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Previous Article | Next Article 
Clinical Microbiology Reviews, July 2007, p. 489-510, Vol. 20, No. 3
0893-8512/07/$08.00+0 doi:10.1128/CMR.00005-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Animal Biotechnology Research Laboratories, Department of Physiology, Monash University, Victoria 3800, Australia,1 VMRD Pfizer Australia, 52 Poplar Rd., Parkville, Victoria 3052, Australia,2 University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush, Roslin, Midlothian EH25 9RG, United Kingdom,3 Publications Department, World Organisation for Animal Health, 12 rue de Prony, 75017 Paris, France,4 National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1790 Copenhagen, Denmark5
SUMMARY INTRODUCTION VETERINARY VIRAL VACCINES Conventional Live and Inactivated Viral Vaccines DIVA Vaccines Molecularly Defined Subunit Vaccines Genetically Engineered Viral Vaccines Live Viral Vector Vaccines DNA Vaccines VETERINARY BACTERIAL VACCINES Conventional Live Vaccines Conventional Inactivated Vaccines Gene-Deleted Vaccines Subunit Vaccines Vaccines against Zoonotic Bacteria Rickettsia Vaccines VETERINARY PARASITE VACCINES Protozoal Vaccines Live protozoal parasite vaccines. (i) Vaccines based on complete life cycle infections. (ii) Vaccines based on drug-abbreviated infections. (iii) Vaccines based on infections with parasites with a truncated life cycle. (iv) Vaccines based on infection with virulence-attenuated strains. Killed or subunit protozoal parasite vaccines. Helminth and Ectoparasite Vaccines VETERINARY VACCINES FOR NONINFECTIOUS DISEASES Allergy Vaccines Cancer Vaccines VETERINARY VACCINES FOR FERTILITY AND PRODUCTION CONTROL Design of Reproduction Control Vaccines Vaccines against Reproductive Hormones Vaccines against Gamete Antigens: Wildlife Control Sperm antigens. Oocyte antigens. Vaccines To Increase Fertility CONCLUSION AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
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While veterinary vaccines comprise only approximately 23% of the global market for animal health products, the sector has grown consistently due mainly to new technological advances in vaccine development, the continuous development of drug resistance by pathogens, and the emergence of new diseases. Apart from improving animal health and productivity, veterinary vaccines have a significant impact on public health through reductions in the use of veterinary pharmaceuticals and hormones and their residues in the human food chain. This will be an increasing impetus for activity with the more stringent requirements of regulatory agencies and consumer groups, particularly in the major markets of Europe and the United States (166). For example, the use of antibiotics in animal production has already been severely restricted, and the European Union has recently banned the use of coccidiostats for poultry. In addition, vaccines contribute to the well-being of livestock and companion animals, and their use is favored by the growing animal welfare lobby.
The process of developing veterinary vaccines has both advantages and disadvantages over human vaccine development. On the one hand, the potential returns for animal vaccine producers are much less than those for human vaccines, with lower sales prices and smaller market sizes, resulting in a much lower investment in research and development in the animal vaccine area than in the human vaccine area, although the complexity and range of hosts and pathogens are greater. For example, the market size for the recently launched human vaccine (Gardasil) against papillomavirus and cervical cancer is estimated to be greater than 1 billion U.S. dollars, while the most successful animal health vaccines (e.g., against foot-and-mouth disease [FMD] virus in cattle and Mycoplasma hyopneumoniae in pigs) enjoy a combined market size that is 10 to 20% of this figure. On the other hand, veterinary vaccine development generally has less stringent regulatory and preclinical trial requirements, which can make up the largest cost in human vaccine development, and a shorter time to market launch and return on investment in research and development. In contrast to human vaccine development, veterinary scientists are also able to immediately perform research in the relevant target species. This is an obvious advantage over human vaccine development, as experimental infections, dose-response studies, and challenge inoculations need not be carried out in less relevant rodent models.
Immunity acquired through natural infection can take on several forms depending on the type and life cycle of the pathogen, as schematically represented in Fig. 1. Vaccines may be used to prevent clinical signs of disease after infection or to help control, eliminate, or even eradicate an infection at the population level (e.g., the expected global eradication of rinderpest virus through vaccination). Both vaccine effectiveness and mechanism of action may vary depending on the required outcome. New technologies to achieve the selective induction of effective immune responses in the development of new vaccines are becoming available to vaccine researchers and have been extensively reviewed in recent papers (33, 136, 163, 166). Notwithstanding the scientific advances, the single factor that determines the success of an experimental vaccine is its successful commercialization and/or use in the field. This outcome requires a combination of basic research, commercial imperatives, local requirements, and global perspectives depending on the particular disease under investigation. These four aspects are represented by the authors of this review, which will concentrate on recent advances in veterinary vaccines that have reached the marketplace or are actively produced by veterinary institutes for use by local farming communities.
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Despite such drawbacks of live viral vaccines, they have played a major role in successful disease control and eradication. For example, the virtual eradication of rinderpest virus from the globe is widely believed to have been critically dependent on the use of the "Plowright" vaccine (12, 150). This is an attenuated vaccine produced from the Kabete O strain passaged 90 times in tissue culture (141). The vaccine virus was recently found to have attenuating mutations in most of its genes, none of which are sufficiently debilitating to induce strong pressure for reversion (11). Although there are examples of stable attenuations from a single point mutation in the polymerase gene, the high rate of spontaneous mutations of RNA viruses increases the risk for reversion to virulence. Safe live viral vaccines are therefore likely to require a number of attenuating mutations distributed throughout the genome.
Whole inactivated or killed viral vaccines are generally more stable and do not pose the risk of reversion to virulence compared to live vaccines, but their inability to infect cells and activate cytotoxic T cells makes them much less protective. Consequently, they generally require strong adjuvants and several injections to induce the required level of immunity and are usually effective in controlling only clinical signs rather than infection (113). Inactivated adjuvanted vaccines also pose a greater risk of causing autoimmune diseases, allergic disorders, and vaccine injection site sarcomas (46). Viral inactivation is commonly achieved through heat or chemicals (e.g., formaldehyde, thiomersal, ethylene oxide, and ß-propriolactone). The higher production cost and need for adjuvants make these vaccines more expensive to manufacture. Inactivated viral vaccines for a wide range of viral diseases have been available for several decades (reviewed in references 113 and 121) and are still being developed for some recently emergent diseases. For example, a one-dose inactivated porcine circovirus type 2 (PCV2) vaccine has recently been licensed in the United States for the prevention of postweaning multisystemic wasting syndrome in pigs (Table 1). Much of the recent research in this area has concentrated on the development of improved adjuvanted formulations to overcome the effects of maternal antibodies on young animals (see, for example, reference 17).
Inactivated vaccines for several viral diseases need to be continuously adapted to contain the appropriate serotypes, as exemplified by equine influenza virus vaccines. Vaccines for equine influenza virus, mostly inactivated, have been available since the 1960s (28). The most important equine subtypes are H7N7 and H3N8, although H7N7 has not been detected for several decades and is no longer included in vaccines, at least in Europe and the United States (130). Conversely, vaccination against H3N8 has been less effective, possibly due to antigenic drift, and there are now considered to be two distinct lineages, European and United States, and vaccines therefore tend to contain both. Over the years, improvements have been attempted, and more potent adjuvants have been used. Several European vaccines now produce high antibody responses that last for up to 1 year (113). Until recently, equine influenza virus vaccines produced in the United States have been considered to be of limited efficacy and sometimes lacking the relevant H3N8 strains (M. Mellencamp and A. Schultze, presented at the Proceedings Quality Control of Equine Influenza Vaccines, Budapest, Hungary, 2001).
The ability to identify and selectively delete genes from a pathogen has allowed the development of "marker vaccines" that, combined with suitable diagnostic assays, allow differentiating infected from vaccinated animals (DIVA) by differentiation of antibody responses induced by the vaccine (no antibodies generated to deleted genes) from those induced during infection with the wild-type virus (e.g., see Fig. 2). Such DIVA vaccines and their companion diagnostic tests are now available or in development for several diseases including infectious bovine rhinotracheitis (IBR), pseudorabies, classical swine fever (CSF), and FMD, as detailed below.
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Deletion of the gE gene has also been used to enable a DIVA approach for an Aujeszky's disease vaccine (137). The gene for thymidine kinase is also deleted in some formulations (e.g., Suvaxyn Aujesky), adding to the degree of attenuation (e.g., see reference 56). These deletion vaccines have been available since the 1980s, and their use has contributed to disease control and eradication in the United States and several European countries (e.g., see reference 24).
CSF is on the World Organization for Animal Health list of notifiable diseases and is one of the most important contagious diseases of pigs worldwide. In its classical clinical form, it is an acute hemorrhagic disease accompanied by high fever, depression, anorexia, and conjunctivitis. Morbidity and mortality are both very high and may reach 100%. However, it can also present as a subacute, chronic, or even subclinical condition. Countries in which the disease is enzootic tend to vaccinate with a very effective live, attenuated vaccine, while those that are free of disease do not (reviewed in reference 13). Two subunit vaccines based on the viral envelope glycoprotein E2 produced in a baculovirus/insect cell system, formulated in a water-in-oil adjuvant, and accompanied by discriminatory ELISA tests are available (116, 181). These vaccines will allow a DIVA approach to emergency vaccination and disease control in the case of new outbreaks, although these have not yet been used widely in the field and appear to be less protective than conventional live, attenuated CSF vaccines (13).
As there are now major doubts about the sustainability of "stamping-out" policies in areas of high animal population density, there is considerable investment in DIVA vaccine approaches for FMD (reviewed in references 8 and 66). Subunit antigen approaches to vaccination have been largely ineffective, as they present only a limited number of epitopes to the animal's immune system, and multiple antigens are generally required for protection. Current research is focused largely on combinations of capsid proteins, including empty capsid delivered by various expression systems, and the development of sensitive tests (ELISA) for antibodies against nonstructural proteins (66). Other diseases for which a DIVA approach is highly desirable but currently unavailable include bluetongue virus in cattle, Newcastle disease virus and avian influenza virus in poultry, bovine viral diarrhea, and equine viral arteritis.
PCV2 is considered to be the major pathogen in the etiology of postweaning multisystemic wasting syndrome (2). A recombinant baculovirus producing the protective ORF2 protein of PCV2 has recently become available as a vaccine for pigs (20).
Dow AgroSciences successfully registered the first plant-based vaccine for Newcastle disease virus in poultry in the United States in 2005. Recombinant viral HN protein was generated in plant cell lines via Agrobacterium transformation and could successfully protect chickens from viral challenge (G. A. Cardineau, H. S. Mason, J. Van Eck, D. D. Kirk, and A. M. Walmsley, 2004, PCT patent application 60/467,998, WO 2004/098533; C. A. Mihaliak, S. Webb, T. Miller, M. Fanton, D. Kirk, G. Cardineau, H. Mason, A. Walmsley, C. Arntzen, and J. Van Eck, presented at the 108th Annual Meeting of the United States Animal Health Association, Greensboro, NC, 2005). This process was a proof-of-concept exercise designed to test regulatory feasibility, and the product is not on the market.
Gene-deleted vaccines (glycoprotein I and/or glycoprotein X) against pseudorabies allowed a DIVA approach and control of Aujeszky's disease in swine (96); however, the potential for recombination between pseudorabies virus strains has raised concern (101). Similarly, thymidine kinase deletion in BHV-1 vaccines has been associated with latency and reactivation after treatment with dexamethasone (194), and deletion of multiple genes has been proposed in order to improve safety (14).
An interesting development in genetically engineered viral vaccines is the production of chimera viruses that combine aspects of two infective viral genomes. A chimera PCV1-2 vaccine has the immunogenic capsid gene of PCV2 cloned into the backbone of the nonpathogenic PCV1 and induces protective immunity to wild-type PCV2 challenge in pigs (55). A further sophistication of this approach is a recently developed vaccine against avian influenza virus (Poulvac FluFend), where the hemagglutinin (HA) gene has been removed from an H5N1 virus, inactivated by removing the polybasic amino acid sequences, and combined with the NA gene from an H2N3 virus onto an H1N1 "backbone" virus (Fig. 2). A vaccine containing the resultant inactivated H5N3-expressing virus administered in a water-in-oil emulsion protects chickens and ducks against the highly pathogenic H5N1 strain.
Similarly, a live Flavivirus chimera vaccine against West Nile virus (WNV) in horses (PreveNile) was registered in the United States in 2006. In this chimera vaccine, the structural genes of the attenuated yellow fever YF-17D backbone virus have been replaced with structural genes of the related WNV. The resulting chimera vaccine express the PreM and E proteins of WNV, while the nucleocapsid (C) protein, nonstructural proteins, and nontranslated termini responsible for virus replication remain those of the original yellow fever 17D virus (114). After a single shot, the vaccine stimulates both cell-mediated and humoral responses without causing any clinical illness or spreading to sentinel horses and provides protection against WNV challenge for up to 12 months (PreveNile package insert). A similar vaccine could be a candidate for a human WNV vaccine (115).
A particular success story has been the development of an oral recombinant vaccinia-rabies vaccine in bait for wild carnivores such as foxes in Europe (26) and foxes, raccoons, and coyotes in the United States (152, 170). Rabies is caused by a negative-stranded Rhabdoviridae RNA virus transmitted mainly via saliva following a bite from an infected animal. The main source of infection for humans is domestic reservoir species including dogs and cats. There are seven rabies virus genotypes, all of which, excluding type 2, produce similar effects in humans. Rabies can infect most if not all mammals. The virus enters the central nervous system, causing an encephalomyelitis that is always fatal once symptoms develop. Worldwide, the disease causes many thousands of human deaths each year. One type of oral vaccine is in the form of a bait containing a recombinant vaccinia virus vector expressing the protective glycoprotein G of rabies virus (100, 135). After several years of vaccination campaigns against fox rabies virus in several Western European countries, rabies could be eliminated from its wildlife terrestrial reservoir, as exemplified by the successful elimination of terrestrial rabies virus from Belgium and France (26, 135, 136).
The canarypox virus vector system ALVAC has been used as a platform for a range of veterinary vaccines including WNV, canine distemper virus, feline leukemia virus, rabies virus, and equine influenza virus (176) (Table 1). Canarypox virus was originally isolated from a single pox lesion in a canary and serially passaged 200 times in chicken embryo fibroblasts and serially plaque purified under agarose (Merial bulletin TSB-4-0019-FTB). Canarypox viruses and fowlpox viruses have the advantage of being more host restricted than vaccinia virus. While they produce an abortive infection in mammalian cells, canarypox virus recombinants still effectively express inserted foreign genes. Several veterinary viral vaccines have been produced using the ALVAC vector system (Table 1). Most notably, a novel equine influenza virus vaccine using the canarypox vector to express the hemagglutinin genes of the H3N8 Newmarket and Kentucky strains has recently been registered in the European Union (Proteq-Flu) (113) and the United States (Recombitek). It contains a polymer adjuvant (Carbopol; Merial Ltd.), and through the induction of both cell-mediated and humoral immunity, it is claimed to produce sterile immunity 2 weeks after the second of two doses. The new vaccine is also designed to protect horses against the highly virulent N/5/03 American strain of equine influenza virus and to prevent the virus from spreading through the elimination of viral shedding.
Trovac AI H5 is a recombinant fowlpox virus expressing the H5 antigen of avian influenza virus. This product has had a conditional license for emergency use in the United States since 1998 and has been widely used in Central America, with over 2 billion doses administered (29). As the vaccinated birds will not develop antibodies against matrix protein/nucleoprotein, this vaccine can also be used with a DIVA approach.
Several vaccines are available based on inactivated adjuvanted formulations for equine herpesvirus type 1 and equine herpesvirus type 4, equine herpesviruses that are major causes of abortion and respiratory disease. None of these vaccines are considered to provide complete clinical or virological protection (113). A canarypox virus-vectored vaccine containing the genes for gB, gC, and gD has been developed, but the latest reports suggested that it did not completely protect against challenge (113).
A further application of vectored vaccines is the use of an attenuated viral pathogen as the vector, with the aim of inducing protection against two diseases, as with the live recombinant vaccine against both Marek's disease virus (MDV) and infectious bursal disease virus (IBDV) in chickens (Vaxxitek HVT + IBD). MDV is a highly contagious neoplastic disease of poultry caused by gallid herpesvirus type 2, while IBDV replicates in the bursa of Fabricius, the primary lymphoid organ in birds, and causes a serious immunosuppressive condition in poultry flocks worldwide. Turkey herpesvirus (HVT) is nonpathogenic in chickens but confers cross-protection against MDV and has traditionally been used in live vaccines against MDV. The new vaccine is based on a recombinant parent HVT virus expressing the VP2 gene of IBDV (44) and can be given to embryonated eggs or 1-day-old chicks without interference from maternally derived antibodies. Data from large-scale field trials for this vaccine have not yet been reported, but those studies may encounter difficulties in maintaining high efficacy. This is because these tightly regulated recombinant vaccines cannot easily adapt to meet the emergence of very virulent strains of both IBDV and MDV, apparently induced by the comprehensive numbers of vaccinations performed against these diseases (81, 146).
Chimera avian influenza virus vaccines have also recently been produced on a backbone of an existing, attenuated Newcastle disease virus vaccine strain. Both Asian H5N1 and the pathogenic H7N7 strain, responsible for the chicken influenza virus outbreak in The Netherlands in 2003, were produced as chimeras with the Newcastle disease virus strain. This chimera vaccine induced strong protection against the respective wild-type influenza virus as well as against Newcastle disease virus (133, 187).
Considerable research into DNA vaccines for fish viruses, where this approach seems to be particularly effective (73, 99), has been ongoing. Notably, the first DNA vaccine for an edible species (Apex-IHN) was registered in 2005 in Canada to protect Atlantic salmon from infectious hematopoietic necrosis (IHN) (167). IHN disease is enzootic in wild salmon populations and can cause devastating outbreaks in farm-raised salmon that have had no prior exposure. The DNA vaccine encodes a surface glycoprotein of IHN virus and is administered intramuscularly (99).
A DNA vaccine to protect horses against viremia caused by WNV (West Nile-Innovator DNA) received a license from the USDA at approximately the same time as the fish DNA vaccine. WNV infection is caused by a flavivirus belonging to the Japanese encephalitis virus complex. It is enzootic in parts of Africa and Asia but was first detected in the United States in 1999 in an outbreak involving birds, horses, and humans in New York, and it subsequently spread rapidly to many states (62). The DNA plasmid codes for the WNV outer coat proteins and is administered with a proprietary adjuvant (143). The vaccine was, however, produced as part of a building-platform technology rather than as a commercial product, as the manufacturer already has a WNV vaccine on the market.
The success of these two DNA vaccines may be due more to good fortune than to any specific technological advances, as DNA uptake into fish muscle seems to be unusually efficient (99), and the WNV viral protein may be particularly effective because it naturally produces highly immunogenic virus-like particles (161). It is likely that a wider application of DNA vaccines will require further improvements and optimization for each host-pathogen combination.
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Many of the established bacterial vaccines are highly efficacious, but since the technology has been available for many years, these conventional vaccines will not be dealt with in this review, and the reader is referred to company websites for information on specific diseases and vaccines. Both live and inactivated autogenous bacterial vaccines are also produced by local veterinary institutions or specialized companies for on-farm specific demands where no commercial vaccines are available.
This section will review some of the more recent additions to bacterial veterinary vaccines (summarized in Table 2), with particular focus on the more molecularly defined vaccines.
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A killed oral vaccine (AquaVac ERM) against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout has been available in United Kingdom since 2001 and is now further approved for a number of European countries. Enteric redmouth disease is a serious infectious disease of farmed rainbow trout in many countries characterized by congestive or hemorrhagic zones in various tissues and organs, particularly around the mouth and in the intestines. The disease has a very high mortality rate, and Y. ruckeri is able to form biofilms on fish tank surfaces and thus persist and remain infective in the aquatic environment, with the possibility of recurrent infections (39). Immersion of fry for 30 s into a vaccine soup at the hatchery provides initial protection for fingerlings but rarely lasts throughout the production cycle. Follow-up booster vaccination by injection is effective, but this is time-consuming and labor-intensive and may be stressful for the handled fish. The oral vaccine protocol recommends a primary immersion vaccination followed 4 to 6 months later by an oral booster of the vaccine, which is mixed and absorbed into the feed pellets. Both the primary and booster vaccine formulations are inactivated bacterial cultures, but for oral vaccination, the bacteria are incorporated into an "antigen protection vehicle," bypassing the acidic environment of the gut and delivering the antigens to the area of the hindgut (64). There are no data available on the nature of the antigen protection vehicle, but the product résumé claims the presence of lecithin and fish oil, indicating that killed bacteria are likely incorporated into liposome structures (61). Similar vaccines against furunculosis caused by Aeromonas salmonicida and vibriosis caused by Vibrio anguillarum have also been developed, and an oral vaccine against infectious pancreatic necrosis virus is registered in Chile for use in salmon. To our knowledge, these are the only licensed inactivated mucosal vaccines against bacterial diseases in veterinary medicine.
Gene-deleted vaccines have been produced against strangles, a highly contagious disease in horses caused by infection with Streptococcus equi subsp. equi. The disease is characterized by fever, profuse nasal discharge, and abscess formation in the lymph nodes of the head and the neck. The pus discharged from bursting abscesses is highly infectious, and the swelling of involved lymph nodes may, in severe cases, cause airway restriction, hence the name. Commercial bacterin or protein extract vaccines for parenteral administration can induce high levels of serum bactericidal antibodies, but the protective effects of these antibodies are questionable (177), and the protective efficacy of inactivated vaccines in the field has been disappointing (174). A live intranasal vaccine based on a nonencapsulated attenuated strain (Pinnacle IN) has been widely used in North America since it was launched in 1998. However, the attenuating mutations of this strain have not been defined, and the vaccine strain sometimes reverts to an aggressive mucoid phenotype indistinguishable from that of wild-type strains. The Pinnacle strain has since been refined into a more stable hyaluronate synthase-defective mutant (191). It is, however, not clear if this new strain has replaced the original vaccine strain in the commercial product. Recently, the Equilis StrepE vaccine, a live recombinant bacterial vaccine prepared from the S. equi TW928 deletion mutant lacking bp 46 to 978 of the aroA gene (80, 84), was licensed in Europe. This mutant was constructed by the electroporation of gene knockout and gene deletion constructs. No foreign DNA such as antibiotic resistance markers was introduced, but the vaccine strain can allegedly be identified by an aroA PCR identifying the partial gene deletion (84). The live gene-deleted attenuated vaccine strain was originally developed for intranasal application, but protection was accomplished only by intramuscular injections, which in turn resulted in the local swelling of muscle tissue and the eventual formation of abscesses at the vaccination site (80). However, submucosal administration of the vaccine in the upper lip was shown to confer protection comparable to that of intramuscular administration but with only minimal local reactions (80), and it is with this unusual route of administration that the vaccine is now licensed.
Chlamydiae are obligate intracellular bacteria with a wide host range and with a wide spectrum of diseases, several of which are zoonotic. The most important veterinary species are Chlamydophila psittaci, causing respiratory infections in poultry (psittacosis/ornithosis), and Chlamydophila abortus (formerly Chlamydia psittaci serotype 1), causing ovine enzootic abortion, one of the most important causes of ovine and caprine abortion worldwide. Both infections are zoonotic. While no vaccines are available for birds and poultry, inactivated vaccines against ovine enzootic abortion have been available for many years (reviewed in reference 145). More recently, a temperature-sensitive mutant strain, TS1B, of the C. abortus reference strain AB7 obtained by nitroguanidine mutagenesis (148) is used to prevent abortion in sheep (Ovilis Enzovax). The temperature-sensitive mutant strain has an optimal growth temperature at 38°C, but at the restrictive temperature, 39.5°C, growth is impaired. The normal body temperature of adult sheep is 38.5 to 40.0°C. The vaccine induces good and long-lasting protection in sheep (34), goats (149), and mice (even though the body temperature of mice is within the permissive growth range). However, the vaccine is licensed only for sheep, not goats, and there is continued research into the development of an effective subunit-based vaccine (59, 190). Temperature-sensitive-mutant vaccines have also been developed and marketed as eye drop, spray, or inhalation vaccines. These include vaccines against Mycoplasma synoviae and M. gallisepticum in chickens (Vaxsafe MS and Vaxsafe MG, respectively) (10, 119) and Bordetella avium rhinotracheitis (coryza) in turkeys (Art Vax) (79).
Gene-deleted live bacteria with well-defined targeted attenuations also offer an attractive option as mucosal vectors for passenger antigens, with many potential advantages over traditionally injectable vaccines. Unlike viral vaccines, there are at present no commercialized vector vaccines based on a bacterial backbone carrier delivering antigens from other pathogens, although several bacterial vectors have shown very promising results (reviewed in references 61 and 108).
It is generally recognized that cell-mediated immunity is more important than humoral responses in protection against Salmonella, and together with the need for local mucosal immunity, this calls for live, attenuated vaccines as the most effective type. This is supported by a comparison of a live double-gene-deleted Salmonella enterica serovar Typhimurium vaccine (MeganVac 1) with an inactivated Salmonella enterica serovar Enteritidis vaccine (Poulvac SE), which showed reduced fecal shedding following live vaccination, while chickens receiving a killed vaccine experienced inhibited cell-mediated immune responses, enhanced antibody responses, and an increased bacterial load (5). The MeganVac 1 organism has recently been reformulated for immunization of laying hens (MeganEgg). The Megan vaccines for broilers and hens were licensed by the USDA in 1998 and 2003, respectively, but worldwide, there are at least 10 other live Salmonella vaccines available for Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, or Salmonella enterica serovar Gallinarum infection in poultry.
Campylobacter jejuni is one of the most important causes of food-borne human bacterial gastroenteritis. Although several vaccines aimed at preventing human disease are in the pipeline, an effective vaccination intervention strategy for infected poultry flocks would be the most effective means of preventing human disease. Similar to the requirements of a vaccine against non-host-specific Salmonella serotypes, such a vaccine must, however, be able to provide a very high degree of protection in the flock to eliminate the subsequent contamination of meat products. Experimental vaccines, mainly killed whole-cell cultures or flagellum preparations, have been tested in poultry but provide only partial protection against a challenge with Campylobacter, and the development of an attenuated live strain may be more promising although not yet commercially available.
Brucellosis continues to be a major zoonotic threat to humans and a common cause of animal disease, especially in developing countries. In many industrialized countries, a test-and-slaughter policy has been effective for the eradication of the disease, while vaccines, although providing a fairly high level of protection, also induce antibodies that interfere in subsequent surveillance programs. Numerous attempts to produce a protective killed vaccine have so far been disappointing, and the most successful vaccines against brucellosis have been those employing live, attenuated Brucella spp. (158). Of these, Brucella abortus strain 19 (first described in 1930) and Brucella melitensis Rev.1 (first described in 1957) vaccines have been widely used in cattle and in small ruminants, respectively. The S19 and Rev.1 vaccines are, however, far from perfect, as absolute protection is not achieved, allowing for subclinical carrier animals, and both strains have retained some virulence and may induce abortions with variable frequency. Furthermore, both of these vaccines are infectious for humans (Rev.1 is also resistant to streptomycin), and they will induce antibodies against smooth lipopolysaccharide, making them incompatible with test-and-slaughter procedures in countries with an ongoing eradication program. More recently, a vaccine based on a stable spontaneous rifampin-resistant rough mutant of B. abortus, named RB-51 (157), has replaced S19 in many countries including the United States. RB-51 carries IS711 inserted into the wboA glycosyl transferase gene (188), but experimental data with other wboA mutants indicate that additional unknown defects are carried in this strain (118). Rough strains do not carry smooth lipopolysaccharide, and therefore, vaccination with RB-51 does not induce antibodies that are detectable in routine serological tests. This is an obvious advantage in many cases but may also result in the late diagnosis of accidental human infections, although RB-51 appears to be much less virulent for humans than S19 and Rev.1 vaccines (3). At present, several million animals have been vaccinated with the RB-51 mutant strain, but the protective efficacy in cattle compared to S19 remains controversial (118, 124, 158), and the protection against Brucella suis in pigs (172) and against B. abortus in elk (89) is very limited, if present at all.
Heartwater is the most important tick-borne disease of domestic and wild ruminants in sub-Saharan Africa and the West Indies, which is caused by Ehrlichia ruminantium. The only commercially available vaccination procedure is based on the controlled infection of animals with cryopreserved infected sheep blood, followed by antibiotic treatments with tetracyclines when fever develops. A nonvirulent strain has recently been generated through in vitro cultivation and shown to confer good protection (198). Progress in developing cost-effective in vitro cultivation processes may lead to the development of inactivated vaccines (103).
Bovine anaplasmosis is another tick-borne disease caused by Anaplasma marginale infection of red blood cells. Transmission can occur by mechanical means via blood contamination or through blood-sucking arthropods and transplacentally from cow to calf. Cattle that survive acute infection are resistant to the disease but develop persistent, cyclic, low-level infections and therefore remain as "carriers." Calves are less susceptible to infection and clinical disease than adult cattle. Infected blood containing a less pathogenic isolate or subspecies of A. marginale, generally referred to as Anaplasma centrale, remains the most widely used live vaccine in Africa, Australia, Israel, and Latin America. Infection with A. marginale followed by treatment of the patent infections with low doses of tetracycline drugs has also been used but requires close supervision for timely treatment. Following large-scale production of A. marginale antigen from infected bovine blood, a killed vaccine was effectively marketed and used in the United States until withdrawal in 1999 (88). Apart from its higher cost and need for yearly boosters, the killed vaccine was generally less effective in inducing protective immunity than live vaccines.
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(i) Vaccines based on complete life cycle infections. Vaccination with low doses of infective organisms has been used extensively in the poultry industry to combat coccidiosis, the major economic parasitic disease of poultry worldwide. Coccidiosis in poultry is caused by the obligate intracellular protozoal parasite Eimeria species, which undergoes a defined number of asexual cycles of merozoite production in gut epithelial cells (three to four merogenic cycles) before the final sexual stages develop and produce the infective oocysts. The infection is therefore self-limiting, and vaccination with small doses of oocysts, while producing minimal pathology, induces solid protection against homologous challenge. More recently developed live vaccines contain oocysts selected from naturally occurring "precocious" Eimeria strains that produce less merogenic cycles and are therefore safer to use. Although there are many problems with this kind of vaccine, including the need for simultaneous administration to prevent infection of susceptible birds by vaccine-produced oocysts and species- and strain-specific immunity, live coccidian vaccines have been used successfully for over 50 years and are produced as a commercial product by many animal health companies (Table 3) (reviewed in reference 164). The commercial success lies primarily in breeder and layer flocks where anticoccidial drugs have to be withdrawn to prevent the carryover of drugs into eggs.
(ii) Vaccines based on drug-abbreviated infections. Hemoprotozoal parasite infections are not self-limiting, and parasites can proliferate continuously in the blood stages if not checked by the immune response or drug treatment. In contrast to most hemoprotozoal pathogens, including the closely related Theileria annulata, Theileria parva causes a highly fatal disease in cattle by transforming infected lymphocytes, while the erythrocyte stage of this parasite is much less pathogenic. Solid, sterile immunity develops after primary infection, and vaccination of cattle by infection with pathogenic wild-type T. parva followed by drug treatment (long-acting tetracyclines) has been used for many years to control East Coast fever. This vaccination regimen confers solid protection against homologous challenge and limited protection against heterologous challenge but is expensive to administer. Resistance is thought to be conferred mainly by cell-mediated immunity, more specifically, CD8+ cytotoxic T cells, against the intracellular schizont (106), and the targets of the protective cytotoxic-T-lymphocyte (CTL) response are currently being defined (65).
(iii) Vaccines based on infections with parasites with a truncated life cycle.
Several protozoal parasites produce cysts within the host that are a persistent source of infection when eaten by carnivores. These cysts can also cause reinfection when the immune system is compromised or can be reactivated during pregnancy, causing congenital disease and abortion. Toxoplasma gondii infects a wide variety of hosts, including humans, and is the major cause of abortion in sheep and goats. As immunity to primary infection develops, the intracellularly replicating tachyzoites become encysted in a dormant stage (zoitocysts), which can persist for several years, containing hundreds of infective bradyzoites. T. gondii parasites that were continuously passaged in mice to produce diagnostic antigens were later found to have lost their ability to form cysts. The "incomplete" S48 strain of T. gondii now forms the basis of a commercial vaccine conferring long-lasting immunity (
18 months) of susceptible ewes against Toxoplasma-induced abortion when administered prior to mating (30).
(iv) Vaccines based on infection with virulence-attenuated strains. Continuous passage of the tick-borne piroplasms Babesia bovis and Babesia bigemina in splenectomized calves was shown to result in attenuated infections while still inducing immunity in young calves. Live vaccines using infected blood collected from acute infections of splenectomized calves were developed in Australia several decades ago (31, 41) and are still used in most countries to protect against babesiosis, usually produced by local departments of agriculture or veterinary institutions (reviewed in reference 48). In some cases, this is supplemented with A. centrale-infected blood where A. marginale is enzootic. To increase shelf life and allow more rigorous safety testing, several veterinary institutes now produce frozen-blood vaccines stored in liquid nitrogen using either dimethyl sulfoxide or glycerol as the cryoprotectant. For reasons that are still unknown, young cattle up to 9 months of age are more resistant to Babesia infections, but vaccination of susceptible adult cattle often requires additional drug treatment even using the attenuated strains. Continuous exposure to natural tick infections is generally required to ensure continuous and long-lasting immunity.
A live, attenuated Theileria annulata vaccine has been produced by continuous in vitro passaging of the intracellular macroschizont stage and is used in many tropical and subtropical countries for the control of tropical theileriosis in cattle. In contrast to T. parva, immunity to the erythrocytic pathogen T. annulata is short-lived and wanes after 6 months in the absence of natural challenge infections (reviewed in references 140 and 165).
Killed or subunit protozoal parasite vaccines. Several inactivated vaccines consisting of crude whole organisms or, more recently, defined antigenic structures have been registered and target mostly the companion animal market (Table 4). In general, these vaccines are not as effective as live organisms but can ameliorate disease or transmission to various degrees. They may also form the basis for the development of recombinant vaccines.
The final host of the coccidial parasite Neospora caninum is the dog, but its economic impact is felt mostly in the intermediate cattle host, where it is a major cause of abortion (51, 78). A crude N. caninum vaccine has been licensed in the United States to aid in the reduction of N. caninum-induced abortion in healthy pregnant cattle and prevent the transmission of the parasite to calves in utero. The vaccine consists of inactivated N. caninum tachyzoites with an adjuvant administered subcutaneously. A large field study in Costa Rica (151), where infection is highly prevalent in diary herds, demonstrated an overall twofold (46%) reduction in abortion rates through vaccination (49/438 versus 91/438 in saline-injected controls). As also reported in a multiherd vaccination trial in New Zealand (C. Heuer, C. Nicholson, D. Russell, and J. Weston, presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology, New Orleans, LA, 2003), there was a high variability in efficacy between farms, which is likely due to abortions being caused by other infections or by noninfectious causes (87). Timing of vaccination is also likely to play a role in preventing abortion and transmission (78).
A vaccine to alleviate a neurological disease in horses caused by infection with Sarcocystis neurona, equine protozoal myeloencephalitis, has recently been released and is being tested under conditional USDA license by Fort Dodge Animal Health. It consists of in vitro-cultured merozoites, originally obtained from the spinal cord of a horse, which are chemically inactivated and mixed with a proprietary adjuvant for intramuscular injection (104).
Giardia lamblia (synonyms, Giardia duodenalis and Giardia intestinalis) is an enteric parasite of many animal species. Infection is generally self-limiting, but severe gastrointestinal disease can develop in young and immunocompromised individuals. Its importance is mainly in animal-to-human transmission, and Giardia is a major cause of outbreaks of waterborne infections. Only one commercial vaccine has been licensed for use in dogs and cats in the United States (GiardiaVax). It is licensed to prevent clinical disease in dogs and significantly reduce the incidence, severity, and duration of cyst shedding. The vaccine consists of a crude preparation of disrupted, axenically cultured G. duodenalis trophozoites (sheep isolate) and has been shown to eliminate most clinical signs of infection and significantly reduce the total number of cysts shed in the feces in puppies and, to a lesser extent, in kittens. Some efficacy in the clearing of chronic infections resistant to chemotherapeutic agents may also be achieved through vaccination, but this requires more extensive testing (128, 129). It is thought that the vaccine acts mainly through the neutralization of parasite toxins by antibodies.
Two subunit vaccines have been developed to protect dogs against canine babesiosis caused by Babesia canis (Table 4). Both vaccines consist of soluble parasite antigens (SPA) released into the culture supernatant by in vitro-cultured parasites, combined with adjuvant. The first vaccine released, Pirodog, contains SPA from B. canis cultures only (117), while the recently released NobivacPiro contains SPA from B. canis and Babesia rossi in an attempt to broaden the strain-specific immunity. The protective effect of this vaccine seems to be based on the antibody-dependent neutralization of a soluble parasite substance that causes hypotension and clinical disease, rather than acting through reducing parasitemia per se (156). This vaccine approach was also evaluated in cattle but did not confer sufficient protection (165).
A killed subunit vaccine has been developed against coccidiosis in poultry by ABIC Veterinary Products, Israel, particularly for use in the broiler industry (192, 193). Interestingly, this vaccine does not target the merozoite stages, as most live vaccines are thought to do, but rather targets the final sexual, macrogametocyte stages that develop to form the disease-transmitting oocysts. The principle behind this vaccine strategy is that it will still allow immunity against the asexual stages to be generated by natural infections while reducing oocyst shedding and parasite transmission. Added advantages of this approach are that the laying hens, rather than the chicks, are immunized, transferring protective immunoglobulins into the egg yolk and subsequently the hatchlings. Considering that each hen lays more than 100 eggs in her lifetime, this considerably reduces the number of vaccinations and animal handling. In contrast to the species and strain specificity of live vaccines, this gametocyte vaccine was shown to confer partial protection across the three major Eimeria species. A major disadvantage of the vaccine is that it is expensive to produce, consisting of affinity-purified native gametocyte antigens derived from infected chickens, and is still a fairly complex preparation (15). Three major components of affinity-purified native gametocyte antigens have recently been cloned and characterized with a view to identifying the protective components and develop a recombinant vaccine (16). It remains to be seen if the translation of native vaccine to recombinant vaccine will be successful, as this has been a major stumbling block for much of the parasite development area (123).
Human visceral leishmaniasis, or kala-azar, is a devastating human disease caused by the intracellular parasite Leishmania chagasi or Leishmania infantum and transmitted through sand flies. Dogs are the principal carriers of the disease and are also clinically affected. Recently, a subunit vaccine against canine visceral leishmaniasis, based on a strongly antigenic surface glycoprotein complex, fucose mannose ligand, or FML antigen, from Leishmania donovani and saponin adjuvant has been developed in Brazil. Vaccine efficacy was reported to be 76 to 80% against both homologous and heterologous challenge with L. chagasi and to last for at least 3.5 years (21, 44). A concomitant reduction in human incidence of the disease was also reported, which is likely due to the transmission-blocking properties of the vaccine (42, 125). The vaccine may also have a therapeutic effect on infected dogs (22).
There are three different families of helminths or worms, nematodes (roundworms), trematodes (flatworms), and cestodes (tapeworms), that infect both animals and humans. At present, only one worm vaccine is on the market in Europe for the cattle lung nematode Dictyocaulus viviparous (Bovilis Lungworm), consisting of irradiated infective L3 larvae that cannot develop into the adult stage (7). Vaccination with irradiated L3 larvae of the economically important gastrointestinal nematodes has been attempted but was not successful due mainly to their lack of efficacy in inducing immunity in young animals (reviewed in reference 6). The increasing drug resistance of gastrointestinal nematodes has renewed intense interest in developing vaccines for these important veterinary pathogens (reviewed in references 19, 123, and 189).
Tapeworms have a larval stage in intermediate hosts that is uniquely susceptible to immune killing after a single infection. Antigens from this early larval stage (oncospheres) were the first to confer protection against a multicellular pathogen as recombinant proteins (82). Commercial or field application of anticestode vaccines is, however, still in progress (reviewed in reference 98).
The most important veterinary trematode species are liver flukes (Fasciola hepatica and Fasciola gigantica). Vaccine development against these parasites is hindered by the fact that they do not seem to induce immunity in their natural ruminant hosts, even after repeated infections. Recently, a unique breed of sheep (Indonesian thin tail) was shown to develop immunity against F. gigantica, and further dissection of this protective mechanism may offer new approaches to vaccine development (109).
Ectoparasitic arthropods would seem to be the ultimate challenge in vaccine development, as they not only are large and complex but also spend most of their life outside or on the surface of the host. Interestingly, the only recombinant parasite antigen vaccine commercially available is against a tick parasite, Boophilus microplus, and was first introduced commercially in Australia in 1994 (TickGUARD; Fort Dodge Australia) and later in Cuba and a few South American countries (Gavac; Heber Biotec SA, Cuba). This vaccine is unique in that it is not based on natural antigens recognized by the immune system during infection but takes advantage of the ferocious blood-feeding habits of the tick. High antibody levels are generated by vaccinating cattle against a tick gut membrane-bound protein, Bm86, using a recombinant protein in a potent adjuvant. These antibodies bind to the tick's gut surface when taking a blood meal, causing the rupture of gut wall and tick death. The vaccine induces significant levels of protection against tick infestation and, in some cases, against tick-borne diseases (195). However, as the molecule is not seen during natural infection ("hidden" or "concealed" antigen), antibody levels are not boosted by infection and need to be maintained at high levels by repeated immunization. The vaccine is best used in conjunction with drug administration, which limits its practical and commercial appeal. The presence of a tick immunoglobulin excretion system seems to hamper the effectiveness of this vaccine approach in other ticks (126).
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), reactive oxygen and nitrogen species, and indoleamine 2,3-dioxygenase (IDO). Toxins released by pathogens (red circles) can be neutralized by circulating antibodies, thereby decreasing clinical signs of infection. Multicellular helminth parasites generally do not reside within host cells and are too large to be phagocytosed; therefore, they usually require alternative immune killer mechanisms mediated by antibody-directed actions of mast cells and eosinophils. Essential secreted proteins and toxins derived from the worms (brown circles) may also be neutralized by antibodies and thereby interfere with parasite growth.