemia are typically overweight and become anorexic. clinical signs include lethargy, dyspnea, and anorexia, usually progressing to death within a few days. therapy of affected sows should center on providing nutritional support, correcting electrolyte imbalances, and preventing opportunistic infections. prognosis is generally considered poor, as many sows fail to respond to treatment. neoplasia of the reproductive tract is not commonly reported in guinea pigs, but several tumor types have been described. of the described reproductive neoplasms, the vast majority occur in female guinea pigs. uterine leiomyoma (often associated with ovarian cysts) and leiomyosarcoma, ovarian teratoma, and granulosa cell tumor are reported neoplasms of the reproductive tract. diagnosis, as in any other patient, should be based on the results of cytologic or histopathologic sampling. benign neoplasms may be resolved with ovariohysterectomy, but further diagnostics to determine the extent of local invasion should be performed before surgery takes place. a thorough diagnostic work-up, including imaging techniques (e.g., abdominal ultrasound, thoracic radiographs), should be performed to look for metastases of malignant tumor types. mycoplasma caviae has been isolated from the reproductive tracts of guinea pigs. these guinea pigs are often unaffected by the organism, but metritis has been suspected to be associated with infection. as with other species, mycoplasma spp. is suspected to cause reproductive problems (e.g., abortion and decreased fertility). respiratory disease is a common presenting complaint in guinea pig patients. clinical signs can vary from sneezing and upper respiratory signs to severe dyspnea and death. bordetella bronchiseptica is one of the most common respiratory bacterial agents associated with pneumonia in guinea pigs. many guinea pigs are carriers of the organism, which will cause clinical disease if the animal is stressed. a thorough history, obtained from the patient's owner, often reveals interactions with other species that are also subclinical carriers of b. bronchiseptica (e.g., rabbits, dogs). clinical signs noted in guinea pigs infected by the bordetella organism include nasal discharge, dehydration, tachypnea, and lethargy. diagnosis should be based on clinical and radiographic signs and history. confi rmation of the diagnosis can be determined with enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence diagnostic tests, or culture of exudates. treatment/preventive options for b. bronchiseptica infections in guinea pigs include an autogenous bacterin vaccine as well as three commercially available vaccines for bordetella spp. (porcine b. bronchiseptica and human b. pertussis), which were found to offer some protection against the development of bronchopneumonia in experimentally infected guinea pigs. streptococcus pneumoniae can cause pleuropneumonia, pleuritis, and peritonitis in guinea pigs. serologic detection of s. pneumoniae antibodies using elisa have been described in previously published papers. on postmortem examination, lesions found to be associated with s. pneumoniae infection included pleuritis, pleural effusion, lung abscessation, otitis media, pericarditis, and others. streptococcus pneumoniae was also identifi ed from septic arthritis lesions in a group of guinea pigs. five guinea pigs in a laboratory colony demonstrated multiple enlarged joints from which pure cultures of s. pneumoniae were isolated. several other individuals in this colony had previously died with typical s. pneumoniae lesions (e.g., pleuritis, pericarditis) and lesions consistent with hypovitaminosis c. the group with septic arthritis also had scorbutic changes on necropsy. streptobacillus moniliformis, the causative agent of rat-bite fever in humans, was isolated from a laboratory guinea pig with pneumonia. this organism is of particular importance because of its zoonotic potential. adenovirus has been shown to cause necrotizing bronchopneumonia in guinea pig populations, although it can also produce a transient, subclinical infection. , clinical signs include depression and dyspnea, but guinea pigs often die acutely without clinical signs. identifi cation of adenovirus dna from diseased lung tissue was achieved using a polymerase chain reaction technique in one study. development of this pcr assay for identifi cation of the virus has determined that the guinea pig adenovirus is distinct. histopathologic examination of affected tissues of animals infected with adenovirus will reveal the presence of characteristic intranuclear inclusion bodies. the most commonly reported neoplasm of the respiratory tract in guinea pigs is bronchogenic papillary adenoma. the prevalence of the tumor is as high as % in guinea pigs over the age of years. given the relatively more common occurrence of pneumonia in guinea pigs, bronchogenic papillary adenoma can often be misdiagnosed. for this reason, thoracic radiographs of guinea pigs with respiratory disease are highly recommended. yersinia pseudotuberculosis can cause septicemic pneumonia in guinea pigs. death usually occurs rapidly, after the development of coughing and dyspnea. at necropsy, severe congestion of the lungs is found grossly and through histologic evaluation of the tissues. trixacarus caviae are sarcoptoid mites that commonly affect guinea pigs (figure - ) . affected guinea pigs are intensely pruritic, sometimes to the extent of seizure development. as with other mite infestations, diagnosis is based on the identifi cation of the parasites on skin scrapings. once defi nitive diagnosis has been made, treatment with ivermectin and selamectin ( mg/kg q - wk) is usually effective. chirodiscoides caviae are fur mites diagnosed in guinea pigs. because it rarely causes clinical disease, treatment is usually unnecessary. , gyropus ovalis and gliricola porcelli are species of lice that are commonly identifi ed in guinea pigs (figure - ) . infested guinea pigs may be pruritic, but are usually unaffected. guinea pigs severely infested with these mites may demonstrate poor coat quality and alopecia. patchy hair loss without associated pruritus may be attributed to dermatophytosis, most commonly trichophyton mentagrophytes (figure - ) . lesions are circular and scaled and usually occur on the face and head. the diagnosis of dermatophytosis is made by a positive fungal culture. because of the zoonotic potential of these fungal organisms, care should be streptococcus zooepidemicus lancefi eld's group c is the causative agent of cervical lymphadenitis. this disease will cause severe swellings of the lymph nodes in the cervical region in guinea pigs. affected guinea pigs will frequently exhibit no other clinical signs but may become septicemic, with lesions affecting the heart, lungs, kidney, and skin. the most effective treatment for cervical lymphadenitis is complete surgical excision of the affected lymph nodes, followed by appropriate antibiotic therapy based on culture and sensitivity testing. lancing and draining the abscesses is often not curative, as the abscesses form thick capsules that harbor organisms, leading to recurrence. yersinia pseudotuberculosis has also been shown to cause cervical lymphadenitis in guinea pigs. although the affected guinea pigs are usually not ill, the concern is that rupture of the abscesses will release large amounts of this potentially zoonotic organism into the environment. another potential causal agent of cervical lymphadenitis is streptobacillus moniliformis. one report exists of a cervical mass, initially believed to be cervical lymphadenitis, which was histologically determined to be a thyroid papillary adenoma. the mass was apparently nonfunctional and the guinea pig appeared otherwise healthy, but surgical resection was curative. guinea pigs housed in cages with wire fl ooring are predisposed to developing ulcerated lesions on the plantar surfaces of their feet. mild lesions may appear as hyperemic, swollen areas of the weight-bearing surfaces. these lesions can progress to ulcerations with secondary infections (figure - ). vitamin c defi ciency has also been considered a predisposing factor for the development of pododermatitis, as affected animals may be in pain and reluctant to move, resulting in the development of pressure sores. ulcerated, infected lesions should be managed with appropriate antibiotics and antiinfl ammatory drugs, but the focus of treatment should be improving husbandry (e.g., providing appropriate fl ooring/bedding, vitamin c supplementation). trichofolliculomas are the most common cutaneous tumor seen in guinea pigs. these benign tumors often occur on the dorsum and are typically round and hairless (figure - ). another cutaneous abnormality that has been described in guinea pigs is cutaneous vascular malformation. the lesion described was a raised, ulcerated plaque on the animal's fl ank, which bled intermittently. ultimately, the cutaneous vascular malformation resulted in fatal hemorrhage. histologically, the lesion was described as an expansile mass extending into the skeletal muscle and consisting of multiple vascular spaces of varying sizes. these vascular spaces were lined with endothelial cells. compared to the incidence of neoplasia in other mammalian species, the incidence of neoplasia in guinea pigs appears low or is underreported. however, there have been several reported cases of neoplasia in guinea pigs. as more guinea pig owners seek quality veterinary care for their pets, reports of neoplasia will increase. lymphoma is the most commonly reported neoplasia in guinea pigs. clinical signs associated with guinea pig neoplasia include lymphadenopathy, splenomegaly, and hepatomegaly. leukemic and aleukemic forms of guinea pig lymphoma have been identifi ed. thyroid carcinoma has been reported in an adult guinea pig that demonstrated multiple masses in the ventral cervical region. as there was no evidence of neoplastic disease elsewhere on postmortem examination, this was considered a primary tumor. mesothelioma has been reported in the abdomen of an adult guinea pig. the guinea pig had died of complications associated with pneumonia, and the mesothelioma was diagnosed through a necropsy examination. the mass consisted of diffuse nodules on the serosal surfaces of numerous organs in the abdominal cavity. guinea pigs are native to cooler regions of south america and are therefore relatively intolerant of temperatures above ° f, lower if the environment is also humid. guinea pigs should be housed in well-ventilated enclosures at temperatures between ° and ° f to prevent heat stress. clinical signs of heat stress include rapid, shallow respirations, lethargy, poor peripheral perfusion, and ptyalism. treatment includes reducing the animal's core body temperature with cool water baths or applying alcohol to the feet and ears; in addition, fl uid therapy (either intravenous or subcutaneous) is recommended to improve perfusion. the prognosis for this condition is guarded. chlamydophila psittaci has been identifi ed as a disease-causing agent in guinea pigs; it usually causes a mild, self-limiting conjunctivitis. this intracellular bacterial disease usually occurs in young guinea pigs to weeks of age but has been reported in adults as well. in one outbreak, the typical conjunctival abnormalities were present with other, more signifi cant, clinical signs, such as rhinitis, abortion, and pneumonia. a defi nitive diagnosis can be achieved through giemsa staining, immunofl uorescent antibody testing of conjunctival scrapings, and serologic testing. guinea pigs have been reported to develop pathologic changes related to ingestion of forssk fern. , in these studies, guinea pigs fed fresh fern developed hematuria and hemorrhage of the bladder wall. one guinea pig fed dried fern developed proliferative urocystica and adenoma of the bladder, a fi nding sometimes considered precancerous in human patients. this guinea pig did not show clinical signs associated with the bladder abnormalities. rabies virus infection is uncommon in rodent species, but it has been described and should be considered a differential diagnosis for an ill guinea pig with suspect contact to wildlife, especially raccoons. a recent report of a rabies in a privately owned guinea pig described abnormal behavior (biting the owner) days after the guinea pig had possible interactions with a raccoon. in this guinea pig, rabies virus antigen was detected by immunofl uorescent antibody testing in the sublingual salivary gland, tongue, and buccal tissues, implying that the guinea pig could have transmitted the virus via a bite wound. sick guinea pigs are often anorexic and therefore dehydrated. restoration of normal hydration status is crucial for the successful treatment of many disease processes. replacement of fl uid defi cit and maintenance of normal hydration can be achieved by administering crystalloids substances through subcutaneous, intraperitoneal, intravenous, or intraosseous routes. because of the diffi culty in placing and maintaining catheters in peripheral veins and bones, the most common route for fl uid administration is subcutaneous. subcutaneous fl uid administration is generally well tolerated. fluids are administered under the skin of the cranial, dorsal thorax (figure - ) . butterfl y catheter needles are useful because they allow the patient to move around without pulling out the injection needle. maintenance fl uid rates for guinea pigs are - ml/kg/day. another important aspect of management for the sick guinea pig is nutritional support. anorexic guinea pigs can experience a change in their normal gi fl ora in as little as to hours. this change of gi fl ora can lead to ileus, colic, overgrowth of pathogenic bacteria, and enterotoxemia. commercial products are available that are palatable and high in fi ber. these products help to maintain gut motility (oxbow critical care for herbivores, oxbow hay company, murdock, ne). patients will often eat directly from a dish or -ml catheter tip syringe. for patients that are more resistant to eating, a technique that is useful, in my experience, is to remove the plungers from -ml or -ml syringes and fi ll them individually using a catheter tip syringe. although this method may seem tedious, it allows the delivery of small boluses of food to be incrementally dispensed. the gi tract of guinea pigs can be very sensitive to the effects of certain classes of antibiotics. the gi fl ora of guinea pigs is primarily gram positive, and administration of antibiotics with a primarily gram-positive spectrum can result in overgrowth of gram-negative and anaerobic organisms. enteral administration of penicillins (e.g., amoxicillin, ampicillin), macrolides (e.g., erythromycin, lincomycin), and fi rst-generation cephalosporins can result in overgrowth of opportunistic pathogens. ampicillin administered subcutaneously at doses of and mg/kg three times a day resulted in mortality rates of % and %, respectively, in a group of guinea pigs. at necropsy, clostridium diffi cile was cultured from the ceca of all fatalities. twenty-fi ve percent of guinea pigs administered mg/kg of cefazolin, a fi rst-generation cephalosporin, intramuscularly died of enterocolitis after several injections. all antibiotics should be used with caution in guinea pigs because of the possibility of disruption of the gi fl ora (table - ). guinea pigs can be induced using isofl urane or sevofl urane administered by mask or induction chamber (figure - ) . for lengthy or potentially painful procedures, preanesthetic medications may be used before induction of the inhalant anesthetic agent. as a preanesthetic, an injectable combination, midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg), has been used with success. the administration of premedications will also decrease the stress of induction. isofl urane and sevofl urane are both acceptable inhalant anesthetics for guinea pigs. the advantage of sevofl urane is that it does not appear to have as noxious a scent as isofl urane, thereby reducing breath holding during induction and producing a smoother anesthetic episode. after induction, the inhalant anesthetic may be switched to isofl urane if cost is an issue. guinea pigs should not be fasted more than to hours before anesthesia. because these animals are hindgut fermenters, withholding food for longer periods of time may disrupt gi fl ora. careful monitoring during anesthesia is essential. the number of respirations must be visually monitored for depth and character. heart rate and rhythm should also be monitored continuously using a pediatric stethoscope or doppler unit, which can be placed on a peripheral artery. changes in heart rate or respiratory rate can occur rapidly, so it is advantageous to have precalculated doses of emergency drugs (e.g., glycopyrrolate, epinephrine, atropine, dopram) drawn and available for use before anesthetic induction. intubation of guinea pigs is generally considered to be very diffi cult. the long, narrow oral cavity makes visualization of the glottis diffi cult. the soft tissues of the tongue and soft palate are continuous in the caudal oropharynx, leaving only a small aperture, the palatal ostium. intubation must be performed through this opening in the mucosal tissues. several techniques have been described for visualizing the glottis to assist with intubation. these usually involve alterations of laryngoscope blades so that they may be introduced into the oral cavity without traumatizing the delicate buccal mucosa. , another possible intubation technique utilizes a stethoscope altered so that an appropriately sized endotracheal tube can be affi xed to the end. the endotracheal tube is then inserted into the caudal oropharynx. the anesthetist will then listen through the ear pieces of the stethoscope for expiration. the tube is then gently introduced into the trachea. this technique requires practice to perfect but, once the procedure has been performed several times, is a reliable way to intubate guinea pigs. application of a small amount of lidocaine to the rima glottis can ease intubation. rigid endoscopes and otoscopes, when available, are also very helpful in visualizing the glottis for intubation. indications for an ovariohysterectomy procedure in guinea pigs include routine sterilization, dystocia, and reproductive tract disease (e.g., neoplasia, muco/hydro/pyometra, ovarian cysts). in general, the surgical approach and technique are similar to those used with domestic species. the patient is placed in dorsal recumbency, and a ventral midline incision is made through the linea alba. care must be taken upon entering the abdomen to avoid incising the large cecum, which is often situated along the ventral body wall. the body of the uterus can be located dorsal to the bladder. the ovaries lie caudally and laterally to the kidneys and are to mm in length. the abdomen of the guinea pig is deep, and the ovaries can be diffi cult to exteriorize. care must be taken to avoid tearing the short ovarian vessels. once the ovaries are removed, the uterus is ligated and transected in a manner similar to that used with other mammals. another method of sterilization that is reported in guinea pigs is ovariectomy. the incidence of uterine disease compared with ovarian disease in guinea pigs is quite low. the approach for removal of the ovaries is through small incisions of the dorsal-lateral body wall caudal to the last rib. the advantage of this approach is that the sensitive gi tract is not manipulated to reach the structures to be excised. orchiectomy is typically performed to prevent reproduction, to decrease undesirable sexual behavior, and to treat reproductive tract disease. the orchiectomy procedure in guinea pigs is performed by making an incision through the scrotum over each testicle. reactions to suture material are relatively common in guinea pigs after surgical procedures. these reactions can vary in severity from mild local irritation to abscessation. suture materials that cause a large amount of infl ammation, such as chromic gut, should never be used in guinea pigs. in general, monofi lament suture materials that are degraded by hydrolysis are preferred. advances in diagnosis and treatment of small exotic mammal dental disease practitioner's guide to domestic rodents biology, husbandry, and clinical techniques hematologic and serum biochemical values of rodents mammalian hematology: laboratory animals and miscellaneous species natural cytotoxicity in the guinea-pig: the natural killer (nk) cell activity of the kurloff cell the kurloff cell radiology equipment and positioning techniques cryptosporidiosis in guinea pigs: a retrospective study tyzzer's disease subclinical infection and transmission of tyzzer's disease in rats disease problems of guinea pigs coronavirus-like virions associated with a wasting syndrome in guinea pigs natural infections of guinea pigs salmonellosis in laboratory animals the guinea pig or cavy, the ufaw handbook on the care and management of laboratory animals macrophage function in vitamin c-defi cient guinea pigs ureterolithiasis and papilloma formation in the ureter of a guinea pig guinea pigs reproductive medicine of rabbits and rodents spontaneous tumors of small mammals airborne-induced experimental bordetella bronchiseptica pneumonia in strain guinea pigs interlaboratory comparison of enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence (iif) for detection of bordetella bronchoseptica antibodies in guinea pigs effi cacy of commercial vaccines for protecting guinea pigs against bordetella bronchiseptica pneumonia serodiagnosis of streptococcus pneumoniae infection in guinea pigs by an enzyme-linked immunosorbent assay isolation of streptobacillus moniliformis from a guinea pig with granulomatous pneumonia polymerase chain reaction for detection of guinea pig adenovirus pathogenesis of guinea pig adenovirus infection adenoviral bronchopneumonia of guinea pigs anorexia and chirodiscoides caviae infection in a guinea pig (cavia porcellus) ilar: a guide to infectious diseases in guinea pigs, gerbils, hamsters, and rabbits, part ii, diseases outlines thyroid papillary adenoma in a guinea pig with signs of cervical lymphadenitis cutaneous vascular malformation in a guinea pig (cavia porcellus) thyroid carcinoma of a guinea pig: a case report abdominal mesothelioma in an aged guinea pig guinea pig inclusion conjunctivitis (gpic) in a commercial colony verlauf einer chlamydienbedingten "meerschweinchen-einschluß körperchen-könjunktivitis" in einer versuchstierhaltung, zeitschrift für versuchsteirkunde proliferative urocystica and adenoma in a guinea pig preliminary studies on christella dentate (forssk) fern toxicity in guinea pigs rabies virus infection in a pet guinea pig and seven pet rabbits an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs pharmacokinetics of cefazolin in guinea pigs exotic animal formulary ferrets, rabbits, and rodents: clinical medicine and surgery plumb dc: veterinary drug handbook endotracheal intubation in guinea pigs by direct laryngoscopy an improved method for direct laryngeal intubation in the guinea pig biology of the guinea pig key: cord- -tsmnkuhw authors: jung, kwonil; wang, qiuhong; scheuer, kelly a.; lu, zhongyan; zhang, yan; saif, linda j. title: pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: tsmnkuhw to understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged us strain, pc a, of the virus. at – hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. these findings confirm that strain pc a is highly enteropathogenic. in june , intestinal contents were obtained from a -day-old pig with diarrhea on a farm in ohio, usa. pedv strain pc a was detected in the sample by reverse transcription pcr (rt-pcr) selective for the nucleocapsid gene ( - nt). the partial nucleocapsid gene sequence of pc a was identical to that of us pedv outbreak strains from colorado, usa: usa/colorado/ (genbank accession no. kf ) and - (genbank accession no. kf ). only coronavirus-like particles were observed in the fecal sample by electron microscopy (figure ). the sample was negative for rotavirus groups a and c and for transmissible gastroenteritis virus/porcine respiratory coronavirus by rt-pcr ( , ) . the sample was bacteriologically sterilized by using . -µm syringe filters and then prepared as inoculum. nearterm gnotobiotic pigs were delivered aseptically by hysterectomy from a specific pathogen-free sow ( ) . six -to -day-old pigs were randomly assigned to a pedv-infected group (pigs - ) or a negative control group (pig ). information about inoculation and inocula pig-passage number is described in table . pigs - and were inoculated orally and/or intranasally with . - . log genomic equivalents (ge) of pedv strain pc a; pig was exposed to the virus by indirect contact with inoculated pig . for each sample, the quantity of pedv rna ge was ≈ times higher than plaque assay results for a cell-adapted pedv strain, pc a. clinical signs were monitored hourly. pig was monitored for longer-term clinical signs and virus shedding. pigs were euthanized for pathologic examination at stages of infection: acute, mid, and later stages (< h, - h, and > h, respectively, after onset of clinical signs). the ohio state university institutional animal care and use committee approved all animal-related experimental protocols. fecal or rectal swab samples were prepared as described ( ) . virus rna was extracted by using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. titers of virus shed in feces were determined by taqman real-time rt-pcr using the onestep rt-pcr kit (qiagen, valencia, ca, usa) as reported ( ) , with modifications in the forward primer and probe to provide a % match to the us strains: forward ′-cg-caaagactgaacccactaac- ′ and probe fam-tgyyaccayyaccacgactcctgc-bhq. a standard curve was generated by using the pcr amplicon (pedn / ) of strain pc a. the detection limit was ge per reaction, corresponding to . log and . log ge/ml of fecal and serum samples, respectively. small and large intestine tissues, lung, liver, heart, kidney, spleen, and mesenteric lymph node were examined grossly and histologically. mean jejunal vh:cd was measured by using pax-it software (paxcam, villa park, il, usa) as described ( ) . the frozen tissues were prepared and tested by immunofluorescence staining, as described ( ) , for the detection of pedv antigen, using monoclonal antibody c - against the spike protein of pedv strain dr (provided by daesub song, korea research institute of bioscience and biotechnology, daejeon, korea). acute, severe watery diarrhea and vomiting developed in all inoculated pigs. clinical signs developed - h after inoculation, regardless of the inoculum dose or number of inoculum pig passages (table ). pig , which was followed longer, also exhibited dehydration, loss of bodyweight, and lethargy, but it consumed most of the milk that was offered. however, ≈ h after onset of clinical signs, pig collapsed after showing signs of disorientation and emaciation. immune electron microscopy, using a gnotobiotic pig hyperimmune serum to pedv, showed only pedv particles in the intestinal contents. for the pig-passaged pc a strain, rt-pcr/pcr results were negative for transmissible gastroenteritis virus/porcine respiratory coronavirus ( ), rotavirus groups a-c ( ), caliciviruses ( , ) , astroviruses ( ) , circoviruses, enterovirus, kobuvirus, and bocavirus. for pigs and , the detection of fecal virus shedding - h after inoculation coincided with the onset of clinical signs; for pigs and , fecal shedding occurred before the onset of clinical signs (table ) . by macroscopic examination, all infected pigs exhibited typical pedv-like lesions, characterized by thin and transparent intestinal walls (duodenum to colon) and accumulation of large amounts of yellowish fluid in the intestinal lumen (figure , panel a) . the stomach was filled with curdled milk, possibly due to reduced intestinal peristalsis. the other internal organs appeared normal. histologic lesions included acute diffuse, severe atrophic jejunitis (figure , panel b) and mild vacuolation of superficial epithelial cells and subepithelial edema in cecum and colon ( figure , panel c). these findings were similar to those in conventional pigs naturally infected with asian or us strains of pedv and in caesarean-derived, colostrum-deprived pigs experimentally infected with cv ( , , , ). the mean jejunal vh:cd of the infected pigs ranged from . to . , probably depending on the stage of infection (table ) , and that of the negative control pig was . (± . ). vh:cd for pig , which was euthanized at a later stage of infection, was . (± . ), a ratio indicative of continued cellular necrosis. neither clinical signs nor lesions developed in the negative control pig during the experiment. immunofluorescence-stained cells were observed mainly in the epithelium of atrophied villi of small (duodenum to ileum) and large intestines (table ; figure , panels d-f), as reported in other studies ( , , ) . the immunofluorescence was confined to the villous epithelial cells (figure , panels d-f). a few immunofluorescence-stained cells were detected infrequently in the peyer patches of pig . lung tissues of the infected pigs did not show immunofluorescence staining, indicating that pedv does not infect figure . electron micrograph of a us porcine epidemic diarrhea virus (pedv) particle detected in a field fecal sample collected during a outbreak of ped on a farm in ohio, usa; the fecal sample from which pedv strain pc a in this study was obtained was from a pig on the same farm during the same outbreak. the sample was negatively stained with % phosphotungstic acid. scale bar = nm. lung tissues under the conditions tested. although pc a strain replicated in cecum and colon epithelial cells, cellular necrosis and villous atrophy were not evident. whether pedv infection of the large intestine contributes to the severity of ped is unclear. all infected pigs tested at acute or later stages of infection had viral rna titers of . - . log ge/ml in serum samples (table ) . these titers were similar to those for field samples tested by real-time rt-pcr; ( %) of acutephase serum samples collected from -to -week-old pigs with diarrhea from ohio had viral rna titers of . - . ge/ml. the early, severe diarrhea and vomiting and the pedv fecal shedding at high titers may be accompanied by viremia. no infected pigs had detectable viral rna in serum samples obtained before inoculation, and no negative control pig had detectable viral rna during the experiment. ( ); -, no cells showed staining. ‡at days of age, noninoculated pig was exposed by indirect contact to pig (at pih ) through small holes drilled into the stainless steel divider panel located between the pigs in the shared pig tub isolator unit. clinical signs and virus shedding were monitored after indirect contact. diarrhea and vomiting developed in pig approximately - h after clinical signs developed in pig (i.e., in pig , signs developed - h after indirect contact with inoculated pig ); pig was euthanized  h after the onset of clinical signs. in , the first us outbreaks of the rapidly spreading porcine virus, pedv, caused a high number of pig deaths and substantial economic losses ( , ); however, little was known about progression of the disease. our data confirm that us pedv pc a is highly enteropathogenic and acutely infects the entire intestine, but the jejunum and ileum are the primary sites of infection. pc a infection causes severe atrophic enteritis accompanied by viremia that leads to severe diarrhea and vomiting. fighting a deadly pig disease: industry, veterinarians trying to contain ped virus, new to the us emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences the pathogenesis of an enteric infection in pigs, experimentally induced by the coronaviruslike agent, cv- pathology of experimental cv coronavirus enteritis in piglets. i. histological and histochemical study an immunohistochemical investigation of porcine epidemic diarrhoea in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs detection and genetic diversity of porcine group a rotaviruses in historic ( ) and recent ( and ) swine fecal samples in ohio: predominance of the g p[ ] genotype in nursing piglets the effects of simvastatin or interferon-alpha on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus the effects of transplacental porcine circovirus type infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in us swine characterization and prevalence of a new porcine calicivirus in swine, united states novel astroviruses in insectivorous bats we thank james e. collins and doug marthaler for kindly providing us pedv sequences for primer design and testing for enterovirus, kobuvirus, and bocavirus; j. hanson, g. meyers, and r. mccomick for assistance with animal care; x. wang, m. lee, s.s. wagner, a. veeramani, and chun-ming lin for technical assistance; andrea kaszas for assistance with electron microscopy; and v. anastasia for helpful discussion.salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported in part by a grant from the national pork board (no. - to q.w. and l.j.s.).dr jung is a veterinary pathologist at the ohio state university. his major research interests include diagnostic molecular pathology, pathogenesis, and immune responses to enteric viral infections, using germ-free animal models. key: cord- -c y m do authors: guo, baoqing; lager, kelly m.; henningson, jamie n.; miller, laura c.; schlink, sarah n.; kappes, matthew a.; kehrli, marcus e.; brockmeier, susan l.; nicholson, tracy l.; yang, han-chun; faaberg, kay s. title: experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: c y m do the pathogenesis of type highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) in -week old swine in the united states was investigated. rjxwn , rescued from an infectious clone of chinese hp-prrsv, replicated in swine with at least -fold increased kinetics over u.s. strain vr- . rjxwn caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to hp-prrsv than to those infected with vr- . novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of hp-prrsv to rapidly transmit between animals. furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in hp-prrsv infected swine, and showed that contact swine differed in the degree of cytokine response. in , investigators from several chinese provinces reported a unique syndrome in growing swine that was highlighted by the predominant clinical signs of high fever, anorexia, listlessness, red discoloration of skin, respiratory distress and very high morbidity and mortality rates (li et al., ; tian et al., ; tong et al., ; wu et al., ; zhou et al., ) . originally known as porcine high fever disease (phfd), this syndrome spread to vietnam in and to cambodia, laos, the philippines, bhutan, myanmar, thailand, south korea and russia in later years feng et al., ) . although there was concern that this syndrome may have been caused by a new disease agent, extensive diagnostic testing revealed only known pathogens. one consistent finding was the detection of porcine reproductive and respiratory syndrome virus (prrsv) with two discontinuous deletions in the replicase polyprotein known as nonstructural protein (nsp ), and two single nucleotide deletions in the and untranslated regions (utrs) (zhou and yang, ) . experimental infection of chinese swine with the initial novel prrsv field isolates reproduced the clinical disease (li et al., ; tian et al., ; tong et al., ; wu et al., ; zhou et al., ) , providing strong evidence for its role as the causal agent of phfd. since the severity of the clinical disease was greater than expected for a typical prrsv infection, there remained the chance that an unknown agent in the prrsv isolates exacerbated the disease symptoms. this question was resolved when phfd was reproduced in chinese swine with virus derived from an infectious clone of the jx prrsv isolate (lv et al., ) . the prior studies demonstrated that prrsv isolates with a common genetic pattern had a causal role in phfd, leading to the new designation of this viral lineage as highly pathogenic prrsv (hp-prrsv). the presence of the unique nsp deletion motif was initially thought to contribute to the severity of disease (tian et al., ) . however, studies showed that the nsp region of hp-prrsv strain rjxwn containing the novel deletion could be replaced with amino acids from low virulence strain hb- / . to result in a chimeric virus with only a minor delay in mortality (zhou et al., ) . there was also concern that there may be some unique aspect that may predispose asian pigs to a severe outcome, e.g., husbandry practices, endemic infections with other pathogens, climate, or host genetics. although a number of experiments have associated genetic changes in prrsv with an attenuation phenotype using point mutations (grebennikova et al., ; nielsen et al., ; storgaard et al., ) or by the construction of chimeric viruses (ellingson et al., ; kwon et al., ; wang et al., ; zhou et al., ), there appears to be no single locus for which mutations confer a predictable change in virulence. collectively, this area of study suggests the factors that contribute to prrsv pathogenicity are complex and viral strain-specific . as part of the single-strand positive-sense rna virus order nidovirales, family arteriviridae, prrsv genomes can vary between kb to . kb, consist of at least open reading frames (orfs) and replicate through a nested set of subgenomic rnas (van hemert and snijder, ) . members of this highly variable virus have been grouped into two genotypes, type (european-like, prototype strain lelystad) or type (north american, prototype strain vr- ), which differ in nucleotide similarity by approximately % (nelsen et al., ) . both genotypes have representatives of varying pathogenicity, and intragenic nucleotide sequence variation in each can be as much as % (shi et al., ) . for this study, we imported a plasmid containing the cdna copy of the hp-prrsv strain rjxwn genome, a type prrsv genome that is , bases in length and has % pairwise nucleotide identity to the prototype genome of vr- (zhou et al., ) . to investigate the pathogenesis of hp-prrsv infection and the potential contribution of climate, host genetics, commensal bacteria, other environmental conditions and husbandry practices to the pathogenicity of hp-prrsv in u.s. swine, we compared and contrasted the pathogenicity of rescued jxwn (rjxwn ) virus to that of vr- in -week old swine. we found that this hp-prrsv strain caused extreme morbidity, as was seen in asia, but novel to this study, resulted in up to x higher abundance of circulating virus when compared to vr- , caused extremely exacerbated thymic atrophy such that the thymus was often difficult to discern, and the host response was assessed in comparison to animals infected with strain vr- for the first time by a swine protein array including innate and adaptive cytokines in serum, bronchoalveolar lavage fluid and lymph nodes. moreover, we completed bacterial speciation and loads after necropsy and specified the degree of weight loss seen. lastly, we showed that hp-prrsv readily transmitted to contact swine causing a different pattern of cytokine responses. as a result of our study, the remaining plausible contributing factors to the high virulence seen in asia were discredited, and thus hp-prrsv strains pose a serious threat to the u.s. swine industry. pigs challenged with chinese hp-prrsv strain rjxwn (group ) began exhibiting clinical signs of disease within - days post exposure (dpe). as a group, the pigs developed fevers, became listless, anorexic, and began shivering and huddling together in a pile. clinical signs became more severe over the next few days, with pigs rapidly losing body weight (fig. ) , becoming dehydrated and weak. respiratory distress, characterized by dyspnea, tachypnea and coughing was common in all pigs. erythema of the skin was present in most of the pigs, and several developed cutaneous hemorrhages and cyanotic extremities (blue ears) (fig. ). on dpe, one pig from group was found dead. two others were euthanized due to severe weakness and moribund condition on dpe and , and one was found dead on dpe . on and dpe, clinical signs of disease in some of the hp-prrsv challenged group began to decrease in severity. while pigs remained huddled in a pile with respiratory signs, approximately half of them were subjectively less listless, would move away when approached and began showing an interest in feed again. during the study, contact pigs displayed similar clinical signs as those challenged with hp-prrsv . pigs challenged with vr- (group ) were clinically normal until dpe , when they began to exhibit slightly increased respiratory rates and became a little less active than the control group. one pig from the vr- group was found dead on dpe ; however, the cause of death was attributed to gastric dilatation and volvulus, rather than to clinical disease typical of prrsv strain vr- . control pigs (group ) remained clinically normal for the duration of the study. postmortem examination revealed severe lesions in the hp-prrsv challenged pigs including: marked interstitial pneumonia, lymphadenopathy and thymic atrophy. necropsy findings in this group include: pulmonary edema, pleuritis, peritoneal and pericardial effusions, renal petechia, and fibrinous peritonitis that are delineated in table . lungs and representative lesions are shown in fig. . no pathologic lesions were identified in control pigs (group ). pigs in the vr- challenge group (group ) had diffuse interstitial pneumonia, characteristic of an uncomplicated prrsv infection. although lung lesions were subjectively more severe in the hp-prrsv and contact groups, a statistically significant difference in lung scores was only found between the rjxwn contact group and the group inoculated with vr- (groups and , po . ) (fig. a) . lymphadenopathy was present in all infected pigs (groups - ), with no statistically significant difference between hp-prrsv and vr- infected groups (fig. a ). thymic atrophy was also a common finding in infected pigs and was significantly more severe in hp-prrsv infected and contact pigs compared to vr- infected pigs (p ¼ . and po . ; fig. b ). table summarizes aspects of the clinical disease seen in this study. shown are adwg and associated standard errors for each of the four groups for days À - and days - . sham inoculated control swine (group ) and those challenged with vr- (group ) gained weight at an expected rate for their age group. group swine challenged with hp-prrsv strain rjxwn and their contacts (group ) lost body weight during the last week of the experiment. the adwg for group on days À - includes only pigs, and on days - includes only pigs. the body weights for the contact animals were taken on the same day as pigs in the other groups. significance values are shown. microscopic lung lesions consisted of histiocytic interstitial pneumonia with increased numbers of macrophages in alveolar septa and lumina (fig. a , c). type ii pneumocyte hyperplasia was also present. these lesions were seen in the hp-prrsv challenge group (group ), the hp-prrsv contact pigs (group ) and the vr- challenge group (group ). mean interstitial pneumonia lesions were . , . , and . for chinese prrsv challenge group (group ), contact chinese prrsv pigs (group ) and vr- challenge (group ), respectively (fig. b ). lesions were significantly (p¼ . ) more severe in the chinese prrsv challenge group (group ) and contact chinese prrsv pigs (group ) as compared to the vr- challenge (group ). two sham inoculated control pigs had mild lesions with a score of , while the remaining pigs had no lesions. immunohistochemistry labeling for prrsv nucleocapsid was positive in % of chinese prrsv challenge and contact pigs (groups and ) and % of vr- challenge pigs (group ), with mean ihc labeling scores of . , . , and . , respectively (fig. ) . positive labeling was present in alveolar macrophages and interstitial macrophages for all infected groups ( fig. b and c). no significant difference was present in the amount of prrsv labeling in the lung among the three treatment groups. immunohistochemistry labeling for prrsv in lung tissue was negative in all sham inoculated control pigs. sera collected at À , , and dpe were tested for prrsv antibody. most swine infected with rjxwn (group ) had seroconverted by dpe (mean s/p ¼ . , sd¼ . ), and all had seroconverted by dpe (mean s/p ¼ . , sd ¼ . ); however, the rjxwn contact animals (group ) had not yet seroconverted by dpe (mean s/p ¼ . , sd ¼ . ) but did by dpe (mean s/p¼ . , sd¼ . ). vr- infected pigs (group ) were seronegative at dpe (mean s/p ¼ . , sd ¼ . ); but positive by dpe (mean s/p¼ . , sd ¼ . ). the sham inoculated control animals (group ) remained negative for prrsv nucleocapsid antibody throughout the study. all serum and balf samples were screened on marc- cells for infectious virus, and vi positive samples were then titered. a significant increase in virus load ( - fold) for the rjxwn challenge and rjxwn contact pigs was found in the serum when compared to the vr- challenge group (fig. a ). no significant differences were found in serum virus titer between the rjxwn challenge and rjxwn contact groups at any time points even though the contact pigs were exposed to rjxwn infected pigs at dpe. at necropsy, no significant differences in balf titers were found among the rjxwn challenge, rjxwn contact, or vr- challenge groups (fig. b ). control serum and balf samples were vi negative throughout the experiment ( fig. a and b) . to examine the levels of prrsv rna and to strengthen the case for the apparent difference in viral loads between the hp-prrsv and vr- inoculated animals seen above, qrt-pcr was completed on all serum samples as well as balf and lymph node tissue (fig. ) . the level of viral rna detected in serum samples mirrors the results obtained by virus isolation on marc- cells (fig. a, pr . at all dpe). the qrt-pcr results from balf showed little or no significant difference between the infected lymph node weight (lnw) to body weight (bw) ratios showed pronounced lymphadenopathy in all infected animals (groups - ) as compared to control animals (group )(p¼ . or higher significance), but no statistical significance between prrsv infected swine. (b) thymus weight (tw) to bw ratios showed thymic atrophy in all infected animals (groups - ) as compared to control animals (p o . ), and statistically significant differences between hp-prrsv inoculated (po . ) and naturally infected (p ¼ . ) animals compared to vr- inoculated swine. groups (fig. b ). tbln samples suggested that swine inoculated with vr- had significantly less viral rna present than those inoculated (p¼ . ) or infected (p¼ . ) with hp-prrsv (fig. c ). sham inoculated control serum, balf, and tbln tissue samples were viral rna negative throughout the experiment ( fig. a -c ). bacteria were isolated from the balf of of the rjxwn inoculated pigs (group ) and of the rjxwn contact pigs (group ) but from only of pigs in each of the sham inoculated control (group ) and vr- inoculated (group ) groups. in the rjxwn challenge group pasteurella multocida was isolated from four pigs (ranging from to cfu/ ml), p. multocida ( cfu/ ml) and actinobacillus suis ( cfu/ ml) were isolated from two pigs, p. multocida, a. suis and streptococcus suis (approximately , , and cfu/ ml, respectively) from one pig, and staphylococcus aureus and klebsiella pneumoniae (approximately and cfu/ ml, respectively) were isolated from one pig. in the contact group, p. multocida and a. suis were isolated from one pig ( cfu/ ml each) and p. multocida, a. suis, and arcanobacterium pyogenes (approximately , , and cfu/ ml, respectively) were isolated from a second pig. s. aureus and escherichia coli ( cfu/ ml each) were isolated from vr- inoculated pig, and bordetella bronchiseptica ( cfu/ ml) was isolated from sham inoculated control pig. results on cytokines measured in serum, balf and tbln homogenates are presented in figs. and and the changes in cytokine levels relative to sham-inoculated controls are listed in table . pigs directly inoculated with rjxwn (group ) had significantly elevated average serum levels of ifna, il- b, il- , il- and ifng, ranging from to times the levels detected in sera of sham inoculated controls (group ). in pigs inoculated with rjxwn , serum ifna levels were significantly elevated at dpe in comparison to both controls and vr- exposed pigs (po . ) and remained significantly elevated at dpe versus both groups. serum levels of il- were elevated by dpe, with il- , il- , il- and ifng all signficantly elevated at dpe compared to both controls and vr- exposed pigs (po . ). compared with sham inoculated controls, contact pigs exposed to rjxwn (group ) developed significant serum level elevations ( to times greater) in out of innate immunity cytokines measured (ifna, tnfa and il- b) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). in pigs exposed to rjxwn by contact transmission, serum ifna levels were signifcantly elevated at , and dpe in comparison to controls (po . ). in addition, contact pigs had significant elevations in il- b at and dpe (po . ). contact pigs also had significantly elevated levels of all adaptive immunity cytokines at and dpe (p o . ) with il- also significantly elevated at dpe. in contrast, none of the cytokines measured had significant elevations in serum levels detected in pigs inoculated with the north american prototype strain vr- (group ) when compared with sham inoculated controls. similarly, when compared to sham inoculated controls, balf of swine inoculated with rjxwn (group ) had significantly elevated ( to times greater) levels of out of innate immunity cytokines measured (tnfa, il- b and il- ) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). compared with sham inoculated controls, contact swine exposed to rjxwn (group ) had significantly elevated balf cytokine levels ( to times greater) in out of innate immunity cytokines measured (ifna, tnfa and il- ) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). in contrast, swine inoculated with vr- (group ) had significantly elevated balf cytokine levels ( times greater) for only out of innate immunity cytokines measured (il- b and il- ) and elevated levels ( to times greater) for of cytokines measured associated with adaptive immunity (il- and ifng). in this study, we compared the pathogenicity of chinese hp-prrsv strain rjxwn to north american prototype vr- in u.s. high health swine under controlled conditions. the clinical disease induced in this study by the hp-prrsv virus was similar to what has been reported in asia for phfd and for experimental infections with the wild-type or rescued virus ( zhou et al., ; zhou et al., ) . likewise, the clinical disease induced by the vr- virus was similar to that observed in previous reports (faaberg et al., ; rossow et al., ) . clinical disease and pathology were much more severe in the rjxwn group and their contacts than in the vr- group. overall, gross pathology lesions in the rjxwn challenged and contact groups were much more extensive and not restricted solely to the respiratory tract and lymph nodes as was the case with the vr- challenged pigs. the high occurrence of bacterial co-infections in the rjxwn challenge and contact swine likely played a prominent role in the difference in pathology and clinical disease between groups. bacterial co-infections in pigs naturally infected with prrsv have been documented, with susceptibility attributed to factors including prrsv strain differences, host genetics, management practices and environmental factors (rossow, ) . in this study, we used swine of high health status in a controlled research environment that were from -and -way crosses of commercial genetic lines. we believe the incidence and magnitude of bacteria isolated from the rjxwn challenge and contact groups when compared to the vr- and control groups suggest the differences in secondary bacterial infection susceptibility are specific to viral strain. in this study, we observed a % mortality rate in rjxwn infected pigs by dpe, which is less mortality than the original report (zhou et al., ) , but similar to other hp-prrsv strains used (li et al., ; lv et al., ; zhou et al., ) . possible explanations for differences may be the route and dose of inoculation, the age of pigs, the hp-prrsv strain utilized, and the time course of study. in addition, since the hp-prrsv lineage may exacerbate subclinical bacterial infections, it is possible different endemic bacterial infections played a role in apparent different mortality rates. in natural infections with chinese hp-prrsv, pulmonary interstitial hyperplasia with hemorrhage and edema is described, which suggests an acute septicemic process due to a secondary bacterial pathogen (tian et al., ; zhou and yang, ) . upon histopathologic examination of tissues from this study, north american pigs directly inoculated with rjxwn , as well as contact pigs, had an interstitial pneumonia that was significantly more severe than the vr- inoculated group, which is consistent with the severity of disease reported in china. although pulmonary lesions were more severe in the rjxwn challenge and contact pigs, the amount of antigen labeling was not significantly different from the vr- inoculated swine. since levels of proinflammatory cytokines, including tnfa, il- b and il- , were significantly increased in the balf of rjxwn -inoculated and contact pigs, the host response to hp-prrsv may play a role in the augmented lung pathology seen. in addition, the increased incidence of secondary bacterial infections in rjxwn challenge and contact pigs may have contributed to increased cytokine production and resultant immunopathology. there are numerous reports about the interplay of prrsv with the swine immune system that describe variable responses to infection at the cellular and cytokine level (miguel et al., ; thanawongnuwech et al., ; thanawongnuwech and thacker, ; wang et al., ) . although a large part of this variability may result from differing methods, challenge viruses, outbred pigs, and experimental designs, there are consistent findings emerging among the studies of increases in levels of selected cytokines associated with both innate and adaptive immunity. here we report a comprehensive assessment of the effects of prrsv infection on levels of cytokines critical to innate (ifna/b, tnfa, il- b, il and il- ) and adaptive (il- , il- , il- , il- and ifng) immune systems in serum, balf and tbln homogenates. it was demonstrated that infection with a highly pathogenic strain of prrsv elicited a significant elevation of all adaptive immunity cytokines measured in balf, as well as a majority of these cytokines in serum and tbln homogenates of the same groups of pigs. this observation is consistent with previous reports of table changes in cytokine levels in sampled tissues relative to non-challenged control pigs (p-values in comparison to non-challenged controls). serum values are an area under the curve overall average level measured in samples collected after exposure to virus. geometric means back-calculated from log means were used to determine relative changes in tbln and balf cytokine levels compared to sham-inoculated controls. (sun et al., ) ]. in contrast, in pigs infected in this study with rjxwn , we observed significant elevations of serum ifna as the first cytokine to peak following exposure to rjxwn in either the directly challenged or contact pigs, with the zenith occurring and dpe, respectively. however, consistent with a previous study (gomez-laguna et al., ), elevated serum ifna had little to no apparent effect on virus clearance as the viremia peaked in the rjxwn challenge and contact pigs on dpe and , respectively, and remained above the serum virus levels of vr- pigs until the end of the study ( and dpe). it is well documented that prrsv infection will increase the susceptibility of swine to co-infection with various bacteria (brockmeier et al., ; thanawongnuwech et al., ; thanawongnuwech and thacker, ; thanawongnuwech et al., ; thanawongnuwech et al., ; xu et al., ) , and based on previous findings with other hp-prrsv strains (xu et al., ) and our current rjxwn findings, it is clear that exposure to hp-prrsv greatly increases the likelihood of secondary bacterial infection due to commensal or pathogenic organisms typically found in the swine upper respiratory tract. whether the significant elevations of multiple cytokines that were measured in serum, balf and tbln following exposure to rjxwn were a direct result of the virus, an indirect effect mediated by the secondary bacterial infections, from extensive host tissue damage, or a combination of all of these events cannot be determined from our experiments. however, the pattern of multiple cytokines being elevated nearly simultaneously in serum ( of cytokines in rjxwn contact pigs and of cytokines in direct inoculated pigs) has not previously been reported and was not detected in vr- infected swine. cytokines are a diverse collection of peptides that elicit a wide range of biological responses and are characteristically understood in the context of an immune response wherein inflammation and immunity are carefully orchestrated by sequential secretion of cytokines that coordinate innate and adaptive immune responses. macrophages and stressed or damaged cells typically initiate a cytokine cascade through secretion of chemokines and proinflammatory cytokines in order to initiate the innate immune response at sites of acute infection or damage. generally, they act locally at nano-to picogram levels with short half-lives and transient activity. however, inflammatory cytokine cascades classically comprise a sequential appearance and disappearance of proinflammatory cytokines (e.g., tnfa, il- and il- ) intended to activate immune cells and their recruitment to generate additional cytokines and chemokines. this initial wave of cytokine production is usually followed by anti-inflammatory cytokine production (mainly the il- family) to moderate or down regulate the pro-inflammatory cytokines. cytokine cascades are therefore usually sequential with transiently detectable levels in peripheral blood; any dysregulation of these cascades can lead to adverse immunopathological responses. while significant elevations of several cytokine levels in tissues such as balf and tbln were expected, near simultaneous elevation of several cytokines in serum was not entirely expected as part of a normal host immune response. moreover, the levels of cytokines detected were in several instances significantly elevated (usually several times more) over levels detected with the low virulence north american prototype prrsv strain, vr- . prolonged elevations of serum ifng levels have been reported in swine infected with prrsv, a finding in contrast to serum ifng in pigs infected with influenza or respiratory coronavirus, where minimal transient detectable levels are observed (wesley et al., ) . in swine infected with rjxwn , it appears the normal sequence of cytokine production (e.g., ifng, tnfa, and il- often being the first cytokines produced in response to a viral infection, followed by il- and il- ) leading to effective virus clearance and a normal immune response was dysregulated given that significant elevations of several serum cytokines levels were still evident at to dpe. two recent studies have reported hp-prrsv infection in swine results in down regulation of a key toll-like receptor adapter gene, sarm (sterile aand armadillomotif-containing protein) (zhou et al., ; zhou et al., ) . sarm normally dampens the proinflammatory immune response by attenuating nf-kb activation and decreasing expression of il , il- and tnfa (carty et al., ) . previous studies in pigs infected with strains of hp-prrsv were reported to have swollen livers and petechial hemorrhages on the kidneys as well as immunohistochemical staining evidence of viral antigen in the liver and kidneys (among other tissues) (li et al., ; tian et al., ) . impaired hepatic and renal function as a consequence of viral or secondary bacterial disease could have a significant effect on clearance of the cytokines detected in serum, and contribute to an apparent severe cytokine release syndrome. whether our findings represent a parallel condition in swine to severe cytokine release syndromes or cytokine storms reported in humans that have been attributed to various causes cannot be proven with our data (descotes and gouraud, ; tarrant, ) . however, given prrsv causes polyclonal b cell activation, autoimmunity, lymphoid hyperplasia and hypergammaglobulinemia in pigs (lemke et al., ) , and it modulates multiple intracellular signaling pathways [reviewed in (sun et al., ) ], it is not surprising to find an exaggerated immune stimulation in swine infected with a particularly virulent strain of prrsv. early studies with cytokine administration to livestock species identified potential toxicities associated with systemic cytokine administration. interferon-g was found to have beneficial activity on immune function but was too toxic for practical usage due to febrile responses observed within h of a single dose of . mg/ kg of body weight (roth and frank, ) . similarly, administration of ng of bovine il- b/kg of body weight every h for days caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis (goff et al., ) . blood cytokines have been proposed as biomarkers of in vivo toxicity associated with new drug development (tarrant, ) . under most disease scenarios where there is rapid resolution of the infection by the host immune response, much of the biological activity of cytokines will occur in the locally infected tissues and elevated levels of the cytokines may not be detected in serum. we cannot definitively state whether the elevated cytokine syndrome reported here contributes to the pathophysiology of the disease caused by this highly pathogenic prrsv isolate. however, the association between the elevated levels of multiple cytokines and the severe morbidity and high mortality reported is consistent with a multiple cytokine toxicity syndrome in humans associated with various therapeutics (tarrant, ) . adverse reactions ranging from mild-to-moderate flu-like reactions to severe cytokine release syndromes have been observed with many therapeutic proteins in current use in human medicine, and some result in severe and even potentially life-threatening syndromes (reviewed in (tarrant, ) ). macrophage activation syndrome and cytokine storm are different names for two syndromes that share many features including a massive inflammatory response, elevated serum cytokine levels, multiorgan system disease and often death (behrens et al., ) . although these syndromes may be clinically indistinguishable, the cytokines that predominate in each may differ with tnfa being dominant in bacterial sepsis and ifng predominate in the macrophage activation syndrome (behrens et al., ) . the exact pathophysiology of the systemic toxicity in these syndromes is not fully defined. in pigs infected by natural contact with rjxwn inoculated pigs, we detected significant serum elevations in ifna, tnfa, il- b, il- , il- , il- , il- and ifng. however, given that prrsv infects the macrophage cell line in pigs, the elevated serum cytokine levels in pigs infected with hp-prrsv may represent both conditions (macrophage activation syndrome and cytokine storm) and contribute to the multiorgan damage and high mortality reported here. marc- cells were cultured in minimum essential medium (mem, safc c) with % fetal bovine serum at c, % co . wild-type (wt) type prrsv strain vr- (genbank u ), passage on marc- cells, was titrated, and used for the swine study. virus (rescued rjxwn ; rjxwn ) was rescued from a cloned cdna of chinese highly pathogenic type prrsv strain jxwn in marc- cells [pwsk-jxwn; genbank ef , (zhou et al., ) ] and passaged times on marc- cells for use in the swine study. the in vivo swine study described here was performed at the national animal disease center under approval from its animal care and use committee. thirty-two -week-old cross-bred pigs were obtained from a u.s. high-health status herd and were found to be free of prrsv antibodies by herdchek elisa, influenza virus antibodies by np elisa, and negative for porcine circovirus type by quantitative real-time pcr (data not shown). one day prior to inoculation, pigs were bled, weighed and randomly assigned to one of four groups. group (n ¼ ) consisted of sham inoculated control pigs, which received an intranasal ml sham inoculum of mem on day . group pigs (n ¼ ) were challenged intranasally with ml of  % tissue culture infective dose (tcid )/ml of chinese prrsv strain rjxwn in animal biosafety level -agriculture (bsl -ag) housing, where they remained for the duration of the experiment. group consisted of naïve pigs (n ¼ ) that were placed in contact with group two days after group was inoculated (dpe). group pigs (n ¼ ) were challenged intranasally on day dpe with ml of  tcid /ml of type prototype strain vr- . groups and were housed in separate isolation rooms in an absl facility. clinical monitoring of pigs was performed daily throughout the study. specifically, observations were made regarding the pig's mental alertness, body condition, appetite, activity level, and clinical signs of respiratory or systemic disease. serum was collected on À , , , , and dpe (sera from group pigs was collected on À , , , and dpe), and pigs were weighed on À , and dpe (group were weighed on À , and dpe). necropsy was scheduled on dpe (dpe for group ), or sooner if pigs died or were euthanized due to severe disease. at necropsy, a thorough post-mortem examination was performed, and a complete set of samples was collected for evaluating disease severity. tracheobronchial lymph nodes (tbln) and thymic tissue were weighed (lnw and tw, respectively) and compared to respective body weights (bw) to derive a tissue mass index (lnw/bw, tw/bw) measuring the effect of prrsv infection on organ weight (mengeling et al., ) . upon removal, lungs were examined and extent of macroscopic lung lesions was estimated, as previously described, and reported as a percentage of lungs affected (halbur et al., ) . sections of tissues (lung, tracheobronchial lymph node, trachea, thymus, heart, tonsil, spleen, iliac lymph node, mesenteric lymph nodes, ileum, bone marrow, kidneys, liver, inguinal lymph node, cerebrum, brainstem, cerebellum and ventral midbrain) were collected into % neutral-buffered formalin for histopathology and immunohistochemistry. tbln were collected for rna extraction and cytokine protein assays. bronchoalveolar lavage fluid (balf) was collected after removing whole lungs from pigs and aseptically lavaging with ml antibiotic-free mem. tissues were processed by routine histopathologic procedures and slides were stained with hematoxylin and eosin. a boardcertified veterinary pathologist blinded to treatment groups evaluated microscopic lesions. lung sections were scored on a -point scale that accounted for distribution and severity of interstitial pneumonia: -no lesions, -mild, focal to multifocal interstitial pneumonia (o % of lung section affected), -moderate, multifocal to coalescing ( - % of lung section affected), -severe, patchy to coalescing and extensive ( - % of lung section affected), and -severe and diffuse ( % of lung section affected). immunohistochemistry prrsv-specific antigen was detected in lung tissues using a previously described immunohistochemical (ihc) method with minor modifications (halbur et al., ) . briefly, tissue sections were deparaffinized and hydrated in distilled water. slides were quenched in % hydrogen peroxide for min, rinsed three times in distilled water and treated in . % protease for min. slides were then rinsed three times in distilled water. a primary monoclonal antibody (mab) cocktail of one part : prrsv sdow (rti, brookings, sd) and one part : prrsv sr (rti, brookings, sd) was applied to the slides and were incubated at room temperature for h. bound mabs were stained with peroxidase-labeled anti-mouse igg followed by chromogen using the dako lsab -hrp detection system (dako, carpinteria, ca) according to the manufacturer's instructions. , -diaminobenzidine (dab; vector laboratories, burlingame, ca) was applied to the slides for min. the slides were rinsed in deionized water and counterstained with gill's hematoxylin. prrsv labeling was graded on a point scale of : none, : mild scattered signals ( to cells in entire section), : moderate scattered signals (less than or equal to % of high power fields (hpf) containing immunolabeling) and : abundant scattered signals (greater than % of hpf contain labeling and/or there are at least - groups of cells or more with staining). virus isolation was attempted on all serum and balf samples as described previously (faaberg et al., ) . those samples that were positive by virus isolation were then titered by serial dilution on marc- cells to determine the quantity of virus present to produce a cytopathic effect in % of inoculated tissue culture cells (tcid ). quantitative rt-pcr (qrt-pcr), as previously described (faaberg et al., ) , was used to determine the amount of viral rna per ml of serum and balf, and per gram of tbln homogenized tissue prepared as described below. serum samples were tested on study days À , and for evidence of seroconversion with the prrs xr enzyme-linked immunosorbent assay (herdchek elisa; idexx laboratories). a sample was considered positive for antibodies to prrsv nucleocapsid protein if the sample-to-positive (s/p) ratio was equal to or greater than . . levels of tnfa, il- b, il- , il- , il- , il- , il- , il- p and ifng cytokine levels (pg/ml) were measured in serum, balf and tbln samples diluted : with dilution buffer supplied by the manufacturer using a searchlight (aushon biosystems, woburn, ma, usa) customized multiplex immunoassay (m-elisa) following the manufacturer's protocol. m-elisa samples were assayed in duplicate. the elisa limits of detection for tnfa, il- b, il- , il- , il- , il- , il- , il- p and ifng were . , . , . , . , . , . , . , . , and . pg/ml respectively. approximately g of tbln was homogenized in ml of lysis buffer containing . % triton x- , mm nacl, mm tris, mm cacl , and mm mgcl , ph . , using a tissue homogenizer (biospec products, bartlesville, ok) (greenberger et al., ) . homogenates were incubated on ice for min, then centrifuged at x g for min. supernatants were collected, passed through a . micron filter (gelman sciences, ann arbor, mi), then stored at À c prior to assessment of cytokine levels. ifna protein was measured with a porcine ifna specific elisa by using f monoclonal antibody (mab) and k mab (r&d systems inc.) as previously described (miller et al., ) . mab k was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). immulux hb flat-bottomed -well plates (dynex technology, chantilly, va) were coated overnight at c with f at a concentration of mg/plate in coating buffer ( mm carbonate buffer, ph . , sigma inc., st. louis, mo). after blocking with % non-fat dried milk, . % tween in phosphate buffered saline (pbs) for h at c, the plates were washed three times with . % tween in pbs. samples ( ml) were added into each well containing ml of % non-fat dried milk, . % tween in pbs and incubated for h at c. following three washes, ml of peroxidase conjugated k was added to each well. after h incubation, at c, and three washes, ml of substrate solution, tetramethylbenzidine (kpl inc., gaithersburg, md), was added to each well. after min, the reaction was stopped with tetramethylbenzidine stop solution (kpl inc., gaithersburg, md) and the optical density was measured at nm by an elisa plate reader. quantified recombinant porcine ifna (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifna concentrations were calculated based upon a standard curve. one unit/ml of rifna is equivalent to pg/ml. bacterial culture was performed by plating ml of balf both on a casman's agar plate supplemented with . % nicotinamide adenine dinucleotide (nad) and % horse serum, and on a % sheep's blood agar plate. both agar plates were incubated for h at c. bacterial identification was performed by s rrnaspecific pcr and dna sequencing. s rrna-specific pcr and dna sequencing whole-cell bacterial lysates, used as templates, were prepared by suspending a colony, $ mm in diameter or the equivalent, in ml of sterile water. the mixture was boiled for min, placed on ice until chilled, and centrifuged at ,  g for min to pellet cell debris and stored at À c. supernatant ( ml) was used as the template in each pcr. the forward primer ( -agagtttgatcctggctcag- ), designated univ s- , is homologous to a highly conserved sequence from the end of the s rrna gene and the reverse primer ( -gcggctgctggcacg- ), designated univ s- , is homologous to a highly conserved sequence between the third and fourth variable regions of the s rrna gene. this previously described primer set generates an amplicon of approximately bp (register and yersin, ) . reactions were carried out in a volume of ml and contained u amplitaq polymerase (applied biosystems, foster city, ca), ml x buffer ii ( mm tris-hcl , ph . , mm kcl), ml dimethyl sulfoxide, . mm mgcl , . mm primers, and mm deoxynucleoside triphosphates. an initial denaturation step of min at c was followed by cycles of s at c, s at c, and min at c, with a final extension step of min at c. ml of each pcr was analyzed by agarose gel electrophoresis and pcr products were purified with spin columns (promega, madison, wi) and sequenced directly by fluorescence-based cycle sequencing with amplitaq and bigdye terminators on an abi sequencer at the national animal disease center genomics unit. sequences were analyzed using geneious . software (biomatters ltd, auckland, new zealand). quantitative virus copy numbers and serum cytokine levels were analyzed using a mixed linear model for repeated measures (proc mixed, sas . for windows, sas institute, cary, nc, usa). linear combinations of the least squares means estimates for each variable were used in a priori contrasts after testing for either a significant (po . ) effect of prrsv challenge strain (vr- , rjxwn , rjxwn contacts or sham inoculated controls). comparisons were made between groups at each time-point using a % level of significance (po . ) to assess statistical differences. log transformed virus copy numbers and cytokine levels in balf and tbln homogenates were analyzed by analysis of variance using a general linear model for unbalanced data (proc glm, sas . for windows, sas institute, cary, nc, usa). a % level of significance (po . ) was used to assess statistical differences. geometric mean back transformations were made for final data presentation in figures and tables. mention of trade names or commercial products in this article is solely for the purpose of providing specific 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characterization of porcine sarm and its role in regulating tlrs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo highly virulent porcine reproductive and respiratory syndrome virus emerged in china the authors would like to recognize ann vorwald, sarah anderson, deb adolphson and amanda burow for their excellent technical assistance, and jason huegel, brian pottebaum, and jason crabtree for their exceptional animal care. we also appreciate the suggestions on manuscript layout and data organization made by crystal loving. key: cord- -d f a ds authors: pensaert, m. b.; de bouck, p. title: a new coronavirus-like particle associated with diarrhea in swine date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: d f a ds coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. in , do yr.e and hutc~i~gs ( ) described a viral diarrhea, in swine and called it transmissible gastroenteritis. until recently, transmissible gastroenteritis virus was the only virus known to be specifically associated with diarrhea in swine of all ages. in , following the discovery of rotaviruses in different, animal species, a porcine rotavirus was detected in the feces of pigs with diarrhea ( ) . diarrhea could be reproduced experimentally in piglets with this virus. in a search for rotaviruses on belgian swine breeding farms with diarrheal problems, a new coronavirus-like particle was detected b y electron microscopic examination of intestinal or fecal samples from sick pigs. the present report describes the morphology of this coronavirus-like particle, and shows that it is distinct from the known porcine coronaviruses and causes diarrhea. up to now, the only coronaviruses isolated from swine have been transmissible gastroenteritis virus {tgev) and hemagglutinating encephalomyelitis virus (hev). tgev has been described as a cause of diarrhea in swine in countries all over the world ( ) . numerous studies have been performed on the v i r u s --a n i m a l interactions of tgev, which is usually detected either by its isolation from fecal these studies were supported by the institute for the encouragement of l~esearch in industry and agriculture (iwonl), brussels, belgium. material in cell cultures or by immunoflu orescence in the smm intestinal epithelium of infected pigs ( , ) . tgev infections can also be diagnosed serologically. j:iev was first described in canada in as a cause of centrm nervous disorders in pigs ( ). the same virus was later associated with a disease syndrome called vomiting and wasting disease in several european countries ( , ) . the virus can easily be detected by cultivation in several porcine cell cultures ( ). both tgev and hev have been classified as coronaviruses mainly on the basis of their specific morphology ( ). in , a sudden outbreak of diarrhea was observed in swine of all ages on belgian swine breeding farms. the morbidity in sows was very variable and the animals recovered after a diarrhea which lasted to days. all the pigs showed a watery diarrhea. death occurred up to the age of days and the overali mortality rate in these piglets was approximately per cent ( ) . it decreased with increasing age. tgev was suspected as the cause of this diarrhea. however, the direct immunofluorescence test for the diagnosis of tgev, which is routinely applied on cryostat sections of the small intestine of sick pigs, was negative for these pigs. the absence of seroneutralizing antibodies to tgev in the blood of sows collected to weeks after the outbreak confirmed that tgev was not involved. in an attempt to arrive at an etiologie diagnosis, fecal material and intestinal contents from pigs of each farm were subsequently processed for examination in an electron microscope by negative staining. they were diluted t to (v/v) in phosphate-buffered saline, ph . and clarified at × g, at ° c, for minutes. the supernatant was layered on top of a per cent sucrose solution and centrifuged at , x g, at ° c, for minutes. the resulting pellet was resuspended in a few drops of distilled water, placed on mesh formvar coated grids, and stained with per cent k-phosphotungstate, ph . . grids were examined using a zeiss em s- electron microscope at an acceleration voltage of kv. micrographs used for particle size measurement were taken at an instrumental magnification of , x, which were then photographically-enlarged to , x or , ×. gotavirus particles were not detected. however, eoronavirus-like particles were observed in specimens of pigs from each of the breeding farms. one of the fecal samples containing these coronavirus-like particles was designated cv and was used for further studies. the etiologic relationship between the corona virus-like particles, cv , and the occurrence of diarrhea was established by oral inoculation of a per cent suspension of the fecal material contmning cv into a one day old colostrumdeprived pig. the experimental pig was killed hours later, at the height of diarrhea, and a virus stock was prepared from an homogenate of its small intestine and contents. a bacteria free filtrate of the supernatant of a per cent suspension of this material was used for inoculation of colostrum-deprived-hysterectomy-derived piglets, kept i n isolation. seven control pigs were used. the pigs were inoculated at the age of to days. all the inoculated pigs developed a watery diarrhea within to hours after inoculation whereas the control animals remained normal. coronavirus-like particles were detected by electron microscopic examination in the watery feces or intestinal contents of each of the experimentally inoculated pigs. such particles were not found in the feces of the same pigs prior to inoculation or in the fecal samples of the control animals. the particles, shown in figure , had typicm coronavirus morphology. they were pleomorphic with a range in diameter of to nm, including the projections, which were approximately nm in length. most particles were between t and t nm in diameter. the projections formed a single fringe radiating from the core. they appeared to be club-shaped. only the dilated distal ends of the projections were seen on the micrographs. the negative stain also appeared to settle on the surface of some particles and an electron opaque central area covered by surface projections was often seen (fig. a --a r r o w s and lc) . no internal structure was observed. it was impossible to distinguish these coronavirus-like particles morphologically from tgev or h e v particles from similar preparations. other particles, different from these coronavirus-like particles, were also observed in the majority of the fecal samples. as seen in figure , they were pleomorphic and very variable in size, ranging in diameter from to nm with an average diameter of to rim. they carried numerous short projections, of approximate length nm, on their surfaces. similar particles of unkno~m identity have been described in human and animal fecal samples ( , , ) . in the present studies such particles have also been found in the solid fecal samples of the control pigs. they appeared, therefore, not to be associated with diarrhea. rotaviruses and other recognizable virus particles were not seen in control or experimentally inoculated pigs. as already mentioned, tgev was eliminated as the cause of the diarrhea on the origina farms. additionmly out of the experimentally inoculated pigs, killed at, the heigth of diarrhea, were negative for tge viral antigens in their small intestinal epithelium by the direct immunofluorescenee test. furthermore, the remaining pigs, inoculated with cv at the age of days, were allowed to recover after a diarrhea which lasted -- days. a serum sample, collected from these pigs weeks later, did not contain neutralizing antibodies against the cell culture adapted purdue strain of tgev. fig. . one eoronavirus-like particle cv (arrow) together with pleomorphie particles of unknown identity. bar represents nm the possibility that the cv particles consisted of h e v was less likely since the latter virus does not cause diarrhea in pigs. cryostat sections of the sma~ intestine of experimentmly inoculated pigs were negative for fluorescence by the direct test using a conjugate directed against the vw isolate of h e v ( ) . furthermore, the pigs that had been allowed to recover did not possess hemagglutination-inhibiting or seroneutralizing antibodies against this t t e v isolate. preliminary attempts were made to cultivate the coronavirus-like particle, cv , in primary pig kidney cell cultures and in secondary porcine thyroid cells. four weekly blind passages were made. the cells were examined for cytopathic effect and hemadsorption with chicken red blood cells, and the cell culture fluids were examined for hemagglutination. no evidence of viral replication in the cell cultures was obtained. it is kno~n that the tiev can easily be isolated in primary pig kidney cell cultures using the same criteria ( ) . the present data suggest that, as well as tgev and hev, another previously unrecognized coronavirus-like virus is prevalent in swine. the results indicate that diarrhea can be reproduced in experimental pigs with this virus and that it is associated with certain outbreaks of epizootic diarrhea on belgian swine breeding farms. more details on the clinical disease in the field and on the results of the experim e n t a l infections will be reported later. vomiting and wasting disease of piglets a transmissible gastroenteritis in pigs the hunt for viruses in infections of the alimentary s y s t e m : a n immuno etectronmicroscopicat approach a hemagglutinating virus producing encephalomyelitis in baby pigs pleomorphic virus-like particles in human faeces virus-like particles in calves' faeces diagnosis of transmissible gastroenteritis in pigs by immunofluorescence characteristics of a coronavirus causing vomition and wasting in pigs a virus isolated from an apparently new epizootic diarrhea in swine. proe. th world int the size and morphology of transmissible gastroenteritis and vomiting and wasting disease viruses of pigs titration of two porcine respiratory viruses in mammalian cell cultures by direct fluorescent antibody staining isolation of the virus of transmissible gastroenteritis (tge) from naturally infected piglets in cell culture transmissible gastroenteritis of swine the isolation of reovirus-like agents (rotaviruses) from acute gastroenteritis of piglets key: cord- -tigj fhc authors: cleveland, christopher a.; denicola, anthony; dubey, j.p.; hill, dolores e.; berghaus, roy d.; yabsley, michael j. title: survey for selected pathogens in wild pigs (sus scrofa) from guam, marianna islands, usa date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: tigj fhc pigs (sus scrofa) were introduced to guam in the ’s and are now present in high densities throughout the island. wild pigs are reservoirs for pathogens of concern to domestic animals and humans. exposure to porcine parvovirus, transmissible gastroenteritis, and leptospira interrogans has been documented in domestic swine but data from wild pigs are lacking. the close proximity of humans, domestic animals, and wild pigs, combined with the liberal hunting of wild pigs, results in frequent opportunities for pathogen transmission. from february–march , blood, tissue and ectoparasite samples were collected from wild pigs. serologic testing found exposure to brucella spp. ( %), toxoplasma gondii ( %), porcine reproductive and respiratory syndrome (prrs) virus ( %), porcine circovirus type ( %), pseudorabies virus ( %), actinobacillus pleuropneumoniae ( %), lawsonia intracellularis ( %), and porcine parvovirus ( %). eleven ( %) samples had low titers ( : ) to leptospira interrogans serovars bratislava (n = ), icterohaemorrhagiae (n = ), pomona (n = ), and hardjo (n = ). kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens. numerous pigs had metastrongylus lungworms and three had stephanurus dentatus. lice (hematopinus suis) and ticks (amblyomma breviscutatum) were also detected. no antibodies to influenza a viruses were detected. in contrast to the previous domestic swine survey, we found evidence of numerous pathogens in wild pigs including new reports of pseudorabies virus, prrs virus, brucella, and leptospira in pigs on guam. these findings highlight that domestic swine-wild pig interactions should be prevented and precautions are needed when handling wild pigs to minimize the risk of pathogen transmission. wild pigs are nearly globally distributed and are hosts for many parasites and bacterial and viral pathogens, some of which are transmissible to agricultural animals, wildlife, and humans (barrios-garcia and ballari, ; bevins et al., ) . numerous studies have investigated the prevalence and distribution of various pathogens in wild pigs and the risk they pose for pathogen transmission in the united states and europe, but there are relatively few reports from southeast asia (baroch et al., ; hill et al., ; pedersen et al., pedersen et al., , pepin et al., ) . wild pig populations are increasing dramatically on several pacific islands, yet data on pathogen exposure are limited or absent. wild pigs were introduced to guam, an unincorporated territory of the united states located in the marianna island chain, in the late 's and despite current liberal hunting regulations, populations continue to increase (conry, ) . this population increase has led to severe ecological damage and agricultural losses. the only report of swine pathogens on guam was limited to domestic pigs (dugies et al., ) . the lack of data on pathogens in wild pigs is a concern as increased swine populations have led to increased pig-human interactions. recent reports of leptospirosis in residents and tourists is one concern and although rodents tend be considered the most common reservoir of leptospira, there is evidence of leptospira in domestic swine on guam (dugies et al., ) , and wild pigs in numerous countries have antibodies to leptospires (jansen et al., ; corn et al., ) . in , there was an effort to decrease wild pig populations within fenced areas of two military bases on guam to minimize the ecological damage caused by the pigs. samples collected from these animals were used to conduct a comprehensive surveillance project on pathogen exposure of wild pigs on guam. andersen air force base (aafb) is a ha military installation in northern guam ( . °n, . °e) . forested portions of the base contain high quality native habitat, some of which is included in the guam national wildlife refuge. the naval base guam naval munitions site (nbg nms) is centrally located on the island and covers approximately acres ( . °n . °e). removal of wild pigs was conducted via strategic sharp-shooting techniques conducted by well-trained shooters using suppressed . caliber rifles mounted with scopes. during removal, shooters only fired if the situation met the following criteria: ) there was certainty that the animal would be dispatched and not escape, ) if other animals were nearby, every animal had a high probability of being dispatched, and ) it was safe to dispatch the animal. the shooting methods followed the american veterinary medical association's guidelines for humane euthanasia of animals (avma, ) . pigs were aged based on tooth eruption patterns and wear. animal and sample collection procedures were reviewed and approved by uga's institutional animal care and use committee (a - ). immediately after euthanasia, blood samples were collected via cardiocentesis and placed into ethylenediaminetetraacetic acid (edta) and plain tubes (greiner bio-one, monroe, nc). clotted blood was centrifuged at g for min and serum was removed and frozen at − °c until diagnostic testing. whole blood was also frozen at − °c until testing. representative ectoparasites were collected and preserved in % ethanol. information on pathogens we screened pigs for as well as diagnostic assays and diagnostic laboratories used are listed in table . most pigs were serologically tested for all of the pathogens listed; however, due to limited sample volume, some pigs were only tested for selected pathogens. small sections of lung were fixed in formalin and processed for routine histology. small sections of kidneys were also fixed in formalin and if antibodies to leptospira were detected, they were tested for leptospira antigens by immunohistochemistry (ihc). if any gross lesions were noted during necropsy, they were also collected in formalin for histologic examination. pigs were not systematically examined for internal parasites, but if any were seen they were collected in formalin for identification. body weights were compared between male and female swine using a two-sample t-test. prevalence of pathogen exposures was compared between males and females and between adult and juvenile swine using fisher's exact test. all testing assumed a two-sided alternative hypothesis and p < . was considered statistically significant. analyses were performed using commercially available statistical software (stata version . , statacorp lp, college station, tx). from the two sites, a total of wild pigs were sampled including six ( . %) juveniles and ( . %) adults; ( . %) were males and ( . %) were females. weight (kilograms) was recorded for individuals: juveniles and adults. all juveniles weighed < . kgs and the mean ± sd weight of the adult males ( . ± . ) was significantly greater than that of the adult females ( . ± . ; p < . ). all pigs were positive for at least one of the pathogens included in the study. exposure to or infection with influenza a virus (iav), brucella, porcine epidemic diarrhea virus, babesia spp., transmissible gastroenteritis/porcine respiratory coronavirus and trichinella spp. was rare or absent (table ). based on antibody testing, a high percentage of pigs were exposed to actinobacillus pleuropneumonia (app; %), lawsonia intracellularis ( %), and porcine parvovirus ( %). exposure to multiple serotypes of app was also common ( table ). based on mat testing, antibodies to four leptospira serovars were detected in pigs (tables and ) , with some individuals having antibodies to more than one serovar. kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens by ihc. juvenile pigs had a significantly higher prevalence of antibodies to porcine circovirus type ( / [ %]) compared with adults ( / ; p = . , respectively). there was no difference in prevalence between males and females for any pathogen. only pigs were examined for ectoparasites; lice (hematopinus suis) and ticks (amblyomma breviscutatum) were found on ( %) and seven ( %) pigs, respectively. lung samples from pigs were examined histologically and eight ( %) were positive for metastrongylus lungworms. stephanurus dentatus were found in abdominal lesions from three pigs and one pig had a liver abscess with intralesional nematode larvae which could not be identified. our results indicate that wild pigs on guam are exposed to multiple pathogens of zoonotic and agricultural importance. guam has many free-range pig operations which have an increased risk of domestic pigwild pig interactions. previous work on guam was restricted to domestic pigs and in contrast to our data, domestic pigs were exposed to relatively few pathogens (duguies et al., ) . clinically, several diseases were noted, but the only pathogen detected by serologic testing was parvovirus ( / , %). although domestic pigs were not serologically tested for app or l. intracellularis, porcine pleuropneumonia and proliferative enteritis were noted. domestic pigs tested negative for antibodies to pseudorabies virus (n = ), porcine reproductive respiratory syndrome virus (n = ), brucella spp. (n = ), leptospira spp. (n = ), swine influenza virus (n = ), transmissible gastroenteritis virus (n = ), mycoplasma hyopneumoniae (n = ), and trichinella spp. (n = ) while we found serologic evidence of the first four pathogens in wild pigs (duguies et al., ) . porcine pleuropneumonia, caused by app, is a highly infectious disease that is economically important for domestic swine. although known to occur on guam among domestic pigs, prevalence was unknown (duguies et al., ; bossé et al., ) . clinical disease and mortality resulting from infection with one or more app serotypes can vary geographically; however, serotype has been consistently associated with morbidity and mortality in domestic pigs (baroch et al., ) . over half of the wild pigs from guam had antibodies for serotypes - - - . reproductive disease in domestic pigs due to porcine parvovirus (ppv) has been reported on guam (duguies et al., ; ruiz-fons et al., ) . in addition to reproductive failure due to ppv infection, coinfection with porcine circovirus type (pcv ) in domestic pigs can result in mortalities (ellis et al., ) . we report in wild pigs the first evidence of pcv on guam; surveillance and vaccination for these pathogens may be warranted. l. intracellularis has been documented worldwide in domestic and wild pigs and is the etiologic agent of proliferative enteropathy (chiriboga et al., ) . the high prevalence of these three pathogens in wild pigs, indicates that transmission to domestic pigs is a risk. exposure to leptospira interrogans and toxoplasma gondii, two important zoonoses, was common in wild pigs on guam. leptospirosis is a growing concern in guam ( mason et al., ) . while exposure to several serotypes was detected, prevalence is likely underestimated because antigens from serotypes readily available for testing in our us laboratory were used rather than those likely present in guam/other pacific nations. regardless, exposure of pigs does not implicate them in human cases due to the diverse reservoir-range, but suggests that proper ppe should be utilized when in contact with pigs. domestic pigs have not been previously tested for t. gondii but evidence of t. gondii was noted in goats (duguies et al., ) . this parasite has a wide host range and can pose a risk to native avian fauna that do not have an evolutionary history with the parasite (work et al., ) . in addition, t. gondii is zoonotic; due to the popularity of wild pig hunting for sport and population management, hunters in guam should properly butcher and cook wild pig meat (hill et al., ; conry ) . several pathogens were not detected or were rare in wild pigs. no evidence of exposure to trichinella spp., iav, or coronaviruses associated with enteric and respiratory disease was found; one pig had antibodies to porcine epidemic diarrhea virus. previously, trichinella was absent from domestic pigs, but because of the zoonotic potential of this parasite, surveillance and education campaigns to ensure pork is thoroughly cooked (which would also kill any t. gondii present) should continue. swine influenza, a zoonosis, is relatively common in domestic pigs worldwide, but exposure of wild pigs is not ubiquitous and varies geographically (smith et al., ) . while prevalence was high in a study in southern china (luo et al., ) , in korea, the us and spain, seroprevalence was generally low (< %) and varied seasonally; it is possible that our sample size was insufficient to detect exposure in guam (hall et al., ; corn et al., ; feng et al., ) . enteric and respiratory coronaviruses, which have been reported in several asian countries, are of significant concern due to high morbidity and mortality in naïve pig populations (song and park, ) . porcine epidemic disease virus is clinically similar to transmissible gastroenteritis; both are coronaviruses that can lead to acute diarrhea in all age groups of swine, often resulting in poor body condition or mortalities (lee, ) . one wild pig had antibodies to brucella; the species could not be distinguished based on serologic testing. serology indicates if brucella is present in a population, but culture and/or advanced molecular assays are required to confirm species (leiser et al., ) . brucellosis, a zoonotic disease, has been documented in wild pig populations globally (leiser et al., ) . lice (h. suis) were common on guam, similar to studies on domestic and wild pigs worldwide (girişgin et al., ) . h. suis can transmit swine poxvirus and classical swine fever virus but there is no evidence that these viruses are present on guam. amblyomma breviscutatum were found on several pigs but little is known about this tick species; it is believed to have been historically present on guam and other islands in greater micronesia (vander velde and vander velde, ) . currently, there are no data on pathogens associated with a. breviscutatum. our testing method for helminth parasites was limited due to time and cleveland et al. veterinary microbiology ( ) - importation restrictions; we only examined small sections of lung tissue (or lesions if noted). confirmed infections with metastrongylus lungworms and s. dentatus were relatively common, and likely underrecognized because of testing method. both metastrongylus lungworms and s. dentatus, previously reported in domestic pigs on guam, can impact domestic pig health (duguies et al., ) . our study highlights the importance of surveillance efforts for pathogens transmitted by wild pigs on guam as many of the pathogens circulating in wild pigs can cause disease in domestic swine. we detected serologic evidence for multiple zoonotic pathogens, necessitating simple yet effective preventative measures. wearing appropriate ppe, practicing good hygiene, washing all utensils and surfaces that have come into contact with butchered wild pigs, and thoroughly cooking meat prior to consumption may assist in preventing infection. management of wild pigs is often controversial as they are seen as a food source for hunters or in some cases may be considered native species. the wild pig populations on guam are not native, harbor pathogens of importance to domestic swine and people, and very importantly, cause major damage to the environment leading to economic losses for farmers and extreme habitat destruction for native species of wildlife. guidelines for the euthanasia of animals, edition. avma, schaumburn, il exposure of feral swine (sus scrofa) in the united states to selected pathogens impact of wild boar (sus scrofa) in its introduced and native range: a review consequences associated with the recent range expansion of nonnative feral swine actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection detection of lawsonia intracellularis in faeces of swine from the main producing regions in brazil management of feral and exotic game species on guam survey of selected diseases in wild swine in texas pathogen exposure in feral swine populations geographically associated with high densities of transitional swine premises and commercial swine production animal health survey for guam coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome influenza a subtype h viruses in feral swine occurrence of haematopinus suis linnaeus, (insecta, anopluridae) on a wild boar (sus scrofa) influenza exposure in united states feral swine populations surveillance of feral swine for trichinella spp. and toxoplasma gondii in the usa and host-related factors associated with infection leptospirosis in urban wild boars porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus feral swine brucellosis in the united states and prospective genomic techniques for disease epidemiology exposure to swine h and h and avian h and h influenza a viruses among feral swine in southern china leptospira interrogans antibodies in feral pigs from new south wales identification of brucella suis from feral swine in selected states in the usa widespread detection of antibodies to leptospira in feral swine in the united states contact heterogeneities in feral swine: implications for disease management and future research seroprevalence of six reproductive pathogens in european wild boar (sus scrofa) from spain: the effect on wild boar female reproductive performance diversity of piroplasms detected in blood-fed and questing ticks from several states in the united states origins and evolutionary genomics of the swine-origin h n influenza a epidemic porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines known and potential ticks and tick-borne pathogens of micronesia fatal toxoplasmosis in free-ranging endangered 'alala from hawaii primary financial support for this project came from the us department of the navy (n - -r- and n - -r- ). additional financial support was provided by the wildlife management agencies of the southeastern cooperative wildlife disease study member states through the federal aid to wildlife restoration act ( stat. ) and by the us department of the interior cooperative agreement g ac . the authors thank the personnel at the diagnostic laboratories for diagnostic support and r.l. poulson for comments on this manuscript. key: cord- - i mk x authors: hryhorowicz, magdalena; lipiński, daniel; hryhorowicz, szymon; nowak-terpiłowska, agnieszka; ryczek, natalia; zeyland, joanna title: application of genetically engineered pigs in biomedical research date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: i mk x progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. the development of transgenesis has created new directions in research and possibilities for its practical application. generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. the further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. numerous studies have shown that the pig (sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species this review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models. pigs have been extensively used in biomedical research due to anatomical and physiological similarities to humans. moreover, progress in gene editing platforms and construct delivery methods allow efficiently, targeted modifications of the porcine genome and significantly broadened the application of pig models in biopharming and biomedicine. target editing is possible through site-specific nucleases, of which the following are most commonly used: zinc finger nucleases (zfns), transcription activator-like effectors (tales), and nucleases from the crispr/cas (clustered regularly interspaced short palindromic repeats/crispr associated) system. introducing modifications in a specific site of the genome is possible due to cellular processes of repairing double-strand breaks induced by site-specific nucleases. double-strand breaks may be repaired in two ways: by non-homologous end joining (nhej) or by homologous recombination (hr). repair provided by nhej may lead to the formation of indel (insertion/deletion) mutations in the table . summary of the most important advantages and disadvantages of the methods for obtaining genetically modified animals. advantages increased efficiency of the transgene integration precise transformation and selection of modified cells used in cloning the number of damaged zygotes do not exceed % the obtained animals do not exhibit mosaicism modification is also revealed in germ cells-transgenic offspring disadvantages low process efficiency ( %- % in pigs) very low efficiency the possibility of random integration of the transgene early fetal mortality high process invasiveness the possibility of genetic defects genetically modified animals as research models for human diseases are a very important tool in searching for and developing new methods of therapy. a suitable model organism should be characterized by rapid growth, a high number of offspring, easy and inexpensive breeding, ability to be easily manipulated, and having a sequenced genome. initially, only rodents were used as a model in biomedical research. experiments on mice contributed to understanding the genetic background of numerous diseases. however, not every genetic disease induced in mice has the same clinical manifestation as in humans. furthermore, the short life span, together with a higher metabolic rate, makes the analysis of some hereditary diseases challenging. currently, pigs are one of the most important large animal models for biomedical research. many human diseases, such as cardiovascular diseases, obesity, and diabetes, have their counterparts in this species. the use of model organisms makes it possible to analyze diseases that occur naturally in animals with specific mutations and those deliberately induced. introduced animal genome changes may reflect mutations occurring in people suffering from specific genetic disorders. moreover, accurate and efficient genome editing can be used in the treatment of monogenic diseases. a slightly different direction of research is the use of genetically modified animal models in toxicological studies for testing drugs. genetically modified pigs are used as model organisms in research into various diseases, including cardiovascular and neurodegenerative diseases, neoplasms, and diabetes. cystic fibrosis (cf) is an autosomal recessive disorder manifested by bronchopulmonary failure and pancreatic enzyme insufficiency. cf is caused by a mutation in the gene responsible for the synthesis of the cftr (cystic fibrosis transmembrane conductance regulator) chloride channel, altering the mucosal function in the respiratory epithelium, pancreatic ducts, intestines, and sweat glands. the most common cystic fibrosis-causing mutation is the deletion of a phenylalanine at amino acid position (df ). cf lung disease is the main cause of morbidity and mortality in cf patients. porcine lungs share many anatomical and histological similarities with humans. it has been shown that pigs in which the cftr gene was inactivated develop all symptoms of the disease occurring in humans, such as meconium ileus, defective chloride transport, pancreatic destruction, and focal biliary cirrhosis. this makes them a very good model species for this disease [ ] [ ] [ ] . cystic fibrosis is a monogenic disease, and the insertion of the functional cftr gene into cf patient cells should theoretically restore the cftr channel function. therefore, pigs have also been used in gene therapy. the treatment with viral vectors successfully improved anion transport and inhibited bacterial growth [ , ] . currently, the research focuses on improving the cf gene therapy with the use of the crispr/cas system. these efforts are focused on increasing the delivery efficiency of crispr/cas elements to target locus and obtaining sustained expression of the cftr transgene [ , ] . it was demonstrated that precise integration of the human cftr gene at a porcine safe harbor locus through crispr/cas -induced hdr-mediated knock-in allowed the achievement of persistent in vitro expression of the transgene in transduced cells. these results can help design effective gene therapy to treat cf patients [ ] . duchenne muscular dystrophy (dmd) is a progressive, monogenic, x-linked lethal disease characterized by degenerative changes in muscle fibers and the connective tissue. it involves the degeneration of subsequent muscles-skeletal, respiratory, and cardiac-and progressive muscular dystrophy. muscular dystrophy is caused by a frameshift mutation in the dmd gene, which encodes dystrophin, a protein in muscle cells that connects the cytoskeleton with the cell membrane. the dystrophin gene contains exons, with exons - and - being the most susceptible to mutations. dmd gene mutations are usually large deletions or duplications of one or several exons, as well as point mutations, leading to a change in the reading frame, the appearance of a premature stop codon, and failure to produce a stable protein. muscular dystrophy is most often diagnosed in early childhood, and patients become wheelchair dependent by years of age. untreated boys die of cardiorespiratory complications around their years. the rapid progress in gene editing gives hope for effective targeted therapies for dmd. moreover, the use of an animal model can facilitate the development of personalized treatment approaches. pigs with a dmd gene mutation (exon deletion) develop human disease symptoms, such as lack of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive muscle dystrophy, and impaired mobility [ ] . however, these animals died prematurely (up to months old at most) what precluded natural breeding. the histological evaluation of skeletal muscles and diaphragm confirmed the presence of excessive fiber size variation, hypercontracted fibers, and segmentally necrotic fibers, resembled that of human dmd patients [ ] . moreover, proteome analysis of biceps femoris muscle was performed. an increased amount of muscle repair-related proteins and reduced amount of respiratory chain proteins was found in tissue from -month-old dmd pigs. this indicated severe disturbances in aerobic energy production and a decrease in functional muscle tissue [ ] . as the deletion of exon in the human dmd gene is a common cause of duchenne muscular dystrophy, pigs can make an accurate research model for gene therapy. another porcine dmd model is genetically modified miniature pigs with a mutation in exon in the dmd gene obtained by the crispr/cas system. in addition, these animals have shown symptoms of skeletal and heart muscle degeneration, characteristic of human patients with duchenne muscular dystrophy. reduced thickness of smooth muscle in the stomach and intestine was also observed in the pigs studied. however, founder pigs died of unreported causes [ ] . although mutations in exon are not reported in human dmd patients, pigs with this deletion constitute another useful animal dmd model. recently, moretti et al. demonstrated the restoration of dystrophin by intramuscular injection of crispr/cas components with the use of adeno-associated viral vectors in a pig model. in this study, pigs with dmd carrying a deletion of dmd exon (d dmd), resulting in a complete loss of dystrophin expression, were used. the restoration of dystrophin expression was possible due to the excision of exon and the restoration of the dmd reading frame. the internally truncated d - dmd sufficed to improve skeletal muscle function, prevent malignant arrhythmias as well as prolonging lifespan of dmd pigs [ ] . in the future, this strategy may prove useful in the clinical treatment of patients with d dmd. alzheimer's disease (ad) is an age-related, progressive neurodegenerative disorder characterized by memory dysfunction followed by cognitive decline and disorientation. ad accounts for %- % of human dementia cases. familial forms of ad are caused by autosomal mutations in the genes encoding presenilin (psen ) and presenilin (psen ) and amyloid precursor protein (app). these mutations are associated with the accumulation of amyloid β (aβ) peptide in senile plaques and phosphorylated tau protein in neurofibrillary tangles (nfts), which leads to synaptic damage and neuronal dysfunction [ ] . the first ad model with the use of transgenic pigs was generated in by kragh et al. they produced göttingen mini pigs that carried a randomly integrated construct containing the cdna of the human app gene with ad causing a dominant mutation known as the swedish mutation (appsw) and a human pdgfβ promoter fragment [ ] . although the transgene was specifically expressed in brain tissue at a high level, no ad phenotype was observed in mutant pigs. the same group also obtained göttingen minipigs with the human psen gene carrying the ad-causing met ile mutation (psen m i) and driven by a cytomegalovirus (cmv)-enhanced human ubic promoter. pigs were generated with the use of a site-specific integration system-recombinase-mediated cassette exchange (rmce) [ ] . the psen m i protein was expressed and tolerated well in the porcine brain, but also in this case, no symptoms of the ad disease were noticed. therefore, this group generated double transgenic göttingen minipigs with both appsw and psen m i mutations. such a solution allowed the increase in intraneuronal accumulation of aβ [ ] . in turn, another group obtained ad transgenic pigs using a retroviral multi-cistronic vector containing three ad-related human genes: app, tau, and psen , with a total of six well-characterized mutations under the control of a fusion promoter: cmve+ hpdgfβ promoter region. they confirmed that transgenes were expressed at high levels in brain tissue and demonstrated a two-fold increase in aβ levels in the brains of transgenic pigs compared to wild-type [ ] . cancer is a genetic disease involving uncontrolled, abnormal cell growth in the blood or solid organs resulting from acquired or inherited mutations. pigs represent a useful animal for the development and validation of new medicines and procedures in human tumor models. there are many resemblances in cancer biology between pigs and humans. these animals can correctly mimic human tumors and show similar pharmacokinetic responses to humans. adam et al. demonstrated that autologous transplantation of primary porcine cells transformed with retroviral oncogenic vectors caused tumorigenesis akin to those found in humans [ ] . in turn, schook et al. induced tumor formation in pigs by introducing random transgenes that encode cre-dependent kras (kirsten rat sarcoma viral oncogene homolog) g d and tp (tumor protein ) r h oncogenic mutations (orthologous to human tp r h) [ ] . moreover, saalfrank et al. reported that porcine mesenchymal stem cells (mscs) resemble human mscs requiring disturbance of p , kras, and myc signaling pathways to become a fully transformed phenotype [ ] . at present, pig models commonly used in cancer research include the tp knock-out model of osteosarcoma and apc (adenomatous polyposis coli) mutations model of familial adenomatous polyposis (fap). tp is a known tumor suppressor gene, and a germline mutation within this gene leads to li-fraumeni syndrome, a rare, autosomal dominant disorder that predisposes carriers to cancer development. the first model of li-fraumeni syndrome using genetically modified pigs has been described by leuchs et al. they generated pigs carrying a latent tp r h mutation that can be activated by the cre-lox recombinase system [ ] . after several years of observation, it was noted that both pigs with homozygous tp knock-out and pigs with heterozygous knock-out of tp showed osteosarcoma development. the heterozygous knock-out caused the development of spontaneous osteosarcoma in older animals, while homozygous tp knock-out resulted in multiple large osteosarcomas in to -month-old pigs [ ] . moreover, sieren and colleagues generated genetically modified yucatan minipigs that carried the tp r h mutation. animals heterozygous for this mutant allele showed no tumorigenesis process, whereas homozygotes that reached sexual maturity developed lymphomas, osteogenic tumors, and renal tumors at varying rates. the tumor formations were validated by computed tomography, histopathological evaluation, and magnetic resonance imaging [ ] . familial adenomatous polyposis is an inherited disorder characterized by the development of numerous adenomatous polyps in the colon and rectum which greatly increases the risk of colorectal cancer. the mutations in the apc tumor-suppressor gene are responsible for fap and may result in a hereditary predisposition to colorectal cancer. flisikowska et al. generated gene-targeted cloned pigs with translational stop signals at codon in porcine apc (apc ), orthologous to common germline apc mutations in human fap. evaluation of one-year-old pigs carrying the apc mutation showed aberrant crypt foci and adenomatous polyps with low-to high-grade intraepithelial dysplasia, similar to tumor progression as in human fap [ ] . the apc pig model resulting in the development of polyposis in the colon and rectum can be useful in the diagnosis and therapy of colorectal cancer. cardiovascular diseases (cvds) are the major cause of morbidity and mortality worldwide. cvd is a group of disorders of the heart and blood vessels that involve coronary heart disease (such as angina and myocardial infarction), deep vein thrombosis and pulmonary embolism, peripheral arterial disease, cerebrovascular disease, and rheumatic heart disease. the dominant cause of cvd is atherosclerosis, which is characterized by the narrowing of arteries due to the accumulation of lipid and plaque formation. the plaque buildup restricts blood flow, and plaque burst can entail blood clots. similarities in heart anatomy and physiology, vessel size, blood parameters, coronary artery system anatomy, and lipoprotein metabolism make pigs a suitable model for the human cardiovascular system. atherosclerosis starts with the buildup of serum low-density lipoprotein (ldl), and mutations in the ldl receptor (ldlr) gene may cause familial hypercholesterolemia (fh). a porcine fh model has been generated in yucatan miniature pigs through recombinant adeno-associated virus-mediated targeted disruptions of the endogenous ldlr gene. ldlr+/− heterozygous pigs exhibited mild hypercholesterolemia, while ldlr−/− homozygotes animals were born with severe hypercholesterolemia and developed atherosclerotic lesions in the coronary arteries. these phenotypes were accelerated by high fat and high cholesterol diets [ ] . the utilization of ldlr-deficient yucatan minipigs in the preclinical evaluation of therapeutics was also demonstrated. ldlr+/− and ldlr−/− pigs were used to assess the ability of novel drug-bempedoic acid (bema)-to reduce cholesterol biosynthesis. long-term treatment with bema decreased ldl cholesterol and attenuated aortic and coronary atherosclerosis in this fa model [ ] . moreover, a model of fa and atherosclerosis was created by using the yucatan miniature pigs with liver-specific expression of a human proprotein convertase subtilisin/kexin type (pcsk ) carrying the gain-of-function mutation d y. pcsk plays important functions in cholesterol homeostasis by reducing ldlr levels on the plasma membrane. gain-of-function mutations in this protein cause increased levels of plasma ldl cholesterol, which in turn may result in more susceptibility to coronary heart disease. pcsk d y transgenic pigs exhibited decreased hepatic ldlr levels, severe hypercholesterolemia on high-fat, high-cholesterol diets, and atherosclerotic lesions [ ] . it is also considered that hypertriglyceridemia is an independent risk factor for coronary heart disease, in which apolipoprotein (apo)ciii is associated with plasma triglyceride levels. the hypertriglyceridemic apociii transgenic miniature pig model was generated for the examination of the correlation between hyperlipidemia and atherosclerosis. transgenic pigs expressing human apociii exhibited increased plasma triglyceride levels with their delayed clearance and reduced lipoprotein lipase activity compared to non-transgenic controls [ ] . diabetes mellitus (dm) is a group of metabolic disorders characterized by hyperglycemia (elevated levels of blood sugar over a prolonged period), which results from deficiency or ineffectiveness of insulin. dm may lead over time to cardiovascular disease, chronic kidney disease, damage to the nerves and eyes. there are two main types of diabetes mellitus, called type and type . type dm, also referred to as juvenile diabetes or insulin-dependent diabetes mellitus, is caused by the pancreas's failure to produce enough insulin. the most common is type diabetes, which is characterized by insulin resistance (reduced tissue sensitivity to insulin) that may be combined with relative insulin deficiency. the anatomical and physiological resemblance to the human pancreas and islets makes pigs excellent animals for metabolic diseases modeling. moreover, the structure of porcine and human insulin is also very similar (differs by only one amino acid). a transgenic pig model for type dm was generated to evaluate the role of impaired glucose-dependent insulinotropic poly-peptide (gip). the main function of incretin hormones gip and glucagon-like peptide- (glp ) is stimulated insulin secretion from pancreatic beta cells in a glucose-dependent manner. in type dm, the insulinotropic action of gip is impaired, which may suggest its association with early disease pathogenesis. the transgenic pigs expressing a dominant negative gip receptor (giprdn) in pancreatic cells were produced by lentiviral vectors. a significant reduction in oral glucose tolerance due to delayed insulin secretion as well as in β-cell mass caused by diminished cell proliferation was observed in giprdn animals [ ] . these observations resemble the characteristic features of human type diabetes, which makes the porcine giprdn model useful for testing incretin-based therapeutic strategies. further analyses revealed characteristic changes in plasma concentrations of seven amino acids (phe, orn, val, xleu, his, arg, and tyr) and specific lipids (sphingomyelins, diacylglycerols, and ether phospholipids) in the plasma of -month-old giprdn transgenic pigs that correlate significantly with β-cell mass [ ] . these metabolites represent possible biomarkers for the early stages of prediabetes. moreover, the porcine giprdn model has been used to test liraglutide, glp receptor agonist, which improve glycemic control in type diabetic patients. ninety-day liraglutide treatment of adolescent transgenic pigs resulted in improved glycemic control and insulin sensitivity as well as reduction in body weight gain and food intake compared to placebo-treated animals. however, the use of liraglutide did not stimulate beta-cell proliferation in the endocrine pancreas [ ] . another type of diabetes, type dm, is maturity-onset diabetes of the young (mody ). mody is a noninsulin-dependent type of diabetes with an autosomal dominant inheritance and is caused by mutations in the human hepatocyte nuclear factor α (hnf α) gene. mutation in hnf α gene leads to pancreatic β-cell dysfunction and impaired insulin secretion. a pig model for mody was generated by expressing a mutant human hnf α gene (hnf α p fsinsc) using intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. the transgenic piglets exhibited the pathophysiological characteristics of diabetes, including high glucose level and reduced insulin secretion from the small and irregularly formed langerhans islets [ ] . furthermore, hnf α p fsinsc pigs revealed nodular lesions in the renal glomeruli, diabetic retinopathy, and cataract, complications similar to those in patients with dm [ ] . mutations in the insulin (ins) gene may result in permanent neonatal diabetes mellitus (pndm) in humans. a pndm large animal model was establish by generated pigs expressing a mutant porcine ins gene (ins c y), orthologous to human ins c y. transgenic animals showed signs of pndm, such as lower fasting insulin levels, decreased β-cell mass, reduced body weight, and cataract development. in addition, ins c y pigs exhibited significant β-cell impairment, including the reduction in insulin secretory granules and dilation of the endoplasmic reticulum [ ] . the porcine ins c y model was further used to perform analysis of pathological changes in retinas and evaluation of the liver of transgenic pigs. the studies revealed several features of diabetic retinopathy, such as intraretinal microvascular abnormalities or central retinal edema [ ] . moreover, the multi-omics analysis of the liver demonstrated higher activities in amino acid metabolism, oxidation of fatty acids, gluconeogenesis, and ketogenesis, characteristic of insulin-deficient diabetes mellitus [ ] . the genetically modified pig models for human diseases described in this review are summarized in table . human-derived proteins have long been used as therapeutics in the treatment of numerous diseases. however, their quantities are limited by the availability of human tissues. thanks to the development of biotechnology and genetic engineering, modified animals can be used as "bioreactors" to produce recombinant proteins for pharmaceutical use. by using adequate regulatory sequences, promoters, the expression of transgenes can be directed to selected cells and organs. the therapeutic proteins can be obtained from milk, blood, urine, seminal plasma, egg white, or salivary gland that can be collected, purified, and used at an industrial scale. moreover, it is possible to generate multi-transgenic animals that produce many biopharmaceuticals or vaccines in a single organism. the use of an animal platform allows for the relatively low-cost production of pharmacologically valuable preparations in high quantity and quality. the mammary gland is considered to be an excellent bioreactor system for pharmaceutical protein production. the advantage of milk is that it contains large amounts of foreign proteins that do not affect the animal's health during lactation as well as the ease of product collection and purification. while cows are the best species for obtaining large amounts of pharmaceuticals in milk, the cost and time necessary to carry out successful transgenesis make rabbits, sheep, goats, and pigs more popular species. although the pig is not a typical dairy animal, a lactating sow can give about l of milk per year. velander et al. generated transgenic pigs that synthesized human protein c in the mammary gland. protein c plays an important role in human blood clotting, which makes it a potentially attractive drug. the collected milk contained g/l of this protein [ ] . other recombinant human proteins involved in the coagulation process, such as factor viii [ ] , factor ix [ , ] , von willebrand factor [ ] , were also successfully obtained in the porcine mammary gland. furthermore, the line of transgenic pigs producing functional recombinant human erythropoietin in their milk was demonstrated. erythropoietin regulates red blood cell production (erythropoiesis) in the bone marrow by binding to a specific membrane receptor and has been used in the treatment of anemia. this bioreactor system generates active recombinant human erythropoietin at concentrations of approximately . ± . iu/ ml [ ] . in turn, lu et al. generated transgenic cloned pigs expressing large quantities of recombinant human lysozyme in milk. lysozyme is a natural broad-spectrum antimicrobial enzyme which constitutes part of the innate immune system. the authors demonstrated that the highest concentration of recombinant human lysozyme with in vitro bioactivity was . ± . mg/l [ ] . biopharmaceuticals can also be synthesized in pigs with the use of alternative systems, such as blood, urine, and semen. the blood of transgenic animals can be a source of human blood proteins, such as hemoglobin. swanson et al. and sharma et al. obtained transgenic pigs that produced recombinant human hemoglobin in their blood cells at a high level, with the ability to bind oxygen identical to that of human blood hemoglobin [ , ] . there remains, however, the issue of obtaining large amounts of animal-generated therapeutics easily and inexpensively, without killing the animal. moreover, blood cannot store high levels of recombinant proteins for a long time, which are innately unstable, and bioactive proteins in the blood may affect the metabolism of the animals [ ] . for this reason, research is being conducted into the production of recombinant proteins secreted into the urine or semen. the advantage of semen is that it is easily obtained and produced in high amounts in species such as pigs (boars can produce - ml of semen - times a week), while the advantage of urine is that proteins can be obtained from animals of both sexes throughout their lives. in addition, urine contains few proteins, which facilitates the purification of the protein product, and the urine-based systems pose a low risk to the animal's health. however, the limitation of protein production in the bladder is low yield [ ] . the recombinant pharmaceutical proteins produced from transgenic pigs are listed in table . genetically modified pigs can also be used as a source of cells, tissues, and organs for transplantation into human recipients. despite the growing knowledge and ability to perform transplants, the shortage of organs means that the number of patients awaiting a transplant is constantly increasing. xenotransplantation is any procedure involving the transplantation, implantation, or infusion of cells, tissues or animal donor organs, and also body fluids, cells, tissues, and human organs (or their fragments), which had ex vivo contact with animal cells, tissues, or organs into a human recipient. organ xenotransplantation would give us an unlimited and predictable source of organs and enable careful planning of the surgery and preoperative drug treatment of the donor. the animal that best meets the criteria for xenotransplantation is the domestic pig (sus scrofa domestica). pig and human organs show great anatomical and physiological similarities. however, the significant phylogenetic distance results in serious immunological problems after transplantation. despite major difficulties, the pig is currently the focus of all research aimed towards eliminating the problem of organ shortage for human transplantation in the future. thus, the challenge now is to overcome interspecies differences that cause xenograft rejection by the human immune system. the solution, therefore, is to modify pigs in such a way that their organs are not rejected as belonging to another species. advances in genetic engineering have brought scientists closer to obtaining modified animals that would be useful for pig to human transplants. a number of studies have reached the preclinical stage, using primates as model organisms. pig organs transplanted into human recipients are immediately rejected as a result of the so-called hyperacute immunological reaction. xenograft rejection is mainly caused by the gal antigen found on the donor's cell surface, which is synthesized by the ggta- enzyme. humans lack both the gal antigen and the ggta- enzyme, but have xenoreactive antibodies directed against the porcine gal antigen, which leads to the so-called enzymatic complement cascade in the recipient. the sequence of reactions results in a formation membrane attack complex, lysis, and destruction of the graft cells. the best possible solution to the problem of hyperacute rejection is to inactivate the gene encoding the ggta- enzyme responsible for the formation of the gal antigen. in , the first heterozygous ggta knock-out pigs were produced [ ] , and one year later, the first piglets with two knock-out alleles of the ggta gene were born [ ] . a series of ggta knock-out pigs has also been generated by using zfns [ ] , talens [ ] , and the crispr/cas system [ ] . moreover, other carbohydrate xenoantigens present on pig cells but absent in humans have been identified and include neu gc antigen (n-glycolylneuraminic acid) catalyzed by cytidine monophosphate-n-acetylneuraminic acid hydroxylase (cmah) and the sda antigen produced by beta- , -n-acetyl-galactosaminyltransferase (β galnt ). pigs with ggta /cmah/β galnt triple gene knock-out were generated using the crispr/cas system. cells from these genetically modified animals exhibited a reduced level of human igm and igg binding resulting in diminished porcine xenoantigenicity [ ] . to prevent hyperacute rejection, it is possible to introduce human genes regulating the enzymatic complement cascade into the porcine genome. as the complement system may undergo spontaneous autoactivation and attack the body's own cells, defense mechanisms have developed in the course of evolution. they regulate complement activity through a family of structurally and functionally similar proteins blocking complement activation and preventing the formation of a membrane attack complex (mac). introduction of human genes encoding complement inhibitors, such as cd (daf, decay-accelerating factor), cd (mcp, membrane cofactor protein), cd (membrane inhibitor of reactive lysis), into the porcine genome may overcome xenogeneic hyperacute organ rejection [ ] . it was demonstrated that the expression of human complement-regulatory proteins can prevent complement-mediated xenograft injury and prolong the survival time of the xenotransplant [ ] [ ] [ ] . studies have shown that the absence of ggta and additional human cd , cd , or cd expression has greater survival rates than just ggta knock-out [ , ] . many genetically modified pigs with human complement inhibitors and other modifications important for xenotransplantation were also generated [ ] [ ] [ ] . the modifications of the porcine genome described above largely resolved the problem of hyperacute rejection. however, xenogenic transplant becomes subject to less severe rejection mechanisms resulting from coagulation dysregulation, natural killer (nk) cells-mediated cytotoxicity, macrophage-mediated cytotoxicity as well as t-cell response. the coagulative disorders result from incompatibilities between pig anticoagulants and human coagulation factors. overcoming coagulation dysregulation in xenotransplantation will require the introduction of human gene encoding coagulation-regulatory proteins into the porcine genome, for example, thrombomodulin (tbm), endothelial cell protein c receptor (epcr), tissue factor pathway inhibitor (tfpi), and ectonucleoside triphosphate diphosphohydrolase (cd ). thrombomodulin binds thrombin and functions as a cofactor for the activation of protein c, which is strongly anticoagulative. porcine tbm binds human thrombin less strongly and cannot effectively activate protein c. it was demonstrated that expressing human tbm (htbm) in porcine aortic endothelial cells (paecs) suppresses prothrombinase activity and delays clotting time [ ] . the endothelial protein c receptor enhances the activation of protein c and decreases proinflammatory cytokine synthesis. in vitro studies revealed the correlation between human epcr (hepcr) expression in paecs and reduced human platelet aggregation [ ] . a meta-analysis of multiple genetic modifications on pig lung xenotransplant showed that hepcr was one of the modifications that had a positive effect on xenograft survival prolongation in the ex vivo organ perfusion model with human blood [ ] . further study demonstrated that kidneys from genetically-engineered pigs (carrying six modifications) functioned in baboons for and days. the authors suggested that prolonged survival time was associated, among others, with the expression of the human epcr gene [ ] . tissue factor pathway inhibitor is the primary physiological regulator of the early stage of coagulation. tfpi binds to factor xa, and then xa/tfpi inhibits the procoagulant activity of the tissue factor (tf)/factor viia complex. it was demonstrated that the expression of human tfpi in paecs can inhibit tf activity, suggesting potential for controlling the tf-dependent pathway of blood coagulation in xenotransplantation [ ] . more recently, multi-modified pigs carrying human tfpi transgene were produced [ , ] . cd is an ectoenzyme that plays a key role in reducing platelet activation. cd converts adenosine triphosphate (atp) and adenosine diphosphate (adp) to adenosine monophosphate (amp), which in turn is further degraded by ecto- -nucleotidase (cd ) to antithrombotic adenosine. transgenic pigs with human cd (hcd ) gene were generated. the study showed that hcd expression protects against myocardial injury in a model of myocardial acute ischemia-reperfusion injury [ ] . another approach to xenograft protection may be introducing a human gene that protects against the inflammatory response into the porcine genome. transgenic pigs expressing antiapoptotic and anti-inflammatory proteins, such as human heme oxygenase- (ho- ) and human tumor necrosis factor-alpha-induced protein (a ), were produced [ , ] . porcine aortic endothelial cells derived from pigs carrying human a transgene were protected against tnf-α (tumor necrosis factor alpha)-mediated apoptosis and less susceptible to cell death induced by cd (fas) ligands [ ] . similarly, overexpression of human ho- ensured prolonged porcine kidney survival in an ex vivo perfusion model with human blood and paecs protection from tnf-α-mediated apoptosis [ ] . furthermore, pigs with a combined expression of human a and ho- on a ggta knock-out background were generated. that transgenic approach alleviated rejection and ischemia-reperfusion damage during ex vivo kidney perfusion [ ] . the cellular immune response is another barrier to xenotransplantation. human nk cells can activate the endothelium and lyse porcine cells through direct nk cytotoxicity and by antibody-dependent cellular mechanisms. direct nk cytotoxicity is regulated by activating and inhibitory receptor-ligand interactions. to prevent nk-mediated lysis through the inhibitory cd /nkg a receptor, pigs with human leukocyte antigens-e (hla-e) were obtained [ , ] . the study showed that the expression of hla-e in endothelial cells from transgenic pigs markedly reduces xenogeneic human nk responses. in addition, it was demonstrated that the introduction of the hla-e gene into the porcine genome may also protect pig cells from macrophage-mediated cytotoxicity [ ] . more recently, ggta knock-out pigs with hcd and hla-e/human β -microglobulin transgenes were produced. the study showed that multiple genetically modified porcine hearts were protected from complement activation and myocardial natural killer cell infiltration in an ex vivo perfusion model with human blood [ ] . another approach to inhibit direct xenogeneic nk cytotoxicity is the elimination of porcine ul- -binding protein (ulbp ), which binds to nkg d activating nk receptors. crispr technology was adapted to create genetically modified pigs with a disrupted ul -binding protein gene. in vitro studies confirmed that porcine aortic endothelial cells derived from ulbp knock-out pigs were less susceptible to nk-cells' cytotoxic effects [ ] . macrophages also play an important role in xenograft rejection and can be activated by direct interactions between receptors present on their surface and donor endothelial antigens as well as by xenoreactive t lymphocytes. the binding cd antigen to macrophage surface signaling regulatory protein (sirp-α) delivers a signal to prevent phagocytosis. however, the interaction between porcine cd and human sirp-α does not supply the inhibitory effect on macrophages [ ] . therefore, the introduction of human cd (hcd ) into the porcine genome can overcome macrophage-mediated responses in xenotransplantation. the overexpression of hcd in porcine endothelial cells suppressed the phagocytic and cytotoxic activity of macrophages, decreased inflammatory cytokine (tnf-α, il- , il- β) secretion and inhibited the infiltration of human t cells [ ] . the pigs with ggta knock-out and hcd were obtained [ ] . it was demonstrated that the expression of human cd markedly prolonged survival of donor porcine skin xenografts on baboons in the absence of immunosuppression [ ] . another challenge in xenotransplantation is the prevention of t cell-mediated rejection. t cells can be induced directly by swine leukocyte antigen (sla) class i and class ii on porcine antigen-presenting cells (apcs) or by swine donor peptides presented on recipient apcs. the main co-stimulatory signals regulating t cell function include cd -cd and cd -cd / pathways. the cytotoxic t-lymphocyte antigen -immunoglobulin (ctla ) can inhibit the cd -cd / co-stimulatory pathway. therefore, the introduction of human ctla -ig (hctla -ig) into the porcine genome may alleviate t cell response in xenografts. it was shown that neuronal expression of hctla -ig in pigs reduced human t lymphocyte proliferation [ ] . moreover, transgenic hctla -ig protein in pigs extended the survival time of porcine skin grafts in a xenogeneic rat transplantation model [ ] . another approach to inhibit t-cell immune response may be the deletion of swine leukocyte antigen class i. reyes et al. created sla class i knock-out pigs using grna and the cas endonuclease. the obtained animals revealed decreased levels of cd −cd + t cells in peripheral blood [ ] . recently, pigs carrying functional knock-outs of ggta , cmah, b galnt , and sla class i with multi-transgenic background (hcd , hcd , hcd , hho , ha ) were produced. in vitro study presented that the four-fold knock-out reduced the binding of human igg and igm to porcine kidney cells [ ] . beyond immune barriers in xenotransplantation, there is also concern about the risk of cross-species pathogens infection. the main problem constitutes porcine endogenous retroviruses (perv), which are integrated into multiple locations in the pig genome. utilizing the crispr/cas technology gives great hopes for the complete elimination of the risk of perv transmission. niu et al. using the crispr/cas system inactivated all copies of functional pervs in a porcine primary cell line and successfully generated healthy perv-inactivated pigs via somatic cell nuclear transfer. what is more, no reinfection was observed in the obtained pigs [ ] . advances in genetic engineering and immunosuppressive therapies prolong organ survival time in preclinical pig-to-non-human primate (nhp) xenotransplantation models. the first xenotransplantation using pig hearts with eliminated gal antigen into immunosuppressed baboons was performed in . the longest surviving heterotopic graft functioned in the recipient for days [ ] , in comparison to - hours of survival time with the use of wild-type pig hearts [ ] . introducing additional modifications extended the xenograft survival time even more. the longest survival was obtained for heterotopic cardiac xenotransplantation-up to days. the authors used hearts derived from genetically multimodified pigs (ggta knock-out, hcd , htbm) and chimeric c r anti-cd antibody therapy [ ] . additional expression of htbm in ggta knock-out, hcd genetically modified pigs prevented early dysregulation of coagulation and prolonged the cardiac xenografts survival time [ , ] . using the same genetic background, orthotopic heart xenotransplantation was performed, resulting in a maximum survival of days [ ] . however, xenograft survival time depends on the types of transplanted organs. in the case of kidneys in pig-to-nhp transplantation models, the longest survival of a life-sustaining xenograft was days. ggta knock-out pigs carrying hcd gene as well as immunosuppression with transient pan-t cell depletion and an anti-cd -based regimen were used in the experiments. moreover, the selection of recipients with low-titer anti-pig antibodies improved the long-term survival of pig-to-rhesus macaque renal xenotransplants [ ] . the success of porcine liver and lung xenotransplantation remains limited, which is mainly associated with the occurrence of coagulation disorders [ ] . the longest survival time for orthotopic liver xenografts ( days) was achieved using ggta knock-out pigs, exogenous human coagulation factors, and immunosuppression, including co-stimulation blockade [ ] . in turn, watanabe et al. demonstrated prolonged survival time of lung xenotransplants ( days) from ggta knock-out, hcd /hcd donor pigs in immunosuppressed baboons [ ] . the authors indicated the important role of hcd expression in reducing immunologic damages and extending lung graft survival in the pig-to-nhp model. however, additional genetic modifications of the porcine genome and immunosuppressive regimen strategy are necessary for the clinical application of xenotransplantation. table summarizes the most important genetic modifications of the porcine genome for xenotransplantation purposes. the anatomical and physiological similarity between pigs and humans makes this species very interesting for biomedical research [ ] . the rapid development of genetic engineering in recent years has allowed for precise and efficient modification of the animal genome using site-specific nucleases. the nuclease-mediated editing of the porcine genome, as well as potential applications of genetically modified pigs in biomedicine, are shown in figure . certainly, the driving force for development is the human mind and ideas that arise in it. one of the factors limiting the possibilities of using the potential of our ideas is the technical aspect. the transfer of new technologies, tested on smaller animal models, for example, is often limited and requires optimization for a large animal model. in the case of crispr/cas technology, a lot of emphasis should be placed on the possibility of reducing the risk of so-called off-targets by improving this system. paired nicking has the potential to reduce off-target activity in mice from - times without compromising on-target performance [ ] . another strategy to limit the number of undesirable off-targets is to increase the specificity of the system. in this situation, one can focus on enhancing or improving cas protein or sgrna modifications. protein cas properties can be modified, or their lifespan can be changed [ , ] . for the future clinical success, it is also important to improve the efficiency of hr-mediated gene correction, especially in the situation of treating disease in which a template sequence is delivered to replace the mutated variant. another important goal to achieve is the possibility of applying hr not only for dividing cells but also for cells in the post-mitotic stage. hopes are placed in the fusion of the crispr/cas technique and aav (adeno-associated virus) as a donor template provider [ ] . considering the low immunogenicity of the aav virus, the ability to transduce a wide spectrum of cells in terms of both type and developmental stage and strong limiting factor-capacity, it should be considered to minimize the crispr/cas system or use more than one separate virus to simultaneously exploit the potential of both technologies. the use of other delivery systems, e.g., nanoparticles, is also worth considering [ ] . the different site-specific nucleases (zfn, talen, crispr/cas ) used for genome editing and two techniques (somatic nuclear transfer and microinjection) to produce genetically modified pigs are shown. biomedical applications for which genetically engineered pigs are generated include modeling human diseases, production of pharmaceutical proteins, and xenotransplantation. genetically modified pigs serve as an important large animal model for studying the genetic background of human diseases, testing novel drugs and therapy methods as well as developing models for gene therapy [ ] [ ] [ ] . pigs can be used as anatomical (e.g., endovascular), surgical, behavioral, and cytotoxic models. ideas for new models of large animals are provided by the reality that shapes current demand. a lot has been done (pig model for influenza a infection), but still there is a need for pig models of other human viral diseases (hepatitis b; human immunodeficiency virus, hiv; severe acute respiratory syndrome coronavirus , sars-cov- ) [ ] . hiv has been modeled in mice, filoviruses (ebolavirus, marburgvirus) have been modeled in small animals (i.e., mice, hamsters), but still, we need large models to investigate vaccines and antiviral drugs [ , ] . transgenic pigs can also be a promising source of recombinant proteins used as pharmacological preparations. actually, the possibility of using pigs for the production of biopharmaceuticals has been slowed in recent years. some studies demonstrated that the pig mammary gland can be used as a complex recombinant protein source with appropriate post-translational modifications [ ] . despite the advantages of pig animal platform (natural secretion, correct posttranslational modifications, constant production), some ethical doubts are probably limiting the boost. finally, the use of genetically engineered pigs for xenotransplantation is becoming an increasingly feasible alternative to standard allogeneic transplants and a potential solution to the problem of organ shortage. the combination of various multi-modified pigs and immunosuppressive therapies is required for overcoming immune rejection and effective xenotransplantation of different solid organs [ ] [ ] [ ] . when it comes to treating end-stage organ failure, biomedical research could go a step further and try to create chimeric genetically modified pigs that would be carriers of human organs [ ] . the publication was co-financed within the framework of a ministry of science and higher education program as "regional initiative excellence" in the years - , 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production of functional human hemoglobin in transgenic swine an isologous porcine promoter permits high level expression of human hemoglobin in transgenic swine production of pharmaceutical proteins by transgenic animals expression systems and species used for transgenic animal bioreactors production of alpha- , -galactosyltransferase knockout pigs by nuclear transfer cloning production of alpha , -galactosyltransferase-deficient pigs production of zfn-mediated ggta knock-out pigs by microinjection of gene constructs into pronuclei of zygotes production of α , -galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology generation of ggta mutant pigs by direct pronuclear microinjection of crispr/cas plasmid vectors evaluation of human and non-human primate antibody binding to pig cells lacking ggta /cmah/β galnt genes expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention 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genetically modified pig endothelial cells and thrombin inhibition meta-analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts atorvastatin or transgenic expression of tfpi inhibits coagulation initiated by anti-nongal igg binding to porcine aortic endothelial cells generation of α- , -galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes transgenic swine: expression of human cd protects against myocardial injury transgenic expression of human heme oxygenase- in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys transgenic expression of the human a gene in cloned pigs provides protection against apoptotic and inflammatory stimuli kidneys from α , -galactosyltransferase knockout/human 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pig-to-human hematopoietic cell transplantation prolonged survival of pig skin on baboons after administration of pig cells expressing human cd transgenic expression of ctla -ig by fetal pig neurons for xenotransplantation transgenic expression of human cytoxic t-lymphocyte associated antigen -immunoglobulin (hctla ig) by porcine skin for xenogeneic skin grafting creating class i mhc-null pigs using guide rna and the cas endonuclease viable pigs after simultaneous inactivation of porcine mhc class i and three xenoreactive antigen genes ggta , cmah and b galnt inactivation of porcine endogenous retrovirus in pigs using crispr-cas galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching months intravenous infusion of galα- gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts chimeric c r anti-cd antibody therapy is critical for long-term survival of gtko.hcd .htbm pig-to-primate cardiac xenograft cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs consistent success in life-supporting porcine cardiac xenotransplantation long-term survival of pig-to-rhesus macaque renal xenografts is dependent on cd t cell depletion overcoming coagulation dysregulation in pig solid organ transplantation in nonhuman primates: recent progress prolonged survival following pig-to-primate liver xenotransplantation utilizing exogenous coagulation factors and costimulation blockade intra-bone bone marrow transplantation from hcd transgenic pigs to baboons prolongs chimerism to > days and promotes increased porcine lung transplant survival production of biallelic cmp-neu ac hydroxylase knock-out pigs the generation of transgenic pigs as potential organ donors for humans a human cd transgenic pig model system for the study of discordant xenotransplantation pigs transgenic for human thrombomodulin have elevated production of activated protein c 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genetically modified pigs as donors of cells, tissues, and organs for xenotransplantation xenotransplantation: current status in preclinical research interspecies chimerism with mammalian pluripotent stem cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -r ev br authors: englund, stina; hård af segerstad, carl; arnlund, frida; westergren, eva; jacobson, magdalena title: the occurrence of chlamydia spp. in pigs with and without clinical disease date: - - journal: bmc vet res doi: . / - - - sha: doc_id: cord_uid: r ev br background: within the genera chlamydia, the development of refined diagnostic techniques has allowed the identification of four species that are capable of infecting pigs. the epidemiology, clinical, and zoonotic impacts of these species are however largely unknown. the study aimed to investigate the presence of chlamydia spp. in the intestines of growing pigs and in conjunctival swabs from finisher pigs, and relate the findings to clinical signs. results: by histology, of pigs had intestinal lesions that may be consistent with chlamydial infection. by pcr, forty-six of the pigs were positive whereas two samples were inhibited. sequencing of dna extracts identified these as chlamydia suis. by immunohistochemistry, of samples were positive and a significant relationship was detected between macroscopically visible intestinal lesions and a high degree of infection. by real-time pcr, a significant difference was detected between pigs with and without conjunctivitis when a ct value of was employed but not when a ct value of was employed. conclusions: chlamydia suis was demonstrated in most samples and overall, no correlation to clinical signs was detected. however, a correlation was noted between samples with a high degree of infection and the presence of clinical signs. it is possible, that the intensive pig production systems studied might predispose for the transmission and maintenance of the infection thus increasing the infectious load and the risk for disease in the pig. the chlamydiaceae family including the genera chlamydia is a well-recognised cause of disease in many animal species including cats, ruminants, birds and humans [ , ] . within the genera, nine distinct species have been identified. four of these have been described in pigs (chlamydia (c.) abortus, c. psittaci, c. pecorum, and c. suis) and related to reproductive disorders, conjunctivitis, enteritis, pneumonia, polyarthritis, pleuritis and polyserositis [ , [ ] [ ] [ ] . the epidemiology, clinical, and zoonotic importance of these species are however largely unknown [ ] [ ] [ ] . however, c. suis seems to be both common and widespread, often occurring in mixed infections with c. abortus and c. pecorum [ ] . in gnotobiotic pig challenge studies from usa, c. suis caused dose-dependent diarrhoea in young piglets. histologically, villi atrophy, tip erosions, necrosis, inflammatory changes and lymphangitis were noted in the distal small intestine. chlamydial antigens were demonstrated in enterocytes and chlamydiae were reisolated from tissue specimens and faecal swabs [ , ] . however, weaned pigs developed microscopical lesions but remained clinically healthy [ ] . similar lesions have also been demonstrated in clinical cases of diarrhoea in weaned pigs [ ] . however, other studies have not been able to confirm a causal relationship [ , ] . a few studies have addressed conjunctivitis [ ] . chlamydiae were seen in eight pigs by ultrastructural examination and were isolated in two pigs [ ] , and subclinical conjunctivitis was experimentally induced in -day-old gnotobiotic piglets [ ] . isolation is the gold standard for diagnosis but many strains are difficult to cultivate [ , ] . other methods includes serology, immunohistochemistry, histology, and pcr on faeces, mucosal swabs or tissue specimens [ ] . because of cross-reactivity, some tests may lack sensitivity and specificity [ , , ] . in a study comparing four diagnostic methods, c. suis were demonstrated in % of the samples by pcr and in % by culture. the sensitivity was . % and specificity . %, whereas the two elisas performed considerably weaker. pcr was concluded to be a reliable, highly sensitive and specific tool for detection of c. suis [ ] . the present study aimed to investigate the presence of chlamydia spp in the intestines of growing pigs and in conjunctival swabs from finisher pigs, and relate those findings to the occurrence of clinical signs. the study was approved by the ethical committee for animal experiments, uppsala, sweden. all herd owners had given an informed consent prior to the study. dna from enteric specimens originated from a previous study on growing pigs with diarrhoea [ ] . the samples originated from the major pig producing areas of sweden, i.e. in the south-western and eastern parts of the country, and included pigs from six herds with poor performance and diarrhoea in growing pigs, and pigs from four herds with good performance and no diarrhoea. from each of the poor performance herds, three case and three control pigs were chosen, and from each of the four good performance herds, three control pigs were chosen. the case pigs had diarrhoea that had commenced within days and no other diseases were evident. the control pigs were matched to the case pigs for age and sex, but showed no signs of clinical disease. the control pigs had a mean age of days and a mean weight of . kg, and the case pigs had a mean weight of . kg. the pigs submitted from the good performance herds had a mean age of days and a mean weight of . kg. all animals were weaned at approximately weeks of age and none of the pigs had been treated with antibiotics. the pigs were transported to the laboratory and euthanized within min prior to necropsy. further, animals from three finisher herds were sampled based on the present occurrence of conjunctivitis. the pigs originated from several piglet-producing herds and were introduced to the finisher herds at - kg. b.w., i.e. - weeks prior to sampling. they were kept pigs per pen in units of pigs each. in two herds, case and control pigs were selected from each of units, and in one herd, case and control pigs from one unit were selected. the case pigs were selected based on clinical signs of moderate to severe conjunctivitis, defined as hyperaemia and chemosis with epiphora and/or muco-purulent secretion. the control pigs were selected from the same pens as the case pigs but showed no or only mild conjunctivitis, i.e. none to slight hyperaemia in the conjunctiva and no chemosis or epiphora. the necropsies were carried out at the department of pathology at the national veterinary institute, uppsala, or at analycen, skara. the animals were stunned with electricity, weighed and exsanguinated, and necropsy was immediately performed. all gross lesions were noted, and specimens for histological examination were taken from ileum and from all macroscopically visible lesions. the samples were fixed in % buffered formalin, embedded in paraffin blocks, sectioned and stained with haematoxylin and eosin according to standard protocols. the finisher pigs were snared and sterile cotton swabs were rubbed against the conjunctiva in the conjunctival sac in the left and right eye, respectively. the swabs were placed in sterile tubes and transported to the laboratory. intestinal samples were prepared from a . cm piece of frozen mucosa from the distal ileum. dna was extracted by phenol/chloroform and precipitated by ethanol [ , ] . dna from the conjunctival swabs was extracted according to k. sachse [ ] and all samples were analysed by real-time pcr according to everett and others [ ] . the primers tqf and tqr targeted the s ribosomal dna and detected all members of the family chlamydiaceae. multiple negative controls were included and dna from cp. abortus and c. suis (kindly provided by k. sachse, friedrich-loeffler-institute, jena, germany) were used as positive controls. to detect false negative pcr results due to inhibiting agents, an internal amplification control (mimic) was constructed and used as previously described [ ] . the primers used in the mimic producing pcr were tqfactin ( 'gaaaagaacccttgttaagggagc-catgtaccctggcattg- ') and tqractin ( '-cttaactccctggctcatcatggatccacacg-gagtacttgc- '). the sequence of the rox-labelled mimic probe used in real-time pcr was '-ccgacag-gatgcagaaggagatca- '. the conjunctival swabs were analysed both undiluted and diluted : in accordance with the standard protocol applied at the nvi, and a positive reaction in at least one of these dilutions was regarded as positive. in the statistical analyses, threshold values below ct were regarded as positive whereas values between ct and ct were regarded as doubtful. the results were calculated separately for the respective thresholds. values above ct were regarded as negative or were excluded in the calculations. the difference in detection rate of chlamydia spp. between pigs with moderate to severe conjunctivitis and the control pigs were analysed by chi-square test. sequence analysis was performed on samples from the intestinal specimens giving a strong positive reaction in the pcr. pcr was performed with primers sf and r according to everett and andersen [ ] on dna samples from seven case pigs and six control pigs from poor performance herds and six pigs from good performance herds. the predicted pcr product of bp as well as one pcr product of larger and one of smaller size was purified prior to sequencing by using the gfx pcr dna and gel band purification kit (amersham bioscience europe, gmbh germany). the purified products were sequenced with the same primers used in the pcr and by using the bigdye terminator v . cycle sequencing kit (applied biosystems, foster city, ca, usa) in combination with ethanol/edta/ sodium acetate precipitation, according to the protocol supplied. thermocycling was performed in a geneamp thermocycler (applied biosystems). the sequencing products were subjected to electrophoretic separation and on-line detection on an abi prism genetic analyzer (applied biosystems), followed by blast search. paraffin-embedded intestinal specimens were available from of the growing pigs. -μm thick sections of the formalin-fixed, paraffin-embedded blocks where cut and placed onto positively charged slides (polysine™, menzel-gläser, braunschweig, germany). prior to immunostaining, deparaffinisation and hydration where done in xylene and graded ethanol to distilled water. after hydration, a blocking for endogenous peroxidase where done in . % h o in tris-buffered saline (tbs) and nonspecific binding sites where blocked with % bovine serum albumin (bsa). antigen retrieval was performed by heat induced epitope retrieval (hier) in retrieval buffer ph = (tbs-edta), using microwave as heat source. hier was performed by heating the polysine™-slides immersed in retrieval buffer for min at w followed by min at w. after completed heating procedure, the slides remained in the retrieval buffer at room temperature for min. the slides where incubated with a mouse monoclonal antibody against chlamydia, clone aci (progen biotechnik gmbh, germany) diluted : . visualisation of the bound primary antibody was achieved by using dako envision™ + system utilising an hcp-labelled antimouse monoclonal antibody (glostrup, denmark), and the presence of the relevant antigen was detected with , '-diaminobenzidine (dab, nvi, sweden). slides were weakly counterstained with haematoxylin. the staining included at least one positive and one negative control section. the positive control originated from previously confirmed routine cases. the results were graded as negative (-), sparse (+), moderate (++) or abundant (++ +) occurrence. no gross lesions were observed in the pigs from the good performance herds. in the pigs from the poor performance herds, five control pigs and pigs with diarrhoea had macroscopic lesions consistent with lawsonia (l.) intracellularis infection and one had a parasitic colitis. by histology, lesions that may be consistent with chlamydial infection [ ] were noted in pigs: villus atrophy was noted in six pigs with diarrhoea and in ten control pigs from the poor performance herds. epithelial exocytosis or necroses were noted in five pigs with diarrhoea, in one control pig from the poor performance herds, and in one pig from the good performance herds (table ) . in the previous study [ ] , brachyspira pilosicoli were demonstrated in eight case pigs and five control pigs, campylobacter jejuni; in one case pig and four control pigs, yersinia enterocolitica in three case and five control pigs, haemolytic escherichia (e.) coli in two case and two control pigs, clostridium perfringens in two case pigs, and l. intracellularis were previously demonstrated in case and eight control pigs from the poor performance herds. in addition, haemolytic e. coli had been demonstrated in two pigs from the good performance herds. rotavirus had been demonstrated in one case pig. coronavirus was not included in the study, since sweden has previously been shown to be free from table the findings in intestinal specimens from growing pigs with diarrhoea (case), clinically healthy control pigs from the same poor performance herds (casecontrol), and from healthy pigs originating from good performance herds (control), examined by immunohistochemistry (ihc), necropsy, and pcr. transmissible gastroenteritis and porcine epidemic diarrhoea. balantidium coli were demonstrated in one case and three control pigs from the poor performance herds, and in one pig from the good performance herds. of the enteric samples analysed, were positive for chlamydiaceae (table ) . two samples could not be evaluated due to amplification inhibition. the major band of b.p. found in all samples was confirmed as c. suis in the samples subjected to sequence analysis. the larger pcr product described in the methods' section was - % identical to escherichia coli whereas the smaller pcr product did not match any sequence in the blast search. in the pcr analyses on the conjunctival swabs, one pig with conjunctivitis and three control animals together with their matched counterparts were excluded from the statistical calculations, since the pcr was partially or totally inhibited. thus, in total, conjunctival swabs were included in the statistical analyses. of these, three samples from pigs with conjunctivitis gave weakly positive reactions (ct > in undiluted samples) and were judged as negative. employing a cut-off value of ct , samples from pigs with conjunctivitis and control pigs were determined as positive by pcr. using a cut-off value of ct , samples from pigs with conjunctivitis and control pigs were determined as positive. a statistically significant difference (p = . ) was detected between pigs with and without conjunctivitis when a ct value of was employed. the difference was mainly related to one of the herds ( positive pigs with conjunctivitis and positive control pigs). when a ct of was employed, no significant differences were noted. the results are shown in table . among the case pigs, ( %) were negative or were carrying a low-grade infection. among the control pigs from good performance herds, ( %) were negative or sparsely (+) infected. the two control specimens in which the pcr analyses had been inhibited were graded as + and ++, respectively, by immunohistochemistry. a significant (p < . ) relationship was detected between the presence of clinical signs and a high degree (++ or +++) of infection. no relationship was detected between the occurrence of histological lesions and the demonstration of chlamydial antigen by immunohistochemistry. the pig intestine is considered as the natural reservoir for c. suis, and the microbe seems generally to be well adapted to its host [ ] . in the present study, chlamydia spp. were demonstrated in high prevalence in all herds investigated. this is consistent with the few studies that have previously addressed the occurrence of chlamydia spp. in pig herds. overall, it was not possible to relate the demonstration of the microbe to the presence of diarrhoea. however, based on the immunohistochemistry, pigs with diarrhoea might have been more heavily infected than the healthy pigs. this is in consistency with the results from another study on pigs submitted for necropsy, where it was not possible to relate the presence of chlamydia spp. to the occurrence of diarrhoea, but in cases, it was the only pathogen found [ ] . similar experiences have also been noted in calves and sheep [ ] [ ] [ ] . in the study by becker et al. [ ] , c. suis was significantly (p < . ) related to conjunctivitis in extensive pig production systems, whereas in intensive farming systems, high prevalences ( - %) were found in both pigs with conjunctivitis and in clinically asymptomatic pigs. this is in accordance with the findings in the present study. becker et al. discussed, that environmental factors might predispose to infection. however, in one of the herds in the present study, factors such as emission of ammonium and carbon dioxide gases, air movements and overcrowding were investigated, but it was not possible to relate any environmental factor to the occurrence of conjunctivitis (data not shown). however, in one stable a high relative humidity ( %) was noted that might facilitate microbial survival. in the present study, the pigs originated from several piglet producing herds and it should be emphasized that intensive farming systems with the mixing of pigs from several sources also imply increased opportunities for microbial spread among animals of different immunological status. in fact, in one herd a significant relationship was noted between conjunctivitis and the presence of chlamydia spp. when the lower threshold value was applied in the real-time pcr. this might further indicate that the infectious load is important in the development of disease. although chlamydia spp. was demonstrated in the ileal sections by immunohistochemistry in the present study, lesions compatible to those described in gnotobiotic pigs [ ] were only noted in % of the pigs. several authors speculate that the development of lesions may depend on different factors such as the virulence of the strain, the infectious dose, the route of infection, or the age and the immunological status of the host [ , , , , , ] . since young, naive pigs seem to develop lesions in response to an experimental infection [ ] , host immunity might be induced at an early age in the field [ , ] . it is also possible that co-infections with other presumptive pathogens might act synergistically to exacerbate the lesions or that lesions induced by one microbe might increase the susceptibility to other infections. several pathogens have been discussed in this respect [ , , , , , ] . most of the intestinal lesions described in the present study were shown to be related to infections with l. intracellularis or b. pilosicoli [ ] . some studies also report the occurrence of mixed infection with several chlamydial species [ , [ ] [ ] [ ] . in wild boars, c. psittaci was the dominating species but c. suis and c. abortus was also demonstrated by sequencing [ ] . in the present study, preliminary data obtained by pcr-rflp indicated the co-infection by other species [ ] . however, sequencing of the amplicons revealed the involvement of c. suis only, whereas other bands detected by pcr originated from e. coli and un-identified bacterial species. the results underline the difficulties involved in the diagnosis and a large variation in reported prevalences between various detection methods exist [ , , , ] . in the present study, the problem was circumvented by the use of an internal probe and a high melting temperature in the real-time pcr, combined with sequencing of the amplicons that assured a high specificity. chlamydia suis was found in high prevalences in growing pigs with or without diarrhoea, and in finisher pigs with or without conjunctivitis. overall, no correlation to clinical signs was detected. however, a correlation was noted between samples with a high degree of infection and the presence of clinical signs. it is possible, that the intensive pig producing systems investigated in the present study might predispose for the transmission and survival of the microbe, thus increasing the infectious load and the risk for disease in the pig. interestingly, no other species of the chlamydiaceae family were detected, as also supported by the findings in other animal species in swedish surveys [ ] . chlamydial 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samples by single, fluorescent and nested pcr based on the is gene identification of nine species of the chlamydiaceae using pcr-rflp behavior of different bovine chlamydial agents in newborn calves pathologic changes in intestinal chlamydial infection of newborn calves high frequency of chlamydial co-infections in clinically healthy sheep flocks an experimentally induced chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation experimental chlamydia psittaci serotype enteric infection in gnotobiotic piglets: histopathological, immunohistochemical and microbiological findings intestinal chlamydia in pigs concurrent infection of enterocytes with eimeria scabra and other enteropathogens in swine occurrence of chlamydiaceae spp. in a wild boar (sus scrofa l.) population in thuringia (germany) mixed infections with porcine chlamydia trachomatis/ pecorum and infections with ruminant chlamydia psittaci serovar associated with abortions in swine detection of all chlamydophila and chlamydia spp. of veterinary interest using speciesspecific real-time pcr assays the occurrence of chlamydia species in intestinal specimens from swedish grower pigs. the th international pig veterinary society congress prevalence of chlamydial infection in breeding sows investigation of chlamydophila spp. in dairy cows with reproductive disorders the occurrence of chlamydia spp. in pigs with and without clinical disease we wish to thank marianne persson for skilful technical assistance. we also wish to thank the herd owners who kindly supplied the pigs and allowed us to enter the herds. authors' contributions se was responsible for the pcr investigation and sequencing of dna from the intestinal specimens, analysis and interpretation of these data, and revised the manuscript. chs made an intellectual contribution by drafting the manuscript and revising it critically. fa was responsible for collecting of the conjunctival swabs, pcr analysis and interpretation of these data, and revised the manuscript. ew was responsible for the immunohistochemical analyses, interpretations of these data, and revised the manuscript. mj was planning the studies, collected and prepared the intestinal specimens, and was responsible for drafting and writing the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -xmbnpawj authors: meekins, david a.; morozov, igor; trujillo, jessie d.; gaudreault, natasha n.; bold, dashzeveg; artiaga, bianca l.; indran, sabarish v.; kwon, taeyong; balaraman, velmurugan; madden, daniel w.; feldmann, heinz; henningson, jamie; ma, wenjun; balasuriya, udeni b. r.; richt, juergen a. title: susceptibility of swine cells and domestic pigs to sars-cov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: xmbnpawj the emergence of sars-cov- has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. the susceptibility of different animal species to sars-cov- is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. in the current study, we determined the ability of sars-cov- to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. sars-cov- was able to replicate in two different porcine cell lines with cytopathic effects. interestingly, none of the sars-cov- -inoculated pigs showed evidence of clinical signs, viral replication or sars-cov- -specific antibody responses. moreover, none of the sentinel pigs displayed markers of sars-cov- infection. these data indicate that although different porcine cell lines are permissive to sars-cov- , five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. pigs are therefore unlikely to be significant carriers of sars-cov- and are not a suitable pre-clinical animal model to study sars-cov- pathogenesis or efficacy of respective vaccines or therapeutics. the emergence of sars-cov- , the causative agent of covid- , has resulted in a global pandemic with over million cases and , deaths as of august , [ , ] . sars-cov- causes a respiratory disease in humans with a broad clinical presentation, ranging from asymptomatic or mild illness to severe fatal disease with multi-organ failure [ ] [ ] [ ] [ ] . sars-cov- is rapidly transmissible via contact with infected respiratory droplets and can also be transmitted by asymptomatic carriers [ ] [ ] [ ] . to curb viral spread, countries have instituted varying levels of social distancing policies, which have significant negative economic and social impacts [ ] . mitigating the effects of this unprecedented pandemic will necessitate the development of effective vaccines and therapeutics, which will require well-characterized and standardized preclinical animal models. sars-cov- is a member of the betacoronavirus genus that includes the pathogenic human viruses sars-cov- and mers-cov [ , [ ] [ ] [ ] . while details of the origin of sars-cov- are unknown, evidence indicates it emerged from a zoonotic spillover event, with bats and perhaps pangolins as probable origin species [ , [ ] [ ] [ ] . the potential for a reverse zoonotic event, i.e. human-to-animal transmission, is possible and of significant concern to animal and public health [ ] [ ] [ ] . instances of natural human-to-animal transmission of sars-cov- have been reported with covid- patients in domestic settings (dogs and cats), zoos (lions and tigers), and farms (mink) [ ] [ ] [ ] . therefore, investigations into the infectivity of sars-cov- in various animal species with human contact are essential to assess and control the risk of a spillover event and to establish the role these animals may play in the ecology of the virus. several studies have determined the susceptibility of different animal species to sars-cov- via experimental infection [ , ] . cats, hamsters, and ferrets are highly susceptible to sars-cov- infection, demonstrate varying clinical and pathological disease manifestations, readily transmit the virus to naïve animals, and mount a virusspecific immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] . dogs are mildly susceptible to experimental sars-cov- infection, with limited viral replication but with clear evidence of seroconversion in some animals [ ] . poultry species seem to be resistant to sars-cov- infection [ , ] . these findings establish the respective utility of different animal species as pre-clinical models to study sars-cov- . several lines of evidence suggest that pigs could be susceptible to sars-cov- infection. pigs are susceptible to both experimental and natural infection with the related betacoronavirus, sars-cov- , and demonstrate seroconversion [ , ] . structure-based analyses predict that the sars-cov- spike (s) protein receptor binding domain (rbd) binds the pig angiotensin-converting enzyme (ace ) entry receptor with similar efficiency compared to human ace [ ] . single-cell screening also indicated that pigs co-express ace and the tmprss activating factor in a variety of different cell lines, and sars-cov- replicates in various pig cell lines [ , , , ] . despite these preliminary data indicating that pigs could be susceptible to sars-cov- infection, two recent studies revealed that intranasal inoculation of three and twelve pigs, respectively, with pfu or tcid of sars-cov- did not lead to any detectable viral replication or seroconversion [ , ] . however, the single route of intranasal inoculation used in these studies suggests that additional investigations are necessary before definitive conclusions can be made regarding susceptibility of pigs to sars-cov- . in the present study, we determined the susceptibility of swine cell lines and domestic pigs to sars-cov- infection. two different porcine cell lines were found to be permissive to sars-cov- infection showing cytopathic effects (cpe). domestic pigs were challenged via simultaneous oral/intranasal/intratracheal inoculation with a tcid dose of sars-cov- . sars-cov- did not replicate in pigs and none of them seroconverted. furthermore, the virus was not transmitted from sars-cov- inoculated animals to sentinels. the present findings, combined with the other studies [ , ] , confirm that pigs seem resistant to sars-cov- infection despite clear susceptibility of porcine cell lines. pigs are therefore unlikely to play an important role in the covid- pandemic as a virus reservoir or as a pre-clinical animal model to study sars-cov- pathogenesis or develop novel countermeasures. sars-cov- usa-wa / isolate (genbank accession # mn ) [ ] was eighteen pigs (mix of males and females, five weeks of age) were used in the study. pigs were acquired from a source guaranteed free of swine influenza virus (siv), porcine circovirus- (pcv- ), and porcine reproductive and respiratory syndrome virus (prrsv) infection. the study outline is illustrated in figure . upon arrival, pigs were acclimated for days prior to sars-cov- inoculation. nine pigs were designated as uninfected negative controls and housed in separate bsl- facilities. three of these uninfected negative control pigs were humanely euthanized at days post challenge (dpc) to provide negative control clinical and tissue samples. the nine principal infected pigs were housed in the same room in two separate groups ( or pigs each; gross pathological examinations on major organs were performed and respiratory tissue samples were collected and either stored in % neutral-buffered formalin or stored as fresh samples at - ˚c. blood and swab samples were all filtered using a . µm filter prior to storage at - ˚c. rna was isolated from blood, swabs, and tissue samples, using a magnetic bead-based protocol in a bsl- + laboratory at the bri at ksu. lung tissue homogenates ( mg per ml dmem; % w/v) were prepared by thawing tissue, mincing it into mm sections, followed by lysis in a ml sure-lock tube containing mm stainless steel homogenization beads using the tissuelyser lt (qiagen, germantown, md, usa) for seconds at hz followed by min of hz while keeping the sample cold. following clarification via a -minute centrifugation ( , xg; room temperature), supernatants were mixed with an equal volume of rlt lysis buffer. blood and clinical swabs were directly mixed with an equal volume of rlt lysis buffer. µl of each sample lysate was used to extract rna using a magnetic bead-based during post mortem examinations, the upper and lower respiratory tract, central nervous system, lymphatic and cardiovascular systems, gastrointestinal and urogenital systems, and integument were evaluated. lungs were removed in toto and the percentage of the lung surface that was affected by macroscopic lesions was estimated by single veterinarian experienced in evaluating gross porcine lung pathology as previously described [ , ] . lungs were evaluated for gross pathology such as edema, congestion, discoloration, atelectasis, and consolidation. tissue samples of interest were collected and either fixed in % neutral-buffered formalin for histopathological examination or frozen at - ˚c for rt-qpcr testing. tissues were fixed in formalin for days, then transferred to % ethanol (thermofisher scientific, waltham, ma, usa) prior to trimming and paraffin embedding following standard automated protocols used in the histology section of the kansas state veterinary diagnostic laboratory. following embedding, tissue sections were cut and stained with hematoxylin and eosin and evaluated by a board-certified veterinary pathologist who was blinded to the treatment groups. to detect sars-cov- antibodies in sera, indirect elisas were performed observed. neutralizing sera from sars-cov- -infected cats from a separate study [ ] was used as a positive control. to determine the consensus sequence of the usa-wa/ / virus and to analyze if there were any nucleic acid substitutions in the sars-cov- virus after passage in porcine cell lines, rna was extracted from cell culture supernatant as described above. the rna was then subjected to rt-pcr amplification using a tiledprimer approach to amplify the entire sars-cov- genome as described previously [ ] . briefly, the pcr amplicons were pooled and subjected to library preparation for next generation sequencing using the nextera xt library prep kit (illumina, san diego, ca, usa). the library was normalized and sequenced using a miseq nano v x sequencing kit. the sequence was then analyzed by mapping reads to the parent sequence (genbank accession # mn ) [ ] to generate a consensus sequence. the sars-cov- usa-wa / isolate, which was isolated from a human patient in washington state, usa, was used as the parent stock for the study [ ] . the to determine the effect of sars-cov- infection in domestic pigs, nine sixweek-old sars-cov- seronegative piglets were inoculated with a total of x tcid of the usa-wa / isolate, which was passaged once in swine st cells ( figure ). the challenge material (total ml) was administered orally ( ml), intranasally ( ml; . ml each nostril) and intratracheally ( ml) after sedation of the animals. at -day post challenge (dpc), six uninoculated sentinel contact pigs were comingled with the principal inoculated animals ( animals per pen). daily rectal temperatures were recorded for each pig and clinical signs were monitored daily, including observations for signs of lethargy, hyporexia, respiratory distress (coughing, labored breathing, nasal discharge), and digestive issues (diarrhea or vomiting). no significant temperature elevation or change in rectal temperature was observed in the principal inoculated nor sentinel contact pigs throughout the study (figure ) . moreover, no obvious clinical signs were observed for any of the principal inoculated nor sentinel pigs throughout the -day observation period. to detect viral replication in the principal and sentinel pigs, clinical samples were subjected to rt-qpcr to detect the sars-cov- n gene ( table ) table ). the only the exception was a low suspect positive result in a nasal swab at dpc in a principal inoculated pig # , for which one of two qpcr replicates yielded a low fluorescent amplification curve with a ct of (table ) . moreover, viral rna was not detected in any lung sample collected at post-mortem examination on , and dpc (table ). in addition, gross and histopathological analysis of trachea and lung from the principal challenged pigs did not reveal the presence of any obvious pathological lesions ( table , figure ). these results indicate that sars-cov- failed to replicate in the respiratory and digestive tract as well as the blood in orally/intranasally/intratracheally inoculated pigs throughout an observation period of days. this is confirmed by the fact that the principal infected pigs failed to transmit sars-cov- to co-mingled sentinel animals. to determine whether the orally/intranasally/intratracheally inoculated pigs sars-cov- is a zoonotic agent, and a detailed understanding of the susceptibility of various animal species to sars-cov- is central to controlling its spread [ , ] . in addition, the development of animal models that emulate covid- in humans is essential for pre-clinical testing of novel vaccines and therapeutics [ ] . in this study, we inoculated nine pigs with a high dose of sars-cov- that was passaged once in porcine cells. simultaneous oral/intranasal/intratracheal inoculation did not result in any detectable viral rna in the blood, the oral/nasal/rectal cavities, or the lungs. also, none of the co-mingled, sentinel contact pigs shed viral rna. moreover, a virus-specific immune response characteristic of infection was not observed within the -day study period in the principal infected or sentinel pigs. the transient nature of the igm and igg response observed in pig # could indicate cross-reactivity of antibodies directed against a porcine coronavirus such as porcine epidemic diarrhea virus [ ] . such antibodies could be maternally derived and therefore transient as the lack of sars-cov- specific reactivity by the end of the study might suggest. in contrast to previous sars-cov- swine studies [ , ] , the present study used a more stringent inoculation procedure (intratracheal and oral, in addition to intranasal) and log higher titer of virus inoculum ( vs ). in addition, the inoculum in the present study was passaged once in porcine st cells. these results, combined with previous intranasal pig inoculation studies [ , ] , indicate that pigs seem to be resistant to sars-cov- infection, are unlikely to be a sars-cov- carrier animal species, and are also not suitable as an animal model for research. the results of the present and previous sars-cov- inoculation studies in pigs are intriguing in light of the findings that the porcine ace receptor seems highly compatible with the sars-cov- rbd, suggesting that pigs could be susceptible to sars-cov- infection [ , ] . pigs are susceptible to both experimental and natural infection with sars-cov- [ , ] . however, the experimental sars-cov- infection was via simultaneous intranasal/oral/intraocular/intravenous inoculation [ ] , thus the actual route(s) of sars-cov infection cannot be determined. recently, several porcine cell lines have been shown to be permissive to sars-cov- infection [ , ] ; in addition, single-cell screening studies showed that porcine ace /tmprss expression are compatible with infection [ ] . in contrast to previous reports that some porcine cell lines are susceptible to sars-cov- infection, but show no cpe [ , ] , we found that both st and pk- cell lines are susceptible to infection and observed cpe after two or four passages, respectively. the absence of sars-cov- replication and transmission in the present and two previous pigs studies [ , ] seems to lessen the need to monitor pig populations for sars-cov- during the ongoing pandemic. however, the evidence described above suggests pig susceptibility should not be disregarded, because all pig studies to date have used rather young pigs and commercially available pig breeds/genetics. we also have to be aware that unforeseen genetic changes in the sars-cov- genome may result in a better compatibility of the virus for pigs in the future. pigs are considered to be an excellent model for studying human infectious diseases based on their relatedness to humans in terms of anatomy and immune responses and they have been found to be much more predictive for the efficacy of therapeutics when compared to rodent models [ ] . however, the results presented here indicate that pigs are not a suitable preclinical model for sars-cov- pathogenesis studies and the development and efficacy testing of therapeutics and/or vaccines. a recently available article indicates that while pigs are not susceptible to sars-cov- infection, neutralizing antibody responses were detected in pigs infected via intramuscular or intravenous inoculation routes [ ] ; this indicates that pigs could be used for immunogenicity studies related to sars-cov- . however, the use of pigs to monitor sars-cov- immune responses must be careful to screen for cross-reactive maternal antibodies derived from other coronaviruses [ ] . alternate pre-clinical animal models, namely non-human primates, syrian hamsters, transgenic or transduced mice expressing human ace , ferrets, or even cats need to be considered to gain additional insights into sars-cov- pathogenesis and virulence. comprehensive characterization of sars-cov- pathogenesis in preclinical animal models and the establishment of standardized infection and testing protocols will be crucial for the development of much-need countermeasures to combat covid- . swabs/blood were tested from samples on , , , , , and dpc. lung tissue was collected , , and dpc. swabs/blood were tested from samples on , , , and dpc. lung tissue was collected on dpc swabs/blood were tested on dpc. lung tissue was collected on dpc for these uninfected controls. *one pig (# ) had a ct signal of . ( . x copy number/ml) for / of rt-qpcr wells on dpc. magnification is x for main images and x for inserts. coronavirus disease (covid- ) situation report - a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical presentation of covid- : case series and review of the literature epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease in china transmission, diagnosis, and treatment of coronavirus disease (covid- ): a review early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia temporal dynamics in viral shedding and transmissibility of covid- the socio-economic implications of the coronavirus pandemic (covid- ): a review coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- . nat microbiol severe acute respiratory syndrome (sars): a review middle east respiratory syndrome coronavirus (mers-cov): a review the proximal origin of sars-cov- probable pangolin origin of sars-cov- associated with the covid- outbreak possible bat origin of severe acute respiratory syndrome coronavirus . emerg infect dis is covid- the first pandemic that evolves into a panzootic? vet ital covid- and veterinarians for one health, zoonotic-and reverse-zoonotic transmissions a critical needs assessment for research in companion animals and livestock following the pandemic of covid- in humans. vector borne zoonotic dis are animals a neglected transmission route of sars-cov- ? pathogens evidence for sars-cov- infection of animal hosts. pathogens infectivity, virulence, pathogenicity, host-pathogen interactions of sars and sars-cov- in experimental animals: a systematic review susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus . science transmission of sars-cov- in domestic cats infection and rapid transmission of sars-cov- in ferrets sars-cov- is transmitted via contact and via the air between ferrets simulation of the clinical and pathological manifestations of coronavirus disease (covid- ) in golden syrian hamster model: implications for disease pathogenesis and transmissibility pathogenesis and transmission of sars-cov- in golden hamsters susceptibility of pigs and chickens to sars coronavirus sars-associated coronavirus transmitted from human to pig. emerg infect dis receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus single-cell screening of sars-cov- comparative tropism, replication kinetics, and cell damage profiling of sars-cov- and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid- : an observational study. lancet microbe severe acute respiratory syndrome coronavirus from patient with coronavirus disease, united states. emerg infect dis division of viral diseases. real-time rt-pcr panel for detection - -novel coronavirus pathogenic and antigenic properties of phylogenetically distinct reassortant h n swine influenza viruses cocirculating in the united states comparison of pathogenicity and transmissibility of influenza b and d viruses in pigs. viruses sars-cov- infection, disease and transmission in domestic cats ncov- sequencing protocol v v. lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts the pig: a model for human infectious diseases pigs are not susceptible to sars-cov- infection but are a model for viral immunogenicity studies emerging and re-emerging we gratefully thank the staff of ksu biosecurity research institute, the the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord- -ov fy b authors: nazki, salik; khatun, amina; jeong, chang-gi; mattoo, sameer ul salam; gu, suna; lee, sim-in; kim, seung-chai; park, ji-hyo; yang, myoun-sik; kim, bumseok; park, choi-kyu; lee, sang-myeong; kim, won-il title: evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ov fy b the host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (prrsv) from infected pigs is currently poorly understood. to better understand the dynamics of host–pathogen interactions, seventy-five of pigs infected with prrsv-ja and control pigs were euthanized at , , , and days post-challenge (dpc). blood, lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected to evaluate the cellular immune responses. the humoral responses were evaluated by measuring the levels of anti-prrsv igg and serum virus-neutralizing (svn) antibodies. consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between and dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by dpc. at peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived dc/macrophage and conventional dc frequencies were increased, and these effects coincided with the early induction of local t-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, bal, and bln as early as dpc. conversely, the systemic t-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between and dpc. taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining prrsv through the early induction of t-cell responses at the sites of virus replication. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded positive-sense rna virus with an approximate . -kb genome, belongs to the genus betaarterivirus of the family arteriviridae (ictv ). in pigs, prrsv causes porcine reproductive and respiratory syndrome (prrs), which is characterized by reproductive failure in breeding sows and severe respiratory distress in young and growing pigs [ ] . prrs results in colossal economic losses in the swine industry worldwide, and these losses are still observed three decades after its emergence in the united states and europe. after the exposure of pigs to prrsv, the virus replicates in alveolar macrophages (am) and further spreads rapidly throughout the body via a lymphohematic route. this viral spread results in acute infection characterized by viremia that lasts for approximately month [ ] , and a few studies have reported a nonviremic persistent infection of secondary lymphoid tissues lasting for approximately days or longer [ ] . in general, the viremia peaks at approximately - days post-infection (dpi) and is almost cleared by dpi depending on the viral strain and age of the pigs [ , ] . additionally, the immune response against prrsv depends on the strain, but the virus usually has immunosuppressive properties [ , ] , which leads to the increased susceptibility of pigs to secondary microbial infections [ ] . the interactions between prrsv and host immune responses have been widely studied, but most studies investigated systemic immune responses using pbmc and/or serum [ ] . previous studies have shown that interstitial pneumonia constitutes the major lung lesions in prrsv-infected pigs and that significantly decreased numbers of alveolar macrophages are found in bronchoalveolar lavage (bal) and lung parenchyma samples from prrsv-infected pigs [ ] . however, to the best of our knowledge, the kinetics of local immune responses in the lungs or lymph nodes during the course of infection compared with those of peripheral immune responses have not been previously studied. this information would provide a more in-depth understanding of the sequential activation of both immune compartments and the correlation between local or peripheral immune responses and virus clearance in infected pigs. as a result, achieving a comprehensive understanding of the immune responses against prrsv infection remains an important goal in prrsv research. during prrsv infection, the pig immune system is capable of escalating an immune response to ultimately clear the virus from the body [ ] . for clearance, proper stimulation of the pig innate immune system is required to direct the development of protective adaptive immunity against prrsv. interestingly, the preferential sites for prrsv replication are alveolar macrophages present in the lungs, which form the major component of the respiratory dc/macrophage network. this network is predominantly involved in sensing foreign antigens, controlling inflammation, and initiating the adaptive immune response [ ] . different dc subsets with specific functional specializations exist in the respiratory dc/macrophage network in the lungs and are reportedly resistant to prrsv infection [ , ] . however, upon activation, these dc travel to lymphoid tissues to present antigen to t lymphocytes and thereby serve as the link between innate and adaptive immunity [ , ] . t cells, in turn, play a critical role in the development of anti-prrsv immunity due to their cytotoxic effector functions in clearing infected cells from the body and developing and regulating antigen-specific immune responses [ ] . however, whether the peripheral virusspecific immune response is appropriately correlated with the local immune response during infection, which could result in the precise use of the peripheral response as a surrogate for scrutinizing the local immune response during viral clearance, remains unclear. therefore, discerning the local and peripheral immune responses during prrsv infection is important for understanding the basic mechanism of viral clearance from the host. cytokines secreted by immune cells act on their targets in an autocrine, paracrine, and/or endocrine manner to prompt local and/or systemic immune responses. in porcine respiratory diseases, proinflammatory cytokines play a key role in activating and synchronizing the adaptive immune responses to clear the virus from the body [ ] . however, the tissue damage caused by excessive production of these cytokines is controlled by the secretion of anti-inflammatory cytokines, which results in the maintenance of homeostasis in the body [ ] . moreover, effective instigation of the local inflammatory response in the lungs accompanied by significant changes in the proinflammatory cytokine levels in serum has been observed in pigs with respiratory diseases [ , ] . nevertheless, whether the local or the systemic cytokine/ chemokine response plays the primary role in the clearance of prrsv from pigs during the acute phase of infection remains unclear. in this context, the present study aimed to investigate the trend of host immune responses against prrsv infection during disease progression and to elucidate the innate and adaptive immunological mediators modulated by the prrsv-ja strain both systemically in peripheral blood and locally in the bronchoalveolar lavage, lung parenchyma and bronchial lymph nodes (bln) of infected pigs. marc- cells, an african green monkey kidney cell line that is highly permissive to prrsv [ ] , were used for virus propagation and assays. these cells were maintained in rpmi- medium (gibco ® rpmi- , life technologies, carlsbad, ca, usa) supplemented with % heat-inactivated foetal bovine serum (fbs, life technologies), mm l-glutamine, and an antibiotic-antimycotic cocktail (anti-anti, life technologies) containing iu/ml penicillin, µg/ml streptomycin, and . µg/ml fungizone ® [amphotericin b] in a humidified chamber with % co at °c. in this manuscript, this medium is designated rpmi growth medium. the north american prrsv- strain ja (genbank: ay . ) was used in the present study. one hundred -week-old piglets purchased from a prrsv-seronegative farm were randomly assigned to two groups and housed in separate animal rooms. after days of acclimatization, the pigs in the infected group (n = ) were intramuscularly inoculated with ml of the prrsv-ja strain ( × tcid /ml) diluted in sterile pbs. the control pigs (n = ) remained uninfected. feed and water were provided ad libitum to all the pigs. five pigs from the control group and , , , and pigs from the infected group were humanely euthanized on days , , , and post-challenge (dpc), respectively. euthanasia was performed by electrocution after the intramuscular injection of ml of azaperone ( mg/ml, stressguard ® , dong bang inc., seoul, south korea). three infected pigs died during the course of the experiment due to high fever and reduced body growth. an overview of the animal study is presented in figure . after euthanasia, the lungs, trachea and bronchi were aseptically extracted and lavaged with ml of sterile pbs. the collected lavage fluid was centrifuged at × g and room temperature for min to separate the bronchoalveolar lavage fluid (balf) and cells (bal), and these samples, along with the lung parenchyma and bln, were also used for immune cell analysis. these tissues and balf were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at − °c for rna extraction and cytokine analysis, respectively. for histopathology, the lung tissues were also collected in % neutral-buffered formalin. blood was collected from the euthanized pigs at , , , and dpc, and the serum and pbmc were separated. in addition, blood samples from pigs, including uninfected and infected pigs that were going to be euthanized on and dpc, were also collected at , , , , , and dpc, and the serum and pbmc were separated. the body weights of all the pigs were measured at dpc, and the body weight gains of the euthanized pigs were measured at , , , and dpc. serum viremia was measured at , , , , , , and dpc, and the viral load in the lungs was quantified in the euthanized pigs at , , , and dpc. viral rna was extracted from serum using a magmax viral rna isolation kit (ambion; applied biosystems, life technologies, inc.) according to the manufacturer's instructions. real-time reverse transcription-polymerase chain reaction (rt-qpcr) employing the prime-q pcv prrsv detection kit (genet bio, daejeon, south korea) was performed for the quantification of serum viremia. one-step rt-qpcr was performed in accordance with the manufacturer's instructions. pcr amplification was performed using a model fast real-time pcr system (applied biosystems, foster city, ca, usa). the cycling conditions were as follows: (i) cdna synthesis for min at °c; (ii) -min predenaturation step at °c; and (iii) cycles of denaturation for s at °c figure overview of the animal study. prrsv-negative piglets (n = ) were purchased and acclimatized for days. seventy-five pigs were then infected, and pigs were used as a negative control (nc). pigs belonging to both the nc and infected (i) groups were euthanized at , , , and days post-challenge (dpc), and lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected. blood samples for sera and peripheral blood mononuclear cells (pbmc) were collected weekly post-challenge. the body weight of the pigs was also monitored from the day of purchase to the end of the experiment. and annealing/extension for s at °c. to calculate the amount of prrsv in each sample, the cq values were converted to virus titres (tcid /ml) by generating a standard curve through the titration of prrsv- strain ja . in addition, marc- cells were used to quantify the virus titres in lung tissues using a microtitration infectivity assay [ ] . briefly, tissue homogenates ( % [weight/ volume]) of finely chopped lung pieces were prepared in dulbecco modified eagle medium (dmem) with antibiotics, and these mixtures were vortexed vigorously for - min and then centrifuged at ~ × g and °c for h. the collected supernatant was filtered through a . -μm sterile syringe filter and used as an inoculum for the measurement of virus titres. the virus titres were calculated at to days post-inoculation based on the cytopathic effect (cpe) and are expressed as tcid /ml [ ] . the lungs of the necropsied pigs in both groups were subjected to pathological evaluation on each day of necropsy. the microscopic lung lesions were given a score on a scale from to to reflect no lesion, mild interstitial pneumonia, moderate multifocal interstitial pneumonia, and severe interstitial pneumonia, respectively. the microscopic lesions were examined from five different lobes of the lungs, and the average value was ultimately utilized for scoring purposes. the serum samples from uninfected and challenged pigs were tested for anti-prrsv antibody (igg) using a commercially available elisa kit (prrs ab elisa . ; bionote inc., hwaseong-si, republic of korea) according to the manufacturer's instructions. samples with an s/p ratio (the ratio of the net optical density of the test samples to the net optical density of the positive controls) ≥ . were considered to be positive for the prrsv antibody. the serum virus-neutralizing (svn) antibodies were measured through a fluorescent focus neutralization (ffn)-based svn assay with marc- cells as described previously [ ] , with some modifications. after heat inactivation at °c for h, the serum samples were serially (twofold) diluted using rpmi- growth medium. two-hundred-microlitre mixtures were prepared by mixing each diluted serum sample with fluorescent focus-forming units per ml (ffu/ml) of prrsv-ja at a ratio of : and were then incubated for h at °c in a humidified atmosphere with % co . each mixture was transferred onto a monolayer of marc- cells in -well plates and incubated for another h at °c. the medium was replaced with µl of fresh rpmi growth medium per well and further incubated for h at °c. the cells were later fixed using ice-cold % (v/v) acetone, air-dried, and stained with mouse anti-prrs nc mab a (median diagnostic, gangwondo, korea) and fitc-conjugated goat anti-mouse igg (h + l) (bethyl laboratories, tx, usa). subsequently, the plates were washed at least three times with pbs and observed under a fluorescence microscope to examine the prrsv-specific ffu. the svn titre is expressed as the reciprocal of the highest dilution at which a % or higher reduction in the number of ffu was observed. pbmc were isolated from the blood samples ( ml) by the density gradient method using leucosep ™ centrifuge tubes (greiner bio-one north america inc., nc, usa) and leucoprep ™ lymphocyte separation media (greiner bio-one north america inc.) according to the manufacturers' instructions. the blood samples were briefly stratified on leucoprep ™ solution at a ratio of : (blood:leucoprep) and centrifuged at × g for min. the purified pbmc were collected, washed twice with sterile pbs (ph . ) and resuspended in . ml of sterile pbs supplemented with % heat-inactivated fbs (gibco, carlsbad, ca, usa). contaminating red blood cells (rbc) were removed by treatment with rbc lysis buffer (ebioscience, ca, usa). for pathological evaluation, the right-sided lobes were clamped to collect specimens for rna extraction and histopathology, and the left lobes of the lungs were used for bal collection according to a previous study [ ] . the lungs were lavaged with - ml of pbs containing µg/ml ampicillin (usb corporation cleveland, oh, usa) and an antibiotic-antimycotic cocktail (anti-anti, life technologies), and the harvested fluid was centrifuged for min at × g. the resulting supernatant was collected as bal fluid (balf), whereas the cell pellet (bal cells) was washed three times with pbs after rbc lysis. the cells were resuspended in facs buffer ( % fbs in phosphate-buffered saline and . % sodium azide). the bln were passed through a -μm cell strainer (spl life sciences, pocheon, korea) in pbs and then washed with facs buffer according to a previous study [ ] . the single cell suspension obtained was used for flow cytometric analysis. mononuclear cells from lung parenchyma were prepared based on a previous study [ ] , with few modifications. briefly, lung tissue was collected, washed in sterile ice-cold pbs and suspended in serum-free rpmi media containing dnase i ( u/ml, sigma, st. louis, mo, usa) and collagenase d ( mg/ml, roche diagnostics, mannheim, germany). single-cell suspensions were prepared using the gentlemacs octo dissociator (miltenyi biotec, san diego, ca, usa) and incubated at °c for min. subsequently, the cells were passed through a -μm cell strainer, washed, and resuspended in facs buffer for flow cytometry analysis after rbc lysis. finally, the cells were counted with a countess ™ automated cell counter (invitrogen, carlsbad, ca, usa), and their viability was tested by trypan blue (sigma-aldrich, st. louis, mo, usa) exclusion [ ] . for cell surface staining, single-cell suspensions were incubated on ice for min with specific antibodies as listed in additional file , and the cells were then washed three times with facs buffer. when necessary, secondary antibodies conjugated with fluorochrome were used. natural killer (nk) cells, dc and macrophages required only cell surface staining, whereas the different subsets of t cells required intranuclear and intracellular staining. two subsets of nk cells have been phenotypically defined based on nkp marker expression: nkp + and nkp − nk cells [ , ] . following pbmc staining, a similar gating hierarchy was followed by excluding the unstained cells, doublets and cd + cells, and the cd − lymphocytes were further analysed for cd α and nkp expression. among cd − cells, two populations were found in the pbmc, namely, nkp + and nkp − nk cells, and both of these cells were cd α + (additional file a). bal cells were subjected to cell surface staining for the cd surface marker, and the viability of these cells was analysed using propidium iodide (pi) staining (additional file b). additionally, dc and macrophages were segregated from the bal cell population based on staining and gating strategies outlined previously (additional file c) [ , ] . from the mhc-ii + cell population, five phenotypically and functionally defined subpopulations were distinguished using the cd and cd a (sirpα) surface markers. among the mhc-ii + cells, cd a + / cd high cells were defined as am, whereas cd a + / cd int , cd a + /cd low , cd a + /cd − and cd a − /cd − cells were defined as monocytederived macrophages (momɸs), monocyte-derived dendritic cells (modc), conventional dendritic cells (cdc ) and conventional dendritic cells (cdc ), respectively, based on a previous study [ ] . regulatory t cells (tregs), which require intranuclear staining of foxp after cell surface staining, were fixed with cold fixation/permeabilization buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min and were then stained for foxp at °c for min. based on a previously described staining and gating strategy [ ] , tregs (cd + foxp + cells) were apparent among the cd + cd − population (additional file d). the t-cell subsets subjected to intracellular staining were stained according to previous studies [ , ] , with few modifications. briefly, single-cell suspensions were treated with a mixture of × cell stimulation cocktail (ebioscience, thermo fisher scientific, seoul, korea) and × brefeldin a (ebioscience, thermo fisher scientific, seoul, korea) in rpmi growth media and incubated at °c in a humidified chamber with % co for - h. the cells were then stained with antibodies for various cell surface markers in cold facs buffer for min at °c, properly washed twice with cold facs buffer, and fixed with intracellular (ic) fixation buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min. for intracellular staining, the cells were washed twice with permeabilization buffer ( μl/well) and stained with cytokine-specific antibodies in cold permeabilization buffer at °c for min. subsequently, the cells were washed twice with permeabilization buffer. the gating strategy employed for obtaining various t-cell phenotypes after the gating of singlet lymphocytes is demonstrated in additional file e. a -μl suspension of the stained cell populations in facs buffer was run on an accuri c flow cytometer (bd accuri ™ c plus, bd biosciences, md, usa). bd accuri ™ c plus software version . . . (bd biosciences, md, usa) was used to analyse the data after setting compensation settings according to monocolour and isotype control stains. the data are presented as percentages of all the cell subsets. the cytokine levels in the sera and balf of the uninfected and infected pigs at , and dpc were measured using a porcine-specific procartaplex ™ multiplex immunoassay (thermofisher scientific, vienna- , austria) according to the manufacturer's instructions. magnetic microsphere technology based on porcine cytokine/chemokine antibody-immobilized magnetic beads was employed in the immunoassay for cytokine quantification [ ] . the concentration of each cytokine was measured by running the samples on the luminex ® ™ system (luminex corporation, austin, tx, usa). appropriate standards provided in the kit were utilized to determine the concentration of each cytokine. the machine was verified and calibrated using a luminex ® / ™ verification kit and a luminex ® / ™ calibration kit (luminex corporation, austin, tx, usa) prior to use. graphical presentations of the data were prepared using graphpad prism . (graphpad, san diego, ca, usa), and the data were statistically analysed using spss advanced statistics . software (spss, inc., chicago, il, usa). a nonparametric t-test (mann-whitney u test) was used to compare the viral loads in the lung tissues, the average daily weight gain (adwg), the phenotypes of various cell subsets and the cytokine responses between two groups. the normalized dead cd + cells were analysed by repeated anova (tukey post hoc test) to determine the overall difference, and pairwise comparisons were also performed at different days post-challenge. spearman rank correlation and linear regression were used to determine the associations between two parameters. differences were considered statistically significant if p < . and are indicated by asterisks and different letters over the bars. the highest viremia and lung viral loads in prrsv-ja challenged pigs were detected between and dpc (figures a, b) . the mean peak virus titre in serum at dpc was recorded as . tcid /ml, whereas the mean peak live viral load in the lungs at dpc, which was measured through the microtitration infectivity assay, was . tcid /ml. the virus was gradually cleared by dpc, at which point, the serum and lung viral loads had decreased to mean values of . and . tcid / ml, respectively. as expected, the control group maintained an uninfected state throughout the experiment. the effect of viremia on body weight gain was observed in the infected pigs by calculating the adwg of the pigs ( figure c ). the adwg in the prrsv-ja -challenged pigs was significantly lower than that in the control pigs at , and dpc. the adwg per control pig was recorded as . kg, whereas the adwg of the infected pigs was reduced to . kg. as expected, the growth rate of the pigs was negatively affected by viremia following infection, and this negative correlation was significant (r = − . ; p ≤ . ) ( figure d ). moderate to severe interstitial pneumonia with alveolar wall thickening due to type pneumocyte proliferation and inflammatory cell infiltration was detected in the infected pigs during the period of peak viremia. thus, the highest microscopic lung lesion score in the infected pigs was recorded at dpc, and the scores of the lung lesions in these pigs decreased at later time points but remained at significantly higher levels compared with those in control pigs (figures a, b) . an elisa based on the nucleocapsid (n) protein was employed to measure the prrsv-specific antibody (igg) response in the infected and noninfected pigs ( figure a ). at dpc, prrsv-ja -specific igg were detected in the serum of the infected pigs. additionally, the sample-to-positive (sp) value gradually increased in the infected group from to dpc, whereas in the control group, the pigs did not produce any prrsv-specific igg at any time point. a low svn titre was observed in the challenged pigs at and dpc, when most of the virus was already cleared from the body ( figure b ). in general, the svn antibody responses predominantly appear at the later stages of infection, which is the phase at which most of the virus is cleared from the body, and might play a minor role in the clearance of virus. nk cells, which are a specialized subpopulation of lymphocytes, are the innate immune cells critically responsible for directly killing virus-infected cells, which ultimately leads to viral clearance in the host [ ] . to observe the effect of prrsv infection on nk cells in pigs, the frequencies of two different nk cell subsets in the pbmc population were analysed. the percentages of nkp − nk cells (cd + nkp − in cd − ) in the pbmc populations of the uninfected and infected pigs were higher than those of nkp + nk cells (cd + nkp + in cd − ) (additional file a). moreover, compared with the control pigs, the infected pigs exhibited a significantly (p ≤ . ) increased frequency of nkp + nk cells at the early time point of dpc, whereas the frequency of nkp − nk cells was slightly increased at dpc and significantly increased at dpc. the frequency of nkp + nk cells returned to normal values at dpc, but the frequency of nkp − nk cells in the infected pigs remained at significantly higher values up to dpc ( figures a, b) . the associations between nk cells in pbmc and serum viremia were evaluated post-infection ( figures c, d) . the nkp + nk cell population revealed a significant (r = . ; p < . ) positive correlation with viremia; however, no statistically significant correlation between the levels of nkp − nk cells and viremia was observed. the receptors of host cells determine the cell tropism of prrsv. among others, cd , a cysteine-rich scavenger receptor (srcr), acts as the determinant receptor for prrsv entry and infection [ , ] . the bal cells were subjected to cd staining, and the results show that although the virus did not affect the cd + cells until dpc, the infected pigs exhibited a significant (p ≤ . ) reduction in the cd + cell percentage at dpc, showing a gradual recovery with the decline in the viral loads at dpc ( figure a ). the reduction in the cd + cell population was attributed to the death of cd + cells, which was confirmed by observing the viability of these cells through pi staining. the mean frequencies of dead cd + cells in the infected pigs, which were normalized to those in the uninfected pigs, was significantly (p ≤ . ) higher at and dpc ( . % and . %, respectively) ( figure b ). the class ii major histocompatibility complex (mhc-ii) is essential for the presentation of antigens to t cells and is constitutively expressed on macrophages and dc [ , ] . the mhc-ii + cells were analysed to observe the changes in the dc/macrophage network in the lungs. the mhc-ii + cells, such as cd + cells, show significant (p ≤ . ) decreases at and dpc ( figure c) . interestingly, the percentage of am (cd a + /cd high /mhc-ii + cells), which constituted the majority of mhc-ii + cells, decreased significantly at dpc after infection (p ≤ . ) (figure d ). in contrast, the cd a + /cd int /mhc-ii + cell (momɸ) and cd a + /cd low /mhc-ii + cell (modc) frequencies were significantly (p ≤ . ) reduced in the infected pigs early during the infection process ( dpc) ( figures e, f) . however, the cd a + / cd int /mhc-ii + cell percentages in the infected pigs were significantly (p ≤ . ) increased at and dpc, and a higher (p ≤ . ) cd a + /cd low /mhc-ii + cell frequency was also detected at , , and dpc in the infected pigs compared with the control pigs. cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells were not frequently detected among bal cells and accounted for less than % of the five subsets ( figures g, h) . at dpc, a significantly higher (p ≤ . ) percentage of these cell populations was observed in the infected pigs, and this value decreased with the reduction in the viral load and the recovery of am. the associations between the lung viral loads and different subpopulations of macrophages and dc in bal were evaluated after pooling the results of infected pigs at each time point (figure ) . during the infection, mhc-ii + cells and am displayed a significant negative correlation (p < . ) with lung viral loads; however, other subsets exhibited a significant (p < . ) positive correlation. during infection, prrsv-ja alters the dynamics of the respiratory dc/macrophage network by destroying am while increasing the populations of antigen-presenting cells (apc), which bridge the gap between innate and adaptive immune systems by presenting the foreign antigen to t cells. the peripheral immune response was measured by analysing the t-cell populations in the pbmc of uninfected and infected pigs at , , , and dpc (additional file a). the th (ifn-γ + in cd + cd − ) response in the infected pigs was significantly higher (p ≤ . ) than that in the control pigs at dpc, and these responses continued to increase until dpc. the cytotoxic t lymphocyte (ctl) (ifn-γ + in cd − cd + ) response in the infected pigs started to increase at dpc and was significantly higher (p ≤ . ) compared with that in the control pigs at dpc ( figure a ). at dpc, the th (il + in cd + cd − ) cell response was significantly higher (p ≤ . ) in the infected pigs compared with the control pigs, and the response was further escalated at later time points. the il- -producing cd − cd + cell population was significantly higher (p ≤ . ) in the infected pigs compared with uninfected pigs at dpc. the delay in the induction of effector t cells was mainly perceived peripherally in blood. to observe the activation of the local adaptive immune responses by innate immune cells at the sites of replication and the persistence of prrsv, the t-cell phenotypes in the bln, bal and lung parenchyma of the euthanized control and infected pigs were analysed at , , and dpc. the percentages of various immune cells in the lungs, bal and bln of uninfected and prrsv-ja infected pigs at different stages of infection are summarized in additional files b, c, and d. overall, various t-cell responses were significantly induced in the lungs as early as dpc, and significant responses in bal and bln were first detected at dpc and were maintained until dpc. the th cell (ifn-γ + in cd + cd − ) frequency in all the local tissues was significantly (p ≤ . ) induced in the challenged pigs as early as dpc, and the frequency in bal cells tended to be higher. the ctl (ifn-γ + in cd − cd + ) frequency was significantly (p ≤ . ) higher in the lungs of the infected pigs compared with that of the control pigs at dpc, whereas the frequency in bln and bal cells was higher at the early time point and significantly higher at dpc. the prrsv-ja -infected pigs also displayed a higher induction (p ≤ . ) of th cells (il + in cd + cd − ) in the lymph nodes and lung tissues at dpc, whereas a slight increase in these cells was observed in bal cells. the il- -producing cd − cd + population was significantly (p ≤ . ) induced in the lungs and bal cells of the infected pigs at dpc, whereas in bln, this cell population showed a slight increase at dpc and a significant increase (p ≤ . ) at dpc (figures b-d) . therefore, compared with the weak and delayed peripheral responses, early and effective cellular immune responses were triggered in local tissues by prrsv-ja infection. tregs are well known for their immunosuppressive activities, and previous studies have demonstrated that prrsv infection induces treg responses that might be responsible for ineffective adaptive immune responses [ ] [ ] [ ] . unlike previous studies, no upregulation of tregs (cd + foxp + in cd + cd − ) was detected in pbmc isolated from prrsv-infected pigs. moreover, tregs were significantly (p ≤ . ) reduced in the bal throughout the course of infection, but no such decline was observed in the lungs (figure ). however, the treg frequencies in bln of the infected pigs initially exhibited a significant decline at dpc but then increased significantly at dpc. the levels of seven different innate and adaptive cytokine/chemokine proteins in the sera and balf at , and dpc were compared between the uninfected and infected pigs ( figure ) . the results reveal that interferon-α (ifn-α) was significantly (p ≤ . ) induced in the sera and balf of the infected pigs at dpc. however, at peak viremia ( dpc), the ifn-α level was reduced peripherally in the sera of the infected pigs, whereas the cytokine level was elevated locally in the balf. nevertheless, significant increases in the level of this cytokine (p ≤ . ) were maintained both locally and systemically in the infected pigs compared with the uninfected pigs. furthermore, proinflammatory cytokines/chemokines, such as tumour necrosis factor-α (tnf-α), interleukin- β (il- β), il- , il- and il- , show a similar pattern of early induction at dpc in the sera of the infected pigs followed by decreases as viremia increased. in the sera, significant (p ≤ . ) changes in il- β were only observed in the infected pigs at and dpc. however, significant (p ≤ . ) induction of il- β and il- locally in the balf was observed in the infected pigs at dpc. in addition, elevations in all the proinflammatory cytokine/chemokine levels were observed in the infected pigs as the viral load increased, and significant increases in the levels of il- β, il- , il- and il- were observed in the infected pigs compared with the uninfected pigs at dpc. moreover, il- , an anti-inflammatory cytokine, was significantly induced (p ≤ . ) at and dpc in the balf of the infected pigs. overall, the clearance of the virus from the pigs also coincided with the increased secretion of anti-viral and proinflammatory figure local and systemic levels of cytokines/chemokines in the pigs. the concentrations of cytokines/chemokines in the a sera and b balf of pigs belonging to both groups at , , and dpc were measured using a multiplex luminex-based cytokine immunoassay. the bars in the graphs represent the mean ± sem of the cytokine levels, and the asterisks (*) indicate a statistically significant difference between the averages found for the uninfected (nc) pigs and those obtained for the infected pigs at each time point (* indicates p ≤ . and ** indicates p ≤ . ). cytokines locally in the balf of prrsv-infected pigs, although no considerable shifts were detected in the serum. despite exhaustive research, the understanding of the protective immune responses against prrsv in pigs remains limited. previous studies have mainly focused on certain aspects of immune responses and/or have used a narrow time window to study the immune responses during prrsv infection; therefore, the available data offer a limited picture of the host defence system. to the best of our knowledge, the present study constitutes the first investigation of various aspects of immune responses, such as local vs. systemic and innate vs adaptive, during the course of prrsv infection to obtain a broader picture of the host defence against prrsv. here, we demonstrate the critical role of local immune responses in the clearance of prrsv from pigs due to the early induction of dc/macrophage subsets, the activation of protective t cells in local lymphoid and lung tissues, and the stimulation of proinflammatory cytokines locally in the lungs. in pigs that were intramuscularly inoculated with the biologically characterized prrsv-ja strain [ ] , the viral titre reached its peak value in the serum and lungs between and dpc. in addition, moderate to severe interstitial pneumonia and a significant decrease in the adwg, which are characteristics of prrsv infection [ ] , were detected in the challenged pigs by dpc. mild histopathological lesions (with a lesion score lower than ) characterized by alveolar wall thickening due to type pneumocyte proliferation were also observed in some of the uninfected healthy pigs that were free of other respiratory pathogens. these observations were likely obtained due to the stress induced by environmental factors, such as housing conditions, weaning, individual space, and ambient temperature variations, as previously reported [ , ] . similar to previous reports [ ] , the prrsv-ja strain induced delayed (≥ dpc) and weak (≤ ) svn titres in infected pigs. an svn antibody titre of offers sterilizing immunity, whereas a titre greater than is considered protective against prrsv infection [ ] . the anti-prrsv antibody response induced by prrsv-ja in the infected pigs crossed the threshold of . (s/p ratio) between and dpc, and this response was in accordance with the response produced by other prrsv strains [ ] , but the role of these antibodies in protection against prrsv infection is unknown [ ] . nk cells, an important component of the innate host defence system, play a critical role in the resolution of viral infections [ ] . in general, the potential roles of nk cells in relation to prrsv immunity are poorly understood [ , ] . in the current study, two defined nk cell subpopulations were distinguished in pbmc based on an approach similar to one previously described [ ] . similar to previous observations, the nkp + nk cell frequency in pbmc collected from both control and infected pigs was lower than that of nkp − cells. nkp + and the nkp − nk cells execute analogous cytolytic activities but produce different levels of ifnγ, with nkp + nk cells producing higher amounts of ifn-γ. moreover, nkp can be expressed in nkp − nk cells after stimulation with interleukins (il)- , il- and il- [ ] . in the current study, early increases in the frequencies of nkp + and nkp − nk cells in prrsv-ja -challenged pigs were clearly detected. previous studies have shown similar increases in the cd − cd − cd + cell population after infection with different strains of prrsv [ , ] . induced proliferation of nk cells has also been perceived after influenza infection, suggesting that this subset of lymphocytes is capable of antigen-specific clonal expansion [ ] . prrsv, however, significantly suppresses nk cell-mediated cytotoxicity to evade the host immune response [ ] , but these previous studies did not consider the subsets of nk cells. the specific action of different subsets of nk cells on prrsv has not yet been explored, and further studies are needed to explain the role of nk cells in the containment of the virus during infection. the main targets of prrsv are cells belonging to the monocyte and macrophage lineages, particularly am. however, dc are also reportedly vulnerable to prrsv infection [ ] . the dc/macrophage network, which senses the foreign antigen and initiates the immune response, constitutes one of the main components of the respiratory immune system [ ] . it is plausible to expect that the viral infection of these cells alters this network and thus affects downstream immune responses. therefore, dissecting how these immune cells respond to prrsv infection is important to better understand the nature of prrs. until now, limited studies have investigated the alterations in the respiratory dc/macrophage network in pigs during disease progression [ , ] , and the association of the alteration in this immune network with the t-cell response has not been reported. here, we attempted to explore the dynamics of this important host defence mechanism in relation to prrsv infection. during infection, prrsv replicates in the cd + cells in the lungs and induces apoptosis at the early stages of infection [ ] . in the current study, a flow cytometric analysis of bal cells revealed that prrsv-ja decreased the cd + cell population in the infected pigs at the time when peak viral loads were detected. after the viral load decreased, the cd + cell population recovered to the normal levels. pi staining of the cd + bal cells revealed that the decrease in the cd + cell population was due to cell death. a similar reduction in the macrophage population after prrsv- infection was previously reported [ ] . following the strategy described previously [ ] , different subsets of dc and macrophages were identified in bal cells, and the changes in the populations of these subsets during disease progression were studied. previous reports verify that prrsv infection impairs dc function directly by downregulating mhc-ii expression [ , ] . intriguingly, among the five cell subsets in mhc-ii + cells, only the cd a + /cd high / mhc-ii + (am) cell population decreased significantly at peak viremia, revealing a significant negative correlation between the population of these cells and the viral loads in the lungs, whereas significant positive correlations were found for the other subset populations. the increases in the cd a + /cd int /mhc-ii + and cd a + /cd low /mhc-ii + cell populations can be attributed to the higher influx of monocytes and their differentiation in the lungs during inflammation. moreover, cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells exhibit strong migration and antigenpresenting capabilities [ ] , which can be credited to the increased influx of these cells into the lungs during the course of infection. our results were in agreement with those obtained in previous studies, which found that the populations of these cells are increased in the bal after prrsv- infection [ ] . interestingly, cdc s activate allogenic naïve t cells and aid induction of the th response, whereas cdc s induce a th response in pigs [ ] . after sensing antigen, the mature migrating dc recruit nk cells to drain ln, thus providing an early source of ifn-γ and thereby promoting th polarization [ ] . thus, dc work together with nk cells to regulate innate immunity and further dictate the direction and intensity of the adaptive immune response [ ] . the effective immune response to counter viral infections is governed by the proper activation of t lymphocytes by apc [ ] . in the current study, we analysed the dynamics of t lymphocytes in pbmc, lung parenchyma, bal, and bln to detect the central cause of the clearance of prrsv from the body. delayed induction of th , th and ctl responses was observed in the pbmc of the infected pigs after dpc, when most of the virus had been cleared from the blood, whereas in bln, the virus persists for a longer period, which leads to early and sustained (> dpc) induction of t cell responses. previous studies also found a delayed induction of effector t cells in the peripheral blood of pigs infected with prrsv [ , ] . intriguingly, significant increases in the frequencies of these t lymphocyte subpopulations were detected in the lung parenchyma and lymphoid tissues of the infected pigs at early stages of infection ( dpc), when the virus levels were at their peak in the body. in agreement with our findings, a higher frequency of t-helper cells and ctl has been observed in local tissues, such as the bal, lymph nodes and lung parenchyma, of pigs after swine influenza infection, and this induction has been linked to viral clearance [ ] . the local cell-mediated immune responses are considered crucial for the clearance of the influenza virus [ ] . moreover, during human respiratory syncytial virus infection, increased populations of cd + and cd + t cells exhibiting effector functions have been observed in the bal of infected patients [ ] . a recent study investigating t-cell proliferation in prrsv- -infected pbmc at dpi and the expression of lymph node-homing receptors revealed that the t-helper cell response plays a main role in viral clearance and that the ctl response is strongest at the site of infection [ ] . in addition, the induction of th cells in local tissues has also been found to be essential for the resolution of respiratory infections [ ] . in the present study, the cell responses in bln and bal were found to be significantly increased from or dpc and were maintained until dpc, when the virus was completely cleared. therefore, it can be concluded that local t-cell responses in the lungs, bln and bal are induced markedly faster than systemic responses and are maintained at significantly high levels, even after virus clearance, substantiating the critical role of local immune responses in the clearance of prrsv from pigs. the treg lineage is responsible for the maintenance of homeostasis in the immune system by suppressing the activation of various immune cells, including other t cells, nk cells and dc [ ] . treg play a vital role in the pathogenesis of some viral infections that result in severe inflammatory lesions, such as influenza [ ] . however, the treg response in pigs after prrsv infection is controversial. although some studies have revealed increased frequencies of tregs after prrsv infection [ , , ] , other studies revealed that the induction of tregs was not changed and even suppressed after infection [ , ] . these conflicting results could be due to the strainspecific response of the pigs to the virus after challenge or to the method used to evaluate the changes in the treg response. several studies have revealed that tregs are induced due to prrsv infection, but most of these studies were performed using in vitro or ex vivo assays in which pbmc and mononuclear cells isolated from the lungs and lymph nodes of pigs were infected or reinfected with prrsv and subsequently observed to monitor the proliferation of various t-cell subsets [ , , ] . however, some ex vivo assays have also demonstrated the inability of certain strains of prrsv to induce tregs [ ] . in the current study, the treg frequencies were directly evaluated in the peripheral blood, bal, lung and lymphoid tissues collected from prrsv-ja -infected pigs. during the acute phase of infection, the treg frequencies largely remained unchanged, but suppression was observed in the bal throughout the infection period and in bln at dpc. comparable results were obtained in a previous in vivo study, revealing that the treg numbers remained unchanged in the pbmc, lymph nodes and tonsils of the infected pigs up to dpc [ ] . similar results were observed in another study, which showed no significant upregulation of tregs in the lymph nodes and pbmc at the acute phase of prrsv infection [ ] . furthermore, the decrease in treg frequencies in ja infected pigs could be attributed to phenotypic plasticity, which resulted in most of the naïve cd + t cells differentiating into effector cells, such as th and th , after being antigenically stimulated during the acute infection [ ] . a previous study revealed that the influenza virusinfected pigs showed a similar suppression of tregs in the bal and lymph nodes and an increased frequency of t-helper cells during the acute phase of infection [ ] . moreover, in some acute infections of mice with lymphocytic choriomeningitis virus (lcmv) and influenza virus, little or no effect on the immune response was observed after the removal of tregs from mice [ , ] . based on the findings from these studies, it can be deduced that tregs play only a minor role during many acute infections, but further studies are needed to understand the ultimate cause of this decline in the treg populations, specifically in bal cells during prrsv infection. cytokine secretion by immune cells plays a major role in protection against invading pathogens or in the induction of pathology [ ] . the levels of proinflammatory cytokines have often been associated with the severity of the disease. however, lower mrna and protein expression levels of proinflammatory cytokines have been associated with protection against viruses in pigs infected with prrsv strains that typically induce mild to moderate clinical signs [ ] . prrsv has been shown to reduce or suppress ifn-α production during infection [ ] . similarly, in the current study, ifn-α was peripherally suppressed in the infected pigs with increasing viral replication; however, in the balf, the levels of ifn-α were maintained, even at peak viremia, possibly playing a role in local viral clearance. similar to our observations, the induction of ifn-α with increasing viremia has also been observed locally in lymphoid tissues and the balf in prrsv-infected pigs [ ] . moreover, local induction of the proinflammatory cytokines il- β and il- in lymphoid tissues is reportedly linked to the clearance of prrsv from infected pigs [ ] . similar induction of the proinflammatory cytokines il- α, il- , il- , and tnf-α figure chronological order of the immune responses elicited in prrsv-infected pigs. this figure shows an overall representation of the immune responses elicited in prrsv-infected pigs during the acute phase of infection, which is the stage when the virus reached its maximum titre. most of the virus is cleared from blood and tissues by dpc due to the pig immune responses. the delayed induction of serum virus-neutralizing antibodies and the t-cell response in peripheral blood at days post-exposure, when most of the virus was cleared from the body, indicate that, in addition to the other factors, the early induced local t-cell response plays a key role in the clearance of the virus. has been observed in local tissues in previous studies [ ] . in the current study, the induction of proinflammatory cytokines locally in the balf at peak viremia could thus be linked to their contribution to the clearance of the virus from the pigs. in contrast, the induction of il- in local tissues during early infection was observed in the present study. the production of il- , which has been previously observed, reportedly contributes to the persistence of prrsv by suppressing the immune response in pigs [ ] . however, il- production also protects pigs from the tissue damage caused by proinflammatory cytokine overexpression [ ] . tregs are thought to produce il- , but in the current study, the treg frequencies did not change significantly. however, in addition to tregs, monocytes, th cells, mast cells and b cells are also known sources of il- [ ] . consequently, measuring the systemic cytokine response might not provide a full picture of the protective events orchestrated by cytokines locally in the lungs of prrsv-infected pigs. in conclusion, the early stimulation of the dc/macrophage frequencies coincided with the induction of a protective t-cell response in local lymphoid tissues and lung parenchyma, which are known sites of prrsv replication. moreover, the local proinflammatory cytokine responses at peak viremia augmented the clearance of the virus from the infected pigs. a temporal sequence of immunobiological events after prrsv infection in pigs is proposed in figure , and the observed schematic suggests that local t lymphocytes might play an important role in the clearance of the virus from pigs during the acute phase of infection. in addition, early nk cell induction and delayed t-cell responses in peripheral blood were also perceived, and these might play a complementary role in viral clearance. future studies are needed to further elucidate and refine the understanding of the anti-prrsv immune response in pigs, and these future studies should primarily focus on obtaining a better understanding of and deciphering the local immune responses with the aim of developing an effective strategy to restrain the virus. characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination duration of infection and 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in gnotobiotic pigs porcine reproductive and respiratory syndrome: neb- prrsv infection did not potentiate bacterial pathogens comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs porcine cd (+) nkp (+) lymphocytes have nk-cell characteristics and are present in increased frequencies in the lungs of influenza-infected animals response of the cdc and cdc subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype prrsv strain characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells induced recruitment of nk cells to lymph nodes provides ifn-gamma for t(h) priming dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity t cell responses to viral infections-opportunities for peptide vaccination resistance to and recovery from lethal influenza virus infection in b lymphocyte-deficient mice cd + t cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections the t-cell response to type porcine reproductive and respiratory syndrome virus (prrsv) th lymphocytes in respiratory syncytial virus infection the development and function of memory regulatory t cells after acute viral infections induction of porcine reproductive and respiratory syndrome virus (prrsv)-specific regulatory t lymphocytes (treg) in the lungs and tracheobronchial lymph nodes of prrsv-infected pigs european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells harnessing the plasticity of cd (+) t cells to treat immune-mediated disease partial depletion of natural cd (+) cd (+) regulatory t cells with anti-cd antibody does not alter the course of acute influenza a virus infection role of regulatory t cells during virus infection proinflammatory cytokines and viral respiratory disease in pigs differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells immunohistochemical expression of il- , il- , ifn-alpha and ifn-gamma in lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs interleukin- , interleukin- beta, and interferon-gamma levels are linked to prrs virus clearance porcine reproductive and respiratory syndrome virus infection activates il- production through nf-kappab and p mapk pathways in porcine alveolar macrophages interleukin- and the interleukin- receptor publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are pleased to acknowledge the undergraduates at the college of veterinary medicine and the laboratory technicians at the veterinary diagnostic center of jeonbuk national university (jbnu) for their constant assistance throughout the study. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . key: cord- -geh aaf authors: wagner, judith; kneucker, annette; liebler-tenorio, elisabeth; fachinger, vicky; glaser, melanie; pesch, stefan; murtaugh, michael p.; reinhold, petra title: respiratory function and pulmonary lesions in pigs infected with porcine reproductive and respiratory syndrome virus date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: geh aaf pulmonary dysfunction was evaluated in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv, isolate vr- ) and compared to clinical and pathological findings. infected pigs developed fever, reduced appetite, respiratory distress and dullness at days post-inoculation (dpi). non-invasive pulmonary function tests using impulse oscillometry and rebreathing of test gases (he, co) revealed peripheral airway obstruction, reduced lung compliance and reduced lung co-transfer factor. prrsv-induced pulmonary dysfunction was most marked at – dpi and was accompanied by a significantly increased respiratory rate and decreased tidal volume. expiration was affected more than inspiration. on histopathological examination, multifocal areas of interstitial pneumonia (more severe and extensive at dpi than dpi) were identified as a possible structural basis for reduced lung compliance and gas exchange disturbances. porcine reproductive and respiratory syndrome virus (prrsv), a member of the family arteriviridae, genus arterivirus, causes porcine reproductive and respiratory syndrome (prrs), an important cause of production losses in pigs (rossow, ) . the consequences of prrsv infection have been well documented for the reproductive system and for systemic infection (christianson et al., ; botner et al., ; mengeling et al., ; lager et al., ; kranker et al., ) . numerous studies have evaluated the clinical, pathological and immunological features of the respiratory form of prrs (labarque et al., ; samsom et al., ; opriessnig et al., ) , but the effects on pulmonary function have not been investigated, even though respiratory dysfunction may have a significant impact on the clinical outcome of the disease. typical clinical signs of respiratory infection with prrsv are tachypnoea or dyspnoea that can be accompanied by an increased respiratory effort, lethargy, fever and occasionally coughing, sneezing and chemosis (rossow et al., ; opriessnig et al., ) . gross lung lesions include failure of the lungs to collapse, as well as moderately well demarcated, mottled, brown areas of pneumonia (opriessnig et al., ) . microscopic lung lesions are charac-terised by type ii pneumocytic hypertrophy and hyperplasia, septal infiltration with mononuclear cells and alveolar exudates (opriessnig et al., ) . no data are currently available on the pathophysiological features and derangements of pulmonary function induced by prrsv. since the lungs are important for oxygen supply to all tissues, pulmonary dysfunction might have significant clinical and subclinical systemic consequences. pulmonary function testing has been applied to pigs with bacterial respiratory infections by our group (reinhold et al., , . a combination of impulse oscillometry and rebreathing of test gases can be used to evaluate lung ventilation, respiratory mechanics and pulmonary gas exchange in spontaneously breathing pigs. since both methods are non-invasive and applicable to conscious animals, changes in pulmonary function can be evaluated over time. in this study, pulmonary dysfunction was characterised in relation to clinical signs and pathological changes in pigs with experimental prrsv infection. twenty-four german hybrid pigs from a closed specific-pathogen-free herd known to be free of prrsv were transported to our institute at - days of age and were enrolled in the study after a quarantine period of days. pigs were housed according to the guidelines for animal welfare and fed twice daily with a - /$ -see front matter Ó elsevier ltd. all rights reserved. doi: . /j.tvjl. . . commercial grower diet without antibiotics. water was supplied ad libitum. pigs were housed in two groups of animals each, with pens of six pigs being separated from each other. virus vr- , the prototype type ii prrsv isolated in in minnesota, usa, has been shown to be virulent for sows and piglets (rossow et al., ; opriessnig et al., ; nielsen et al., ) . sixth passage vr- , propagated and titrated on ma- cells, was prepared at a concentration of log % tissue culture infectious doses/ml for use as the inoculum. the study had a randomised, negatively controlled design (table ) and was performed at biosafety level . ethical approval was obtained from the commission for the protection of animals of the state of thuringia, germany (registration number - / ). twelve pigs were exposed to prrsv and pigs served as controls. on the day of challenge, pigs exposed to prrsv had an age of . ± . days (mean ± standard deviation, sd) and a body weight of ± kg, while controls were . ± . days old and weighed ± kg. pigs were inoculated intranasally ( ml per nostril) and intramuscularly ( ml per pig) with either prrsv vr- or . % saline (controls). for intranasal inoculation, a ml syringe was connected to a tube ( cm  . mm inner diameter feeding tube, rüsch sterile, ref , size no. . willy rüsch gmbh) which had been inserted approximately cm into the nose. one millilitre of virus solution, along with ml air, per syringe was administered into each nostril with manual pressure, followed by im injection of ml into the gluteal muscle. clinical observations were recorded twice daily and included general behaviour, feed intake, appetite, rectal temperature, respiratory rate (rr) and the presence or absence of clinical signs of respiratory disease or diarrhoea. to monitor for the presence of prrsv by pcr and for seroconversion, blood samples were collected three times before challenge (À , À days and À h) and six times after challenge ( , , , , and days post-inoculation, dpi). blood samples, nasal swabs, rectal swabs and tracheal swabs were also collected to exclude concurrent infections (table ) . pulmonary function tests (pfts) were performed twice before challenge (À and À days) and seven times after challenge ( , , , , , , and dpi) in eight pigs exposed to prrsv and in eight controls (table ) . body weight was deter-mined day before each pft and at postmortem examination. four pigs per group (without pft) were sacrificed at dpi and eight pigs per group (with pft) were sacrificed at dpi for gross pathological, histopathological and immunohistochemical examination (table ) . to quantify the prrsv load in blood, serum samples were analysed by real-time pcr for prrsv strain vr- and its attenuated form, ingelvac mlv. rna was extracted using qiaamp viral rna mini kit (qiagen). reverse transcription was carried out using the multiscribe rt enzyme kit (applied biosystems). reverse transcription conditions were °c for min, °c for min and °c for s. amplification was carried out in triplicate using the taqman universal pcr master kit (applied biosystems) and the prrs vr- -specific primers mlv f ( -gcagctcccatctacagctgatt- ) and mlv r ( -agacaatgtgagtca aaacgggaaagat- ). the probe was tet- -ttggctagctaacaaatttgattgggc agtggagagttt- -tamra. thermal cycling conditions were °c for min, °c for min, then cycles of °c for s and °c for min. prrsv cdna quantification was achieved by comparison of the unknown sample with a standard curve derived from known amounts of plasmid dna. a commercial prrsv elisa (herdcheck prrs elisa, idexx) was used to detect anti-prrs antibodies in the blood serum of pigs before and after challenge. samples with sample-to-positive (s/p) ratio p . were considered to be positive for antibodies against prrsv, as recommended by the manufacturer. routine bacteriological culture was performed on nasal, tracheal and faecal swabs for bordetella spp., pasteurella spp., haemophilus spp., actinobacillus pleuropneumoniae (app) and salmonella spp. (table ). samples were tested for mycoplasma spp. by indirect immunofluorescence and for chlamydia spp. by pcr. paired serum samples from each pig (blood collected at the beginning of the study and at postmortem examination) were used for serology (table ) . commercial elisa test kits were used to detect antibodies against mycoplasma hyopneumoniae (dako m. hyo elisa, oxoid), app (cypress diagnostics), swine influenza virus (siv; idexx), transmissible gastroenteritis virus (tgev; svanova) and porcine table study design for prrsv monitoring, additional microbiological analysis, pulmonary function testing and postmortem examination. m. hyopneumoniae, mycoplasma hyopneumoniae; app, actinobacillus pleuropneumoniae; pcv- , porcine circovirus type ; prcv, porcine respiratory coronavirus; siv, swine influenza virus; tgev, transmissible gastroenteritis virus; pft, pulmonary function tests ( pigs per group examined postmortem at dpi). a n = pigs per group. b n = pigs per group (with pft). c n = pigs per group (without pft). respiratory coronavirus (prcv; svanova). testing for antibodies against porcine circovirus type (pcv- ) was performed by an indirect fluorescent antibody test (fachinger et al., ) . approximately min prior to pft, each pig was sedated with diazepam ( . - . mg/kg body weight im; faustan, weimer pharma). the sedated animal was restrained in a canvas sling with openings for the limbs and wore a tightly fitting face mask, allowing spontaneous breathing. after an adaptation period of approximately min, two non-invasive lung function techniques were applied consecutively: ( ) impulse oscillometry system (ios; masterscreen ios; jaeger); and ( ) rebreathing system (masterscreen diffusion; jaeger). both systems were originally produced for human medicine and have been successfully applied to pigs previously (reinhold et al., , . ios was used to measure variables of respiratory mechanics based on a forced oscillation technique, as previously validated for pigs (klein and reinhold, ; klein et al., ) . externally generated test impulses given by a loudspeaker were superimposed on spontaneous airflow of the breathing animal. variations in pressure and flow signals were analysed using fast fourier transformation to calculate the complex respiratory impedance, which consists of both respiratory resistance (rrs) and respiratory reactance (xrs) (smith et al., ) . airflow was registered during spontaneous breathing using a lilly-type pneumotachograph with a mesh resistance of pa/(l/s) and used to calculate spirometric variables of spontaneous breathing (rr and tidal volume, vt). each test per day and animal consisted of three consecutive ios measurements free of any artefacts (e.g. coughing or irregular breathing pattern). each measurement lasted for s. three impulses were generated per second, leading to independent results per min. the sampling rate was set at hz (period between two sampling points of ms), selecting sampling points after each impulse. results of the three measures per pig and per time point were averaged for statistical analysis. the following variables of ventilation and respiratory mechanics were analysed: ( ) rr; ( ) vt; ( ) volume of minute ventilation (mv); ( ) both vt and mv related to body weight (vt/kg, mv/kg); ( ) rrs; ( ) xrs, each at , , and hz, and separated for inspiration and expiration at each frequency (rrs,in hz . . . rrs,in hz ; rrs,ex hz . . . rrs,ex hz ; xrs,in hz . . . xrs,in hz ; xrs,ex hz . . . xrs,ex hz ); ( ) resistance of proximal airways (r prox ); and resistance of distal airways (r dist ). using different test gases and a multiple breath approach (i.e. steady state method), the rebreathing system permitted the simultaneous measurement of two additional pulmonary function variables. the functional residual capacity (frc) of the lungs was measured by the helium (he) dilution technique (washin). the transfer factor of the lungs for carbon monoxide (tl co) was determined in order to evaluate the transfer of oxygen from the lungs into the blood. for assessing both frc and tl co, each pig inhaled the test gas mixture ( % he, . % co in synthetic air) from a reservoir bag. since animals were growing, the volume filled in the reservoir bag was l in the pre-challenge period and was adjusted for the increasing lung volume with growth to l from to dpi. the time to perform this test (i.e. rebreathing time) increased from ± s (mean ± sd) week before challenge (body weight of ± kg, mean ± sd) to ± s at dpi (body weight of ± kg) due to a significant correlation with increasing body weight (r = . ; r = . %; p = . ) or increasing frc (r = . ; r = . %; p = . ), respectively. frc was calculated per kg body weight (frc/kg) to avoid body weight being a confounding factor. data for tl co were corrected for the individual concentration of haemoglobin in the blood (tl co hb ), as determined before each pft using a haemoxymeter that allows species-specific analysis of haemoglobin fractions for pigs (osm , radiometer). to correct for body weight and metabolism, tl co hb was calculated per kg body weight (tl co hb /kg), as well as per kg metabolic body weight (tl co hb /kg . ). pigs were euthanased by intravenous injection of pentobarbital sodium (release, wdt) at p mg/ kg body weight. the trachea was exposed, severed distal to the larynx and the lungs were instilled with neutral buffered formalin (nbf) at a pressure of mm water column. after min of intrathoracic fixation, the trachea was closed and the lungs were removed from the thoracic cavity and placed in nbf for h. samples collected from proximal and distal parts of the cranial and caudal pulmonary lobes and from one area of the accessory lobe were embedded in low-melting paraffin ( - °c). paraffin sections were stained with haematoxylin and eosin for histological examination. immunohistochemistry prrsv antigen was detected by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase (apaap) method. paraffin sections collected on charged slides were pretreated with proteinase k ( . % in phosphate buffered saline, ph . ) for min. the monoclonal antibody sr -a (rural technologies) was used as the primary antibody and rabbit anti-mouse ig (dianova) was used as the secondary antibody to bind the apaap complex (dako). alkaline phosphatase was visualised using neufuchsin. slides were counterstained with methylene green. positive control sections (bioscreen evdmc) were included with each reaction. statistical analyses were performed to determine the relationship between prrsv infection and clinical signs, body weight or lung function variables. for statistical evaluation of clinical signs, the study period from days before inoculation until dpi was separated into nine sub-periods, each of three consecutive days. within each sub-period, data obtained per day and per animal were averaged. for calculating baseline data obtained by pft, all measures per pig before inoculation were averaged. numeric data are presented as medians, minima and maxima. the mann-whitney-wilcoxon-test (w-test; comparison of medians) was used to determine significant differences between groups at p . . for regression analyses a linear model was used (y = a + bx) and both coefficient of linear correlation (r) and coefficient of determination (r ) were calculated. pcr for quantification of viral load and elisa for antibody detection demonstrated that all pigs were negative for prrsv before challenge. controls remained free of prrsv infection throughout the trial. in pigs exposed to prrsv, infection was confirmed by increasing viral loads in serum. viraemia was detected in all pigs at dpi and reached a maximum dpi (fig. a) . antibody titres against prrsv were first observed at dpi in one pig and at dpi in all other pigs, reaching a maximum at dpi (fig. b) . none of the control pigs exhibited any clinical signs of respiratory disease during the study. pigs exposed to prrsv developed clinical signs initially observed at - dpi, including reduced appetite, dullness and fever. body temperature was significantly increased in challenged pigs compared to controls from to dpi and this increase showed a biphasic character, with maxima at - dpi and - dpi ( fig. a) . most prrsv challenged pigs remained dull until the end of the trial, whereas reduced appetite and/or diarrhoea was identified only sporadically from to dpi. the daily weight gain was ± g (mean ± sd) in controls and ± g in pigs exposed to prrsv; there was no significant difference between groups. coughing, dyspnoea and ocular discharge were detected in pigs exposed to prrsv. coughing was present from to dpi in / pigs and was most prominent from to dpi. rr increased significantly in prrsv challenged pigs compared to controls starting in the period to dpi and reached maximal values at - dpi (fig. b) . dyspnoea developed in / pigs in the period - dpi, with a maximal intensity at - dpi. one prrsv challenged pig exhibited ocular discharge in the period - dpi. interestingly, no nasal discharge and no vomiting was observed. compared to controls, vt/kg was significantly reduced in pigs exposed to prrsv after challenge (fig. a) . this decrease was most prominent in the period - dpi. in contrast, the volume of mv in relation to body weight (mv/kg) increased significantly in prrsv challenged pigs. compared to controls, the most marked increase in mv was seen between and dpi (fig. b) . in pigs experimentally exposed to prrsv, marked changes in respiratory impedance occurred at - dpi; the changes were more prominent during expiration than during inspiration. spectral curves of rrs and xrs within the frequency range of - hz at dpi are shown in fig. . rrs at and hz measured during expiration was significantly elevated in comparison with controls, leading to a significantly larger difference between inspiratory and expiratory resistance values. this increase in rrs,ex was statistically significant on days and after challenge for both rrs,ex hz and rrs,ex hz , as well as for rrs,ex hz alone on days and post-challenge (supplementary table ). in contrast, rrs at and hz measured during inspiration was not significantly different between groups (fig. , supplementary table ). both rrs,ex and rrs,in assessed at and hz were lower in pigs exposed to prrsv compared to controls (supplementary table ). due to significantly increased expiratory resistance data at low frequencies ( and hz) and significantly lower rrs,ex data at hz, the resistance curves during expiration became inversely frequency dependent after prrsv challenge, i.e. resistance decreased with increasing frequency (fig. ) . xrs at all frequencies ( - hz) was significantly lower in prrsv infected pigs compared to controls (fig. ) . this observed negativity of the total xrs curves was more prominent during expiration compared to inspiration, mainly from to days after exposure (supplementary table ). proximal and distal airway resistances are shown in table . after challenge, pigs exposed to prrsv had significantly higher distal airway resistance compared to controls. in contrast, r prox was significantly lower in prrsv challenged pigs than in control pigs. as shown in table , volumes of frc increased in growing pigs of both groups during the study (linear correlation between body body temperature (a) and respiratory rate (b) of pigs exposed to prrsv and controls. box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). days À to : n = per group; days - : n = per group. # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). weight and frc: control pigs: r = . ; r = . %; p . ; prrsv infected pigs: r = . ; r = . %; p . ). neither absolute volumes of frc nor frc/kg body weight (data not shown) differed significantly between groups at any time point. in controls, tl co hb ranged from . mmol/min/kpa (median; minimum-maximum: . - . ) days before challenge to . mmol/min/kpa (minimum-maximum: . - . ) at dpi. due to the increase in body weight of pigs during the trial (r = . ; r = . %; p . ) and because gas exchange in the lung physiologically adapts to metabolism, groups were compared on a basis of tl co hb data corrected for either body weight (fig. a ) or metabolic body weight (fig. b ). compared to controls, reduced gas transfer became evident in pigs challenged with prrsv from dpi until the end of the study. this decrease was statistically significant for tl co hb /kg at and dpi and for tl co hb /kg . at dpi. there were no gross changes in the lungs of prrsv infected or control pigs. systemic enlargement of lymph nodes (including the tracheobronchial lymph nodes) was evident at and dpi in the prrsv inoculated group. histopathological examination of the lungs of inoculated pigs revealed mild to moderate multifocal interstitial pneumonia characterised by thickening of interalveolar septa, septal infiltration with mononuclear cells, hyperplasia and hypertrophy of type ii pneumocytes and alveolar exudates of macrophages, necrotic debris and occasionally multinucleated cells (fig. a) . pulmonary lesions were more frequent and more severe in cranial lobes compared to caudal lobes and in the proximal part of lobes compared to the distal part. at dpi, mild to moderate interstitial pneumonia was evident in / pigs. at dpi, lesions were milder, but all eight pigs were affected. hyperplasia of the tracheobronchial lymph nodes was evident in / pigs at dpi. in the lungs of control pigs, there were mild to moderate peribronchiolar and perivascular lymphocytic infiltrates, along with mild lymphocytic infiltrates in interalveolar septa and mild multifocal clusters of alveolar macrophages in alveolar spaces. there was no thickening of interalveolar septa (fig. b ). prrsv antigen was detected only in pigs inoculated with prrsv. there was a close correlation between pulmonary lesions and the presence of viral antigen. prrsv antigen was present in the cytoplasm of alveolar macrophages and occasionally in multinucleate cells within alveoli (fig. c) . it was also seen in mononuclear cells within thickened interalveolar septa and in type ii alveolar epithelial cells. prrsv antigen was also detected in one pig which did not develop interstitial pneumonia. at dpi, the highest amount of viral antigen was found in two pigs with moderate pulmonary lesions. there was less prrsv antigen present in the lungs at dpi compared to dpi. all pigs were free of app, bordetella spp., mycoplasma spp., pasteurella spp., siv and tgev. chlamydia spp. were detected in rectal swabs of all pigs. all animals had antibodies against pcv- and prcv. haemophilus parasuis was detected in a nasal swab from / pigs in the control group before challenge. salmonella spp. were detected sporadically in rectal swabs before and after challenge in both groups. this study characterised respiratory dysfunction induced by the well-characterised prototypical na-type prrsv isolate vr- (rossow et al., ; opriessnig et al., ; nielsen et al., ) . moderate clinical disease was induced that allowed consecutive pulmonary function testing until weeks after challenge without any spontaneous deaths before the end of the study. this is the first simultaneous within-subject study of clinical, functional and morphological changes in pigs experimentally infected with prrsv. in the absence of other major infectious causes of respiratory disease, changes in clinical status, pulmonary dysfunction and pathology in pigs in this study can be attributed to prrsv. prrsv viraemia and seroconversion were evident in all challenged pigs. fig. . tidal volume per kg body weight (vt/kg; a) and minute ventilation per kg body weight (mv/kg; b) of pigs exposed to prrsv (n = ) and controls (n = ) as measured with the impulse oscillometry system. box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (wtest, p . ). challenge with vr- induced reproducible and representative respiratory disease consistent with previous studies using the same strain (rossow et al., ; opriessnig et al., ) . prrsv viraemia was detected at dpi and coincided with an increase in rectal temperature. an increase in rr was evident at dpi and lasted until the end of the study, i.e. until at least days after infection. other clinical signs included dyspnoea and coughing and were most prominent from to dpi. pulmonary dysfunc- fig. . medians of spectral respiratory impedance within the frequency range of - hz separated for respiratory impedance during inspiration (a) and respiratory impedance during expiration (b) of pigs exposed to prrsv (n = ) and controls (n = ) dpi. rrs, respiratory resistance; xrs, respiratory reactance, both during expiration (rrs,ex, xrs,ex, respectively) and inspiration (rrs,in, xrs,in, respectively) . # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). distal airway resistance (r dist ) and proximal airway resistance (r prox ) in pigs exposed to prrsv (n = ) and controls (n = ). control prrsv a.i. represents data measured prior to inoculation with prrsv (averaged per pig). # indicates significant difference between pigs exposed to prrsv and controls (w-test, p . ). functional residual capacity (frc) in pigs exposed to prrsv (n = ) and controls (n = ). tion was evident earlier ( dpi) and was most prominent at dpi. microscopic lung lesions were more severe at dpi than at dpi which is in accordance with opriessnig et al. ( ) . some authors have reported coughing as a clinical sign of prrs (done et al., ; beyer et al., ) , whereas others have attributed coughing to secondary infections (e.g. m. hyopneumoniae, b. bronchiseptica, pcv- ) (thacker et al., ; brockmeier et al., ; thacker and thanawongnuwech, ) . we observed a spontaneous dry cough in most of the pigs exposed to prrsv, but not in controls. due to the absence of other major respiratory pathogens in the present study and because there was no evidence of tracheitis, bronchiolitis or airway mucus on pathological examination, we interpret the observed cough as being induced by bronchospasms as a component of the respiratory illness caused by prrsv. this interpretation is supported by the presence of peripheral airway obstruction, as evaluated by pulmonary function testing. the pattern of ventilation, as indicated by spirometric data determined during spontaneous breathing, includes rr, vt and mv. in our study, rr increased significantly in pigs exposed to prrsv, with maximal values at - dpi ( % compared to baseline data). increased rr may be a response to hyperthermia, as well as an attempt to compensate for reduced vt and/or arterial oxygen deficiency caused by reduced gas transfer from the lungs into the blood. relative vt (vt/kg) was significantly reduced (< ml/kg) in pigs exposed to prrsv, while vt/kg was always > ml/kg in healthy controls. reduced vts indicate changes in the pattern of breathing caused by obstructive or restrictive disorders and/or by disorders in gas exchange. pulmonary function tests in prrsv challenged pigs indicate both obstructive and restrictive disorders (confirmed by increased rrs at frequencies hz and decreased xrs), as well as disorders in gas exchange (confirmed by decreased tl co hb ). the latter correlates with the pathological findings of thickened interalveolar septa and the presence of alveolar exudates. the observed increases in mv in prrsv challenged pigs were caused by increases in rr and indicate compensation for reduced vts. pigs infected with prrsv had shorter breathing cycles and shallower inspiration. this pattern of breathing, however, is pathophysiologically linked to a higher percentage of dead space venti-lation compared to alveolar ventilation and consequently carries the risk of alveolar hypoventilation. in addition, the energy requirement for breathing increases due to increased effort of respiratory muscles. impulse oscillometry measures complex respiratory impedance, which consists of rrs and xrs (smith et al., ) . while rrs reflects mainly airway resistance, xrs is determined by inertive and capacitive components of the respiratory system. pressure impulses generated by a loudspeaker in a frequency range of - hz are used as test signals. low frequencies ( - hz) penetrate deep into the respiratory tract and provide a representation of the peripheral airway system, whereas high frequencies ( - hz) provide a representation of the upper and central airway systems. results at frequencies > hz were disregarded, since they are mainly influenced by mechanical properties of the face mask, which acts as a confounding factor for respiratory impedance measurements in animals (reinhold et al., ; klein et al., ) . in addition to the spectral curves of rrs and xrs, two model-derived resistances (i.e. r dist and r prox ) were analysed to differentiate between the effects of prrsv on either proximal airways (nasal cavities, larynx and pharynx) or distal airways (lung periphery), respectively. all variables of respiratory mechanics were significantly different in pigs challenged with prrsv compared to controls. rrs at - hz, r dist and xrs are important variables with respect to the lung periphery, including peripheral airways. rrs at frequencies hz was significantly elevated in pigs exposed to prrsv and this increase was only evident during expiration. this phenomenon indicates airflow limitation predominantly in the peripheral airway system, since peripheral airway obstruction affects expiration much more than inspiration. the presence of peripheral airway obstruction is emphasised by significant increases in model-derived resistance of distal airways (r dist ). due to the absence of mucus and bronchial wall oedema, peripheral airway obstruction was most likely to have been caused by bronchospasms. morphological correlates for bronchospasms, however, are lacking, since contractions or spasms of airway muscles do not persist after euthanasia and the fixation method used in this study (intratracheal instillation with formalin) opens airways and fills alveolar regions, irrespective of the ventilation status . transfer factor of carbon monoxide corrected for haemoglobin in relation to body weight (tl co hb /kg; a) and metabolic body weight (tl co hb /kg . ; b) in pigs exposed to prrsv (n = ) and controls (n = ). box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). during normal breathing. decreases in xrs indicate reduced compliance of the lung. this might be caused by restrictive disorders due to both stiffness of obstructed airways and pulmonary tissue components. the lungs of pigs challenged with prrsv had thickened interalveolar septa and septal infiltration with mononuclear cells, which may be responsible for reduced compliance. findings with respect to central and/or upper airways are reflected by both rrs at - hz and r prox . after prrsv challenge, proximal airway resistance in exposed pigs was significantly lower compared to controls. this phenomenon might be interpreted as 'upper airway opening', an attempt to compensate for peripheral airway obstruction by increasing the diameters of upper airways (nasal cavities, larynx and pharynx). due to the functional character of this phenomenon, no morphological equivalent is visible at postmortem examination. in humans, enlargement of the aperture of the glottis is recognised during a high frequency, low vt breathing pattern (stȃnescu et al., ) . a further approach to enlarge the upper airway diameter is reduction of mucosal thickness by changes in blood flow. in rats, cervical sympathetic nerve stimulation induces a reduction in upper airway resistance, which is most likely induced by a-adren-ergic vasoconstriction of the upper airway mucosal vasculature, resulting in reduced mucosal thickness (o'halloran et al., ) . stimulation of cervical sympathetic nerves, however, can be caused by systemic hypoxia and hypercapnia (matsumoto et al., ) . prrsv challenged pigs showed a significantly increased partial pressure of co compared to controls in venous blood gas analysis (data not shown). other factors contributing mainly to proximal airway resistance are anatomical peculiarities in the upper airways and head position. while performing pfts, head position was standardised as recommended for pigs (klein et al., ) . the anatomical structure of the nasal cavities, however, might contribute to different upper airway resistances between individuals and could explain significantly different rrs data p hz between groups even before exposure. influence of prrsv on functional residual capacity frc represents the volume present in the lung at the end of spontaneous expiration. therefore, it might be an indicator of 'trapped air', hyperinflation or emphysema. in our trial, no signifi- cant changes in frc could be identified, indicating that airway obstruction was not severe enough to induce 'air trapping' or obstructive emphysema. this is in accordance with a lack of emphysema on pathological examination in our study, as well as studies of others (pol et al., ; halbur et al., ; rossow, ) . in the present study, both tl co hb in relation to body weight (tl co hb /kg) and metabolic body weight (tl co hb /kg . ) revealed significant decreases in the diffusion of oxygen from the lungs into the blood in pigs exposed to prrsv compared to controls. limiting oxygen diffusion from the alveoli to the blood can be explained by increased diffusion distance due to infiltration of alveolar walls by mononuclear cells. in addition, hyperplasia and hypertrophy of the alveolar epithelial cells, as well as the presence of alveolar exudates, decreases the amount of gas within alveoli. alveolar hypoventilation due to a rapid and superficial pattern of breathing may also contribute to decreased oxygen exchange. in the pig, which has no collateral airways (mclaughlin et al., ) , each airway obstruction results in regional heterogeneity of alveolar ventilation (robinson, ) . consequently, obstruction of ventilation in pigs exposed to prrsv most likely resulted in imbalances between ventilation and perfusion, leading to reduced oxygen transfer from the lungs into the blood. this would result in respiratory acidosis characterised by reduced ph and increased partial co pressure in venous blood, indicating reduced alveolar ventilation. this is the first study evaluating disorders of lung function caused by prrsv in pigs. significant imbalances in respiratory mechanics, lung ventilation and pulmonary gas exchange were correlated with clinical signs and pathological findings. prrsv isolate vr- alone was capable of inducing respiratory distress for at least weeks after exposure. studies using other prrsv strains would be useful to identify differences in the pathogenesis of infections caused by different prrsv isolates. evaluation of therapeutic and vaccination strategies will be supported by combining the functional approach of this study with knowledge generated from animal studies focussing on inflammatory markers and the immune response. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. porcine reproductive and respiratory syndrome virus (prrsv): kinetics of infection in lymphatic organs and lung isolation of porcine reproductive and respiratory syndrome (prrs) virus in a danish swine herd and experimental infection of pregnant gilts with the virus effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs experimental reproduction of swine infertility and respiratory syndrome in pregnant sows porcine reproductive and respiratory syndrome (prrs): a review, with emphasis on pathological, virological and diagnostic aspects the effect of vaccination against porcine circovirus type in pigs suffering from porcine respiratory disease complex comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus analysis of respiratory mechanics by impulse oscillometry in non-sedated and diazepam-sedated swine respiratory mechanics in conscious swine: effects of face mask, head position and bronchoconstriction evaluated by impulse oscillometry experimental inoculation of swine at various stages of gestation with a danish isolate of porcine reproductive and respiratory syndrome virus (prrsv) effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine a study of the subgross pulmonary anatomy in various mammals cervical preganglionic sympathetic nerve activity and chemoreflexes in the cat comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure generation of an infectious clone of vr- , a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus influence of cervical sympathetic nerves on ventilation and upper airway resistance in the rat comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr- ), atcc vr , and two recent field isolates of prrsv pathological, ultrastructural and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) validation of impulse oscillometry in friesian and blue belgian calves with respect to changes in extrathoracic upper airway resistance evaluation of lung function in pigs either experimentally or naturally infected with chlamydiaceae an experimentally induced chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation some functional consequences of species differences in lung anatomy experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs porcine reproductive and respiratory syndrome changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for cd (+) cells forced oscillation technique and impulse oscillometry glottis opening and airway resistance mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia porcine respiratory disease complex (prdc) the authors are very grateful to annelie langenberg, sylke stahlberg, ines lemser and to all colleagues working in the team of the animal house (fli jena, germany) for their skilful assistance during the study. furthermore, the authors are thankful to colleagues of the institute of bacterial infections and zoonoses (ibiz) in the 'friedrich-loeffler-institut' (jena, germany), namely dr. ulrich methner and his team for salmonella spp. diagnosis and dr. astrid raßbach and co-workers for performing bacterial screening for pasteurella spp., bordetella spp., haemophilus spp. and app. in addition, the authors thank dr. konrad sachse and staff of the oie reference laboratory for chlamydiosis for chlamydia spp. testing and renate haß for assaying mycoplasma spp. this work was funded by the non-profit organisation 'akademie für tiergesundheit e.v.' (germany), which provided a scholarship for judith wagner. supplementary data associated with this article can be found, in the online version, at doi: . /j.tvjl. . . . key: cord- -hksmt i authors: mclean, rebecca k.; graham, simon p. title: vaccine development for nipah virus infection in pigs date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: hksmt i nipah virus (niv) causes a severe and often fatal neurological disease in humans. whilst fruit bats are considered the natural reservoir, niv also infects pigs and may cause an unapparent or mild disease. direct pig-to-human transmission was responsible for the first and still most devastating niv outbreaks in malaysia and singapore in – , with nearly human cases and over fatalities. pigs can therefore play a key role in the epidemiology of niv by acting as an “amplifying” host. the outbreak in singapore ended with the prohibition of pig imports from malaysia and the malaysian outbreak was ended by culling % of the country's pig population with costs exceeding us$ million. despite the importance of niv as an emerging disease with the potential for pandemic, no vaccines, or therapeutics are currently approved for human or livestock use. in this mini-review, we will discuss current knowledge of niv infection in pigs; our ongoing work to develop a niv vaccine for use in pigs; and the pig as a model to support human vaccine development. nipah virus (niv) is an enveloped, single stranded, negative sense rna paramyxovirus, genus henipavirus. the natural hosts and wildlife reservoirs of niv are old world fruit bats of the genus pteropus ( ). both nipah and the related hendra virus possess a number of features that distinguish them from other paramyxoviruses. of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-b and -b as cellular receptors ( ) . the niv attachment glycoprotein (g) is responsible for binding to ephrin-b /-b ( ). following receptor binding, the g protein dissociates from the fusion (f) protein. subsequently, the f protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry ( ) . during viral replication, the f protein is synthesized and cleaved into fusion active f and f subunits. these subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells ( ) . this cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of niv as it allows spread of the virus, even in the absence of viral budding ( , ) . niv infection is currently classed as a stage iii zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission ( , ) . niv outbreaks have been recognized yearly in bangladesh since as well as occasional outbreaks in neighboring india (figure ). these outbreaks have been characterized by person-to-person transmission figure | previous locations of henipavirus infection outbreaks. nipah and hendra virus distribution map highlighting the range of the natural wildlife reservoir, pteropus spp. bats [adapted from ( ) ]. and the death of over % of infected people ( , ) . in may , the first ever outbreak in southern india was reported. a total of niv cases, of which resulted in death, were reported in the state of kerala. pteropus giganteus bats from areas around the index case in kozhikode, kerala, were tested at the national high security animal diseases laboratory at bhopal. of these, % were found to be niv positive by rt-pcr ( ) . characteristics of niv that increase the risk of it becoming a global pandemic include: humans are already susceptible; many niv strains are capable of person-to-person transmission; and as an rna virus, niv has a high mutation rate ( ) . niv has been found to survive for up to days when subjected to various environmental conditions, including fruit bat urine and mango flesh ( ) . whilst survival time was influenced by fluctuations in both temperature and ph, the ability for niv to be spread by fomites could play a role in outbreak situations. the first and still most devastating niv outbreak occurred in peninsular malaysia from september to may ( , ) . the link to pigs in this outbreak was obvious as % of the infected patients had contact with pigs ( ) . if a niv strain were to become human-adapted and infect communities in southeast asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic ( , , ) . in september , there was an outbreak of severe febrile encephalitis among pig farmers in the state of perak, malaysia, that was associated with a high mortality rate. a total of cases of encephalitis, of which resulted in death, were confirmed. these deaths were initially thought to be due to japanese encephalitis (je), an endemic disease in malaysia. however, with most cases occurring in men who worked with pigs, the epidemiological characteristics of this disease were distinct from those of je, where ∼ % of cases occur in children aged - years ( ) ( ) ( ) . the epidemiological link was from fruit bats infecting pigs that then served as amplifier hosts, resulting in transmission to humans through close contact ( ) . as a result of movement of infected pigs and humans to other states in malaysia, by february similar diseases were recognized in both pigs and humans in new outbreak areas ( ). in the following month, there were cases of respiratory illness and encephalitis amongst singapore abattoir workers who had handled pigs imported from the outbreak areas in malaysia ( ) . due to this, the importation of pigs from malaysia ceased which in turn ended the outbreak in singapore. the outbreak in malaysia ended when . million pigs ( % of the country's pig population) were culled from outbreak and surrounding areas ( , ) . the niv outbreak incurred significant economic costs and long-term damage to the malaysian pig industry: us$ million in direct costs and lost market revenue, including us$ million in compensation to farmers for the . million pigs slaughtered and , jobs lost ( ) . to this date, malaysian pig farming is only permitted in "identified pig farming areas." pigs also suffered during the / malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. the severity of symptoms of niv infection in pigs varied with age. in suckling pigs (< weeks old), mortality could be high (up to %) and labored breathing and muscle tremors were evident. in growing pigs ( to months), an acute febrile (> . • c) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis ( ) . in some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. pigs of this age had high morbidity and low mortality (< %) ( ) ( ) ( ) . some animals over months of age died rapidly (within h) without signs of clinical disease. respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. first trimester abortions were also reported ( ) ( ) ( ) . in an experimental infection study, pigs were inoculated subcutaneously with a niv isolate from the central nervous system of a fatally infected human patient. infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough ( ) . pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. in a second study, piglets were inoculated oronasally with a human niv isolate ( ) . all infected animals showed a transient increase in body temperature between and days post-infection. two of these animals developed transient respiratory signs, mild depression and a hunched stance. both these studies concluded that niv infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. this can make field diagnosis of niv infection in pigs difficult, as observed in the outbreak in malaysia ( , ) . the name proposed for the disease caused by niv infection of pigs was "porcine respiratory and neurological syndrome" (also known as "porcine respiratory and encephalitis syndrome"), or, in peninsular malaysia, "barking pig syndrome" ( ) . niv infection was included as the sixth pig disease notifiable to the oie world organization for animal health ( ) . the oie approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases. despite the importance of niv as an emerging disease with the potential for pandemic, no therapeutics or vaccines are approved for use in humans or livestock species. due to the lethal nature of niv infection, producing a safe, live attenuated vaccine with no potential for reversion is difficult. however, recombinant niv mutants, attenuated in hamster and ferret models, have been shown to generate strong neutralizing antibody responses ( , ) . more commonly, niv vaccine approaches have focused individual candidate antigens delivered as subunit vaccines or using viral vectors. the most studied vaccine candidate is the soluble form of the g protein (sg) from the related hendra virus (hev). hev and the niv malaysia strain share between and % amino acid homology between their proteins; with f and g proteins sharing and % homology, respectively ( ) . both f and g envelope glycoproteins are regarded as vaccine candidate antigens since they are the targets of niv neutralizing antibodies ( ) . an adjuvanted hev sg protein subunit-based vaccine (equivac r hev, zoetis) has been licensed in australia to protect horses against hev and to reduce the zoonotic risk to humans ( ) . equivac r hev protects ferrets and african green monkeys (agms) after experimental challenge with niv, as well as hev ( , ) . surprisingly, this vaccine failed to protect pigs from experimental niv challenge ( ) . since the vaccine induced cross-neutralizing antibodies but not measurable t cell responses, the authors concluded that both arms of the adaptive immune response may be required for protection against niv and hev. these studies also potentially highlight that adjuvants can have species specific effects and tailoring of adjuvants to the target species may be required or considered in the context of preclinical models. the experimental viral vectored vaccine candidates for niv include vesicular stomatitis virus, rabies virus, canarypox virus (alvac strain), adeno-associated virus (aav), measles virus, newcastle disease virus (ndv) and venezuelan equine encephalitis virus ( ) . alvac expressing niv g or f (alvac-g and alvac-f) was found to protect pigs against niv challenge weeks after the second immunization ( ) . high titres of niv neutralizing antibodies were induced with the alvac-g vaccine, while despite the low levels of neutralizing antibodies induced by the alvac-f; all vaccinated pigs were protected against virulent niv challenge. recombinant attenuated ndv expressing niv glycoproteins have been shown to induce long lasting niv-specific nabs in pigs, with the vector expressing niv g performing better than niv f ( ). however, no challenge was performed in this study and it remains to be determined whether these paramyxovirus-based vaccine candidates are efficacious. compared to canarypox vectors, ndv-based vectors have a number of advantages including their high titer propagation in chicken eggs removing the requirement for cell culture ( , ) . despite these encouraging results and the continued threat posed by niv, no vaccine candidate has progressed toward market for either pigs or humans. the promising performance of experimental niv and hev vaccines in animal models and the licensure of equivac r hev, as a "one health" vaccine to safeguard animal and human health, strongly support the proposition that a safe and effective niv vaccine may be developed for pigs to reduce the severe economic consequences of niv outbreaks and the threat to public health. with partners, we have initiated a project that aims to develop such a vaccine. we are systematically analyzing the immunogenicity and protective efficacy of three niv vaccine candidates in pigs: ( ) an adjuvanted niv sg protein (orthologous to the equivac r hev vaccine), ( ) niv g protein delivered by a replication-deficient simian adenoviral vector (chadox niv g), and an adjuvanted, molecular clamp stabilized niv f (mcsf) protein. chadox is a multispecies vector with an established human and livestock safety profile ( ) . chadox offers the potential for both single dose efficacy and thermostabilization ( , ) . the molecular clamp is a proprietary stabilization domain that preserves the f protein in its native "pre-fusion" form, which should enhance immunogenicity and thermostability. in depth analyses of t cell and antibody responses are being conducted to identify correlates of vaccine-induced protection. we will examine the durability of niv-neutralizing antibodies and other immune responses associated with protection, including a comparison of a singleshot vs. homologous prime-boost immunization regimes. incontact animals will be introduced to assess transmission of challenge virus from vaccinates or unvaccinated control animals. the sporadic nature of niv outbreaks means that the commercial development of niv vaccines for use in pigs (other livestock or humans) is limited and animal health companies are of the opinion that niv vaccines will have limited marketability. our ongoing studies should help facilitate this by developing a safe and efficacious prototype niv vaccine that is amenable to "surge production" and discrimination of infection in vaccinated animals (diva) capability. subsequent development and licensure of this vaccine will require engagement with international, regional, and national agencies and the creation of dependable markets via the establishment of niv vaccine banks. the oie world fund manages vaccine banks and the delivery of vaccines for avian influenza, rabies, foot-andmouth disease, and peste de petit ruminants ( ) . vaccine banks ensure the procurement and delivery of high quality vaccines mass-produced in line with oie intergovernmental standards. critically these vaccine banks can be rapidly deployed when required and this model appears most appropriate in the context of reactive emergency vaccination programmes to aid niv outbreak control. vaccines can play a major component in an emergency response against emerging infectious disease, with the main aim to reduce virus spread between susceptible hosts ( ) . the precise decisions on control strategies will be complex and vary for different regions. factors such as: herd density, production systems, the presence of susceptible wildlife, the impact on export trade and current opinions on economic vs. ethical factors will likely play a role. one strategy to halt a niv outbreak would be to deploy a stockpiled vaccine for ring vaccination around the niv affected area. this approach was utilized in the ebola outbreak in guinea and showed great promise in terms of disease containment and elimination ( ) . for such a strategy, a vaccine with single-dose efficacy and a rapid onset of immunity preventing virus transmission would be preferential. this is likely to be best achieved with a viral-vectored ( ) or mrna vectored vaccine ( ) . the highly unpredictable nature of niv outbreaks means that it is highly unlikely that niv vaccines would be used routinely by pig producers. one strategy that could help ensure that immunity to niv is maintained in pig herds could involve the engineering of niv g into a live attenuated viral vaccine, such as pseudorabies, which are widely used in countries at-risk. the recent ebola and zika epidemics highlighted how poorly prepared we were to deal with these new and emerging diseases. there has therefore been a global drive to develop vaccines against these diseases and improve preparedness. the coalition for epidemic preparedness innovation's (cepi's) was established in with a mandate of financing and coordinating the development of new human vaccines to prevent and contain infectious disease epidemics. cepi selected niv, lassa virus and middle east respiratory syndrome-coronavirus, three pathogens from the who's list of priority diseases needing urgent r&d attention as its initial focus ( , ) . the who's list of priority diseases is part of the r&d blueprint, which identifies priority diseases and addresses gaps in the global scientific community to increase preparedness for future outbreaks. the main aim of the blueprint is to fast-track the availability of effective tests, vaccines, and medicines that can be used to save lives and avert large scale crises ( ) . in , the us food and drug administration (fda) established the "animal rule" for regulatory approval of vaccines and therapeutics for which efficacy testing in humans is impossible, therefore requiring relevant animal models that represent a disease model similar to that of the human disease ( ) . vaccine efficacy studies in animal models aim to identify specific vaccine-induced correlates of protection including neutralizing antibodies or cell-mediated responses ( ) . in , a vaccine to protect against anthrax was the first to be approved through the "animal rule" ( ) . the licensing pathway for the "animal rule" requires that immunogenicity results from clinical trials must be consistent with previously identified immune correlates associated with protection ( ) . therefore, identifying reliable markers of vaccine-generated immunity becomes critically important for pathogens such as niv. large animal models have been shown to more accurately predict vaccine outcome in humans in comparison to small animal models ( ) therefore defining correlates of vaccineinduced protection in pigs, may play an important role in supporting subsequent human vaccine licensure under the "animal rule." animal models can be validated for a particular disease according to a number of different criteria, which include "face" and "predictive" validity. for face validity there must be similarities in the pathology and clinical symptoms between the animal model and the human disease ( ). as discussed above, niv infection of pigs causes a similar respiratory and neurological syndrome as seen in human infections. although, disease severity in pigs may be considered lower than in humans. the predictive validity of a model means that clinically effective interventions demonstrate a similar effect in the animal model ( ) . no clinical trials of niv vaccine candidates have been reported to compare with vaccine performance in animal models, including the pig. as noted above, the success of the equivac r hev vaccine in horses and other animal models was not replicated in swine ( , ) , highlighting a potential issue of predicative validity when comparing niv vaccines between animal species, which may extend to humans. on the other hand, pigs have been used successfully as models to study many human infectious diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) , including niv infection ( ) . there is also a growing appreciation that pigs provide a superior animal model for influenza a virus infection and immunity and should play a more prominent role as a model for human influenza vaccine development ( ) . the success of the pig as an experimental animal model is partly due to their similarities with humans in terms of anatomy, immunology, and physiology, but also due to their manageable behavior and size, and by the general ethical acceptance of using pigs for experimental purposes instead of non-human primates ( , , ) . the niv outbreaks in malaysia and singapore demonstrated that pigs can play a key role in the epidemiology of niv by acting as an amplifier host. the region most at risk of niv infection has some of the highest pig population densities found anywhere in the world, which are rising fast due to the demand of a growing human population. this increases the risk of niv transmission to pigs and humans. the development of a niv vaccine for use in pig populations would decrease the major risk niv poses to the developing pig industries, as well as to the livelihoods of poor livestock keepers in southeast asia. the use of non-human animal models is crucial for vaccine development against diseases such as niv since efficacy testing in humans is impossible. the pig model may therefore contribute to human vaccine development, supporting human vaccine licensure under the animal rule. pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission functional studies of host-specific ephrin-b ligands as henipavirus receptors structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b unraveling a three-step spatiotemporal mechanism of triggering of receptorinduced nipah virus fusion and cell entry organ-and endotheliotropism of nipah virus infections in vivo and in vitro role of endocytosis and cathepsinmediated activation in nipah virus entry origins of major human infectious diseases the pandemic potential of nipah virus virus 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recombinant hendra virus g glycoprotein subunit vaccine protects nonhuman primates against hendra virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response status of vaccine research and development of vaccines for nipah virus recombinant nipah virus vaccines protect pigs against challenge newcastle disease virusvectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity potency of a thermostabilised chimpanzee adenovirus rift valley fever vaccine in cattle chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever available online at emerging and neglected infectious diseases: insights, advances, and challenges efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial zika virus protection by a single low-dose nucleosidemodified mrna vaccination available online at research and development blueprint for action to prevent epidemics new drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. final rule can ebola virus vaccines have universal immune correlates of protection? first vaccine approval under the fda animal rule large animal models for vaccine development and testing animal models in translational medicine: validation and prediction staphylococcal wound infection in the pig: part i a pig model of acanthamoeba keratitis: transmission via contaminated contact lenses animal models for gastric helicobacter immunology and vaccine studies the benefits of using diverse animal models for studying pertussis animal models of ventilator-associated pneumonia swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus the pig: a model for human infectious diseases animal models of henipavirus infection: a review swine as a model for influenza a virus infection and immunity contribution of the swine model in the study of human sexually transmitted infections all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -trwa grl authors: costa vallés, cristina; máñez mendiluce, rafael title: transgenic organs and xenotransplants date: - - journal: stem cell transplantation doi: . / - - - - _ sha: doc_id: cord_uid: trwa grl a dvances in immunosuppressive treatments reached in the last decades of the th century have made solid organ transplantation the treatment of choice for cases of irreversible organ failure. however, the availability of human cadaver organs is limited and the demand for transplants is still on the rise. also, there is a recognised lack of cells and human tissues for generalised use in transplantation for the treatment of diseases that are characterised by failure of specialised cells (such as pancreatic cells to cure diabetes). xenotransplantation, which is the transplant of cells, tissues or organs from other species, became the focus of attention in the nineteen-nineties as a solution to the lack of organs and tissues for transplantation. previous clinical studies using nonhuman primates produced poor outcomes (survival from days to a few months) and confirmed the difficulty of obtaining organs from these species. since then, progress in xenotransplantation has been slow and still now various immunological and non-immunological barriers need to be overcome. these barriers are reviewed in this chapter and the various approaches explored to date to overcome them, in particular those based on the genetic modification of pigs. also, cell transplant studies such as those of pancreatic islets in monkeys have led to even more hopeful results. the range of possibilities offered by this technology will be unlimited, making it possible for xenotransplantation to be a clinical reality in a not very distant future. advances in immunosuppressive treatments reached in the last decades of the th century have made solid organ transplantation the treatment of choice for cases of irreversible organ failure. however, the availability of human cadaver organs is limited stem cell transplantation, edited by carlos lópez-larrea, antonio lópez-vázquez and beatriz suárez-Álvarez. © landes bioscience and springer science+business media. and the demand for transplants is still on the rise. in spain, the world leader in cadaver organ donation, the greatest achievement has been to avoid a year-on-year growth in waiting lists, as occurs in most countries. it has not been possible to reduce them, despite the notable increase of live donor transplantation; rather these have only served to slow the growth of waiting lists, not to mention the risks of live donor transplantation for the donor. also, there is a recognised lack of cells and human tissues for generalised use in transplantation for the treatment of diseases that are characterised by failure of specialised cells (such as pancreatic cells to cure diabetes). xenotransplantation, which is the transplant of cells, tissues or organs from other species, became the focus of attention in the nineteen-nineties as a solution to the lack of rujdqv dqg wlvvxhv iru wudqvsodqwdwlrq $w wkdw srlqw diwhu wkh surgxfwlrq ri wkh ¿uvw wudqvjhqlf pigs, this species was selected as the best source of organs and xenogeneic tissues. [ ] [ ] [ ] previous clinical studies using nonhuman primates produced poor outcomes (xenograft survival iurp gd\v wr d ihz prqwkv dqg frq¿uphg wkh gli¿fxow\ ri rewdlqlqj rujdqv iurp wkhvh species. moreover, the risk to public health of using the organs from nonhuman primates was quite high, as vividly demonstrated by the aids epidemic. on the other hand, pigs are domesticated animals, which produce large litters, and that, apart from their interest in the food industry, have also had some medical uses (production of insulin, heart valves). finally, they combine a physiology and anatomy that is similar to primates and they can eh jhqhwlfdoo\ prgl¿hg kxv lw lv qrw vxusulvlqj wkdw wkh srvvlelolw\ ri ³kxpdqlvlqj´ slj organs and tissues should be talked about with a view to using them in clinical practice. lqfh wkhq vljql¿fdqw dgydqfhv kdyh ehhq pdgh kh prvw zhoo vwxglhg slj rujdqv for their potential for xenotransplantation are the kidney and heart, followed by the lung and liver. indeed, the liver is considered as the potential bridge for allotransplantation, either through transplant or by ex vivo perfusion. research is also being carried out on cell xenografts of pancreatic islets, hepatocytes, chondrocytes and neural cells, among others. despite the great impact that their clinical application would represent, progress in xenotransplantation has been slow, mainly due to technological problems. a decade later, we are still facing various barriers that prevent the clinical application of pig organs, tissues and cells. these barriers are reviewed below and the various approaches explored to date wr ryhufrph wkhp lq sduwlfxodu wkrvh edvhg rq wkh jhqhwlf prgl¿fdwlrq ri sljv duh ghvfulehg the main obstacle for xenotransplantation to be used in clinical practice is the strong immune response caused by the pig organ in the recipient. cell, tissue and organ xenografts are subject to a variety of rejection mechanisms that include humoral and cellular immune responses. the cellular immune response seems to play a key role in the rejection of cell grafts, such as hepatocytes and pancreatic islets, while rejection of vascularised organs mainly involves humoral immunological mediators. various types of rejection have been described in solid organ xenotransplantation, depending on the time elapsed following the transplant and the immune elements involved: hyperacute, acute humoral and acute cellular rejection. [ ] [ ] [ ] +\shudfxwh uhmhfwlrq +$ lv wkh ¿uvww\sh wr rffxu ehwzhhq plqxwhv dqg krxuv after the xenotransplant. har is triggered by a humoral immune response in which xenoreactive natural antibodies (xna) (already present in the recipient) are deposited rq wkh hqgrwkholxp ri wkh [hqrjudiw dfwlydwlqj wkh frpsohphqw v\vwhp dqg fdxvlqj Àxlg extravasation from the intravascular space into the interstitium (oedema). [ ] [ ] [ ] this process also causes the activation of the coagulation cascade and thus thrombosis, ischemia and necrosis of the xenograft in a short period of time following the transplant. the main xenoepitope recognised by xna is the disaccharide galactose-alpha- , -galactose (gal), widely expressed in pig tissues and synthesised by the alpha , -galactosyltransferase (_ , -gt) enzyme. humans and old world primates lack functional _ , -gt and have high titre of anti-gal antibodies. acute humoral xenograft rejection (ahxr), also known as acute vascular rejection or delayed xenograft rejection occurs in a period of days to months after the transplant. it has not been possible to prevent it using currently available immunosuppression regimens. - ahxr shows notable similarity to har since it involves a strong humoral immune response with the participation of antibodies, the deposition of complement proteins and thrombosis in xenografts. however, in other respects ahxr differs vljql¿fdqwo\ iurp +$ hvshfldoo\ lq wkh ruljlq ri wkh dqwlerglhv uhvsrqvleoh iru uhmhfwlrq in this case, it involves a response in which anti-gal antibodies participate, but it also includes antibodies targeting other epitopes. further, ahxr is characterised by the suhvhqfh ri lqqdwh lppxqlw\ fhoo lq¿owudwhv . fhoov dqg pdfurskdjhv dqg dq dfwlydwlrq ri hqgrwkholdo fhoov wkdw surprwhv lqwudydvfxodu wkurpervlv dqg ¿eulq ghsrvlwlrq acute cellular rejection occurs some days after the transplant and is a response mediated mainly by t cells against the donor antigens. the activation of these cells during xenotransplant rejection is mediated by a primary signal through the t receptor and secondary costimulatory signals conserved across species. currently, it is thought that it is possible to control the cellular immune response against the xenotransplant using the various immunosuppressive protocols currently available, since no failure of pig xenograft caused by this type of rejection has been demonstrated. however, this point cannot be properly judged until ahxr can be prevented in an effective and systematic way. avascular tissue and xenogeneic cells are also subject to rejection of the graft, similar to the process that occurs in solid organ rejection, but without the vascular component and with special features for each cell and tissue. pancreatic islets have drawn great attention due to their potential clinical application for the treatment of diabetes. first, adult pig beta cells do not express the gal antigen, thus reducing the humoral component of rejection. potent immunosuppressive protocols focussed on averting the activation of t cells have led to long-term survival of pig pancreatic islets in diabetic monkeys ( days). the problem for the clinical application of cellular xenotransplantation lies in the fact that, while they present fewer immunological barriers than solid organs, they vwloo uhtxluh vwurqj lppxqrvxssuhvvlrq ri wkh uhflslhqw wkdw fdqqrw eh vxvwdlqhg lqgh¿qlwho\ in any case, the strategies developed for this type of transplant may be very useful for their application in other organs of tissues of interest, especially regarding genetic prgl¿fdwlrq ri wkh grqru slj wkdw fdq lqyroyh d zlgh ydulhw\ ri fhoo dqg wlvvxh w\shv therefore any progress made in this area has an impact that goes beyond xenografts, be it to cells, tissues or organs. the various techniques have been developed to prevent har can be summarised in two groups: those focused on modifying the xenograft, and those that use systemic wuhdwphqwv wr dowhu wkh lppxqh uhvsrqvh ri wkh uhflslhqw kh prvw uh¿qhg phwkrgv duh edvhg rq wkh jhqhwlf prgl¿fdwlrq ri sljv xvhg dv wkh vrxufh iru rujdqv vlqfh wkh\ duh less harmful for patients and their objective is to decrease the need for immunosuppression or other conditioning treatments. however, these are complex techniques and involve slow and expensive procedures. systemic treatments, on the other hand, may help us to identify molecules or key rejection processes and to speed up the clinical application of [hqrwudqvsodqwv shfl¿fdoo\ wkh nh\ urohv ri ; $ dqg frpsohphqw lq +$ zhuh frq¿uphg e\ wkh hiihfwlyhqhvv ri dqwlerg\ devruswlrq e\ sodvpdskhuhvlv dqg v\vwhplf complement inhibition to prevent this type of rejection. kh ¿uvw dssurdfk wr qhxwudolvh +$ e\ jhqhwlf hqjlqhhulqj zdv wkh lqklelwlrq ri complement activation through the expression of human complement regulatory proteins in transgenic pigs. , the reason for this was the notable restriction of the function of the complement regulatory proteins between species, which led to the suggestion that pig molecules were unable to control the activation of the human complement. subsequent studies showed that pig complement regulatory proteins were also able to regulate, at least in part, the activity of the human complement. klv vxjjhvwv wkdw wkh ehqh¿w ri wkh h[suhvvlrq ri wkh kxpdq frpsohphqw surwhlqv lq slj fhoov lv qrw rqo\ gxh wr vshfl¿flw\ but also to the increase in expression of these proteins. initially in vitro studies demonstrated that the expression of human cd (hcd ) or cd (hdaf) in pig cells notably protects these cells from cytolysis triggered by the human serum. ex-vivo perfusion experiments with human blood also demonstrated that kidneys and hearts from pigs transgenic for human cd functioned for longer than control animals. later it also demonstrated the almost systematic prevention of har and longer survival rates of transgenic pig organs that expressed human complement inhibitors in transgenic pig-to-primate models. , however the use of organs from these transgenic pigs did not achieve full protection against humoral xenograft rejection, since all the organs were subject to ahxr sooner or later, the various immunosuppressive protocols being unable to modify this response (tables , and ). this led research to focus on wkh surgxfwlrq ri sljv zlwk jhqhwlf prgl¿fdwlrqv wkdw zrxog uhgxfh wkh uhdfwlylw\ ri wkh organs to xenoantibodies present in human serum. before homologous recombination and "knockout" techniques were available in pigs, one of the most widely investigated strategies was based on the transgenic expression of human alpha , -fucosyltransferase (h transferase, ht). ht produced fucosyl residues (h antigen of the o blood group) that are universally tolerated (fig. + zdv vkrzq wr frpshwh hi¿flhqwo\ zlwk doskd* iru wkh vdph vxevwudwh n-acetyl-lactosamine, preventing the transfer of the terminal galactose, the residue which gives rise to the production of the gal antigen. the reduction in the expression of the gal epitope in ht transgenic pig cells leads to a decrease in their reactivity with human antibodies and in cytolysis caused by human sera. further, the hearts of ht-transgenic mice have been shown to have higher survival rates after being perfused with human serum or transplanted to , gt knockout mice, which produce anti-gal antibodies. as ht and other competitive enzymes are not able to completely inhibit the expression of gal epitopes, this technique was combined with the expression of human complement inhibitors. thus, peripheral blood mononuclear cells and aortic endothelial cells of double transgenic pigs that co-express ht and hcd are better protected from lysis by human serum than controls or single transgenic cells for each of the genes. in addition, double transgenic cells maintained their resistance to xna dqg frpsohphqw diwhu ehlqj wuhdwhg iru krxuv zlwk sruflqh surlqÀdppdwru\ cytokines. despite these advances, these pigs were developed to be used in cellular xenotransplantation and the organs have not been transplanted to nonhuman primates, so it has not been possible to study their effectiveness in this experimental model in frpsdulvrq wr slj rujdqv fduu\lqj rwkhu w\shv ri jhqhwlf prgl¿fdwlrqv the description of the process of nuclear transfer of a somatic cell into a germinal cell by the team led by ian wilmut in opened the door to the generation of "knockout" pigs. from that moment, several groups started the race to produce the ¿uvw doskd* gh¿flhqw sljv khuh zdv d frqfhuq wkdw sljv surgxfhg e\ wklv whfkqltxh zrxog qrw eh yldeoh exw wkh ¿uvw olwwhuv ri doskd * ³nqrfnrxw´ sljv zhuh khdowk\ and developed well. subsequent studies showed survival times of up to days for kidneys of alpha , -gt knockout pigs transplanted in baboons, meaning that the organs of these transgenic pigs were also protected against har, as occurred in animals transgenic for human complement regulatory proteins. despite this progress, the majority of human sera show reactivity towards pig cells that lack for alpha , -gt gene, suggesting the existence of antibodies that recognise other antigens apart from the gal epitope. therefore, the introduction of a human complement inhibitor is still necessary to completely block human serum-mediated cytotoxicity. another carbohydrate in pigs of interest to prevent har in organ xenotransplantation is the antigen of the type a blood group. as in the human, these antigens are present in some pigs and can be recognised by human antibodies directed against this blood-type. the reactivity of human serum igm against this epitope was discovered in a kidney from a type a group pig whose kidneys were extracorporeally connected to a volunteer dialysis patient. nevertheless, these antibodies should not be an obstacle to clinical xenotransplantation, since blood groups can be selected for the animals used as source of organs, in the same way as with allotransplantation. the methods described above, alone or in combination, have successfully and routinely manage to prevent har. however the same strategies have not proved successful to avoid ahxr. the studies carried out to date suggest that this type of rejection is not caused by a single immunological element, but a collection of them. we will go on to describe research carried out so far into the control of humoral as well as cellular xenograft uhmhfwlrq ,w lv gli¿fxow wr vhsdudwh wkhvh wzr surfhvvhv dv wkh\ ryhuods lq wkh wlph ri progression and possibly also in the immunological response mechanisms. however, it should be highlighted that cellular rejection has not been an insurmountable obstacle and that the various immunosuppressive protocols investigated have managed to prevent this type of rejection systematically. most of the information currently available on preclinical xenotransplantation comes from studies using hdaf transgenic pigs as a source of organs and cynomolgus monkeys or baboons as recipients. results of these studies are summarised in the tables to , irfxvlqj rq ruwkrwrslf wudqvsodqwv ri nlgqh\ dqg khduw iurp jhqhwlfdoo\ prgl¿hg sljv given their particular preclinical importance. the transgenic expression of hdaf, alone or in combination with hcd , protects against har and confers some protection against haxr. , one aspect that is worth highlighting is that the species of recipient primate dovr vhhpv wr lqÀxhqfh wkh vxuylydo ri wkh [hqrjudiw vxuylydo udwhv ehlqj kljkhu lq cynomolgus monkeys than in baboons, perhaps due to the fact that the latter being a model that is closer to humans (tables and ). without immunosuppression ahxr occurs or days after the transplant of hdaf transgenic organs in baboons, there being no similar results from cynomolgus monkeys. the use of an immunosuppressive regimen that includes cyclophosphamide (cyp), cyclosporin (csa) and corticosteroids (cs) increases the mean survival of the renal transplant recipient to . days in baboons and approximately days in cynomolgus monkeys. , parallel studies of orthotopic heart xenotransplants in baboons show mean survival rates of - days in similar conditions of immunosuppression. the addition of other immunosuppressants, such as rapamycin, mycophenolate mofetil or methotrexate, to the aforementioned protocol and performing splenectomy during the transplantation procedure, increased the mean survival rates for renal xenografts to up to - days in cynomolgus monkeys and to - days in baboons , (tables and ). the temporary use of systemic complement inhibitors such as c inhibitor or soluble complement receptor (scr , tp ), was also found to be effective, both in prolonging kidney xenograft survival and to revert ahxr, once diagnosed. however, the toxicity of these products in the form of increased susceptibility to infections does not allow a long-term treatment with complement inhibitors. initially it was thought that anti-gal antibodies also have an important role in ahxr, which led to the development of a series of polymers containing many gal epitopes, following the failure of repeated immunoadsorption for neutralising these antibodies. the most widely tested was gas , a trisaccharide composed of _ , gal with a molecular weight of kda. using injections of gas it is possible to continuously neutralise anti-gal antibodies (fig. ) . this also reduces the intensity the arrows indicate the days on which gas was injected, with black corresponding to doses of mg/kg and grey to mg/kg. the levels of anti-gal (circles), anti-gal igg (triangles) and porcine haemolytic (squares) antibodies are shown compared to a standard human serum, from a pool of different human sera, which has been arbitrarily assigned a value of . of the ahxr, although without improving xenograft survival even with the addition of potent immunosuppressive treatments. the presence of antibodies in ahxr despite continuous depletion of anti-gal antibodies in the blood suggested that the rejection was caused by antibodies directed against other pig epitopes. these results have later ehhq frq¿uphg zlwk wkh surgxfwlrq ri _ , -gt "knockout" pigs, that do not express wkh dqwljhq *do . lgqh\v wudqvsodqwhg iurp wkhvh jdogh¿flhqw sljv lqwr ederrqv kdyh reached mean survival rates of days under mild immunosupression, and using higher-dose immunosuppression a mean of days and a maximum of days. , however these organs also suffer from ahxr mediated by antibodies other than anti-gal that cannot be avoided with immunsuppressive treatments. the best renal xenotransplantation results in baboons have been obtained using kidneys of _ , -gt knockout pigs, in combination with protocols that use chimerism to promote graft tolerance. mean survival rates of . days, with a maximum of days, have been attained by transplanting the pig kidney together with vascularised thymic lobe of the same animal, previously grafted under the renal capsule, with a conditioning regimen that included the temporary depletion of complement and t cells and maintenance with anti-cd monoclonal antibodies, mycophenolate mofetil and corticosteroids. the results represent an improvement compared to the mean and maximum survival rates of . and days, respectively, achieved with similar experiments using kidneys from hdaf-transgenic pigs. this suggested that _ , -gt knockout pigs offer advantages with respect to those that are transgenic for complement regulatory proteins in the ahxr, possibly due to the fact that the immune response is a feature that has arisen in all the preclinical studies carried out to date is that treatments that inhibit the production of antibodies, such as high doses of cyclophosphamide or anti-cd monoclonal antibodies, do decrease or prevent ahxr. however, they all are very aggressive and lead to excessive immunosuppression, accompanied by severe side effects (gastrointestinal lesions, anaemia, infections, etc.) which in themselves jeopardise the life of the recipient. for this reason, the experiments carried out by the group led by dr david cooper are of great importance. these researchers performed a heterotopic heart xenograft transplantation in baboons from _ , -gt knockout pigs, using immunosuppressive levels that were acceptable for clinical practice. most of these hearts were rejected with signs of ahxr or thrombotic microangiopathy. non-etheless, they reached average and maximum survival times of days and months respectively, the longest survivals of porcine xenografts in nonhuman primates described to date. the thrombotic microangiopathy described in these experiments is considered to be another manifestation of ahxr, closely linked to the antibody-mediated response. thus, recipients dying after long survival times due to causes not associated with rejection duh irxqg wr kdyh [hqrjudiwv zlwk plqlpdo ru qrqh[lvwhqw sdwkrorjlfdo ¿qglqjv )ru this reason, although it is not possible to completely rule out an effect of clotting incompatibilities between pigs and humans, as we will see in the following section that looks at the physiological barriers between these species, the control of the response mediated by anti-nongal antibodies has become the biggest challenge for clinical xenotransplantation. our group has preliminary data suggesting that these antibodies are not directed against porcine proteins, but rather the targets, though not the gal antigen, are also carbohydrates. although it would be reasonable to expect that the antibodies are directed against many epitopes, their characterization would open the possibility of developing new treatments to prevent or treat ahxr. given that one of the principal advantages of xenotransplantation is the possibility of modifying the donor organ, the solution for ahxr would come from genetically engineering the donor animal, to avoid the expression of those elements that cause a uncontrollable production of xenoantibodies. together with these measures intended to decrease the reactivity of anti-nongal [hqrdqwlerglhv dqg srwhqwldo forwwlqj lqfrpsdwlelolwlhv rwkhu jhqhwlf prgl¿fdwlrqv pljkw decrease the immunogenicity of pig organs. key potential targets are the immunological responses induced by the porcine cells, such as those mediated by the cd -cd /cd sdwkzd\ zklfk lv frqvhuyhg dfurvv wkh vzlqhwrkxpdq vshflhv eduulhu shfl¿fdoo\ cd expressed in porcine aortic endothelial cells provides strong costimulatory signals to human t and nk cells. , porcine cd , in contrast to its human counterpart, is expressed in a wide variety of cells and tissues, and the signal mediated by cd is resistant to immunosuppression by calcineurin inhibitors. other elements which have a potential role in therapeutics are cytokines such as tnf_ and `. the use of strategies that block the tnf may be useful in the development of xenografts resistant to ahxr, as has been demonstrated in rondent xenotransplantation models. in general, the objective would be for these techniques to allow clinical xenotransplantation using the minimum possible level of immunosuppression in the recipient. it is well established that xenogeneic proteins such as porcine insulin can work correctly in humans. however, it is not clear whether xenografts are able to perform their functions in an environment other than that for which they have been genetically programmed, and, if so they are, for how long this functioning can be sustained. pig kidneys have maintained the life of nonhuman primates for several months, , xenograft function apparently failing due to rejection rather than to the existence of physiological incompatibilities between the species. recipients of _ , -gt knockout kidneys required continuous treatment with human albumin to maintain protein levels within the normal range, as a consequence of the proteinuria produced after the transplant and that continued throughout the three months that the xenograft survived. the clinical symptoms of the proteinuria (oedema) were different depending on the immunosuppression protocol used, suggesting that proteinuria was the consequence of the xenograft rejection, and not of a mutual physiological incompatibility. furthermore, nonhuman primates transplanted with _ , -gt knockout kidneys did not suffer from the anaemia previously described zlwk wkh lppxqrvxssuhvvlyh surwrfrov wkdw lqfoxghg f\forskrvskdplgh frq¿uplqj wkdw it had been a consequence of the treatment toxicity rather than due to the inability of porcine erythropoietin to maintain erythropoiesis. ,w kdv ehhq frq¿uphg wkdw sruflqh khduwv dqg nlgqh\v duh deoh ri pdlqwdlqlqj d vlplodu sk\vlrorj\ wr kxpdq iru orqj shulrgv ri wlph dqg wkh\ duh fdqglgdwhv iru wkh ¿uvw solid organ clinical xenotransplants. in contrast, xenografts of other organs such as the lung and liver have not survived more than a few days, though even this has demonstrated that they can maintain the life of the recipient for short periods of time. nevertheless, vrph vljql¿fdqw sk\vlrorjlfdo gliihuhqfhv fdq eh h[shfwhg ehwzhhq wkh grqru dqg wkh recipient, especially in the case of the liver due to its complex metabolic system. in the case of porcine heart and kidney, the existence of some long-term minor incompatibilities cannot be ruled out. an example of this is the clotting abnormalities described in ahxr. currently it seems that these changes may be dependent of the deposit of xenoantibodies in the xenograft, but the existence of some kind of physiological incompatibility cannot be completely ruled out. in vitro, porcine cells have an inherent tendency to clot spontaneously in human plasma, an effect that seems to be dependent on some molecular incompatibilities between porcine and human blood-clotting regulators. shfl¿fdoo\ sruflqh wkurperprgxolq d nh\ dqwlfrdjxodqw h[suhvvhg e\ endotelial cells) hardly works in the human system. if clotting abnormalities after [hqrwudqvsodqwdwlrq rffxuuhg lqghshqghqwo\ ri dqwlerglhv wkh gli¿fxowlhv irxqg lq preventing and treating ahxr in procine xenotransplants to nonhuman primates would be explained. nevertheless, accumulated experience suggests that phyisology should not be an insurmountable obstacle to clinical xenotransplantation of porcine organs. also some methods are currently under investigation to solve the potential clotting incompatibilities between pigs and humans, including the genetic engineering of pig organs. if these problems were to persist once the problem of ahxr has been overcome, it is very likely that they could be also treated by the introduction of suitable human genes/proteins to the donor pig. the success of organ xenotransplantation depends on the balance between the immunosuppressive treatment necessary to avoid rejection and the risk of opportunistic infections or cancer such treatment may cause. in the case of xenotransplantation, experimental data available to date suggest that more immunosuppression is required to prevent ahxr, and, therefore, that the theoretical risk of opportunistic infections is greater in xenotransplantation than in allotransplantation. furthermore, the use of nonhuman cells, tissue or organs will increase the spectrum of opportunistic infections, since it will include diseases from the animal species used as a source of organs. the possibility that a new pathogen agent could be transferred to the recipient through xenotransplantation, and the possible passing of this to the general population, as happened with aids, is cause of concern among scientists and those responsible for public health. the terms of "xenosis" and "xenozoonosis" have been proposed to describe infections produced by microorgansims of other animal species, that do not cause infection in humans in normal circumstances, but that might be transferred from a xenograft. the suredelolw\ ri d vshfl¿f plfurrujdqlvp ri dq dqlpdo vshflhv fdxvlqj d glvhdvh lq kxpdqv is unknown. in theory it may be fairly high in the cases of microorganisms that are zoonotic under normal conditions (for example, toxoplasma gondii), similar to others that cause infections in allotransplantation (such as cytomegalovirus, cmv), and capable of infecting a wide spectrum of species (such as pneumocystis carinii), as well as those microorganisms that can replicate in vitro in human cells. however, it is likely that xenosis, also known as xenozoonosis, caused by bacteria, fungi and parasites, arising iurp erwk frpprq dqg vshflhvvshfl¿f sdwkrjhqv gr qrw lpso\ d sduwlfxodu ulvn iru wkh recipient of the xenograft and even less for public health. the reason for this assertion is that this type of infections should be prevented in the animal that is to be the source of organs. kh surgxfwlrq ri dqlpdov lq frq¿qhg dqg lvrodwhg duhdv iurp zklfk rqo\ dqlpdov that are negative to all known pathogens are prospectively selected, can minimise the number of infections that these animals carry. accordingly, it is important that strict protocols are established for clinical and microbiological assessment, using both immunocompetent and immunodepressed animals, to enable animals with any traces of infection to be detected. this applies, be they carriers of latent microorganisms, similar to those that cause infection in allotransplants, or of pathogens of other species. would rule out any animal as potential source for a lung xenograft, or the virus coxasckie for heart xenografts. the ease of obtaining spf pigs is another of the advantages of using wklv vshflhv dv d vrxufh ri rujdqv d surfhvv wkdw orjlvwlfdoo\ zrxog eh yhu\ gli¿fxow wr carry it out with nonhuman primates. the risk of transmission of the xenosis may be further reduced by the production of pigs totally free of germs (gnotobiotic). although there are currently no facilities that allow the production of mammals in this state, their construction is totally feasible, with wkh kljk frvw ehlqj wkh prvw vljql¿fdqw glvdgydqwdjh +rzhyhu jqrwrelrwlf dqlpdov duh less robust than those which have been subject to normal microbiological colonisation, and at present they do not seem to offer any advantages over spf animals in terms of minimising the risk of transmission of xenosis. therefore, the production of gnotobiotic pigs has been put on hold until clinical experimentation demonstrates its necessity. with the production of spf animals, the risk of transmission of diseases would be limited to some pathogens capable of producing latent infections in the source animal and caused by vertically transmitted viruses that cannot be prevented in the source animal by early weaning and/or caesarean birth. all species have viruses that persist within the host cells in a latent state. while the best known are the herpes viruses and retroviruses, these also include the hepatitis viruses, adenoviruses, rabies and pseudorabies viruses, reoviruses, and papovaviruses among others. it is still not known whether nonhuman latent viruses represent an infection and disease risk for humans. in vitro, it has been possible to infect human neurons with the porcine pseudorabies virus and to transmit various viruses between species in experimental models. nevertheless, it is hard to believe that in the case of a species such as the pig, with which humans have been in contact for many thousands of years, there are many infections with capacity of being transferred from person to person that have not yet manifested themselves. on the other hand, the infection caused by nipah virus in pig slaughterhouse workers in asia, after contact with infected animals, and the recent epidemic of severe acute respiratory syndrome (sars), caused by a coronavirus, transmitted through the consumption of exotic animals in china, are good examples of the ability these zoonoses to spread. the potential re-activation of latent herpes virus infections after xenotransplantation of a porcine organ has been subject of particular attention in preclinical research on [hqrwudqvsodqwdwlrq kuhh khushv yluxvhv kdyh ehhq lghqwl¿hg lq vzlqh ruflqh cytomegalovirus (pcmv), and porcine lymphotropic herpes virus -and -(plhv- , - ) which, in pigs that have been subject to bone marrow transplantation, are associated with a lymphoid proliferative syndrome similar to post-transplant proliferative disease (ptpd) vhhq lq doorwudqvsodqwdwlrq +hushv yluxvhv duh vshflhvvshfl¿f vr li lqihfwlrq rffxuv diwhu the xenotransplant, it should be restricted to the xenograft. the replication of pcmv has been described in porcine xenografts in nonhuman primates, causing an infection that damages porcine endothelial cells and tissues. the elimination of pcmv from swine litters has been possible through early weaning of newborn animals, and the absence of pcmv in xenografts has been associated with the reduction of clotting disorders and improvement of survival rates of pig xenotransplants in nonhuman primates. activation of plhv- has not yet been demonstrated in any solid organ xenotransplant. however, unlike pcmv, this virus cannot be eliminated from source animals by early weaning of newborns, so remains a potential pathogen in porcine organ xenotransplantation. other infections that cannot be prevented using spf pigs are those caused by vertically transmitted viruses. these include porcine endogenous retroviruses (perv) which are viruses that have been permanently integrated into the genome of the host during the evolution of mammals, and are transmitted vertically from mother to offspring. although they are not pathogens in the host, these retroviruses can be xenotropic, that is, capable of infecting other species. two perv have are known to have the capacity to infect human cells in vitro, which leads to us consider the possibility of recombination or complementation of xenograft endogenous retroviruses with viruses present in human wlvvxhv dqg wkh srwhqwldo ulvn ri lqgxfwlrq ri wxprxuv dqg lppxqrgh¿flhqflhv fdxvhg by viruses. research undertaken to date, on humans in contact with living pig tissue, workers in pig slaughterhouses, human patients who have received transplants in contact with pigs and nonhuman primates who have received pig organs and severe immunosuppressive treatment, has not shown the existence of perv replication in humans or nonhuman primates. , however work is ongoing to characterise perv, to optimising systems for their detection, and to develop approaches to avoid or minimise the associated risks. on the one hand, certain families of miniature swine, "mini pigs", that do not transmit perv wr kxpdq fhoov kdyh ehhq lghqwl¿hg uhfhqwo\ q wkh rwkhu kdqg wudqvjhqlf whfkqltxhv such as sirna expression technique are being applied to inhibit the expression of perv, raising the prospect that this infection may be avoided by manipulating the animals to be used as sources for organs. given all this, on the basis of the currently available information, the risk of xenosis, also known as xenozoonosis, should not be an obstacle for clinical xenotransplantation of pig organs. in contrast to the situation existing a few years ago, risks have now been lghqwl¿hg lqyhvwljdwhg dqg whvwv kdyh ehhq ghyhorshg wkdw hqdeoh wkhp wr eh dvvhvvhg lq preclinical and clinical studies. although the possibility of an infection arising from the [hqrjudiw wkdw zrxog diihfw sxeolf khdowk fdqqrw eh uxohg rxw wkh ulvn vhhpv lqvljql¿fdqw and avoidable by close supervision of the source animals and recipients of xenografts. xenotransplantation has great potential to solve the problem of the lack of human tissues and organs for transplantation and continues to be a possible alternative to allotransplantation. progress has been slow in research in this area due to technological sureohpv vxfk dv wkh gli¿fxow\ ri surgxflqj ³nqrfnrxw´ sljv dqg wkh hydoxdwlrq ri [hqr]rrqrvlv ulvnv +rzhyhu wkh nh\ wrrov kdyh qrz ehhq hvwdeolvkhg dqg vr wkh ¿hog can now develop much faster. the main barrier to its clinical application is immune rejection, especially the humoral uhvsrqvh wuljjhuhg e\ ydvfxodulvhg [hqrjudiwv kh lghqwl¿fdwlrq ri wkh nh\ v\vwhpv dqg molecules that are involved in the process of rejection, and the development of strategies to overcome them is just a matter of time. the use of porcine organs that have been vxemhfw wr ydulrxv jhqhwlf pdqlsxodwlrqv kdv douhdg\ vkrzq vljql¿fdqw lpsuryhphqwv lq the xenotransplantation of organs to nonhuman primates. cell transplant studies such as those of pancreatic islets in monkeys have led to even more hopeful results. then the range of possibilities offered by this technology will be unlimited, making it possible for xenotransplantation to be a clinical reality in a not very distant future. discordant organ xenotransplantation in primates life-supporting pig-to-primate xenotransplantation using genetically prgl¿hg grqruv udqvsodqwdwlrq xenotransplantation and the future of renal replacement xenotransplantation-how far have we come? delayed rejection of porcine cartilage is averted by transgenic expression of alpha , -fucosyltransferase prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection human complement regulatory proteins protect swine-to primate cardiac xenografts from humoral injury role and regulation of pig cd and membrane cofactor protein/ cd expressed on pig aortic endothelial cells life-supporting pig-to-primate xenotransplantation using genetically prgl¿hg grqruv udqvsodqwdwlrq expression by _ixfrv\owudqvihudvh lq wudqvjhqlf sljv prgl¿hv wkh cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis transgenic pigs designed to express human cd and h-transferase to avoid humoral xenograft rejection production of _- , -galactosyltransferase knockout pigs by nuclear transfer cloning acute rejection is associated with antibodies to nongal antigens in baboons using gal-knockout pig kidneys allosensitized humans are at no greater risk of humoral rejection of gt-ko pig organs than other humans long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts pig kidney transplantation in baboons: anti-gal(alpha) - gal igm alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation failure to deplete anti-gal_ - gal antibodies after pig-to-baboon rujdq [hqrwudqvsodqwdwlrq e\ lppxqrdi¿qlw\ froxpqv frqwdlqlqj pxowlsoh *do_ - gal oligosaccharides the effect of soluble complement receptor type on acute humoral xenograft rejection in hdaf-transgenic pig-to-primate life-supporting kidney xenografts removal of anti-galalpha , gal xenoantibodies with an injectable polymer improvement in human decay accelerating factor transgenic porcine kidney xenograft rejection with intravenous administration of gas , a polymeric form of alphagal marked prolongation of porcine renal xenograft survival in baboons through the use of alpha , -galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue heart transplantation in baboons using alpha , -galactosyltransferase gene-knockout pigs as donors: initial experience human nk cell-mediated cytotoxicity triggered by cd and gal_ , -gal lv lqklelwhg lq jhqhwlfdoo\ prgl¿hg sruflqh fhoov use of porcine tumor necrosis factor receptor -ig fusion protein to prolong xenograft survival disordered regulation of coagulation and platelet activation in xenotransplantation xenotransplantation and xenogeneic infections porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation lack of cross-species transmission of porcine endogenous retrovirus (perv) in pig-to-baboon xenotransplantation with sustained depletion of anti-_ gal antibodies lack of cross-species transmission of porcine endogenous retrovirus (perv) to transplant recipients and abattoir workers in contact with pigs production of transgenic pigs that express porcine endogenous retrovirus small interfering rnas key: cord- - kxgu t authors: oem, jae-ku; an, dong-jun title: phylogenetic analysis of bovine astrovirus in korean cattle date: - - journal: virus genes doi: . /s - - - sha: doc_id: cord_uid: kxgu t bovine astrovirus (bastv) belongs to a genetically divergent lineage within the genus mamastrovirus. the present study showed that bastv was associated with the gastroenteric tracts of cattle in nine positive fecal samples from cattle, whereas no positive samples were found in the brain tissues of downer cattle. interestingly, the positive diarrheal samples were obtained mainly from calves aged days– months. bayesian inference tree analysis of the partial orf ab and capsid (orf ) gene sequences of bastvs identified four divergent groups. eleven bastvs, four porcine astroviruses, and two deer astroviruses (dastvs; ccastv- and - ) belonged to group ; group contained two bastvs (bastk – and bastk – ) with another two in group (bastk – and bastk – ); and group comprised the bastv-neuros strain derived from a cattle brain tissue sample and an ovine astrovirus. the same divergent groups were obtained when the pairwise alignments were produced using both amino acid and nucleotide sequences. the korean bastvs isolated from infected cattle had a nationwide distribution and they belonged to groups , , and . electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. astroviruses are single-stranded positive-sense rna viruses that measure approximately . - . kb in length. the family astroviridae comprises two genera: mamastrovirus infects mammals and avastrovirus infects birds [ ] . human astrovirus was first reported in children with diarrhea in [ ] and mamastroviruses were found subsequently in a variety of wild hosts, including sheep, cow, pig, dog, cat, red deer, mouse, mink, bat, cheetah, brown rat, roe deer, sea lion, dolphin, and rabbit [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . bovine astrovirus (bastv) was one of the first astroviruses [ ] to be discovered and it has been isolated in the usa and the uk. two bastv serotypes were established based on the results of a virus neutralization assay [ ] . the genomic characterization and sequence analysis of astroviruses in bovine fecal specimens collected in hong kong provides evidence of potential recombination in orf [ ] . recently, the complete genome of a novel bastv associated with neurological disease in cattle was sequenced, i.e., boastv-neuros , which was phylogenetically related to an ovine astrovirus (oastv). a previous study suggested that boastv-neruos infection was a potential cause of neurological disease in cattle [ ] . however, genetically diverse lineages of bastvs have not been identified in many countries because there have been few studies of astroviruses derived from cattle. thus, the present study investigated the genetic groupings of korean bastvs and examined their relationships with the age of cattle infected with bastvs. in total, fecal samples were collected from cattle with certain or suspected diarrheal disease at cattle farms throughout korea between january and december . the cattle comprised calves aged \ days and cattle aged [ days. based on the fecal condition, samples were from animals with diarrhea and from nondiarrheic animals. the cattle comprised korean cattle and holstein cattle. nonambulatory cattle which are commonly referred to as ''downer'' cattle are unable to stand or walk. cattle brain tissue samples with a histopathological diagnosis of encephalitis were also collected from downer cattle between and . out of brain samples collected from downer cattle, were found to be positive for akabane virus and two were bovine viral diarrhea virus (bvdv) whereas no pathogenic agent for encephalitis was detected from the other. viral rna was extracted from the feces using trizol ls b , according to the manufacturer's instructions. bastv was detected in fecal specimens by rt-pcr using a specific primer set for the orf ab and orf regions of bastv (bastv-f, -gtgtttggcatgtgggtyaarcc- and bastv-r: -rtcvyybktggtggt- ), which were designed based on known strains deposited in genbank (accession no. hq -hq ). the rt-pcr process amplified a -nt long fragment at °c for min, °c for min, °c for s, °c for s, and °c for min, followed by cycles using virus-specific conditions. the bastv associated with neurological disease in cattle was also detected in brain tissue specimens by rt-pcr, as described previously [ ] . products with the expected size were cloned using the pgem-t vector system ii tm (promega, cat. no. a , usa). the cloned gene was sequenced with t and sp sequencing primers using an abi prism Ò xi dna sequencer at the macrogen institute (macrogen co. ltd). the sequences of all the bastv-positive samples were submitted to genbank under accession numbers kf -kf . nine of fecal samples from korean cattle were positive for bastv and all of the bastvs were related to diarrhea. however, bastv was not detected in cattle brain tissue samples. although bastv was first reported in england in [ ] , the association between bovine astroviruses and gastroenteric diseases in cattle is still not clear. a recent study reported that bastv is not associated directly with severe diarrheic disease in calves under natural conditions [ , , , ] . in the present study, nine korean bastvs were associated with clinical diarrhea in cattle where calves aged \ month accounted for . % of cases (table ) . a previous study shows that bastvs were excreted by - % of calves on farms [ ] while a recent study of rectal swab samples from asymptomatic adult cattle showed that only . % ( / ) contained bastv [ ] . this must be because bastvs are more frequent in young calves than adult cattle. to investigate the relationships between astroviruses and other bovine viruses that cause diarrhea in cattle, a screening test was conducted using specific primers for the detection of bovine rotavirus (brv) [ ] , bovine coronavirus (bcv) [ ] , bvdv [ ] , and bovine kobuvirus (bkv) [ ] , as described previously. co-infections with other viruses were associated with the clinical symptoms of diarrhea in only two cases: the bastk / strain derived from a -day-old calf was coinfected with brv and the bastk / strain from a -day-old calf was co-infected with both brv and bvdv (table ) . although the association between bkv and diarrhea or gastroenteritis is unclear, it was co-infected with six korean bastvs, except for bastk / , bastk / , and bastk / (table ) . in cattle, two astrovirus serotypes have been recognized based on serological investigations, i.e., boastv- and boastv- infections [ ] , and recent phylogenetic analyses support the classification of bastvs and the newly discovered astroviruses in roe deer (ccastv) under the proposed mamastrovirus genocluster gi [ , ] . all of the astrovirus sequences were aligned using the clustal x alignment program [ ] . the nucleotide sequences were translated and the shared nucleotide and amino acid sequence identities among the astrovirus strains were calculated using bioedit . [ ] . the analysis of the diversity of bastvs in the present study identified four table) . bayesian trees were generated with mrbayes . . [ , ] using best-fit models, which were selected with mrmodeltest . [ ] for nucleotide sequences and prottest . [ ] for amino acid sequences. markov chain monte carlo analyses were run using , , generations for each nucleotide and amino acid sequence. the best-fit model of the orf ab nucleotide sequence selected by mrmodeltest . software was trnef?g, according to the results of a hierarchical likelihood ratio test. the likelihood parameter was set to nst = and rate = gamma for the datasets, and the gamma distribution shape parameter was . . the substitution model rmat was . and - [ ] . recently, the boastv-neuros strain was detected in the brain tissues of cattle and the analysis of its genetic diversity showed that it was most closely related to the oastv prototype, which was identified in [ ] , whereas it was phylogenetically distant from a recently reported oastv [ ] and the hong kong bastvs [ ] . this suggests the occurrence of multiple cross-species transmission events among hosts and other animal species. however, it appears that the histopathogenic findings of encephalitis in korean downer cows were not associated with the detection of boastv-neuros in brain tissue. the bi analysis of the partial orf ab and/or orf genes also showed that all of the known bastvs could be separated into four groups (fig a, b) , in the same way as the diversity analysis. group of the bi tree contained six hong kong bastvs and five korean bastvs, groups and included only korean bastvs, and the boastv-neuros strain was the only member of group . in conclusion, the present study identified four bastvs groups based on the phylogenetic analysis and their shared pairwise amino acid sequence identities. the bastv detection rate in cattle feces was higher in calves aged \ month compared with adult cattle. thus, continuous surveillance of novel diversity in bastvs should be conducted on many cattle farms throughout the world because of the risk of emerging astroviruses associated with neurological disease in cattle. pastk- pig (jq ) pastk- pig (jq ) b /hk cow (hq ) b /hk cow (hq ) ccastv- deer (hm ) ccastv- deer (hm ) poastv - pig (hm ) poastv - pig (hm ) pastk- pig (jq ) pastk- pig (jq ) b /hk cow (hq ) b - cow (jf ) cow pastv- / /hun pig (gu ) wbastv- wildboar (jq ) pastv/ /mn pig (jf ) pastk- pig (jq ) pastk- pig (jq ) pastk- pig (jq ) pastv/ /pa pig (jf ) human astroviurs human (dq ) virus taxonomy. eighth report of the international committee on taxonomy of viruses acknowledgments we are grateful to dr. soo-kyung joo for technical assistance. key: cord- - pclxek authors: cohen, liza miriam; grøntvedt, carl andreas; klem, thea b.; gulliksen, stine margrethe; ranheim, birgit; nielsen, jens peter; valheim, mette; kielland, camilla title: a descriptive study of acute outbreaks of respiratory disease in norwegian fattening pig herds date: - - journal: acta vet scand doi: . /s - - -z sha: doc_id: cord_uid: pclxek background: respiratory diseases are major health concerns in the pig production sector worldwide, contributing adversely to morbidity and mortality. over the past years there was a rise in reported incidents of respiratory disease in pigs in norway, despite population wide freedom from aujeszky´s disease, porcine reproductive and respiratory syndrome, porcine respiratory corona virus and enzootic pneumonia. the main objective of this study was to investigate acute outbreaks of respiratory disease in conventional norwegian fattening pig herds. the study included herds. in seven herds with reported outbreaks of acute respiratory disease, data on clinical signs was recorded and samples for laboratory examination were collected. diagnostic protocols were compared by parallel analysis of clinically healthy pigs from seven non-outbreak herds. results: the most commonly reported clinical signs were sudden deaths and dyspnea. an average compartment morbidity of %, mortality of % and case fatality of % was recorded in the outbreak herds. post-mortem examinations revealed acute lesions resembling porcine pleuropneumonia in all pigs investigated from the outbreak herds and in of the ( %) pigs from the non-outbreak herds. chronic lesions were recorded in another pigs ( %) from the non-outbreak herds. actinobacillus pleuropneumoniae serovar was isolated from lungs and/or pleura from all tested pigs (n = ) in the outbreak herds, and from out of pigs ( %) in the non-outbreak herds, one pig with an acute and another pig with a chronic infection. no other significant bacterial findings were made. seroconversion to a. pleuropneumoniae antibodies was detectable in all outbreak herds analyzed and in six out of seven non-outbreak herds, but the risk ratio for seroconversion of individual pigs was higher (risk ratio . [ . - . % ci; p < . ]) in the outbreak herds. all herds tested positive for porcine circovirus type and negative for influenza a viruses on oral fluid rt-qpcr. conclusion: the main etiological pathogen found during acute outbreaks of respiratory disease was a. pleuropneumoniae serovar . all pigs from outbreak herds had typical lesions of acute porcine pleuropneumonia, and only a. pleuropneumoniae serovar was identified. co-infections were not found to impact disease development. are however hard to determine in field conditions. studies show that coinfections with different respiratory agents are common in pigs [ , ] . viral infections often predispose for secondary bacterial infections. this has been studied under experimental conditions, i.e. coinfections of porcine reproductive and respiratory syndrome virus (prrsv) and mycoplasma hyopneumoniae [ ] , prrsv and actinobacillus pleuropneumoniae [ ] , swine influenza virus (siv) and bordetella bronchiseptica [ ] . moderate to marked fever, lethargy, coughing, sneezing and dyspnea are common clinical signs during disease outbreaks [ , ] . the presence of multiple pathogens often increases the severity of disease and occurrence of lesions in the respiratory tract [ , , ] . there are differences in occurrence and distribution of pathogens between countries, regions and herds [ , ] that contribute to the complexity of respiratory disease. due to strict import regulations in norway, there is negligible import of live pigs to the commercial pig population [ ] . the national yearly yield was approximately . million slaughtered pigs in , originating mainly from registered fattener pig herds with a concession limit of maximum slaughtered pigs per year [ , ] . the norwegian pig production is also characterized by stringent regulation of antimicrobial drug use and a tradition of eradicating diseases from animal populations [ , ] . the commercial pig population in norway has documented freedom from several important respiratory pathogens including aujeszky's disease virus, prrsv, siv (apart from influenza a [h n ]pdm ) [ ] and m. hyopneumoniae [ ] . after the pandemic in / , antibodies to siv (h n )pdm have been detected regularly from to % of examined herds in norway [ ] , but siv (h n )pdm infections in the norwegian pig population has been considered to have limited clinical impact [ ] . in cases of respiratory disease in norwegian herds, a. pleuropneumoniae has regularly been isolated from lungs of carcasses submitted for routine diagnostics [ ] . several studies from other countries conclude that a. pleuropneumoniae is normally present in most conventional pig herds, having a main reservoir in the tonsils of carrier pigs [ , ] . accordingly, outbreaks in conventional herds are most often triggered by factors related to animal housing, management and environment rather than an introduction of the bacteria in a naïve herd [ ] . preceding infection with a primary viral pathogen is also a possible triggering factor [ ] . in the years between and there was an increase in reported acute cases of respiratory disease requiring veterinary treatment in norway [ ] . a systematic investigation of porcine respiratory disease outbreaks in norway has not recently been performed, and updated knowledge is needed for appropriate disease prevention and intervention. the main objective of this study was to investigate clinical outbreaks of acute respiratory disease in norwegian fattening pig herds, using a group of non-outbreak herds to compare diagnostic procedures. the source population was the conventional fattening pig herds located in central and southern parts of norway in the period between september and october . the conventional herds are not part of the norwegian specific pathogen free (spf) sub-population, in which herds are free from e.g. toxin producing pasteurella multocida and all serotypes of a. pleuropneumoniae. seven conventional fattening pig herds with acute outbreaks of respiratory disease (outbreak herds) and seven pig herds without respiratory disease outbreaks (nonoutbreak herds) were included in this study. the inclusion criteria for outbreak herds were; three or more pigs displaying acute signs of respiratory disease including fever and coughing and/or dyspnea, and/ or otherwise reduced general condition e.g. lethargy or inappetence. non-outbreak herds inclusion criteria were; absence of acute clinical signs of respiratory disease at the time of sampling, situated in the same geographical area as the outbreak herds. the non-outbreak herds were not matched to the outbreak herds by means of other parameters. herds were included only if there were more than three weeks until planned slaughter, due to follow-up sampling per protocol. two herds were excluded, due to treatment with antimicrobial drugs before sampling could be carried out, and insufficient time from outbreak to planned slaughter, respectively. descriptive herd data are listed in table . a network of veterinary practitioners was established to collect samples and herd data. the practitioners were contacted through emails, letters, meetings and announcements in relevant journals and national newspapers. the veterinarians contacted the project group immediately upon being called out to examine pigs with symptoms of acute respiratory disease. outbreak herds were recruited for participation by the veterinary practitioners after meeting the inclusion criteria. non-outbreak herds were then recruited by the veterinary practitioners contacting herd owners meeting the matching criteria, asking their participation and arranging a visit. complete kits containing materials and detailed instructions for sample collection, preservation and transport were pre-distributed to designated pick up points at abattoirs and veterinary practice offices and sent to veterinarians across the country upon request. each outbreak herd was visited on three occasions ( fig. , green boxes) ; the first visit was conducted as soon as possible during the reported outbreak for initial sampling. the second visit was performed to days later to conduct interviews and register herd demographic data. during the third and final visit two to four weeks after the first, follow-up samples were collected, as described in fig. . non-outbreak herds were visited on two occasions, once for initial sampling, farmer interviews and herd registrations, and secondly for follow up sampling. details about the diagnostic sampling are shown in fig. . diagnostic sampling in outbreak herds was performed the day the veterinarian was notified about the disease. the veterinary practitioner reported observed clinical signs on a standardized submission form. in these herds, three to five pigs were selected for organ collection, pigs were sampled in total. the selection was made from pigs with clinical signs of respiratory disease prior to death or euthanasia by captive bolt and exsanguination. short time from death to sampling was considered, no additional criteria for sampling were applied. in non-outbreak herds three to five pigs were haphazardly selected, pigs were sampled in total. lungs and mediastinum (including pericardium, excluding the heart) and trachea caudal to the thoracic inlet were collected. within each herd, care was given not to sample pigs treated with any antimicrobial drugs up to days prior to the sampling. blood sampling was performed on a total of pigs per herd by haphazard selection from as many pens in the compartment as possible, up to pens. a total of pigs were sampled. the pigs were selected regardless of clinical presentation and restrained by snaring the upper jaw. during restraint the pigs were ear-tagged for individual identification at follow-up sampling during the final visit. rectal temperature was measured, and blood samples were collected (details in fig. ). pooled oral fluid (of) samples were collected from two haphazardly selected pens (n = pooled of samples from pens) using chewing rope as described by prickett et al. [ ] . care was given to keep the stress of the animals during sampling to a minimum. demographic data sampling was obtained by interviewing the farmers using a purpose-built questionnaire, see details in fig. . relevant information regarding the disease outbreaks including information about the first days after noticing the first clinical signs was registered in table overview of descriptive data in both outbreak and non-outbreak herds (n = ) a herd type: finishers, farrow-to-finish. herds: one compartment affected and tested. herd: two compartments affected and tested, compartment average presented b herd type: finishers, farrow-to-finish. one compartment tested per herd fig. overview of the timeline and procedure of the study outbreak herds. the following data was registered: dates of the pigs' arrival to compartment, a description of earliest observed clinical signs, onset of disease, time to veterinary contact, time from the first clinical signs to the initial sampling, numbers of pigs displaying clinical signs, applied antibiotic treatment, and number of sick and dead pigs from the start of the outbreak until the time of the interview. during the final visit, second blood samples were collected from individually ear tagged pigs, and rectal temperature measured in the same pigs. procedures for sample handling are presented in fig. . organs from pigs were subject to post-mortem examination. the pericardium, pleura, trachea, bronchi, lung parenchyma and tracheobronchial lymph nodes from to pigs from outbreak herds and non-outbreak herds respectively, were examined at the norwegian veterinary institute (nvi) according to a standardized protocol (additional file ). tissue samples from the lungs, pleura and lymph nodes were fixed, processed, sectioned and stained for histological examination (additional file ). in total, histological sections from the outbreak herds and sections from non-outbreak herds were examined following a standardized protocol (additional file ). sampling (on charcoal transport swabs) for bacterial cultivation was performed during postmortem examination of lungs and pleurae, see details in table . the lung surface was flamed and aseptically incised before swabbing of lung tissue. the swabs were cultivated as a part of the routine diagnostics at nvi (additional file ). serovar identification of cultured a. pleuropneumoniae (n = isolates) was performed on sequence data, generated through whole genome sequencing of the a. pleuropneumoniae isolates at statens serum institut (ssi), copenhagen, denmark. the serovar was determined based on the presence of the serovar specific cps operons [ , ] . details regarding the method are described in additional file . the serum samples (n = ) were analyzed using commercial diagnostic kits for antibodies to a. pleuropneumoniae, influenza a virus, prrsv, prcv and m. hyopneumoniae. the analyses were performed as described by the manufacturers; details are given in additional file . interpretation of the test results were categorical, based on the cut-off values recommended by the test manufacturers. presence of antibodies to prrsv, porcine respiratory corona virus (prcv) and m. hyopneumoniae were tested in the second serum sample (n = ). serum elisa was conducted on paired serum samples (n = ) from individual pigs for antibodies to influenza a virus and a. pleuropneumoniae. the presence of influenza a virus and porcine circovirus type (pcv ) nucleic acids in pooled oral fluids (n = ) were analyzed with real time polymerase chain reaction (pcr) by in-house procedures (additional file ). a cycle threshold (ct) value for influenza virus below was considered positive. pcv quantitative pcr (qpcr) is a quantitative test where results are given as measured nucleotide copies in µl sample, calculated from repeated measures at different ct values and results are reported as low (< copies), moderate ( - copies) or high (> copies). our sample size of serum samples per herd was chosen based on an estimate of at least one positive animal if the prevalence of our disease in question is around % at a % confidence level. the same sample size was used for agents not present in the population, that we did not expect to find, due to practical reasons. statistical analyses of the data were performed using the software stata (stata se/ for windows; stata corp., college station, tx, usa). descriptive numeric results are presented as average values and the standard deviation (sd) for data with a normal distribution, or median value followed by the interquartile range (iqr) for data that was not normally distributed. rectal temperatures from the first visit and from the final visit to the herd were compared. the variable "fever" was defined as a rectal temperature above . °c. odds ratios for fever during the outbreak sampling compared to fever during follow-up visits, were calculated using a stata case-control odds-ratio calculator. morbidity was measured as the proportion of pigs with clinical signs of respiratory disease of the total number of pigs in the herd (herd morbidity) and in the compartment (compartment morbidity). mortality was measured as the proportion of pigs dying during the outbreak, out of the total number of pigs in the herd (herd mortality) and in the compartment (compartment mortality). case fatality, an indicator of pathogen virulence and disease lethality, was measured as the proportion of pigs that died during the outbreak and displayed clinical signs of respiratory disease prior to their death, out of the total number of pigs displaying respiratory disease. a herd was classified as seroconverted if at least one pig shifted from negative to positive status and no pigs shifted from positive to negative status. the proportion of seroconverted pigs in each herd was calculated. samples from pigs that could not be identified by ear tags (one herd, n = ) were excluded. when calculating the incidence proportion and risk ratios for seroconversion to a. pleuropneumoniae, pigs that were seropositive on the first serum sample were excluded from the population at risk. incidence proportion was defined as the proportion of the seronegative pigs that seroconvert during the time at risk. time at risk was defined as time between paired serum samples. the risk ratio (rr) for a pig to seroconvert in outbreak herds, compared to nonoutbreak herds, was calculated using a stata cohort study risk-ratio calculator the % confidence interval (ci). the statistical significance of the calculated association, whether it was likely that the rr was different from , was indicated by the reported p value. median number of days from the farmers noticed clinical signs of respiratory disease until calling the local veterinary practitioner was day. onset of outbreak was days (median, iqr ) after the pigs arrived at the compartment. the severity of the clinical signs varied between outbreak herds. clinical signs reported by the veterinary practitioner were mainly sudden deaths (four herds) and dyspnea (three herds). signs such as fever, bloody froth from oronasal openings, cough and lethargy were also reported, and it was observed that sick pigs were reluctant to chew on the cotton ropes used for of sampling. in all herds, intramuscularly administrated procaine benzylpenicillin was used to treat sick pigs over to days. in one herd, tiamulin was additionally administered in the drinking water for days. treatments were started by the veterinary practitioner during the first visit after the outbreak of disease. all herd owners reported the treatment to effectively reduce acute clinical signs and stop the further spread of disease. the average compartment morbidity during the outbreak was % (sd , range - %), while herd morbidity was % (sd , range . - %) in the outbreak herds. case fatality rate during the disease outbreaks was on average % (sd , range - %) over days, suggestive of a low virulent agent. during the outbreaks, compartment mortality was % on average (sd , range - %), while herd mortality was % (sd , range - %). proportion of pigs in the outbreak herds measuring a rectal temperature above . °c was . % (n = ) and % at the first and final visit, respectively. for the non-outbreak herds the proportion of pigs with a rectal temperature above . °c was . % (n = ) and % at first and final visit, respectively. the odds for a temperature above . °c were higher (odds ratio = . , % ci . - . ), during outbreak than during follow-up in the outbreak herds. there were no dropouts among the study animals, the number of animals tested at the visits was the same. median number of days between first and final visit was days (iqr ) in outbreak herds and days (iqr ) in non-outbreak herds. results from the pathological examinations of organs are presented on herd level in table . gross pathology of the lungs was detected in all pigs (n = ) from the outbreak herds. acute pleural lesions were reported in of these pigs ( %) and chronic pleural lesions, were found in one. typical lesions of acute pneumonia were found in all the pigs. the acute lesions were principally dorsally distributed in all lung lobes, but the caudal lobes were the most affected. chronic lung lesions were observed in one pig. moderate to severe enlargement of the tracheobronchial lymph nodes was a prevalent finding (n = , %) in the pigs with pneumonia. characteristic gross lung lesions are shown in fig. . in the non-outbreak herds various gross lung lesions were detected in seven of the pigs ( %). pleuritis was observed in two of pigs ( %), where one had an acute pleuritis, and the second pig focal chronic pleuritis. pneumonia was observed in four other pigs. mild, focal, acute lesions were seen in two of them, while similar acute lesions and abscess formation was seen in another. multifocal, necrotizing, chronic pneumonia was diagnosed in the fourth pig. a single pig from a non-outbreak herd had gross lung lesions of multifocal bleeding and mottled grayish green areas indicative of larval migration by ascaris suum. diagnostic results for individual herds, including the gross findings are summarized in table . histopathological changes agreed with the acute macroscopic lesions observed. histological examination revealed fibrin and neutrophil deposits on the pleura. in the lung parenchyma there was alveolar filling with necrotic leukocytes, neutrophils and fibrin. interstitial edema and hemorrhage, peribronchial and peribronchiolar leukocyte infiltration was observed. subacute to chronic, necrotic lesions of varying sizes were demarcated by macrophages, lymphocytes and plasma cells table results from gross pathology, bacteriology, serology and virology from seven outbreak herds (from to ) and seven non-outbreak herds (from to ) of on the pleura or in alveolar lumen, and areas of interstitial bleeding. actinobacillus pleuropneumoniae was cultured from all sampled pigs (n = ) from outbreak herds (n = ). abundant growth of a. pleuropneumoniae was present in lung tissue in all pigs and on pleura in pigs. in samples from of the lungs and pleurae, a. pleuropneumoniae was the sole microbial species detected. in the remaining samples, a range of bacteria were detected in addition to a. pleuropneumoniae and the results are shown in table . swabs from non-outbreak pigs' lungs produced mostly negative bacteriology. from non-outbreak herds, a. pleuropneumoniae was isolated from lung parenchyma in two out of pigs. the a. pleuropneumoniae isolates originated mainly from areas with acute gross pathology ( table ). in one non-outbreak pig a. pleuropneumoniae was cultured from a chronic lung lesion. serotyping of a. pleuropneumoniae on genome level revealed that all sampled isolates belonged to serovar . the serum samples were successfully analyzed in one session. antibodies to a. pleuropneumoniae were detected in samples from six ( %) outbreak herds and four ( %) non outbreak herds. at the first serum sample, % ( of ) of the pigs in the outbreak herds were seropositive, and % ( of ) in the non-outbreak herds. at the second serum sample, % ( of ) and % ( of ) of the pigs were positive in the outbreak and non-outbreak herds respectively, details are listed in table . six outbreak herds and six non-outbreak herds were considered seroconverted, indicative of an active infection in the period from the first to the second visit. seroconversion in the seventh outbreak herd could not be assessed due to missing ear tags. proportion of seroconverted pigs in each outbreak herd ranged from to %, and from to % in non-outbreak herds ( table ) . incidence proportion was . (sd . ) in outbreak herds over the median time at risk of days. incidence proportion in the non-outbreak herds was . (sd . ) over the median time at risk of days. the risk for seroconversion was more than double compared to pigs from non-outbreak herds (rr . [ . - . % ci; p < . ]). antibodies to influenza a virus were detected in one outbreak herd, where one pig seroconverted during the sampling period, and two pigs were found to have a reduced antibody titer to below cutoff. influenza a-antibodies were not detected in the remaining six outbreak herds or the non-outbreak herds. the proportion of siv seropositive herds was % out of the herds combined. antibodies to m. hyopneumoniae, prcv and prrsv were not detected in samples from any herds. the pooled of samples from pens, median number of pigs per pen was (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , were all negative for influenza a viruses. quantification of pcv by rt-qpcr turned out low or moderate in all samples, results per herd are shown in table . field outbreaks of acute respiratory disease in norwegian fattening pigs were investigated and a. pleuropneumoniae serovar was the main pathogen detected, with negligible presence of co-infections. clinical signs reported were in agreement with previous reports of a. pleuropneumoniae infections, which are described to have a diverse clinical presentation [ ] . even with the large variation in morbidity and mortality rates, the results from this study were in line with observations from other studies, as research on outbreak characteristics of respiratory disease show that morbidity can range from to % [ ] . mortality during outbreaks of acute porcine pleuropneumonia is usually reported to be between and % [ ] . case fatality rates are not commonly included in this research literature but is a more precise measure of the lethality of a disease, especially if little information about other illnesses is available. disease that affects mortality are likely to have common risk factors [ ] and the use of case fatality rate is a more robust measurement and less subjected to confounders such as that of other illnesses. even as a single infectious primary agent, a. pleuropneumoniae can cause severe clinical signs. during acute porcine pleuropneumonia, high fever is common [ , ] . for pigs in the age range from to months, body temperatures normally span from . to . °c [ ] , and the proportion of pigs displaying a fever can be indicative of an outbreak. in the present study, the pigs were restrained by snaring the upper jaw during clinical examination and blood collection, which is stressful for the animal [ ] . the cutoff for fever at . °c + . was used in the study to compensate for this stress. higher odds for displaying fever in the herds during outbreak than at the final visit were found among the pigs in this study. this signified body temperature as a disease characteristic during outbreaks of porcine pleuropneumonia, although technical biases like personnel and thermometers used might have influenced our results. this coincided with results from a recent study from finland [ ] . there are acknowledged a. pleuropneumoniae serovars, of which some were recently described [ ] . from the norwegian pig population, serovars , , , and have previously been reported [ ] . serovar has been most commonly associated with clinical disease in recent years, followed by type [ ] . however, these previous findings were all based on antibody agglutination tests which are prone to cross-reactions, for instance between serovars , , and [ ] . all a. pleuropneumoniae strains in this study belonged to type , raising questions about the importance of serovar . underestimation of serovar has occurred in canada [ ] , england and wales [ ] . serovar is typically viewed as low virulent and is less often associated with clinical disease globally. in a study describing clinical presentation of different serovars in experimentally infected pigs [ ] , serovars that were less commonly associated with disease were able to produce severe clinical signs, including high fever. this could perhaps be a result of absence of other respiratory agents including more virulent serovars of a. pleuropneumoniae. the macro-and histopathologic findings were typical for acute pleuropneumonia caused by a. pleuropneumoniae [ ] [ ] [ ] , supporting that a. pleuropneumoniae was the main etiologic pathogen in these outbreaks. direct agent detection, primarily by bacteriological culturing in affected lung tissue obtained during necropsy, is considered the most adequate method for diagnosing porcine pleuropneumonia [ ] . direct pcr is a method that would be expected to yield similar results but would not allow for storing of the bacterial isolates for further molecular testing, as was done in this study. we observed a low incidence of pathological lesions in non-outbreak herds, and a. pleuropneumoniae was only isolated from lesions resembling porcine pleuropneumonia. other bacteria, including p. multocida and streptococcus spp., were also detected in a few samples in this study. both are known opportunistic bacteria that colonize the upper respiratory tract of healthy pigs [ ] . streptococcus suis is the most important streptococcal swine pathogen found to contribute to bronchopneumonia [ ] . it is not unlikely that the bacteria could colonize areas already infected with a. pleuropneumoniae. the lesions might then be hard to distinguish from the primary pathogen, particularly if large parts of the lungs are affected. in one outbreak herd all five lungs had growth of other bacteria. they could have been contaminated during collection, transport or sampling. alternatively, these pigs were all colonized by secondary bacterial pathogens. the number of herds included in this study was too low to investigate whether the presence of these bacteria was linked to any differences in outbreak characteristics or diagnostic results. the low occurrence of common secondary invaders could have been explained by the short time span between registered disease and sampling. it has been questioned whether the actions that led to the eradication of m. hyopneumoniae from the norwegian pig population [ ] also significantly reduced the occurrence of other pathogens. this has not yet been investigated. treatment with procaine benzylpenicillin was in line with the therapeutic guidelines published by the norwegian medicines agency as the drug of choice for acute porcine pleuropneumonia [ ] . similar recommendations have been published in finland and sweden [ , ] . in denmark, tilmicosin and tulathromycin have been commonly used against acute pleuropneumonia [ ] partly due to the convenience of peroral administration, not due to reduced susceptibility to benzylpenicillin. national surveillance programs for antimicrobial resistance in these countries have recently reported a high proportion of a. pleuropneumoniae isolates being susceptible to benzylpenicillin [ ] [ ] [ ] . nevertheless, there are no recently published studies on the efficacy of procaine benzylpenicillin for porcine pleuropneumonia in norway. such knowledge of causative pathogens is the fundament for correct and prudent use of antimicrobials. the details to antimicrobial resistance patterns of a. pleuropneumoniae in norway are currently being studied further. seroconversion to a. pleuropneumoniae had occurred in most of the herds, in many cases in absence of clinical disease. the risk for seroconversion to a. pleuropneumoniae for pigs in outbreak herds was more than double compared to pigs from non-outbreak herds, despite small within-herd populations at risk due to many seropositive pigs in the first serum samples. seroconversion to less virulent strains might have happened without resulting in a cross-protection to the outbreak-causing serovar. in finland, haimi-hakala et al. observed no difference in either prevalence of seroconverted herds or proportion of seroconverted pigs per herd in the outbreak case group and non-outbreak control group [ ] . they discuss that neither single or paired serum sampling for the diagnosis of acute respiratory disease in field conditions is of much value due to both a lack of details concerning the initiation time of infection and a high prevalence of subclinical infections with a. pleuropneumoniae. the risk for seroconversion was not addressed in their paper. a danish study from investigated correlations in seroconversion to a. pleuropneumoniae and concluded that variation in seroconversion was mainly explained by a common batch level factor, that varies between farms and batches within a farm [ ] . outbreaks of disease might be viewed as a batch level factor in this sense. in cases of all-in-all-out rearing by compartment, which is common, batches of pigs are usually housed separately. as we observed, the outbreaks were often restricted to single compartments. risk factors can be related to animal housing, management and environment [ ] , and infection pressure might be increased during clinical disease and is a likely trigger for seroconversion. risk factor analyses were beyond the scope of this paper due to a lower number of herds in our study than what was expected. the seeming decrease in outbreak occurrence might have resulted from of a collective effort in the norwegian pig production system to increase the health status of herds with reoccurring problems with respiratory disease prior to our sampling. when investigating siv antibody titers we found that only one outbreak herd was seropositive. even though one pig seroconverted during the sampling period, two pigs were found to have reduced antibody titer. since a single false-positive serological reactor could not be excluded, the true status of these animals was uncertain. there being multiple false-positive reactions in one herd, which would have been the case here, was perhaps less likely. the proportion of seropositive herds in this study was less than what is found on a national level, where approximately % of the herds are reported positive [ ] . the virology results from our study suggested that neither siv nor pcv contributed to the disease outbreaks in the study population. the absence of siv in all of samples supported the lack of pathological lesions and serological results indicative of siv infection. no difference was detected in pcv levels between the outbreak-and the non-outbreak herds. reluctancy of sick pigs to chew on the ropes could have resulted in unrepresentative pcv levels. since pcv levels was tested on pooled samples we have no information on the individual pig's contribution to the sample. the health status of the norwegian pig population is very good and have many similarities to the one of finland in the sense that they are free from m. hyopneumoniae, prrsv and until recently prcv [ ] . in finland, a more diverse outbreak etiology has been observed [ ] . in the finnish study, a. pleuropneumoniae was found to be the most likely cause of disease in of the sampled herds. in most of these herds, a. pleuropneumoniae was the only etiologic pathogen identified. similarly, outbreaks of respiratory disease were studied in the netherlands [ ] concluding that five of these were most likely caused by a. pleuropneumoniae, while seven were caused by siv (h n ) and (h n ). like in our study, they did not find any clear evidence of specific dual infections. the main etiological pathogen of acute outbreaks of respiratory disease in the included norwegian fattening pigs was a. pleuropneumoniae. all pigs from outbreak herds were found to have typical lesions of acute porcine pleuropneumonia, and only a. pleuropneumoniae serovar was identified. the clinical presentation and pathology of a. pleuropneumoniae was in line with previous reports on field outbreaks internationally. co-infections did not seem to be of impact on disease development. supplementary information accompanies this paper at https ://doi. org/ . /s - - -z. additional file . protocol for postmortem sampling. a scheme for a standardized postmortem evaluation and sampling of pigs' lungs. the scheme was compiled at the pathology department at the norwegian veterinary institute to be used in the study of acute respiratory disease outbreaks. additional file . histology protocol. a scheme for a standardized histologic evaluation of sections from pigs' lungs, pleura and tracheobronchial lymph nodes, including a description of section preparation. the scheme was compiled by members of the project group grisefine lunger to be used in the study of acute respiratory disease outbreaks. additional file . details of sample handling and diagnostics. a document containing extended details of sample handling and laboratory diagnostic methods performed in the study of acute respiratory disease outbreaks. we would like to thank professor mari heinonen at the university of helsinki and tijs tobias (dvm, ph.d., msc) for their advice and contributions to the planning of this study. at the norwegian veterinary institute several people at different departments have contributed significantly to the diagnostic work that is presented in this study. we would especially like to thank pathologists Øyvor kolbjørnsen and helene wisløff, bacteriologist marianne gilhuus, molecular biologist torfinn moldal. in preparation for the field trial, there had been a substantial marketing campaign for the project, reaching out to pig farmers, veterinarians and farm advisors throughout the country. we would like to thank everyone involved in the sampling and data collection for their time and attention. results presented in this article have not been previously published. authors' contributions lmc, cag, tbk, smg, br and ck planned this study, lmc, ck and br performed the outbreak investigations/collected materials. lmc, mv and ck analyzed and interpreted the data. lmc and ck prepared the tables and figures. lmc had the primary responsibility of writing and revising the manuscript. all authors contributed to revising the manuscript. all authors read and approved the final manuscript. we are grateful for the funding provided by the agricultural agreement research fund and the foundation for research levy on agricultural products, grant number nfr- . thanks to our collaborators who also funded the research: animalia, nortura and klf. the norwegian veterinary institute covered all costs related to administration of sampling equipment and sample analysis for this study. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. to be able to perform sampling from live animals, the norwegian food safety authority approved the study design for 'grisefine lunger' in september , maintaining compliance of ethical guidelines and the three r's. fots norwegian food safety authority reference id . diseases of swine effect of pneumonia on growth rate and feed efficiency of minimal disease pigs exposed to actinobacillus pleuropneumoniae and mycoplasma hyopneumoniae a tool to assess the economic impact of pleurisy in slaughter pigs porcine 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online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from evaluation of actinobacillus pleuropneumoniae diagnostic tests using samples derived from experimentally infected pigs detection of actinobacillus pleuropneumoniae in pigs by real-time quantitative pcr for the apxiva gene herd evaluation restraint, but not frustration, induces prostaglandinmediated hyperthermia in pigs etiology of acute respiratory disease in fattening pigs in finland proposal of serovars and of actinobacillus pleuropneumoniae based on serological and genotypic analysis occurrence of lung lesions and antibodies to serotypes and of actinobacillus pleuropneumoniae and haemophilus parasuis in slaughter weight pigs from elite herds in norway quantitation of serotype-specific and crossreacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating actinobacillus (haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes , , and the challenge of detecting herds sub-clinically infected with actinobacillus pleuropneumoniae actinobacillus pleuropneumoniae serovar predominates in england and wales diseases of swine an abattoir survey of pneumonia and pleuritis in slaughter weight swine from selected herds. i. prevalence and morphological description of gross lung lesions an abattoir survey of pneumonia and pleuritis in slaughter weight swine from selected herds. iii. serological findings and their relationship to pathomorphological and microbiological findings diseases of swine docum ents/veter in%c %a rme disin /terap ianbe falin ger/ terap ianbe falin g_bruk% av% ant ibakt eriel lt% mid ler% til % pro duks recommendations for the use of antimicrobials in the treatment of the most significant infectious and contagious diseases in animals dling srek-vet/rek% -% dos ering % av% ant ibiot ika% til l% gri s% b % d national food institute. danmap -use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in denmark finres-vet : finnish veterinary antimicrobial resistance monitoring and consumption of antimicrobial agents. finnish food authority publications swedres-svarm : consumption of antibiotics and occurrence of resistance in sweden intra-unit correlations in seroconversion to actinobacillus pleuropneumoniae and mycoplasma hyopneumoniae at different levels in danish multi-site pig production facilities springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.