A Review of Host Response to the Inactivated Polyomavirus Vaccine in Experimental and Field Settings

Branson W. Ritchie, DVM, PhD; Kenneth S. Latimer, DVM, PhD; Denise Pesti, MS; Raymond Campagnoli, MS; Phil D. Lukert, DVM, PhD

Psittacine Disease Research Group, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

Abstract. This manuscript reviews the use, safety, and effectiveness of an inactivated, commercially-available, polyomavirus vaccine in experimental settings and in field settings.

Key Words: Avian, Disease, Host immune response, Polyomavirus, Inactivated vaccine.

Introduction

From its initial description in the early 1980's, avian polyomavirus infections have caused emotional and economic burdens for the companion bird industry and a frustration to aviculturists and veterinarians who, until 1995, had no vaccine available for reducing the spread of this virus. In non-budgerigar psittacines, avian polyomavirus-induced disease is most common in young birds (< 150 days old) with a reported mortality rate of 10% to 93% in at-risk neonates. ^1-5 Adults are readily susceptible to infection, can become ill, and some may die. ^6-11 Epizootiologic data suggest that avian polyomavirus is a leading cause of mortality in young psittacine birds. ¹² Virus exposure through direct contact with infected birds or through contact with virus contaminated environments is considered important in the transmission of the environmentally stable polyomavirus^.1,13 Polyomavirus epornitics have been linked to: 1) inadequate quarantine procedures, 2) virus contaminated nest boxes, 3) virus contaminated incubators, 4) transfer of unvaccinated or incompletely vaccinated birds to brokers or pet retailers, 5) mixing unvaccinated birds from numerous locations and 6) exposing unvaccinated flock residents or neonates to infected birds or a contaminated environment and returning them to the aviary without quarantine. ¹

Until the avian polyomavirus vaccine^a was registered by the USDA, control of polyomavirus epornitics was problematic because of the prevalence of virus activity in psittacine birds^5,12-15 and the inherent difficulties in reducing potential exposure to this environmentally stable virus by maintaining closed aviaries, practicing extraordinary hygiene and attempting to detect and isolate transiently-infected birds. Techniques developed to facilitate this latter task include assays to detect anti-polyomavirus antibodies and a DNA probe test to detect polyomavirus nucleic acid. ^b Both of these types of detection assays were developed and initially evaluated for their accuracy in controlling polyomavirus infections by researchers at the University of Georgia College of Veterinary Medicine. Both types of assays have inherent limitations.

In non-budgerigar psittacine birds, which, as a rule, are thought to develop transient polyomavirus infections followed by either death or seroconversion with apparent clearance of the virus, the detection of anti-polyomavirus antibodies merely indicates a previous infection. ¹⁶ The demonstration of a progressively decreasing antibody titer in multiple groups of naturally infected non-budgerigar psittacines suggests that most infected birds are able to mount an effective immune response and clear the infection. Thus, unless paired serum samples are collected, the data generated by an antibody assay is of limited value in managing an individual patient or a flock of non-budgerigar psittacine birds. ¹³ For example, in 6 aviaries containing mixed species of non-budgerigar Psittaciformes, the seroprevalence of polyomavirus ranged from 11% to 63% of the at-risk birds. ^16-19 Theoretically, polyomavirus infections in these flocks could be prevented by screening all of the birds for antibodies, eliminating those that are seropositive, repeatedly disinfecting all potentially contaminated surfaces in the aviary and reducing the potential for virus exposure by maintaining a completely closed aviary. However, this management scheme seems unrealistic when, if to be of value, one must decide how to remove the up to 63% of seropositive birds from the flock. Additionally, a test and "eradicate" type program in lieu of vaccination does not protect immunologically naive neonates produced at a seronegative farm from virus exposure when the chicks are shipped from the nursery and enter the pet trade, and it does not protect the selected seronegative population of adults from being infected if the virus is inadvertently introduced to the aviary.

Polyomavirus nucleic acid can be detected in cloacal swabs taken from non-budgerigar psittacine birds during an epornitic. ^13,20,21 This allows birds that are passing nucleic acid to be isolated from the remainder of the flock until the infection has resolved in the positive birds. Polyomavirus-specific DNA probes also can be used to detect viral nucleic acid in fresh tissues (blood, liver, spleen, etc. ), or in environmental samples collected from areas (hospital, nursery, incubators, etc. ) that may have been contaminated with the virus. ^{1,13,14,20-22} Birds that are DNA probe negative and seronegative could be susceptible to infection as discussed above.

Recently, several clinicians have suggested that polyomavirus could be controlled by doing nothing and depending on survival of the fittest to naturally select a polyomavirus-disease-resistant population of companion birds. Prior to the availability of antibiotics and vaccines, only the fittest survived. Within the confines of laws and regulations established to protect the masses, one could always choose not to use antibiotics, vaccines, diagnostic tests, surgical therapies or any medical or surgical procedure that alters the natural progress of a disease or injury. However, when faced with the dangers of uncontrolled infectious diseases in their pets, livestock, children or themselves, most individuals choose to actively alter the natural selection process. To this end, vaccines have been used as an effective modality for controlling viral-induced diseases in many companion and production animals, particularly for environmentally stable viruses with an unrestricted geographic distribution. As has been the case for many other viral-induced diseases, it is likely that the widespread use of the safe, effective avian polyomavirus vaccine will provide an important tool for controlling the spread of polyomavirus within vaccinated flocks, thus reducing the unnecessary loss of companion birds.

Strategies for Polyomavirus Vaccine Use

The strategies for using the avian polyomavirus vaccine are similar to those used to control infectious diseases of other companion animals, like, parvovirus in dogs. By vaccinating the adults, the population of birds at-risk for infection is substantially decreased, and the likelihood of a progressive cycle of transmission among the mature birds is reduced. This in turn lessens the chances that the adult population will serve as a source of virus exposure for the neonates in the nursery. If virus activity in the breeding aviary is reduced through vaccination, then careless avicultural practices (i.e. , no quarantine procedures, bringing birds from other aviaries into the nursery, allowing visitors with direct or indirect contact with birds access to the nursery) become the only route by which the virus enters the nursery. ¹⁶ Additionally, vaccinating the adults is considered important because even if polyomavirus is introduced to a nursery from an outside source, once an outbreak has started it is likely that this environmentally stable virus will spread through the unvaccinated adults. The likelihood that polyomavirus will spread from chicks in the nursery to adults in the breeding aviary, or vice versa, is favored when the nursery is in the middle of the breeding facility, the food preparation and storage areas are in the same airspace with the nursery and when the same aviary personnel care for the neonates and adults.

Vaccine Safety, Immunogenicity, and Efficacy

Data from experimental and field settings suggest that the inactivated avian polyomavirus vaccine is safe, immunogenic and efficacious in psittacine birds that vary in age, species, and immunologic status. ^1,16,19 Vaccination has been shown to be effective and valuable in helping to control polyomavirus epornitics and has not been associated with clinically recognizable adverse reactions even when used in the face of an outbreak. ¹ Epornitics controlled with the aid of vaccination have varied in severity; in one flock polyomavirus reportedly caused the death of approximately 90% of at-risk neonates during a 2-month period, while in another flock the reported outbreak spanned 2 consecutive breeding seasons and caused the death of approximately 30% of the at-risk neonates. ¹

Safety (i.e. , lack of unacceptable local or systemic reactions) of the polyomavirus vaccine was established by vaccinating birds that were seronegative or seropositive prior to vaccination and then evaluating the birds for clinically detectable systemic or local reactions for up to 3 years after vaccination. In experimental and field studies, the safety of injecting inactivated avian polyomavirus has been evaluated in more than 80 species including cockatoos, macaws, Amazon parrots, African grey parrots, cockatiels, conures, eclectus parrots, lovebirds, rosellas, Poicephalus spp. , Pionus spp. , lories and caiques. ^{1,16,19,23,24} Vaccinates have ranged in age from 10-day-old neonates to > 10-year-old adults. In addition to these studies, to date, more than 50,000 doses of inactivated avian polyomavirus vaccine have been administered with no reported immediate or delayed systemic reactions associated with the vaccine. Extreme caution should be used to ensure that a vial of the multidose polyomavirus vaccine is not contaminated with bacteria, fungi or other viruses during the vaccination process. The type and quantity of local reactions noted to date have been similar to those reported in experimental and field studies. ^{1,16,19,23,24}

Proper vaccination technique is critical to minimize severity of local reactions. Reactions at the site of a properly administered vaccine are minimal. In experimental and field settings, it was found that accidental intradermal injection of vaccine caused more severe reactions than proper subcutaneous delivery. ¹⁶ In one study involving the evaluation of 2955 vaccination sites, 78% had no reaction, 11.5% developed hyperemia or skin discoloration, 6.8% developed a small scab or mild thickening of the skin and 3.5% developed a mass at the site of injection. ¹⁶ Tenting of the skin at the site of injection, as is used in dogs and cats, appears to be the best method to insure an injection is administered subcutaneously.

In evaluating a vaccine intended for widespread use, it was important to establish what effect, if any, vaccination would have on birds that had survived a natural polyomavirus infection as detected by virus-neutralizing antibodies in serum prior to vaccination. In several studies involving the injection of inactivated avian polyomavirus, 215 of 518 (41%) mixed species psittacines were found to have survived a naturally acquired polyomavirus infection as indicated by the detection of virus-neutralizing antibody titers prior to vaccination. None of these seropositive birds developed any clinical signs suggestive of an adverse systemic reaction either immediately following or for up to 4 years after vaccination. ^16,19 Additionally, multiple injections with inactivated avian polyomavirus has not been shown to cause recognizable clinical changes or adverse systemic or severe local reactions. ^16,23 In addition to these studies, to date, more than 50,000 doses of inactivated avian polyomavirus vaccine have been administered with no reported immediate or delayed systemic reactions associated with the vaccine. Seroprevalence studies would suggest that between 10% and 63% of the adult birds that have been vaccinated were infected with avian polyomavirus prior to vaccination. ^{3,5,16,17,19,23} It is important that a vaccine intended for widespread has been shown to be as safe for previously infected birds as it is for their immunologically naive conspecifics.

Based on studies of birds that have died from naturally acquired infection, it has been suggested that an immune-mediated process is involved in the pathogenesis of polyomavirus-induced disease and that vaccination may precipitate this process. ²⁵ The authors and others have noticed that polyomavirus-associated lesions are rarely demonstrated in non-budgerigar psittacine birds that have died from causes other than an acute polyomavirus infection. ^c Given the infrequent detection of such lesions in non-budgerigar psittacine birds, and the prevalence of virus demonstrated by serologic studies, it seems likely that any role an immune-mediated process plays in causing disease occurs only in birds incapable of mounting a complete and effective immune response, and that nonfatally affected birds develop an appropriate immune response and recover. If vaccination were to induce an immune-mediated process, it seems likely that some of the seropositive vaccinates in experimental and field studies would have been fatally affected by vaccination. Additionally, the vaccine has now been in use for more than 2 years. With more than 50,000 doses used, adverse clinical changes have not been reported in any vaccinates. Collectively, these findings would suggest that any role an immune-mediated process plays in polyomavirus-induced disease is not a concern when using the inactivated vaccine as a prophylactic.

Irrespective of whether or not polyomavirus-associated disease is associated with an immune-mediated process, the inactivated avian polyomavirus vaccine is designed to prevent virus that enters the body from inducing a systemic infection. Without a systemic infection, there should not be sustained viral replication in a tissue, which together with an incomplete immune response would be necessary to precipitate an immune-mediated process. Thus, vaccination would be expected to protect uninfected vaccinates from any immune-mediated disease process. Using a similar strategy, vaccines have been developed to prevent disease associated with virus infections that are known to induce immune-mediated processes including dengue virus, yellow fever virus and hepatitis-B virus. ^26-31

Immunogenicity (i.e. , capacity to stimulate a measurable immune response) has been evaluated by testing for virus-neutralizing antibodies, vaccinating birds according to published recommendations, and then testing for a significant change in antibody titer. ^16,19 In several trials, immunogenicity using inactivated avian polyomavirus was evaluated in 518 birds. The pre-and post-vaccination VN antibody titers in the 303 vaccinates that were seronegative prior to vaccination are provided in Figure 1. The pre-and post-vaccination VN antibody titers in the 215 vaccinates that were seropositive prior to vaccination are presented in Figure 2. Actual seroconversion (greater than 4-fold increase in VN antibody titer) by percentage is provided in Table 1. As would be expected, not all birds with a pre-existing antibody titer developed a significant increase in antibodies following vaccination. However, to prevent an infection in an individual bird, and to reduce the amplification of virus within a flock, it would seem reasonable to assume that it is most important for seronegative birds (i.e. , those at risk of infection) to respond appropriately to the vaccine.

Efficacy (i.e. , capacity to induce an immune response that protects a vaccinate from experimental challenge with viable virus) was evaluated by vaccinating birds, followed in 2 to 4 weeks by IM or IV challenge-exposure. After challenge-exposure, protection was evaluated by attempts to recover virus from tissues, or by evaluating birds for clinical signs of disease and testing for a significant change in titer. Reported efficacy results in 104 birds are summarized in Table 2. ¹⁶ In this study, a bird was considered protected from infection if, following challenge, it did not develop clinical signs of disease or a significant increase (four-fold or greater) in virus-neutralizing antibodies. A bird was considered susceptible to infection if, following challenge, it developed clinical signs of disease or a significant increase (four-fold or greater) in virus-neutralizing antibodies. The vaccine protected 100% of birds that seroconverted from infection. By comparison, 96% of unvaccinated birds, as well as the vaccinated birds that did not seroconvert following vaccination, were susceptible to infection.

It has been observed that clinically stable birds experimentally infected with either cell culture derived or wild-type polyomavirus do not develop the severe disease that is common in naturally infected birds. ^16,24 A similar reduction in the severity of experimentally induced infections occurs with many viral-induced diseases in companion animals, for example, canine parvovirus and feline panleukopenia virus. ^32-35

Field Efficacy of Polyomavirus Vaccine

An expected field efficacy has been established by vaccinating flocks while an epornitic is occurring (Table 3). ¹ In 9 flocks, the cumulative mortality rate in at-risk chicks prior to and during the vaccination process was 422 of 1,474 (29%). After the original epornitics were controlled, the cumulative mortality rate in chicks which were vaccinated (all the chicks which died were incompletely vaccinated) and then potentially exposed to polyomavirus was 21 of 2,081 (1%).

The cumulative number of the type of birds that died during these epornitics is provided in Table 4. While vaccinating during a polyomavirus outbreak has been shown to be advantageous, it is possible for mortality to continue in the neonates until flock immunity has been increased, generally 2 to 3 weeks after the last booster vaccination. These deaths are unfortunate, however, they were not unexpected. It would be considered unusual for any inactivated vaccine to alter the pathogenesis of an infection that occurred prior to vaccination.

While it is not recommended to vaccinate a flock during breeding season, vaccination can be used to help stop an epornitic even while birds are breeding. ¹ In field studies, the polyomavirus vaccine has been used to help control polyomavirus outbreaks during the breeding season when adults were incubating eggs, feeding chicks or preparing the nest box. These adults resumed their parental duties immediately following vaccination. Vaccinated birds have produced fertile eggs within 2 weeks after vaccination.

When vaccinating during an outbreak, it is important that the veterinary staff and aviary personnel exercise extraordinary care to prevent handling and injection procedures from serving as methods of virus transmission from bird to bird. While it is recommended that neonates be at least 35 to 40 days of age before being vaccinated, chicks from flocks experiencing an outbreak can be vaccinated from 10 to 20 days of age. Chicks vaccinated when less than 35 to 40 days of age should receive two additional boosters with a 2 to 3 week interval between doses. A bird should receive the last vaccination at least 2 weeks prior to leaving the aviary. ¹⁶

Figure 1: VN antibody titers before and after vaccination in various psittacine birds that were seronegative prior to vaccination. Two-fold dilutions of serum were used starting with an initial dilution of 1:2 in one trial and no dilution in another trial. 16,19

Figure 2: VN antibody titers before and after vaccination in various psittacine birds that were seropositive prior to vaccination. Two-fold dilutions of serum were used starting with an initial titer of 1:2 in one trial and no dilution in another trial. 16,19

Acknowledgments

Development of the avian polyomavirus vaccine was made possible by sustained contributions from the Cowan Avian Health Foundation, the International Avian Research Foundation, Joe and Sue Still, Terry Clyne, Richard and Luanne Porter, Veterinary Medical Experiment Station, Knick Enterprises, Kathleen Szabo, Bobbi Brinker, International Aviculturist's Society, Midwest Avian Research Exposition, National Aviary, Puerto Rican DNR, Ann Arbor Cage Bird Club, Aviary and Cage Bird Club of South Florida, Avicultural Society of Puget Sound, Central Indiana Cage Bird Club, Charlotte Metrolina Cage Bird Society, Cream City Feathered Friends, Dallas Cage Bird Society, Feathered Friends Society, Gateway Parrot Club, Greater Brandon Avian Society, Hookbill Hobbyists of Southern California, Kentuckiana Bird Society, Louisiana Aviculture Society, Northwest Ohio Exotic Bird Club, South Jersey Bird Club, Wasatch Avian Education Society, West Valley Bird Society, Lafeber Inc. and Zeigler Brothers Inc. Hundreds of aviculturists, bird clubs and veterinarians have also made significant contributions. A special thanks to Dr. Frank Niagro for his years of dedicated service to improving the health of companion birds.

Sources and Manufacturers

a. Polyomavirus vaccine, Biomune, Lenexa, KS (913-894-0230).

b. Infectious Diseases Laboratory, University of Georgia, Athens, Ga (706-542-5812) and Avian/Wildlife Laboratory, University of Miami, Miami, FL (800-232-1056).

c. Dr. Robert Schmidt, California Veterinary Diagnostic Laboratory: Personal communication, (1996).

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