key: cord-0930794-r5va3y1n
authors: Wilson, Heather L.; Gerdts, Volker; Babiuk, Lorne A.
title: Mucosal Vaccine Development for Veterinary and Aquatic Diseases
date: 2019-10-25
journal: Mucosal Vaccines
DOI: 10.1016/b978-0-12-811924-2.00048-1
sha: 28a1d5f0ab7ef183e07269f2f70c35d8ccd9cdea
doc_id: 930794
cord_uid: r5va3y1n

Because most pathogens and food antigens enter the host via the mucosal surfaces, effective mucosal immunity is critical for maintaining homeostasis through immune regulation, tolerance, and induction of effective immune responses when needed. Thus the mucosa-associated lymphoid tissues represent an important target for vaccination. Indeed, more than 20 years of research have clearly demonstrated the benefits of mucosal vaccination versus systemic vaccination. Such benefits include local induction of secretory immunoglobulin A (SIgA) as well as activation and maturation of mucosal dendritic cells, homing of effector cells to the mucosal surfaces, expression of specific host defense peptides, and other innate effector molecules. In addition, mucosal vaccination offers the opportunity to induce colostral and lactogenic immunity during pregnancy and the possibility of avoiding neutralization of early life vaccines by maternal antibodies, both of which are critical for protecting the most susceptible from infectious diseases. Moreover, mucosal administration offers the advantage of inducing both effective systemic immunity and mucosal immunity, enhancing vaccine efficacy and providing improved protection. A number of animal vaccines are already administered via the mucosal surfaces, with many more to come over the next few years. It is gratifying to see that veterinary vaccine development has yet again taken a leadership role in exploring innovative approaches and technologies to mucosal vaccination. For the veterinary field, considerations for mucosal vaccine development and use necessarily include costs (often pennies per dose), mass delivery that preferably avoids animal restraint, and economic and trade considerations. In this chapter, we provide an overview of some of the existing vaccine technologies and discuss their advantages and disadvantages.

Vaccines are essential for controlling disease in livestock, companion, and zoo animals and wildlife and for controlling fertility and disease in pest species. Effective mucosal vaccines have myriad advantages over parenteral vaccines that are shared across human and veterinary species. For instance, mucosal vaccines stimulate both the mucosal and systemic immune systems, meaning that mucosal vaccines can reduce pathogen colonization and shedding and thus protect the population or herd against infection. Major challenges for mucosal immunization are to generate effective immunity instead of immunological tolerance as well as overcoming interference from passively acquired maternal antibodies. Oral tolerance is a suppressive mechanism designed to prevent the host immune system from overreacting to innocuous antigens such as those present in feed or commensal flora [1] . Once oral tolerance has been induced, subsequent exposure to that antigen from mucosal or systemic routes will prevent induction of a robust immune response [2] . The vast majority of mucosal vaccines use live attenuated forms of a pathogen that can replicate in the target species, avoiding induction of oral tolerance, but under very rare circumstances may revert back to virulence. Other forms of live vaccines include viruses such as replication-deficient adenovirus [3] , canarypox virus carrier vaccines [4] , and others that act as a vector to express genes of interest from various pathogens. These carrier viruses have no ability or limited ability to replicate in the immunized species; nor is reversion to a replication competent, highly infectious virus possible, making them a safer vaccine choice. There are some reports that mucosal delivery of live viral vaccine to the upper respiratory tract may overcome maternal antibody interference [5] . Previous chapters provided detailed analyses of the numerous advantages of mucosal vaccines, and these will not be discussed in depth here. Instead, aspects of mucosal vaccine development that are particularly important for the veterinary field, such as mass delivery, vaccines that differentiate between infection and vaccination (which has important trade implications), and economic considerations, will be discussed. Examples of the numerous commercially available mucosal veterinary vaccines and discussion of novel experimental vaccines will be provided. Vaccine development for administration in veterinary species to protect human health will also be discussed.

Mass delivery methods for mucosal immunization, such as administration of the vaccine in drinking water or in feed, in sprays (for avian species, which are ingested at preening), in ovo (for avian species), or for immersions (for aquatic species), allow vaccination of hundreds or thousands of animals over a short period of time. Most important, mass delivery of mucosal vaccines means that each individual animal does not need to be restrained, which can be extremely stressful for animals (especially those that are not routinely handled by humans), and restraint may be potentially hazardous to the person or teams of people administering the vaccines. A major drawback to mass delivery is that a uniform dose per animal is not always given across the population. Other mucosal routes that require restraint are vaccines administered intranasally, by drenching (oral immunization by syringe), or through eyedrop (conjunctival) administration. These delivery methods have the advantage that they promote a mucosal immune response and the dose per animal is easily controlled, but they do require animal restraint, which makes them more expensive to deliver.

With today's global economy, trade in animals and in animal products such as meat, eggs, and fur occurs locally, nationally, and internationally. For trade purposes, it is advantageous to be able to distinguish animals that have been infected by disease from animals that have been vaccinated. Vaccines that allow for the immunological differentiation between animals that have been infected and those that have been vaccinated are called DIVA vaccines. DIVA vaccines often lack one or more proteins present in the wild-type microorganism, which can be determined through immune assays. The first-generation DIVA vaccines were developed when it was discovered that pigs vaccinated with the attenuated strains of Aujeszky's disease virus did not develop antibody against select protein epitopes [6] , whereas animals infected by wild-type viruses did have these protein-specific antibodies in their serum. These first-generation marker vaccines were soon improved upon by using genetically modified live vaccines that lacked selected glycoproteins [7] , and enzyme-linked immune sorbent assays were developed that could determine which pigs were vaccinated because they lacked antibodies against the glycoprotein but had antibodies specific for other PrV proteins [8] . By using these strains as vaccines or genetically engineered live viral vaccines, researchers could track the success of eradication efforts; this resulted in effective eradication programs of the disease in many parts of Europe [8] . Without the option of using DIVA vaccines, producers may choose not to vaccinate so as to avoid the presence of antibodies in the herd that they cannot guarantee are due to the vaccine and not the presence of the diseasecausing agent in their herd. At the same time, vaccines made from live viruses, such as replication-deficient adenovirus that express genes of interest from various pathogens or modified viruses with select genes deleted, are considered genetically modified organisms (GMOs). Animals vaccinated with GMOs may face regulatory hurdles that must be overcome before the products from these animals can be sold. Indeed, some countries are reluctant to license GMO vaccines.

Livestock production is a large-scale business and mucosal vaccination will not be implemented unless the disease causes significant mortality and/or morbidity (such that production metrics are negatively affected). Further, the vaccine will be used only if it is sufficiently affordable such that its use does not significantly reduce profitability. Depending on the disease, how easily it spreads, and how well the infectious agent survives in the environment, culling of a barn rather than mass vaccination may be a more economically feasible option to clear an infection. However, from a welfare point of view, such a practice is highly problematic and often results in public outcry. Moreover, as antimicrobialresistant pathogens continue to emerge, there is increasing pressure to raise livestock with reduced levels of antibiotics. Vaccines offer the potential of controlling such infections and represent effective alternatives to antibiotics.

Together, adaptation of mucosal vaccines for mass delivery to livestock, DIVA vaccination and its impact on trade considerations, and the cost of vaccine development and implementation all influence whether vaccines will be used in a veterinary setting.

Whether an effective vaccine can be generated against an infectious agent requires extensive knowledge of disease pathogenesis and epidemiology across all target species. Livestock (cattle and other ruminants, pigs, avian species such as chickens and turkeys, etc.), companion animals (horses, dogs, cats, rabbits, guinea pigs, etc.), wildlife (deer, bison, koala, etc.), pest species (skunks, raccoon, mice, rats, etc.), zoo animals, and aquatic species have unique economic, social, and immunological characteristics that affect whether a mucosal vaccine will be sought for development and/ or whether existing mucosal vaccines will be used.

The growing human population has lead to an increased need for protein from food animals and for animal by-products, which has led to a steady increase in the number of intensive livestock operations worldwide. Such operations can include hundreds or thousands of animals contained in a pasture or barn, and their close proximity to one another can facilitate spread of disease to vulnerable members within the population. Because mucosal vaccination is superior to systemic vaccination in preventing colonization and shedding of pathogens, mucosal vaccination is especially important in controlling infections in livestock. Tremendous strides have been made in veterinary mucosal vaccine development and commercial availability, many of which are listed in Table 48 .1.

The following sections provide examples of mucosal veterinary vaccines under development against selected pathogens using animals from the target species (rather than mice or other experimental animals where possible). diarrhea in newborn piglets. A DNA vaccine expressing S proteins from both viruses delivered by attenuated Salmonella Typhimurium was constructed as a potential vaccine. and its immunogenicity was assessed [10] . Twenty-one-dayold piglets were orally immunized with the attenuated S. Typhimurium with empty DNA vaccine or DNA vaccine expressing the S proteins at a dosage of 1.6 3 10 11 CFU per piglet and then booster immunized with 2.0 3 10 11 CFU after 2 weeks. Virus-neutralizing S-proteinspecific immunoglobulin G (IgG) and secretory immunoglobulin A (SIgA) as well as systemic cellular immune responses (interferon gamma, interleukin 4, and lymphocyte proliferation) was significantly higher in the vaccinated group than in the control and empty DNA vaccine cohorts. These data show that S. Typhimurium can be used to carry DNA vaccines and, when delivered orally, may promote a protective immune response.

A. BRUCELLA OVIS Ovine epididymitis caused by Brucella ovis infection has been reported in the Americas, European countries, Australia, New Zealand, and South Africa. This disease can lead to genital lesions and reduced fertility in rams, placentitis and abortions in ewes, and increased perinatal mortality in lambs [11] . While safer than subcutaneous vaccination, conjunctival vaccination with live Brucella melitensis Rev 1 vaccine can causes abortions, is highly virulent, and is not a DIVA vaccine; therefore it is not recommended in countries that are free from B. melitensis [12] . Alternatively, conjunctival immunization in rams using a thermoresponsive and mucoadhesive in situ gel composed of poloxamer 407 (P407) and chitosan (Ch) could effectively deliver recombinant BLS-OMP31. (BLS is part of the enzyme lumazine synthase from Brucella spp. that is both highly immunogenic and a carrier of foreign peptides and B. ovis antigen OMP31 [13] .) Serum and preputial, saliva, lacrimal, and nasal secretions showed significant antigen-specific IgG antibody, and the levels remained elevated in serum only for several months. Relative to unvaccinated rams, the rams from the vaccinated cohort showed significant induction of antigen-specific SIgA after the first and second immunization in lacrimal, preputial, or nasal secretions (but not in nasal secretions or in serum), but antibodies levels declined rapidly [14] . Further, conjunctival immunization induced a significant BLS-OMP31-specific hypersensitivity response to intradermal injections relative to the control rams, which indicates induction of cell-mediated immunity. Conjunctival administration of BLS-OMP31-P407-Ch may be a promising alternative to current B. ovis immunization strategies.

Bovine herpesvirus 1 (BoHV-1) is responsible for infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, conjunctivitis, abortion, encephalomyelitis, and mastitis in cattle. Parenteral BoHV-1 glycoprotein E deleted mutant viral DIVA vaccines used in conjunction with diagnostic testing and targeted culling of animals infected with field strains has led to eradication of this disease in some European countries [15, 16] . Additional DIVA mucosal vaccines are under development. For example, a small trial showed that calves vaccinated intranasally with BoHV-1 glycoprotein E deleted mutant virus or BoHV-1 triple mutant virus (BoHV-1 tmv), which incorporates mutation for three genes, including glycoprotein E within a single virus, were protected against infectious challenge [17] . While both DIVA vaccines were protective against clinical disease, only BoHV-1 tmv-vaccinated calves generated significantly higher virus-neutralizing titers after challenge relative to the sham controls, and they showed a more rapid cellular immune response onset and a more rapid viral clearance. Although this virus has worldwide distribution, use of marker vaccines will continue to contribute to eradication efforts.

Water buffalo, cattle, and bison are affected by hemorrhagic septicemia (HS), which is an acute, highly fatal form of pasteurellosis. This economically important bacterial disease affects Asia, Africa, and the Middle East, and sporadic outbreaks occur in Southern Europe. An HS vaccine containing avirulent Pasteurella multocida strain B:3,4 (fallow deer strain) has been used in Myanmar to control HS in cattle and water buffaloes [18] . Earlier intranasal vaccines failed to protect against subcutaneous challenge, and the efficacy of this vaccine for primary vaccination of young buffaloes was brought into question [19, 20] . However, a later study showed that an intranasal vaccine containing live gdhA-derivative P. multocida B:2 that was boosted 2 weeks later was protective against a subcutaneously administered challenge with live wild-type P. multocida [21] . Importantly, the vaccine was also effective in protecting in-contact buffalo against a virulent parental strain and has been recommended by the Food and Agriculture Organization of the United Nations as an effective vaccine in Asia.

Calves do not receive maternal antibodies in utero; instead, they receive antibodies through colostrum in the neonatal period. However, while maternal antibodies are critically required to protect the vulnerable neonate against infectious diseases, circulating maternal antibodies interfere with the neonate's ability to develop its own immunity (referred to as maternal interference). Because colostrum is composed mainly of IgG1 (which is not transported across the mucosal epithelium of the upper respiratory tract) and although IgA does cross the mucosa, dimeric IgA makes up only 10% of antibodies in colostrum, it was suggested that the upper respiratory tract may not be affected by maternal interference. To test this hypothesis, cows were vaccinated with modified live bovine viral diarrhea virus (BVDV) vaccine composed of BHV-1, BVDV-1, BVDV-2, PI-3, and BRSV antigens. Calves were shown to have high circulating maternally derived IgG antibodies serum but extremely low titers of maternally derived IgG in nasal secretions [22] . Maternally derived IgA in nasal secretions were present but at much lower levels than in serum. Calves (3À8 days old) either were not vaccinated against BVDV or received one or two (day 0 and day 35) immunizations by the intranasal route. Within 5À7 days after birth, maternally derived IgA in nasal secretions were not detected. Calfderived (i.e., endogenous) BVDV1-and BVDV-2-specific IgA production was detected within 10 days after vaccination. A secondary intranasal vaccination after 5 weeks induced a strong memory antibody response with sustained IgA levels in nasal secretions. Collectively, these studies demonstrated that the mucosal immune system in newborn calves is functional and responsive to vaccination without being affected by maternal interference.

Chicken, turkey, duck, and other avian barns and houses are populated by very large numbers of birds for egg production or for production of meat. Standard laying houses are reported to hold from 100,000 to 500,000 hens, and broiler houses routinely house 20,000 birds. With these numbers, it is not surprising that the industry has actively sought vaccines that could be administered by mucosal routes rather than by parenteral routes, which generally rely on injection with needles. The majority of mucosal avian vaccines are live viruses administered by eyedropper into the eye (intraocular or conjunctival), orally into the drinking water, as a coarse spray whereby birds consume the vaccine during preening, and through the in ovo route. In-feed oral vaccination and spray cabinet (intranasal) routes are also used for some commercial vaccines. In ovo injection has become widely used as a means to deliver precise, uniform doses with the capacity to inject up to 60,000 eggs per hour. In ovo immunization has the added benefit that this method avoids stress to chicks, is sanitary, and has an earlier exposure time than any other immunization method.

Many experimental mucosal avian vaccines are under development to combat avian influenza. It was reported that an intranasally delivered bioadhesive liposome using tremella or xanthan gum and containing the experimental inactivate avian H5N3 virus as a model antigen elicited high mucosal SIgA and serum IgG in chickens [23] . Even the low pathogenic strains such as H9N2 avian influenza virus (AIV) can affect the economic success of commercial poultry industry by causing mild respiratory disease and decreased egg production. Immunizations for multiple forms of avian influenza are under way as experimental vaccines. For instance, Lactobacillus plantarum NC8 strain was engineered to express select peptides from H9N2 AIV. Both oral and intranasal vaccination of 3week-old white leghorn layer chickens succeeded in inducing immunity, but the intranasal route induced stronger immunity and showed less body weight loss, lung virus titers, and pathology after challenge with the H9N2 virus [24] . These nontraditional mucosal vaccine delivery platforms showed that they may be good choices for commercial avian influenza vaccine development.

Rabbit hemorrhagic disease (RHD) is a lethal disease of adult rabbits caused by rabbit calicivirus [25] . Oral immunization of rabbits with recombinant vaccinia virus [26] or recombinant myxoma virus [27] coding for VP60, the major structural protein of RHD virus (RHDV) induced protection against challenge with virulent RHDV. However, little horizontal transfer was achieved. Other researchers showed that oral immunization with VP60 protein expressed in transgenic potatoes generated partial protection against viral challenge [28] .

Rabbits have been used to investigate whether the uterus is a suitable mucosal vaccination site. An experimental vaccine consisting of ovalbumin (OVA), recombinant truncated glycoprotein 1 from bovine herpes virus, and a fusion protein of porcine parvovirus VP2 and bacterial thioredoxin (rVP2ÀTrX) was formulated with poly I:C, host defense peptide and polyphosphazene as adjuvants. Surgery was performed to isolate each uterine horn, and this triple antigenÀtriple adjuvant vaccine was injected into the lumen of the uterine horns (referred to as intrauterine immunization) [29] . Significant induction of OVA and tGD-specific serum IgG and IgA was observed over time in intrauterine-immunized animals. Uterine, lung, and vaginal tissues obtained 1 month after the single immunization showed significant OVAspecific IgG and IgA response relative to sham treatment. Significantly increased tGD-specific and rVP2-TrX antigen-specific IgG titers (but not IgA titers) were observed in lung, vagina, and uterine tissue relative to controls. The results indicate that a subunit vaccine formulated with appropriate adjuvants can trigger both systemic and mucosal immunity when administered into the uterine lumen.

Many wild koalas in Australia are known to have Chlamydia pecorum, which causes debilitating ocular and urogenital infections in koalas with clinical signs that include conjunctivitis and infertility. A single-dose anti-C. pecorum vaccine formulated to contain three major outer-membrane proteins (MOMPs) or polymorphic membrane proteins (PMPs) (an antigenic membrane bound surface-exposed adhesion protein that is important for attachment to the cell membrane [30] ) and a 1:2:1 ratio with PCEP poly[di(sodium carboxylatoethylphenoxy)phosphazene], immune defense regulatory peptide (IDR1002), and poly I:C was tested in wild koalas. Although the vaccine was administered subcutaneously, anti-MOMP IgA increased 10-to 100-fold at ocular and upper genital tract (UGT) sites in 50% and 40% of the koalas, respectively. The PmpG vaccine also triggered a 10-to 100-fold increase post vaccine IgA antibodies at the UGT or the ocular sites in 40% and 50% of koalas, respectively, which suggests that the vaccines elicited a mucosal response in at least some of the koalas. The cohort vaccinated with MOMP vaccine showed decreased chlamydia loads with no new occurrence of infection, but the other vaccination group and the control group showed increased loads with incidences of new infections, suggesting that the MOMP vaccine may be superior [31] . Further development must be undertaken to improve vaccine uptake to more members of the population, but these results suggest that a parenteral vaccine may succeed in promoting mucosal responses when properly formulated.

Sylvatic plague caused by Yersinia pestis and carried in fleas can significantly affect the population dynamics of prairie dogs (Cynomys spp.). In turn, reduced prairie dog numbers can affect the population dynamics of ferrets, burrowing owls, and several canine and avian predators. Administration of insecticides can control the fleas and reduce transmission of Y. pestis, but there is evidence that the fleas can develop resistance [32] . A vaccine that can be used as an alternative to the use of insecticides is actively sought. The orthopoxvirus raccoonpox (RCN) was genetically modified to express two protective Y. pestis antigens (designated RCN-F1/V307) and mixed with bait for oral vaccination of prairie dogs in a lab setting. Sixty percent of prairie dogs that consumed bait containing RCN-F1/307 and were then challenged at 270 days post-vaccination survived, which was a significantly higher percentage than that in the placebo group [33] . Rates of survival were improved if two oral baits were consumed months apart.

Rabies virus, a member of the Rhabdoviridae family, causes neuroinvasive rabies disease in many wild animals, including bats, possums, raccoons, skunks, foxes, coyotes, groundhogs, wolves, and monkeys. It can also infect companion animals such as dogs, cats, rabbits, and horses. It is spread through saliva and can be transmitted through bites and scratches. Symptoms include fever, violent movements, uncontrolled excitement, fear of water, aggressive behavior, and death. Most human cases of rabies come from contact with an infected domestic dog [34] .

Raboral V-RG is an oral vaccine composed of a live vaccinia virus encoding the rabies virus glycoprotein [35] . It is encased in a packet with fish meal and set out as bait for raccoons, foxes, coyotes, and the like; the packet can have a flavor coating to attract target species [36] . An alternative oral vaccine in Canada is ONRAB, a live adenovirus vector encoding the rabies glycoprotein that is administered as an oral vaccine in Ultralite bait matrix [37] . A comparative study showed that when Raboral V-RG or ONRAB was distributed by aircraft at a density of 75 baits/km 2 and sera from raccoons and skunks were collected 5À7 weeks later, skunks showed no significant difference in the proportion of antibody-positive animals, regardless of the vaccine used [38] . In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB-baited areas than in the RABORAL V-RG-baited areas, suggesting that ONRAB may be a better choice to vaccinate more species.

The aquaculture industry is growing faster than any other farmed animal industry in the world, with an increase of more than 10% between 2011 and 2016 to 70 million tons, and an increasingly high proportion of high-quality protein used to feed the world's growing population comes from aquaculture. As with any farming industry, the risk of infectious diseases increases as the density of animals increases, and excellent fish health management practices, such as controlling stocking densities, maintaining adequate oxygen levels and water quality, and reducing pathogen loads, are critical to control disease.

Improved understanding of mucosal immunity in farmed fish and crustaceans may lead to the development of cost-effective mucosal vaccines. It is estimated that there are 25,000 fish species in the world, and they are extremely diverse to accommodate living in warm or cold climates, in fresh or salt water, and in depths at high or low pressure [39] . Teleosts (bony fish) lack bone marrow; instead, their B lymphocytes mature within the kidney [40] . Mucosaassociated lymphoid tissue in teleost fish is composed of skin-associated lymphoid tissue, gill-associated lymphoid tissue, and a diffuse gut-associated lymphoid tissue [40] . Antibodyproducing cells have been identified in the cutaneous dermis and mucus, which may indicate a "mucosal" immune system in fish [41] . It is not yet clear whether fish B and T cells home back to mucosal sites upon mucosal infection after immunization [42] . For instance, oral or anal immunization of carp with formalin-killed Vibrio anguillarum followed by a booster immunization by the same route resulted in slightly enhanced antigen-specific Ig titers detected in skin mucus and bile. Serum antibody titers were elevated after anal intubation but not in response to oral immunization [43] . How the route of immunization affects immunity in fish warrants further study.

Fish body temperature takes on the local temperature, which can have a significant impact on the metabolism and rate of growth of fish as well as on their immune system. For instance, development of antibodies in fish adapted to low temperatures (,15 C) may require at least 4À6 weeks, whereas the time period to develop antibodies may be a few weeks in fish adapted to warmer temperatures. This time frame suggests that even if fish have a functional immune system, they may not develop immunity in a timely manner to protect them from infection. The major causative agents of infectious diseases in finfish aquaculture include bacteria, viruses, parasites, and fungi. Infectious agents can infect fish at some developmental stages and not at others. Although some fish may have a functional immune system in the larva or fry stage, others may not, which means that proper biosecurity rather than vaccination may be critical to protecting them against infection. Farmed fish that are routinely vaccinated include Atlantic salmon (Salmo salar), rainbow trout (Onchorhynchus mykiss), and Atlantic cod (Gadus morhua). With effective vaccine development, there has been a decline in the use of antibiotics along with improved health and increased growth of the fish.

Currently, there are three main forms of vaccination for aquatic species: immersion, oral delivery, and injection.

For immersion immunization, gills are likely the main site of antigen entry, but uptake by the skin, lateral line, and gut have also been suggested and may, in fact, contribute to induction of mucosal immunity [44] . Immersion vaccines are effective for a number of bacterial pathogens, and they are practical, cheap, and easy to batch-administer, especially to small fish. A disadvantage to this vaccination route is that it requires large amounts of vaccine, and levels of protection and duration of immunity may vary across vaccines.

An experimental vaccine for immersion of catfish 10À30 days posthatch with modified live Edwardsiella ictaluri vaccine was shown to produce a protective immune response against Enteric septicemia [45] . Other researchers showed that introducing several small lesions in the skin and then immersing the fish in a vaccine suspension containing formalin-killed Streptococcus iniae produced a protective immune response against these bacteria, and they suggest that the response was equal in effectiveness to that produced by intraperitoneal injection [46] .

Oral delivery can be accomplished with fish of any age. It is relatively cheap and non-laborintensive, it is not stressful for the fish, and it is the only option to deliver vaccine to fish in the seawater growth stage. Disadvantages include the fact that large quantities of the antigen are required, it is impossible to ensure equal distribution among the farmed animals, and the duration of immunity is generally less than that observed with injection or immersion. Oral vaccines can be made to adhere to finished feed. The challenge is to maintain antigen stability in countries with high heat and humidity as well as in the high-acid environment of the stomach once consumed. Bioencapsulation has been used, wherein feed was incubated in a vaccine suspension prior to feeding the fry. Different encapsulation techniques, including formulation with liposomes [47] or alginate beads [48] , have been used to protect the antigens from the destructive environment in the gut.

As with any animal, oral administration may lead to induction of tolerance, which may be compounded by the young age of the animal and repeated low-dose administration. Some researchers believe that oral vaccines may be more suited to act only for booster immunization to avoid induction of oral tolerance. However, studies have shown that primary oral vaccination of salmon in the seawater growth stage with an oral salmonid rickettsial septicemia vaccine formulated with a bioadhesive cationic polysaccharide protected the salmon against a lethal pathogen challenge [49] . Other researchers showed that oral vaccination with a DNA vaccine, wherein the vector expressing a gene from infectious pancreatic necrosis virus encapsulated in alginate microspheres, protected salmonid fish against infectious challenge [50] . Oral vaccination of rainbow trout with an experimental vaccine bacterin of Yersinia ruckeri O1 failed to protect against enteric redmouth disease (yersiniosis), but the same dose administered anally was protective against infectious challenge. These data suggest that the oral vaccine needs to be protected from degradation in the stomach to be effective [51] .

Rainbow trout (O. mykiss) vaccinated intranasally as early as 24 days posthatch with a live attenuated infectious hematopoietic necrosis virus vaccine, killed enteric red mouth bacterin, or saline. Upon challenge with the respective pathogen 28 days later, vaccinated groups were significantly more protected than their age-matched mock control groups [52] . These data suggest that the intranasal route may be amenable for vaccine targeting if it becomes adapted for mass vaccination.

Many farmed fish are vaccinated by an intraperitoneal injection, a route that is very labor intensive. This method can also be stressful for the fish and must be performed on fish of sufficient size, which means that vaccination of fry is difficult. No commercial mucosal vaccines are available against viruses that infect fish, so these vaccines are administered by injection.

With the rise in aquaculture comes an increased need to protect the livestock against infectious diseases. Increased efforts to elucidate the immunology of the target species of fish and pathogenesis of the parasite, virus, or bacteria that target them will undoubtedly lead to development of new vaccines suitable for mass delivery.

Population management has different considerations, depending on whether the species of interest are wildlife, pests, companion animals, or zoo animals. Generally, for all but pest species, an ideal immunocontraceptive would be reversible, safe, long-lasting, and cost-effective. While in some species, reduction in sexual or aggressive behavior may be a beneficial side effect of contraception, some species may require such behavior to maintain the herd hierarchy. Therefore species-specific needs should be a consideration before vaccination [53] .

For decades, two reliable parenteral immunocontraceptives against gonadotropin-releasing hormone (GnRH; also known as luteinizing hormone releasing hormone) or zona pellucida proteins (ZP) have been used to reversibly control fertility in many animal species. Antibodies generated against GnRH neutralize this pituitary hormone, which in turn inhibits steroidogenesis and gametogenesis in male and female mammals. Porcine zona pellucida vaccines prepared by using ZP isolated from pig ovaries or recombinant ZP antigen (SpayVac from ImmunoVaccine Technologies, Canada) are one of the most studied immunocontraceptive vaccines in wildlife. Anti-ZP antibodies bound to sperm impede binding and penetration of the ovum, and anti-ZP antibodies interfere with follicle development in some species [54, 55] .

The overwhelming majority of immunocontraceptive vaccines are delivered parenterally, but some experimental research has focused on delivery by mucosal routes. Delivery of an effective mucosal vaccine (i.e., not relying on injections or darting) for wildlife or zoo animals would be ideal for administration, as it would be less stressful to the animal and present less risk to the person administering the vaccine. Experimental work in rabbits showed that rabbit ZP glycoprotein B delivered by infection with myxoma virus resulted in infertility in 25% of female rabbits [56] . Brushtail possums (Trichosurus vulpecula) were vaccinated with bacterial ghosts (BGs) expressing ZP protein introduced through oral, intranasal/conjunctival, parenteral, and intraduodenal routes. Anti-ZP antibodies were detected in the serum and the ovarian follicular fluid after intranasal/conjunctival immunization [57] . Intraduodenal, but not oral administration of the vaccine, elicited significant systemic immune responses, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. Superovulation and artificial insemination was used to assess the effect of the immunization with BG-delivered ZP. Immunization by the nasal/conjunctival route resulted in induced antibody-mediated and cell-mediated immune responses, and significantly fewer eggs were fertilized in immunized possum females [58] . Field trials will need to be performed to determine whether mucosal immunization with BG containing possum ZP antigens is suitable for fertility control of wild possum populations.

To be adopted for use, mucosal immunocontraceptive vaccines must be effective in the target animals and have limited or no effect on nontarget animals which may include humans who consume the meat, eggs, or milk from the targeted species. For zoo animals, such as captive African and Asian elephants, altering sex hormone levels in male or females may be advantageous in that they reduce aggression during musth season, but the alterations can also interfere with dominance hierarchy in a herd, which may not be advantageous [59, 60] . Further, use of oral bait delivery systems should, if possible, be designed in such a way as to reduce consumption by unintended target species.

The economic practicality of vaccine development such as costs associated with manufacturing and licensing as well as costs associated with treatment, including labor, equipment, and population dynamics, will all determine whether controlling fertility with a vaccine is an effective means to control a population. For instance, research shows that an annual control campaign using baits to sterilize female foxes would reduce the red fox population density by about 30%, but an annual campaign of poisoning would reduce fox density by about 80%. Vaccination would, of course, be the better choice when animal welfare issues are taken into consideration, although it is a less effective means to control population growth [61] . Whether a population can be controlled by immunocontraception or culling depends on the species and the availability of an effective mucosal vaccine and mucosal delivery system.

In addition to contraception, immunization against select targets may be used to improve fertility. Active immunization of cows against inhibin, a protein whose major action is negative feedback regulation of pituitary follicle-stimulating hormone (FSH) secretion, via the subcutaneous route neutralized endogenous inhibin levels, which resulted in increased FSH secretions during the estrous cycle. The immunized cows had a greater number of follicular waves and a greater number of follicles during the estrous cycle, which could be used as a potential source of oocytes for use in in vitro fertilization and embryo transfer programs [62] . Advances have been made with mucosally delivered inhibin vaccines. In buffalo, nasal immunization with a DNA vaccine coding for inhibin and delivered by attenuated Salmonella Cholerasuis has been shown to improve follicle development and fertility [63] . However, the buffalo in this trial underwent estrous synchronization, which would not be feasible in the wild and will have to be investigated further to establish its feasibility as a fertility vaccine. While immunization against select targets may affect fertility, care should be taken that targeting a natural protein may affect other pathways that are important for the animal's health. Further, should ovulation rates be affected, it must be established that an increased number of dams do not suffer from complications associated with multiple offspring per parturition before it is known that the vaccine is safe to use.

Veterinary vaccines can also be used to reduce food-borne illness by targeting bacteria that cause no illness or only mild illness in animals, but that can be harmful to humans upon consumption. For example, chicks orally immunized on the first day of life then boosted orally or via the intramuscular route at 6 and 16 weeks of age with a novel attenuated Salmonella Enteridis vaccine candidate, showed significantly higher plasma IgG and intestinal SIgA levels as compared to those in the control group [64] . The lymphocyte proliferation response and CD45 1 CD3 1 T cell number in the peripheral blood of the vaccinated groups were significantly increased. When the birds were challenged intravenously with the virulent S. enteritidis strain in the 24th week, the egg contamination rates were significantly reduced in both vaccinated groups relative to the controls, but total protection was not achieved. These results indicate that this vaccine may reduce incidences of egg contamination and therefore reduce the risk of human salmonellosis.

While rare, Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) can have serious consequences in the young and in the aged human population, including hemorrhagic colitis, renal failure, and death. Cattle are widely recognized as an important reservoir of STEC O157 for human exposure, making contaminated beef a potential source of food-borne infection. Parenteral vaccination with a combination of antigens associated with type III secretion system-mediated adherence results in significantly reduced shedding in orally infected animals [65] . As yet, no mucosal vaccines have been developed that significantly reduce colonization in cattle, potentially because of poor cross-protection across STEC strains.

Veterinary mucosal vaccines that protect humans from food-borne infectious diseases have tremendous One Health implications. We anticipate that the number of these vaccines will continue to grow in the future.

As the examples in this chapter demonstrate, mucosal vaccines are part of routine immunization practices in veterinary medicine and have been for many years. Research is underway to further improve those vaccines, be it through the use of novel adjuvants, better delivery systems, or effective targeting to the site of uptake at mucosal surfaces. However, it is important to note that most of these vaccines are extremely cost-effective, at pennies per dose, and are used as part of mass vaccination in poultry and fish. Thus one would hope that human vaccine manufacturers and regulators recognize the benefits and potential this technology can offer and start to develop mucosal vaccines for humans at a cost-effective price. While some vaccines are already available for mucosal administration in humans, mucosal vaccination has, unfortunately, not yet become part of routine immunization practices in humans.

Immune responses to dietary antigens: oral tolerance

Oral tolerance

Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1

Evaluation of the response to an accelerated immunisation schedule using a canarypoxvectored equine influenza vaccine, shortened interdose intervals and vaccination of young foals

Priming for local and systemic antibody memory responses to bovine respiratory syncytial virus: effect of amount of virus, virus replication, route of administration and maternal antibodies

Pseudorabies virus avirulent strains fail to express a major glycoprotein

Construction and characterization of deletion mutants of pseudorabies virus: a new generation of 'live' vaccines

Differentiation of serum antibodies from pigs vaccinated or infected with Aujeszky's disease virus by a competitive enzyme immunoassay

A. Manual of diagnostic tests and vaccines for terrestrial animals

Construction of a bivalent DNA vaccine coexpressing S genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated Salmonella Typhimurium

Ovine brucellosis: a review of the disease in sheep manifested by epididymitis and abortion

Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy

Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides

Immune response induced by conjunctival immunization with polymeric antigen BLSOmp31 using a thermoresponsive and mucoadhesive in situ gel as vaccine delivery system for prevention of ovine brucellosis

Efficacy of a live glycoprotein E-negative bovine herpesvirus 1 vaccine in cattle in the field

An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field

A triple gene mutant of BoHV-1 administered intranasally is significantly more efficacious than a BoHV-1 glycoprotein E-deleted virus against a virulent BoHV-1 challenge

Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine

Haemorrhagic septicaemia vaccines

Field use of live haemorrhagic septicaemia vaccine

Efficacy of intranasal vaccination of field buffaloes against haemorrhagic septicaemia with a live gdhA derivative Pasteurella multocida B:2

Mucosal immune response in newborn Holstein calves that had maternally derived antibodies and were vaccinated with an intranasal multivalent modified-live virus vaccine

Mucoadhesive liposomes for intranasal immunization with an avian influenza virus vaccine in chickens

Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens

Identification and characterization of the virus causing rabbit hemorrhagic disease

Protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-RHDV recombinant virus

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus

Oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein

Intrauterine delivery of subunit vaccines induces a systemic and mucosal immune response in rabbits

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis

Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: new insights into immune response, protection and clearance

Xenopsylla cheopis (Siphonaptera: Pulicidae) susceptibility to Deltamethrin in Madagascar

Sylvatic plague vaccine partially protects prairie dogs (Cynomys spp.) in field trials

MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES? REFERENCES

Transmission dynamics and prospects for the elimination of canine rabies

The development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies; a link between Jenner and Pasteur

A new flavor-coated sachet bait for delivering oral rabies vaccine to raccoons and coyotes

High-density baiting with ONRAB rabies vaccine baits to control Arctic-variant rabies in striped skunks in Ontario

Comparing ONRAB AND RABORAL V-RG oral rabies vaccine field performance in raccoons and striped skunks

Vaccines for fish in aquaculture

Mucosal immunoglobulins and B cells of teleost fish

Evidence for a cutaneous secretory immune system in rainbow trout (Salmo gairdneri)

Phenotypic and functional similarity of gut intraepithelial and systemic T cells in a teleost fish

Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system

Antigen uptake and immune responses after immersion vaccination

Development and use of modified live Edwardsiella ictaluri vaccine against Enteric septicemia of catfish

Development of a new vaccine delivery method for fish: percutaneous administration by immersion with application of a multiple puncture instrument

Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes

Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads

Oral vaccination of Atlantic salmon (Salmo salar) against salmonid rickettsial septicaemia

Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish

Oral and anal vaccination confers full protection against enteric redmouth disease (ERM) in rainbow trout

Nasal vaccination of young rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis and enteric red mouth disease

The impact of male contraception on dominance hierarchy and herd association patterns of African elephants (Loxodonta africana) in a fenced game reserve

Fertility control in the bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles

Effect of alloimmunization and heteroimmunization with zonae pellucidae on fertility in rabbits

Infertility in female rabbits (Oryctolagus cuniculus) alloimmunized with the rabbit zona pellucida protein ZPB either as a purified recombinant protein or expressed by recombinant myxoma virus

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula)

MUCOSAL VACCINES BE APPLIED TO OTHER INFECTIOUS AND NONINFECTIOUS DISEASES? against GnRH may suppress aggressive behaviour and musth in African elephant (Loxodonta africana) bulls--a pilot study

Supression of testicular function in a male Asian elephant (Elephas maximus) treated with gonadotropin-releasing hormone vaccines

Fertility control is much less effective than lethal baiting for controlling foxes

The effect of active immunization against inhibin on gonadotropin secretions and follicular dynamics during the estrous cycle in cows

Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in crossbred buffaloes

Efficacy for a new live attenuated Salmonella Enteritidis vaccine candidate to reduce internal egg contamination

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge