Prevention of Avian Polyomavirus Infections through Vaccination (January 1996)

Dedicated to the Birds of the World

Excerpted and modified from the Proceedings of the International Aviculturists Society, January 1996, Orlando FL

Prevention of Avian Polyomavirus Infections through Vaccination (January 1996)

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

University of Georgia

College of Veterinary Medicine

Athens, Ga 30602

Reprinted in part with permission from Avian Viruses: Function and Control

Wingers Publishing, Lake Worth, FL, 1995.

Introduction

Budgerigar fledgling disease (BFD), caused by an avian polyomavirus, was first reported in the United States and Canada in 1981.¹ Subsequently, polyomavirus infections have been described worldwide in various nonbudgerigar psittacine birds, finches and gallinaceous birds. While the viruses that infect these differing species appear to be morphologically and antigenically similar, the clinical presentation, distribution of lesions and epizootiology of infections vary dramatically in differing species.^2-8

Peracute death with no premonitory signs is the most common clinical finding in young, nonbudgerigar psittacine birds affected by avian polyomavirus. Acute infections are characterized by death following a 12-to 48-hour period of clinical changes that may include depression, anorexia, weight loss, delayed crop emptying, regurgitation, diarrhea and subcutaneous hemorrhage.^{3, 6, 8-10}

Neonates with polyomavirus infections may bleed profusely, or for a prolonged period, from intramuscular injection sites or from follicles where feathers have been removed. Subcutaneous hemorrhage over the crop and across the skull is common.¹¹ While a propensity to bleed abnormally is suggestive of a polyomavirus infection, this clinical finding is not diagnostic for the disease. Any disease involving vasculitis, clotting disorders or damage to the liver can cause similar hemorrhaging.

All species of psittacine birds or finches should be considered susceptible to the virus. It has been suggested that polyomavirus-induced disease occurs most commonly in young budgerigars, macaws, conures, eclectus parrots, lovebirds, ring-necked parakeets and caiques.^{3, 8, 12, 13} In Australia, polyomavirus infections are considered particularly common in lovebirds.¹³ In comparison to the species listed above, polyomavirus-induced disease is considered less common in young cockatoos, grey-cheeked parakeets, lories, African grey parrots, hawk-headed parrots and Amazon parrots.^{3, 6, 8, 13} It should be cautioned, however, that reported variances in susceptibility may represent a skew in the population of exposed birds and not an actual difference in susceptibility.

Gallinaceous birds also appear to be susceptible to polyomavirus. A virus that morphologically resembles a polyomavirus was recovered from the intestinal contents of asymptomatic turkeys. The recovered virus did not cause a discernible disease in experimentally infected birds.¹⁴ A polyomavirus-like agent was identified in the feces of an ostrich located in the southeastern United States (Georgia Vet Diagnostic Laboratory). A polyomavirus with similarities to BFD virus was recovered from the drinking water and feces associated with a chicken layer replacement farm in Germany. It was not determined if the virus in this poultry house originated from chickens, or was a contaminant from another source,¹⁵ but, serologic studies suggest that chickens are exposed under natural condition. Polyomavirus-specific antibodies have been demonstrated in broiler chickens from central Europe and the United States.¹⁵ (Ritchie, et al unpublished) During a polyomavirus epornitic in a mixed species aviary, virus-neutralizing antibodies were detected in 2 golden pheasants and a Lady Amhurst pheasant that had been naturally exposed to affected psittacine birds, while a potentially exposed bantam chicken and 2 toco toucans remained seronegative. (Niagro, et al, unpublished data) Inclusion bodies suggestive of polyomavirus have been described from Australia in a Kakariki, a peaceful dove, a brown pigeon and a canary. (Reece, personal communication)

Experimental infections

Both budgerigar and nonbudgerigar psittacine birds have been shown to be susceptible to experimental polyomavirus infections; however, the characteristic disease has only been induced in some budgerigars. Experimentally infected 3-to 10-day-old budgerigar neonates died 11 days after being given BFD virus intramuscularly.¹⁶ When 25-day-old budgerigars were exposed intranasally to virus collected from the skin of diseased birds, they developed microscopic lesions characteristic of a polyomavirus infection but remained clinically normal. Budgerigars of a similar age that were given the same virus preparation subcutaneously, also remained clinically normal and did not develop any gross or microscopic changes suggestive of a polyomavirus infection.¹

In one trial, young seronegative budgerigars seroconverted within 16 days after being placed in the same enclosure with seropositive birds. Seronegative budgerigars also seroconverted when they were placed in enclosures adjacent to those containing seropositive birds. These findings suggest that direct and indirect transmission resulting in subclinical infections can occur in budgerigars.⁵

Avian polyomavirus isolated in cell culture from budgerigars and administered orally or intramuscularly to blue and gold macaw chicks induced infections, but the birds remained subclinical.¹⁷ When blue-crowned conure chicks, 40-to 50-days-old, were exposed to avian polyomavirus by intramuscular inoculation, they seroconverted, shed virus intermittently for 7 to 14 days and remained clinically normal. (Ritchie, et al, unpublished data) Mature Amazon parrots, African grey parrots and cockatoos exposed to avian polyomavirus by the intramuscular or intravenous routes will seroconvert and shed virus intermittently in the feces. Birds infected by the intravenous route develop transient diarrhea 5 to 10 days after inoculation, then recover. (Ritchie, et al, unpublished data) Birds experimentally infected with liver homogenates containing avian polyomavirus have been found to respond in a similar fashion to birds that are experimentally infected with cell-culture derived virus. (Ritchie, et al, unpublished data)

The effect of avian polyomavirus (BFDV) on experimentally infected chickens varies dramatically with the age of exposure. Chicken embryos infected at 10 days of age died 10 days later, and had gross and histologic lesions characteristic of the disease. In contrast, chicken embryos infected at 11 and 12 days of age remained normal, developed precipitating antibodies that could be detected 2 weeks after hatching, and did not develop gross or microscopic changes suggestive of an infection.¹⁸

Two-week to four-month-old broiler and specific-pathogen-free (SPF) chickens injected with avian polyomavirus by the intramuscular or intravenous routes developed virus-neutralizing antibodies, suggesting that they had been infected. Some experimentally infected chickens developed transient diarrhea but otherwise remained clinically normal. None of the experimentally infected chickens developed gross or histologic changes suggestive of a polyomavirus infection. (Ritchie, et al, in preparation)

Age susceptibility

Nonbudgerigar psittacine neonates are considered to be highly susceptible to polyomavirus infection and the diseases it can cause. Infections may occur in either parent-or hand-raised neonates;^{3, 8, 11} however, disease may be more common in hand-raised chicks.¹⁹ In nonbudgerigar psittacines, clinical signs are most common at the time of weaning; however, neonates from 10 to 150 days of age have been reported to be susceptible to naturally induced avian polyomavirus infections and disease.^{3, 11, 12, 15} Reported mortality (death) rates vary from 31% to 41% of the at-risk young birds.^{3, 11} However, clinical experience suggests that 80 to 100% of the young birds in some highly susceptible populations may die.

In most cases, older psittacine birds (greater than 1 month in budgerigars and greater than 5 months in nonbudgerigar psittacine birds) exposed to avian polyomavirus seroconvert and remain clinically normal. However, occasionally, adult psittacine birds may die acutely with lesions suggestive of a polyomavirus infection.^{9, 13, 20, 21}

No one has determined why most adult birds exposed to polyomavirus seroconvert following infection by this virus, while some develop clinical abnormalities and die. Factors that govern the susceptibility of young and adult birds to avian polyomavirus-induced disease might include the route of virus exposure, the quantity of virus to which the bird is exposed, a pre-existing resistance to disease based on previous exposure to the virus, a selective immunosuppression in the affected birds or the occurrence of strains of polyomavirus with increased virulence for certain species.

Incubation

A definitive incubation period for avian polyomavirus infections has not been confirmed in nonbudgerigar psittacine birds because experimentally infected individuals do not develop the clinical signs of disease that are characteristic in naturally acquired infections. Based on clinical observation, the incubation period of polyomavirus in nonbudgerigar psittacine birds has been estimated to be as long as 14 days but may be as short as 2 days.^{3, 11, 22} Budgerigar fledglings with naturally acquired infections show peak mortality rates between the 15th and 19th day of life, suggesting that the incubation in this species may be less than 15 days. Budgerigar neonates experimentally infected by intramuscular inoculation died 11 days after being exposed to the virus.¹⁶

Prevention

Detecting nonbudgerigar psittacine birds that are subclinically infected with avian polyomavirus is difficult, at best. Neither normal appearance nor the presence of antibodies correlate in a simple manner with the likelihood that these birds will shed virus. Of 106 serum samples collected from a group of breeding nonbudgerigar psittacines, 33% were positive (titer greater than 1:10), 20.7% were suspect (titer of 1:10) and 46.3% were negative (titer less than 1:10). Adults from one flock that were exposed to diseased birds seroconverted (developed antibody titers) and raised seronegative, normal young in two subsequent breeding seasons.³ In another aviary, antibody titers were detected in 10 of 15 (67%) blood samples taken from birds ranging in age from 6 weeks old to adults.¹¹ During a polyomavirus outbreak, 32 of 76 (42%) of the birds in the aviary had virus-neutralizing antibody titers that ranged from 1:20 to 1:1280. All of the seropositive birds were clinically normal, yet 5 of the seronegative birds and 3 of the seropositive birds excreted polyomavirus in their feces.²³

Polyomavirus nucleic acid can be detected in cloacal swabs taken from nonbudgerigar psittacine birds during a flock outbreak.^{23, 24} During one outbreak in a group of mixed Psittaciformes, 41 of over 200 birds of 35 different species were found to be shedding polyomavirus in their excrement during the peak of an epornitic. However, only 3 of these 41 birds were still shedding detectable quantities of virus when they were retested 60 days later, indicating that viral shedding is transient. Nucleic acid also has been detected in the excrement of experimentally infected psittacine chicks starting from 2 to 7 days following intramuscular inoculation.¹⁷ The recovery of viral DNA from the cloaca suggests that the virus could be shed from gastrointestinal, renal or reproductive tissues.^{17, 23, 24}

Vaccination

Because of the difficulty in identifying birds that are subclinically infected with avian polyomavirus, use of an effective vaccine is the best way to prevent infections. The is a body of findings derived from observations during natural outbreaks and from experimental trials that support the value of vaccination. During epornitics in mixed psittacine bird collections, infected survivors and asymptomatic birds exposed to them have been shown to develop polyomavirus-neutralizing antibodies.^{3, 10, 11} Seronegative young adult birds will seroconvert when housed adjacent to seropositive breeding adults, indicating that an antibody response does occur following natural exposure to the virus. The detection of virus-neutralizing antibodies in flocks of birds in which individuals are clinically normal suggests that many infections are subclinical.^{3, 5, 10, 11, 25} Collectively, these findings suggest that some exposed birds are able to mount an effective immune response.

If a natural immunity to disease occurs, then it should be possible to induce a similar protective immunologic response through vaccination. Experimental studies have indicated that vaccination does induce an immunologic response that is protective. In one study using blue and gold macaw chicks, an inactivated avian polyomavirus vaccine elicited polyomavirus-neutralizing antibodies in all the vaccinates. The induced immunologic response protected the vaccinated chicks from subsequent challenge with live virus.¹⁷ In other studies, an inactivated avian polyomavirus vaccine was shown to protect Amazon parrots, cockatoos, African grey parrots, lovebirds and chickens from infection.

The safety and immunogenicity of avian polyomavirus vaccines, administered either intramuscularly or subcutaneously, have been evaluated in several flocks. In one study, a group of 233 mixed species Psittaciformes that ranged in age from 12 weeks old to greater than 5 years old were vaccinated. In another flock, a group of 169 adult, mixed species Psittaciformes were vaccinated. Vaccination stimulated a marked virus-neutralizing antibody response, particularly in birds that had been seronegative prior to vaccination. The results of two immunogenicity trials are listed in Table 1.

Table 1. Pertinent data from 2 avian polyomavirus vaccination field trials.

Over 5000 doses of an inactivated avian polyomavirus have been administered to numerous different species of Psittaciformes, and serious reactions have not been observed in any vaccinates. Three types of reactions, yellowish discoloration of the skin, thickening of the skin or formation of a mass are expected at the site of subcutaneous vaccination. These reactions are a response to some protein in the vaccine (hopefully the polyomavirus proteins), and the majority of changes resolve without treatment 3 to 6 weeks post-vaccination. Similar reactions undoubtedly occur in many mammals vaccinated with products containing adjuvants, but the reactions are difficult to visualize because of the fur and thickness of the skin. In one field trial, some cockatoos and macaws experienced a heavy molt of up to 10 days duration that started 3 to 5 days after the second vaccination. It could not be determined if this molt occurred in response to vaccination, the stress associated with handling or climatic and other external factors. The molt was uneventful and appeared to have no adverse affect on the vaccinates.²⁷

The safety of a vaccine intended for widespread use has been established by performing field trials in flocks in which the virus-neutralizing antibody titers to avian polyomavirus were not determined prior to vaccination. From previous studies, it was expected that many of the birds in otherwise stable flocks would have pre-existing neutralizing antibodies.²³ In one flock, 63% of the vaccinates were considered to have been previously exposed to avian polyomavirus because of the detection of virus-neutralizing antibodies prior to vaccination; in another flock, 26% of the vaccinates were seropositive. None of the vaccinates with pre-existing neutralizing antibodies developed an adverse reaction following vaccination.²⁷ A similar vaccine was shown to be safe in naturally infected birds, even when the birds were vaccinated 5 times in a 49-day period.²⁶

It has been suggested that some of the histologic changes that occur in the kidneys of nonbudgerigar psittacine birds are caused by an immune-mediated process.²⁸ However, these observations were based on uncontrolled field cases and are not supported by experimental data. After some nonbudgerigar psittacine birds with pre-existing polyomavirus-neutralizing antibodies are vaccinated, they will develop an increase in antibody titer. Some vaccinates have been found to develop antibody titers that are extremely high (greater than 1:16,000) yet remain clinically normal, even when followed for over 2 years after vaccination. If neutralizing antibodies to polyomavirus were involved in the disease process, some of these experimentally vaccinated birds might be expected to be adversely affected, but in fact, they all remain clinically normal.^{26, 27} Additionally, blue and gold macaw chicks, blue-crowned conure chicks, Amazon parrots and chickens that are experimentally infected with BFD virus will seroconvert and some of these individuals develop high neutralizing antibody titers (greater than 1:640).¹⁷ Experimentally infected blue and gold macaw chicks remained clinically normal 3 years after infection. Experimentally infected blue-crowned conure chicks and adult Amazon and African grey parrots remained clinically normal 18 months after infection. (Ritchie, et al, in preparation)

Given the prevalence of polyomavirus infections in companion birds, as indicated by the detection of virus-neutralizing antibodies, it is noteworthy that an inactivated avian polyomavirus vaccine intended for widespread use does not cause adverse reactions in vaccinates. Aviculturist can find receive information about the vaccine or find a veterinarian in their are that is using the vaccine by calling the manufacturer (Biomune, Lenexa, KS. 913-894-0230). Briefly, it is recommended that all of the birds in an aviary or home be vaccinated. This vaccination regime will help break the cycle of virus transmission by decreasing the number of new birds that are susceptible to infection. Adult breeding birds should be vaccinated twice with a 2 to 3 week interval between doses. It is best to vaccinate these birds several months before the onset of breeding. Chicks should be vaccinated twice at a 2 week interval. It is important that young birds receive their last booster at least 2 weeks before they are shipped, or exposed to other birds. Companion birds are vaccinated twice with a 2 to 3 week interval between doses.

Aviculturists with large breeding facilities rarely handle or evaluate the overall health of their adults. The infrequent attention provided to these adults allows some problems such as liver disease, kidney disease, heart disease, cancers and proventricular dilatation disease to slowly progress in what appears to be clinically normal birds. Many of these hidden problems will be detected or exacerbated during the handling procedures necessary for vaccination. In the best managed aviaries in which adult birds are rarely handled, experience suggests that during the vaccination process pre-existing medical problems will be identified in from 2% to 4% of the adults.

As is the case with many other 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 compensate for the deleterious effects of poor management or hygiene. The techniques recommended for decreasing the occurrence of avian polyomavirus infections are listed:

Techniques for preventing or controlling polyomavirus outbreaks in the nonbudgerigar psittacine nursery.

Prevention

• Vaccinate susceptible adults and neonates.
• Never ship or accept an unvaccinated bird.
• Do not maintain budgerigars, cockatiels or lovebirds in the same airspace with unvaccinated psittacine neonates.
• Clean and disinfect the nursery environment regularly.
• Ship only weaned birds.
• Use biosecure shipping containers to prevent virus exposure during transport.
• Maintain a closed aviary, and strictly limit visitations by non-aviary personnel.
• Never return a neonate to the nursery if it has been exposed to other birds.
• If new birds must be added to the flock, vaccinate and quarantine them for a minimum of 60 to 90 days.
• Never mix neonates from multiple sources in the same airspace.
• Use separate feeding instruments for each bird.
• Never use a feeding utensil and place it back in a common food container.

Control

• Isolate clinically affected birds.
• Carefully vaccinate exposed birds making sure that virus transmission is not facilitated by the handling procedure.
• Never place a clinically ill bird in the same airspace with birds in the nursery.
• Isolate birds in direct contact with clinically ill birds or those that are shedding.
• Completely clean and disinfect the nursery environment.
• Use the DNA probe to test the nursery environment for viral-contamination.

Given the extremely high prevalence of persistent polyomavirus infections, and the frequency with which budgerigars can shed the virus,²⁹ one should never maintain young nonbudgerigar psittacines in the same airspace with budgerigars. The potential for intraspecies transmission of polyomavirus may be a particular problem for pet retailers that maintain both large and small psittacine birds.

Controlling an outbreak

For maximum security, birds vaccinated for avian polyomavirus should not be considered to be protected from infection until 2 weeks after they have received a booster. Thus, vaccination alone cannot be expected to control an outbreak. A DNA probe-based test (Infectious Diseases Laboratory, Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7386) is extremely 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 prevent further transmission, while vaccinated birds are developing antibodies to the virus. By testing cloacal swabs of a bird at the time of death one can determine whether it is shedding virus, which in turn will help determine whether its environment may have been contaminated. If the environment is contaminated, then there is a potential for viral amplification in a susceptible population. If an infected bird dies soon after infection, it may not be shedding virus at the time of death, and thus the bird's environment may not be contaminated with virus. Birds that are clinically ill, are found to be shedding polyomavirus, or are in direct contact with birds that are clinically ill or shedding polyomavirus should be isolated (placed in a separate geographic location) from birds that are clinically normal and not shedding virus.^{8, 9, 22}

Acknowledgements

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, Terry Clyne, Richard and Luanne Porter, Knick Enterprises, Ted Lafeber, Kathleen Sazbo, Allen Berk, Bobbi Brinker, International Aviculturists Society, Avian Research Associates, Midwest Avian Research Exposition, Gateway Parrot Club, Kentuckian Bird Society, Hookbill Hobbyists of Southern California, Greater Brandon Avian Society and Zeigler Brothers Inc. Hundreds of aviculturists, bird clubs, and veterinarians have also made significant contributions. The authors thank Sam Vaughn, Don Harris, Dave Rupiper, Ray Anderson, Greg Harrison, Heather Wilson, Joel Murphy, Sue Sattler, Sue Toper, Diane and Micheal Perry, Terry Clyne, Richard and Luanne Porter, Jeanie and Scott Anderson, Bill Bennett, Brett Blanchard, Mary Ervin, Kathy Murphy, Kristen Johnson, Sherri and Aaron Jones, Will Pace, Don Sanders, Debbie Seaman, Pat Terry, Donna Travers, Bridgett Trulove, Marcus Valentine, Cynthia Webb and Diane Wolff for technical assistance in testing this vaccine.

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