Branson W. Ritchie, DVM, PhD, Kenneth S. Latimer, DVM, PhD, Cheryl B. Greenacre, DVM,
Denise Pesti, MS, Raymond Campagnoli, MS, Phil D. Lukert, DVM, PhD
Psittacine Disease Research Group

Introduction

From its initial description in the early 1980's, avian polyomavirus infections have caused frustration to aviculturists and veterinarians who, until 1995, had no vaccine available for reducing the spread of this virus. Epizootiologic data suggest that avian polyomavirus is a leading cause of mortality in young psittacine birds (< 150 days old), with a reported mortality rate of 10% to 93% in at-risk neonates. ^1-5 In addition to chicks, adult psittacine birds are readily susceptible to infection, can become ill, and some may die. ^6-11

Some affected birds die without developing any clinical signs of disease, while others die 12-to 48-hours after developing clinical signs that may include depression, loss of appetite, weight loss, delayed crop emptying, vomiting, diarrhea and bleeding under the skin. ^3-5,12,13 Many affected young birds die, while most infections in adult birds are unrecognized or birds manifest subtle clinical changes such as transient lethargy, a poor appetite and diarrhea with the surviving birds developing antibodies to the virus. Infections classified as "subclinical" (not obvious upon typical examination) are common in adult and young birds.^3,5,13-16 In most aviaries and pet retail establishments, it is these subclinically infected birds that initiate a cycle of infection, and create an opportunity for the virus to be spread from bird to bird.

Virus exposure through direct contact with clinically or subclinically 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) shipment 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

Diagnostic dilemmas

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 ^5,13,17-19 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 originally developed at the University of Georgia College of Veterinary Medicine to facilitate this latter task include assays to detect anti-polyomavirus antibodies and a DNA probe test to detect polyomavirus nucleic acid.^b,c Both types of assays have inherent limitations.

In non-budgerigar psittacine birds, detection of anti-polyomavirus antibodies in a single serum sample merely indicates a previous infection or vaccination. ²⁰ 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. ^16,20-22 Theoretically, polyomavirus infections in a flock 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, a test and "eradicate" type program in lieu of vaccination does not protect immunologically naive neonates produced at a seronegative facility from virus exposure when the chicks are shipped from the nursery, 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 psittacine birds during an epornitic.^{b 13-15} This allows birds that are excreting 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. ^{1,13,14,18,23} Birds that are DNA probe negative and seronegative could be susceptible to infection as discussed above. It should be stressed that swabs of the cut surface of the liver, spleen and kidney should not be used for confirming the presence of polyomavirus nucleic acid in vaccinated birds. Polyomaviral nucleic acid, in the absence of lesions suggestive of viral replication, can be detected in some vaccinates. This finding would be expected given that vascular tissues contain the cells responsible for immunologic processing of the foreign materials (proteins and nucleic acid) present in the inactivated avian polyomavirus vaccine. In situ hybridization^c can be used to confirm the presence of polyomavirus nucleic acid in the tissues of birds that have died with suggestive lesions.

Strategies for Vaccination

The strategies for using the avian polyomavirus vaccine are similar to those used to control other common viral diseases, like parvovirus in dogs, panleukopenia virus in cats or poliovirus in humans. Preventing polyomavirus infections within a flock requires the vaccination of two crucial groups of birds: the breeding flock, and the young birds before they leave the nursery.

To reduce polyomavirus infections within the aviary, it is essential to vaccinate the adult birds. There are only two known sources for polyomavirus entering a nursery: either it is transported into the nursery during periods when infections (which usually go unnoticed) are occurring in the breeding population, or it is introduced to the nursery by people who bring the virus in from an outside source. Once the breeding flock is vaccinated, the population of birds at risk (those that could be infected) is substantially decreased, and the likelihood of a progressive cycle of transmission among these adult birds is reduced. This in turn lessens the chances that the adult population will serve as a source of virus for the exposure of 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 aviary.²⁰

If the flock is vaccinated and people do not inadvertently introduce the virus to the nursery, it is unlikely that any neonates will be exposed to the virus. This logic is substantiated by observations of polyomavirus outbreaks. In several large aviaries, control of polyomavirus-induced disease occurred when the breeding adults and neonates were vaccinated. By contrast, aviculturists who attempted to control outbreaks by vaccinating only young birds decreased, but did not eliminate, the incidence of disease.¹ These findings emphasize the importance of vaccinating the adults to prevent polyomavirus infections in the nursery. Once polyomavirus has been controlled within the flock, then it is crucial for young birds that will be leaving the aviary to be protected before they are exposed in the pet trade to birds (particularly budgerigars) which may be shedding the virus.

Recommendations for Vaccination

When using a vaccine, or any therapeutic agent, the manufacturer's recommendations should be followed. The suggestions for vaccination described below have been developed during 5 years of evaluating an avian polyomavirus vaccine in experimental and field situations, and are provided to supplement the recommendations of the vaccine's manufacturer.^a

Adults - Breeding birds should be vaccinated twice, with a two week interval between vaccinations. It is best to vaccinate breeding birds in the non-breeding season. However, in flocks that are experiencing an outbreak of disease, vaccination can be performed during the breeding season.^1,20

Immature Birds - An avian veterinarian may adjust the schedule for vaccinating neonates based on the risk of virus exposure in the nursery. These suggestions are based on the fact that the older a young bird is when it is vaccinated, the more likely it's immune system will respond. The original certificate of vaccination, provided by the manufacturer,^a should be sent with the young bird when it leaves the aviary.

1. In the ideal aviary, where the aviculturist has vaccinated the breeding birds, does not have a history of polyomavirus-induced disease and sells only weaned birds, vaccination of young birds can be started about 4 weeks prior to weaning. These birds should be vaccinated twice with a 2 to 3 week interval between doses. A bird should receive the last vaccination at least 2 weeks before leaving the aviary.

2. If an aviculturist has vaccinated the breeding birds, does not have a history of polyomavirus-induced disease but sells birds prior to weaning, vaccination of young birds should be started between 40 to 50 days of age. These birds should be vaccinated twice 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.

3. If necessary, young birds can be safely vaccinated starting at 10 to 20 days of age. These birds 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.

Companion Birds - If a companion bird is maintained in complete isolation, which is not a very realistic scenario, it has minimal risk of being exposed to polyomavirus. However, isolation means that the bird, and its keeper, never leaves the home to go to the veterinarian, groomer, club meetings or any location where direct or indirect exposure to other birds might occur. Complete isolation also means that all of the food, toys, perches and enclosures for the bird are purchased from mail-order catalogs that ship from warehouses that do not house birds. These items may be contaminated with polyomavirus if they are kept in the same airspace with birds. It seems more logical to vaccinate companion birds rather than attempt to maintain such rigorous isolation, although isolation is the only method to reduce a bird's exposure to the infectious agents for which vaccines are not yet available. It is interesting to note that companion dogs and cats that are maintained in relative isolation (those that are confined to indoor living) are routinely vaccinated to protect them from common infectious diseases. Why would one elect to afford a companion bird a lesser level of protection?

Evaluating a Vaccine

Prior to USDA registration of the avian polyomavirus vaccine, it was evaluated for:

(a) safety - the lack of unacceptable local or systemic reactions.

(b) immunogenicity - the capacity of the vaccine to stimulate a measurable immune response.

(c) efficacy - the capacity of the vaccine to induce an immune response that protects a vaccinate from experimental challenge with viable (live) virus.

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. ^1,20,22 Additionally, 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 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 (Table 1). ^20,22 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.

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. ²⁰ 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. ²⁰ 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.

Immunogenicity 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. ^20,22,24 Seroconversion (greater than 4-fold increase in VN antibody titer) by percentage from several studies is provided in Table 2. 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 (ie, those at risk of infection) to respond appropriately to the vaccine.

Efficacy was evaluated by vaccinating birds, followed in 2 to 4 weeks by IM or IV challenge-exposure. In these studies, 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. ²⁰

An expected field efficacy has been established by vaccinating flocks during an outbreak. 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%).¹ The cumulative number of the type of birds that died during these epornitics is provided in Table 3. The economic impact of the outbreak, cost of vaccination and post-vaccination production are summarized in Table 4.

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. ²⁰

On the basis of studies conducted on birds that have died from naturally acquired infection, an immune-mediated process has been suggested to be involved in the pathogenesis of polyomavirus-induced disease, and it has been further suggested that vaccination may precipitate this process.²⁵ Seroprevalence studies indicate that up to 60% of nonbudgerigar psittacine birds maintained in some aviaries have survived polyomavirus infections.^{3,5,15,16,22,24} The authors and others have noted that polyomavirus-associated lesions are rarely demonstrated in nonbudgerigar psittacine birds that have died from causes other than an acute polyomavirus infection. Given the infrequent detection of such lesions in the nonbudgerigar psittacine birds, and the prevalence of virus activity 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 studies, would have been fatally affected by vaccination. In fact, clinically recognizable systemic reactions have not been documented in any experimentally evaluated vaccinates. Additionally, the vaccine has now been in use for more than 2 years. With more than 50,000 doses used, adverse systemic reactions have not been reported in any vaccinates. This 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 caused by 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

The next generation vaccine

While the inactivated avian polyomavirus vaccine continues to function at its research established level of efficacy, it, like all inactivated vaccines, has an inherent group of limitations. In the future, most inactivated vaccines will undoubtedly be replaced with recombinant and plasmid-mediated vaccines. In comparison to inactivated vaccines, this next generation of vaccines reduce the quantity of proteins to which a vaccinate is exposed, in some cases can be engineered so that an adjuvant is not necessary and by design stimulate cellular immunity. Because of these advantages, both recombinant and plasmid-mediated vaccines have been developed for avian polyomavirus and, when eventually approved by the USDA, it is anticipated that one of these vaccines will replace the currently available inactivated polyomavirus vaccine.

In experimental studies, recombinant VP-1 (the major polyomavirus capsid protein) was found to induce an immune response in chickens that altered the natural progression of infection that occurred in non vaccinates.³² A similar response has been noted in chickens vaccinated with plasmid constructs containing only VP-1 or plasmid constructs containing all 3 structural proteins (VP-1, VP-2 and VP-3). (Poet, et al unpublished data).

Controlling a Polyomavirus Outbreak

As is the case with many viral-induced diseases in companion animals, vaccination will play a pivotal role in reducing the incidence of avian polyomavirus infections. However, because no vaccine is 100% effective, vaccination should not be expected to completely combat the deleterious effects of poor management or hygiene.

Controlling polyomavirus in an outbreak requires vaccinating the adults and neonates to stimulate flock immunity, as well as cleaning and disinfecting the contaminated facility. ^15,30 While vaccinating during a polyomavirus outbreak has been shown to be advantageous, it should be stressed that deaths may continue in neonates until flock immunity has been increased, generally 2 to 3 weeks after the last booster vaccination.¹ Once an outbreak has occurred, it is important that the nursery be thoroughly cleaned and disinfected to prevent virus contaminating this environment from infecting neonates before the time that their immune systems will respond to vaccination. Contaminated nest boxes must be replaced. It is crucial during an outbreak that the adults be vaccinated to reduce the spread of the virus among the adults, thus decreasing the chances of the virus entering the nursery.

A DNA probe-based assay^b is valuable for identifying birds that are shedding virus in their excrement during an outbreak. Birds that are shedding the virus can be separated from others in a nursery to reduce further virus transmission, while vaccinated birds are developing antibodies to the virus.

Answers to Commonly Asked Questions Concerning Polyomavirus

Below are some questions submitted by a group of aviculturists concerning the management of polyomavirus. The answers to all the submitted questions will be based on controlling polyomavirus in non-budgerigar psittacine birds.

If a bird has already been infected with polyomavirus, does the vaccine kill the polyomavirus in the bird?

As a general rule, a non-budgerigar psittacine bird that is infected with avian polyomavirus will either die or its immune system will mount an appropriate response and clear the virus from the body. This clearance response is similar to what one would expect to occur with parvovirus in dogs; a dog infected with parvovirus either dies or is able to clear the virus from the body and recovers.

To answer the specific question, experimental data suggest that the vaccine does not hurt nor help birds that are already infected, just as a "flu" vaccine would not help you if you were infected with influenza virus before you were vaccinated. Remember that vaccines are designed to help the body's natural defenses prevent the uncontrolled spread of a virus within the body.

If a bird is fed a nutritional diet, housed in an enclosure that is cleaned regularly and is otherwise healthy, can it still be infected with polyomavirus?

Absolutely! Most aviculturists would consider themselves to be relatively healthy and feel that they live in a clean home yet each of us is susceptible to infection by influenza virus (the "flu"), rhinovirus (a common cause of "colds"), rabies virus or herpesvirus simplex (the cold sore virus). Were it not for widespread use of vaccines, these same "healthy" aviculturists would be readily susceptible to infections with poliovirus and human poxvirus (human poxvirus is now considered eradicated, largely due to a worldwide vaccination program). With this said, a good plane of nutrition and a clean living environment should help strengthen any animal's immune system and may reduce the severity of a virus infection, or may help an animal resist secondary invaders (bacteria, fungi, parasites) that can take advantage of a weakened defense system. For example, an aviculturist who consumes a balanced diet, gets plenty of exercise and obtains a normal amount of sleep can still be infected with influenza virus, but they may develop a less severe disease of shorter duration than an aviculturist that eats poorly, smokes and is sleep deprived. As an observation, aviculturists that experience polyomavirus outbreaks frequently mention that their largest, most robust, "healthiest chick" was the first to die.

If birds experimentally infected with polyomavirus do not develop the same signs of disease as those that are naturally infected, then how can a vaccine be developed and tested?

Disease is actually a complex process that involves an interaction between all the microbes found in an animal's body and the animal's response to these potentially infectious agents. There is an in-depth discussion of the factors that control when an infection might cause a disease, and how a bird responds to viruses in Avian Viruses: Function and Control. As a brief answer to the question, many infectious agents (bacteria, parasites and viruses) that cause severe disease in naturally infected animals will cause mild or inapparent disease in experimentally infected animals. For example, parvovirus can cause severe diarrhea and death in naturally infected dogs, yet the same virus recovered from these severely affected dogs may cause mild or inapparent infections in experimentally infected dogs. As another example, panleukopenia virus causes severe disease in naturally infected cats but may cause mild disease in experimentally infected cats. The same situation occurs with polyomavirus in non-budgerigar psittacine birds; virus recovered from birds that have died from polyomavirus causes only lethargy, anorexia and transient diarrhea in experimentally infected birds.

How then was a vaccine developed to control parvovirus in dogs, panleukopenia virus in cats and polyomavirus in birds?

When a virus infects a cell, it begins the process of replication with subsequent production of thousands to millions of new virus particles that are then released from the originally infected cell and move to new cells. When a sufficient number of new cells have been damaged, disease occurs. Vaccines are designed to prevent a virus that does enter the body from starting the process of uncontrolled replication and thereby prevent disease. A virus that does not cause severe disease in experimentally infected animals can still be detected by examining the tissues for the presence of the virus, by detecting a rise in antibody titer indicating that an active infection has occurred or by documenting any mild clinical changes that do occur. The polyomavirus vaccine was evaluated for efficacy by exposing vaccinated and unvaccinated birds to viable (live) virus. Unvaccinated birds exhibited lethargy, anorexia and mild diarrhea, developed a rapid increase in antibody titer and the virus was recovered from their tissues. By comparison, successfully vaccinated birds remained clinically unaffected, there was no significant change in antibody titer and the virus was not recovered from their tissues.

If the polyomavirus in the vaccine is killed, why do some aviculturist discuss side effects?

I must assume that "side effects" is referring to the local response (swelling, etc) at the site of vaccination that can occur when the vaccine is administered intradermally (within the skin) rather than subcutaneously (under the skin). ALL vaccines are designed to cause a reaction. When your dog or cat is vaccinated with an inactivated (killed) rabies vaccine, your veterinarian will probably tell you that your animal may limp for several days after the injection or that you may notice a lump under your animal's skin where the injection was given. When you or your child is vaccinated, it is likely that your doctor will tell you that the injection site may be tender or swollen for several days to several weeks but that the reaction should subside without incidence. Any soreness, redness or swelling that may occur at the vaccination site is the body's normal response to the foreign protein (inactivated virus) that has been injected. The key to developing a good inactivated vaccine is to combine an inactivated virus with an adjuvant (a mixing agent designed to stimulate the immune system) and inject them into an animal in a site that causes a mild reaction which stimulates the immune system, without causing any severe reaction that will adversely affect the vaccinate. In developing the avian polyomavirus vaccine, we tested 8 different adjuvants and evaluated the vaccine for intramuscular and subcutaneous use. I have now been using a polyomavirus vaccine in experimental or field settings for more than 6 years. I have vaccinated thousands of birds, one group was vaccinated 5 times during a 7 week period, with no severe reactions. Additionally, a polyomavirus vaccine has now been commercially available for more than 2 years and there have been no reports of severe reactions. If the vaccine is administered subcutaneously, the reaction that the vaccine stimulates in the body will remain undetected. If the vaccine is injected intradermally, a swelling or knot may form at that site of injection. These knots generally resolve without treatment.

If you would like to speak with others who have experience in using the avian polyomavirus vaccine and can provide additional information on the safety and efficacy of this vaccine, you can contact:

Dr. Marg Wissman 813-973-3044

Sue Still 706-855-1373

Elaine Boxdorfer 419-693-7439

Dr. Sam Vaughn 502-245-7863

Dr. Greg Rich 504-455-6386

Why don't we test for polyomavirus before we vaccinate?

The advantage to testing for polyomavirus is that it provides you with more information about your birds. However, after you have performed all the testing, any birds that have not previously been infected with the virus are still susceptible to infection. The advantage to vaccination is that a successfully vaccinated bird will be protected from infection. As an example, the test that is currently available for HIV (the virus that causes AIDS) is quite good, yet we are spending hundreds of millions of tax dollars each year to develop a vaccine that will prevent HIV infections. Why? Because you could be tested for HIV today and be negative; however, if you are exposed to the virus any time after being tested you would still be susceptible to infection. If you were exposed to the HIV virus 1 month after being vaccinated, you would have minimal concern. The same is true for polyomavirus; testing provides information at the time of sampling, vaccination is designed to alter the future by protecting birds when they are exposed to the virus.

As for specifics on whether to test or vaccinate for polyomavirus, there are two types of tests that can be used to provide you with varying information about your bird's status with respect to avian polyomavirus. These two types of tests (a test to detect antibodies to the virus in serum or the DNA probe test to detect virus nucleic acid in excrement or tissues) were developed by members of our research group.^b The DNA probe test that we developed can be used to demonstrate the presence of polyomavirus nucleic acid in the excrement, blood or other tissues. We recommend the use of excrement samples because detecting the virus nucleic acid in the feces provides one with more clinically important information than detecting it in the blood. If you choose to use a test to determine whether or not you need to vaccinate a bird for polyomavirus, then the test you need to use is one that detects the presence of antibodies to the virus.^c If a bird has antibodies to the virus, then it has already been infected and need not be vaccinated.

Having been involved in developing and or evaluating the virus-neutralizing antibody test, the fluorescent antibody test, the DNA probe test and the vaccine for polyomavirus, I can tell you that the best and most economical way to control this virus is through vaccination. As a researcher interested in collecting as much information as I can about the health status of companion birds, I suggest that one test and vaccinate. However, if you are an aviculturist with financial restraints, it is preferable to vaccinate rather than test.

If you are not recommending that budgerigars be vaccinated for polyomavirus, then why is it discussed that budgerigars spread polyomavirus? Is the polyomavirus that infects budgerigars the same as the virus that infects non-budgerigars?

The polyomavirus that infects budgerigars is the same virus that infects non-budgerigars. However, budgerigars respond differently to polyomavirus infection than do non-budgerigars. Non-budgerigars infected with polyomavirus will either die or recover. During the infection and recovery period, non-budgerigars may shed the virus for days to weeks (in rare cases several months) exposing other birds in the aviary to the virus. By vaccinating, you create a barrier that prevents new birds from being infected which reduces the uncontrolled spread of the virus within a particular population of birds.

Controlling polyomavirus infections in budgerigars presents a different set of problems. A budgerigar infected with polyomavirus has been considered (correctly or incorrectly) to be infected for "life" and can shed the virus for long periods (months). In some flocks, it has been shown that 100% of the budgerigars have been infected with polyomavirus.¹⁸ Fortunately, not all budgerigar flocks have such a high level of polyomavirus activity. However, the high prevalence of polyomavirus in some budgerigar flocks, and the cost of the vaccine makes it difficult to economically justify in commercial budgerigar flocks. None-the-less, it is important for the avicultural industry to control polyomavirus in budgerigars because this group of birds is probably serving as a reservoir for the virus. For the past 2 years, we have been evaluating an economically feasible vaccination program to control polyomavirus in budgerigars. We are currently testing this program in flocks of budgerigars and our data is encouraging. If our data continues to be positive, we could have a vaccination program that would allow budgerigar producers to establish and maintain polyomavirus vaccinated budgerigars within several years.¹⁹

Will the vaccine ever be less expensive?

According to the vaccine's manufacturer, the wholesale cost of a vaccine is completely dependent on the volume of vaccine used. The cost of producing, testing and distributing a vaccine is relatively fixed. Basically, each dose of the vaccine that will be used is divided into the fixed cost of production to determine the price per dose of vaccine. If we as a community were using as many doses of the polyomavirus vaccine as the chicken industry (tens of millions of doses per week) the vaccine would be pennies. If we were using as many doses of the vaccine as dog and cat owners (tens of millions of doses per year) the vaccine would cost dollars. As it is, we are only using tens of thousands of doses of vaccine per year. While I cannot speak for the manufacturer of the vaccine, I feel certain that the cost per dose would be substantially less if a group of veterinarians or aviculturists contracted with the manufacturer for the production of 500,000 doses of the vaccine per year.

If I vaccinate all of my birds, is there a 100% chance that I will never have polyomavirus in my closed aviary?

We do not have a 100% chance that a mechanic will properly repair a dysfunctional car, and repairing a car is a "cake walk" compared to predicting the interaction of a vaccine, an animal and a particular virus. My 1984 Blazer has been to the mechanic (an excellent mechanic I might add) 3 times over the past year to repair a clutch problem. What were the chances that the clutch would be repaired with the first attempt? Certainly, there is no such thing as 100% when dealing with biologic systems (birds, viruses, vaccines, etc). However, we have not seen a polyomavirus outbreak in a properly vaccinated aviary (adults and neonates vaccinated) and that includes aviaries in which we vaccinated to stop an ongoing polyomavirus problem.¹ We used three aviaries in the field evaluation of the polyomavirus vaccine that were experiencing severe polyomavirus outbreaks at the time of vaccination. One aviary lost approximately 30% of their neonates in two consecutive breeding seasons. The affected aviculturist was preparing to leave aviculture and I convinced them to try one more breeding season using the vaccine. Following vaccination, they have had two very successful breeding seasons without a single loss to polyomavirus. In another aviary, polyomavirus killed 90% of the neonates in a 4 month period. In the two subsequent breeding seasons after vaccination, the flock has produced more than 500 chicks with no deaths from polyomavirus.¹

Can a vaccinated bird "test positive" for polyomavirus?

Yes or no depending on the type of test that is used. To understand the answer to this question, it is critical that the reader comprehend that the polyomavirus vaccine contains whole virus (the viral proteins and its nucleic acid) which has been chemically inactivated (similarly, most rabies vaccines contain whole rabies virus which has been chemically inactivated). Because the virus in the polyomavirus and rabies vaccines have been chemically inactivated, they will not cause disease in a vaccinated animal. However, vaccination can cause any test which is designed to demonstrate virus exposure to be positive (remember a vaccinated bird is exposed to inactivated polyomavirus and a vaccinated dog or cat is exposed to inactivated rabies virus, thus a test designed to detect exposure can be positive). Vaccination will not cause a positive test in any assay (culture, histopathology) designed to document an active virus infection.

Any bacteria, virus, antibiotic, toxin, chemical, etc. which enters the body (entering the body would be defined as passing through a mucosal barrier) is taken by various components of the blood and filtered out of the body through the liver, spleen or kidneys. While an agent is present in the blood and is being filtered out of the body, it can be detected by various types of tests. DNA probe-based tests are particularly sensitive at detecting small quantities of target nucleic acid in a sample. For example, the DNA probe test for polyomavirus can detect as few as 10 copies of the small segment of viral DNA it is designed to detect. When correctly used, a test which can detect such a small quantity of target is extremely useful. When improperly used, a DNA probe-based test is subject to misinterpretation. DNA probe tests indicate only that the small segment of nucleic acid they are designed to detect is present in a sample, they do not indicate whether or not viable virus is present in the sample.^14,15,18 When a bird is vaccinated, it is injected with more than 1,000,000 virus particles, each of which can contain inactivated nucleic acid. A DNA probe test will detect this inactivated nucleic acid. The immune response that the vaccine is designed to elicit occurs when defensive cells in the blood "engulf" the inactivated virus and are then filtered out of the body primarily through the spleen. While these cells are being cleared out of the body, a DNA probe test performed on blood or the cut surface of the liver, spleen or kidney could be positive because it has properly detected the presence of target nucleic acid, even though the nucleic acid is inactivated.

Vaccination should not cause a DNA probe test performed on excrement to be positive. However, if a vaccinated bird swallows polyomavirus and then a swab of the excrement is tested for viral nucleic acid, then the swab could be positive. Consider that the gastrointestinal tract is a long hose which starts at the mouth and ends at the cloaca. If you were to swallow polyomavirus (for analogy purposes only, do not try this at home) and test your feces for the presence of polyomavirus nucleic acid using a DNA probe test, your feces might be positive. Vaccines are designed to stimulate the immune response and any assay designed to detect antibodies to the virus should be positive for a defined period after vaccination.

Is an aviculturist more likely to have problems with polyomavirus if they overbreed their birds?

Continued or excessive breeding would be expected to place increased stress and nutritional demands on a hen. Any stress or malnutrition might decrease the natural resistance of a bird to any infectious agent. However, as discussed above, polyomavirus is capable of infecting a "healthy" bird suggesting that any stresses associated with breeding would be of minimal importance in the epizootiology of the virus within a breeding population. For the general health of your birds, it is best to prevent overcrowding, maintain excellent air quality (no cigarette smoke, no disinfectant fumes, plenty of fresh air) and provide a high quality formulated diet.

Is a polyomavirus infection the result of a "broken down" weakened immune system?

Polyomavirus is capable of infecting a "healthy" bird, just as parvovirus is capable of infecting a "healthy" dog and HIV is capable of infecting a "healthy" person. In a broad simplified manner, the immune system can be considered responsible for protecting an animal from all infectious agents. Excepting this generalized view of the immune system, all disease involves a failure of the immune system to properly do its job. Vaccines are used widely in humans, dogs, cats, horses, cattle, chickens etc, to help the immune system protect the body from infectious agents.

How widespread is polyomavirus in companion birds?

As mentioned above, budgerigars are considered to be the reservoir for polyomavirus and any area in which budgerigars are housed should be considered contaminated with polyomavirus. The reported seroprevalence of polyomavirus in non-budgerigars varies from 10% to 63% depending on the flock. Considering all the data in the literature, the average seroprevalence rate of polyomavirus in non-budgerigars is about 30%. By comparison, the seroprevalence of parvovirus in dogs in 1984 was about 50%, there were less than 600 cases of rabies reported in domestic animals in 1987 (there are about 100 million dogs and cats in the United States), and HIV is thought to infect 1 in 1000 (0.1%) people in the United States. Thus, polyomavirus activity is at least as common as parvovirus in dogs, less common than rabies activity in dogs and cats and less common than HIV infections in people residing in the United States. In a study of necropsy submissions designed to determine the most common problems in psittacine birds, polyomavirus was found to be a leading cause of death in psittacine birds.¹⁷

Acknowledgments

Major sustained contributions that have made this work possible have been provided by the Cowan Avian Health Foundation, the International Avian Research Foundation, Veterinary Medical Experiment Station, Joe and Sue Still, Terry Clyne, Richard and Luanne Porter, Knick Enterprises, Bobbi Brinker, Kathleen Szabo, International Aviculturists 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, Georgia Cage Bird Society, Greater Brandon Avian Society, Hookbill Hobbyists of Southern California, Kentuckiana Bird Society, Kenosha Exotic Bird Club, Louisiana Aviculture Society, Northwest Ohio Exotic Bird Club, South Jersey Bird Club, Wasatch Avian Education Society, West Valley Bird Society and Zeigler Brothers Inc. Hundreds of aviculturists, bird clubs and veterinarians have also made significant contributions.

a. Polyomavirus vaccine, Biomune, Inc., Lenexa, KS 66215, 913-894-0230.

b. DNA probes for PBFD virus and avian polyomavirus, Infectious Diseases Laboratory, University of Georgia, Athens, GA 30602, 706-542-8092, Avian/Wildlife Laboratory, University of Miami, Miami, FL, 33101, 800-232-1056, and Zoo and Aquatic Veterinary Group, Winchester, S0237LS, England.

c. Virus neutralizing assay for avian polyomavirus, Infectious Diseases Laboratory, University of Georgia, Athens, GA 30602, 706-542-8092.

Table 1 - Common name³³ of psittacine birds in which a polyomavirus vaccine has been evaluated for safety.

 Table 2: Seroconversion (greater than 4-fold increase in VN antibody titer) by 2 weeks after the second vaccination with inactivated avian polyomavirus in birds that were seronegative (titer < 10) or seropositive (titer > 16) prior to vaccination.^20,22

Table 3: Number of various types of birds that died in 9 flocks experiencing polyomavirus epornitics. Note: not all deaths that occurred are reported by genera. ¹

Macaws 89

39 blue and gold macaws,

18 military macaws,

3 scarlet macaws

6 greenwing macaws

12 severe macaws

10 Hahn's macaws

Cockatoos 24

8 umbrella cockatoos

10 Moluccan cockatoos

6 lesser sulphur-crested cockatoos

Amazon parrots 16

6 double yellow-headed Amazon parrots

2 orange-winged Amazon parrots

3 yellow-naped Amazon parrots

2 red-lored Amazon parrots

3 blue-fronted Amazon parrots

African grey parrots 4

Conures 60

45 sun conures

6 blue-crowned conures

Cockatiels 8

Lovebirds 8

Rosellas 6

Bourke's parakeets 6

Eclectus parrots 54

Princess of Wales parakeets 4

Senegal parrots 2

Table 4: Summary of pre- and post-vaccination mortality, costs and production in flocks experiencing polyomavirus epornitics.¹

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Genetic Vaccine For Metastatic Breast Cancer Shows Promise In Mice Studies

Scientists at the University of Nebraska Medical Center have developed a genetic vaccine for metastatic breast cancer and other tumors, which shows great promise in early studies in mice. The findings are reported in the Nov. 15 issue of Cancer Research, a leading cancer research journal.

The vaccine uses a combination of components that are being used in existing clinical trials for other diseases. The approach they developed combines DNA and adenovirus gene delivery mechanisms. Adenovirus is a common virus naturally found in the lungs. Together, the combination can deliver the p53 gene as a vaccine for breast cancer. P53 is a tumor suppressor factor that changes in 50 percent of all cancers, including lymphoma, leukemia, breast, lung, colon and prostate cancer.

"This is a unique genetic vaccine," said James Talmadge, Ph.D., professor of Pathology and Microbiology at UNMC and principal investigator on the study. "The results we saw in our mice studies were quite dramatic and provide encouragement that we are potentially on to something that could have significant implications in humans."

The vaccine uses a combination of components that are being used in existing clinical trials for other diseases. The approach they developed combines DNA and adenovirus gene delivery mechanisms. Adenovirus is a common virus naturally found in the lungs. Together, the combination can deliver the p53 gene as a vaccine for breast cancer. P53 is a tumor suppressor factor that changes in 50 percent of all cancers, including lymphoma, leukemia, breast, lung, colon and prostate cancer.

"This is a unique genetic vaccine," said James Talmadge, Ph.D., professor of Pathology and Microbiology at UNMC and principal investigator on the study. "The results we saw in our mice studies were quite dramatic and provide encouragement that we are potentially on to something that could have significant implications in humans."

Dr. Talmadge is director of the Laboratory of Transplantation Immunology at UNMC. First author of the study was Prahlad Parajuli, Ph.D., a post-doctoral fellow in Dr. Talmadge's lab who now works at the Karmanos Cancer Institute, Wayne State University, Detroit.

The study involved multiple groups of mice all with metastatic breast cancer. Metastasis is the process by which cancer spreads beyond the organ in which it originated. One group received no vaccine. The second and third groups received individual components of the vaccine and the fourth group received both vaccine components.

The non-vaccinated mice all died within 30 days. Mice receiving individual components of the vaccine lived for up to 60 days, while mice receiving both components of the vaccine were cured 40 percent of the time.

"The work by Dr. Talmadge is very promising," said Kenneth Cowan, M.D., Ph.D., director of the UNMC Eppley Cancer Center. "The development of a vaccine to prevent breast cancer recurrences would represent an important addition to clinical therapy for breast cancer, a disease that will affect more than 180,000 women in the U.S. this year. Since p53 is commonly altered in many other cancers, this vaccine could have very far reaching implications for cancer prevention." According to the National Cancer Institute, 1 in 8 women in the United States (approximately 12.8 percent) will develop breast cancer during their lifetime. For those who develop metastatic breast cancer, only 35 percent will survive two years or more.

Tumor associated antigens, such as p53, are molecules found on the surface of tumor cells. They can stimulate a unique subset of white blood cells to respond to and kill tumor cells.

"Using the combination of DNA and adenovirus is critical," Dr. Talmadge said. "The adenovirus can stimulate a host response to itself and can deliver a large amount of the p53 gene, but it can't be used to boost the immune response. In contrast, while the DNA portion of the vaccine does not induce a strong immune response, it can boost the immune response initiated by the adenovirus delivered vaccine."

Dr. Talmadge said the adenovirus used in these studies is "safe," as it is unable to reproduce in humans or mice. The adenovirus is produced by Canji, a biotech company in San Diego, which is affiliated with Schering-Plough Corporation.

The breast cancer vaccine also used a growth factor, Flt3L ligand, to induce a strong immune response, he said. This growth factor is being studied clinically by Immunex Corporation, a biopharmaceutical company in Seattle. The additional benefit of Flt3L administration in this study is consistent with the potency of this cytokine as an immunological adjuvant.

"While additional work is needed to further improve this therapeutic approach in both the preclinical and clinical settings, the overall approach of using genetic vaccines is quite promising," Dr. Talmadge said. "By taking advantage of the unique attributes of each delivery system, in combination with growth factors, it appears to improve the immune response to a tumor antigen and ultimately extend survival.

"The availability of the individual vaccine components suggests that there is the potential for a rapid translation to clinical studies." Dr. Talmadge said he hopes clinical trials in humans can