Avian Disease

The avian polyomavirus is one of the most significant viral pathogens of cage birds. It results in substantial economic losses for aviculturalists and pet store owners each year. The biology of this virus is complex and as a result veterinarians and aviculturalists alike are often very confused about how to best prevent this virus infection and once confronted with it, how to minimize its impact. This confusion is exacerbated by the current debate that is on going in the research community about the nature of this virus and its control. The two sides of this debate are represented by Dr. Branson Ritchie and members of his research team at the University of Georgia, on the one hand and on the other by Dr. Jack Gaskin at the University of Florida, Drs. Bob Dalhausen and Steve Radabaugh at Research Associates in Ohio, and myself. Dr. Ritchie has used many forums to discuss his views and feels strongly that vaccination is an important and economically feasible means of control of this disease. Dr. Gaskin, in a letter to the editor of the Journal of Avian Medicine and Surgery has expressed some serious reservations about the usefulness of a polyomavirus vaccine.15 The issue of testing and its value and which test to use has also be a source of contention. It is the purpose of this document to address these issues, both for the aviculturalist and the veterinarian. I feel that his article is timely, as our knowledge biology and behavior of this virus has grown significantly in the past few years.

The avian polyomavirus was first recognized in the early 1980's in the southeastern and southcentral United States4,9,10 and in Ontario, Canada in budgerigars.2,3 It was called the Budgerigar Fledgling Disease Virus. It was found to be a nonenveloped, DNA virus and based on its size, shape, and DNA content it was classified as a papovavirus.2,3,4,9,10,49 The Papovaviridae contain two very different virus families, the papillomaviruses and the polyomaviruses. With further investigation, it was determined that the Budgerigar Fledgling Disease Virus is a polyomavirus. 26,29,57 Subsequently, the virus was found to infect many different species of psittacine birds (parrots) and thus it is generally the convention to call it the avian polyomavirus (APV).5,17,20,35,45 APV is wide spread and can be found in most countries of the world where psittacine birds are raised.29,31,32,59,61 As will become clearly apparent, generalizations about this virus cannot be made and over simplification about the issues of infection and disease, while convenient, are often misleading.

AVIAN POLYOMAVIRUS DISEASE

Budgerigars In the budgerigar, disease and death is confined to nestlings between 10 and 25 days of age.2,3,4,9 Budgerigar breeders first detect this problem in their flocks when there is a sudden increase in the number of dead nestlings in the nest boxes. The signs of APV disease in budgerigar nestlings are somewhat variable. Most often, the young birds experience an abbreviated course of disease. At death, birds are found to be stunted, to have abnormal feather development, skin discoloration, abdominal distension, ascites (fluid in the abdomen), enlargement of the liver with localized areas of hepatic necrosis (cell death), and scattered areas of hemorrhage. In some outbreaks, the virus attacks the cerebellum (a portion of the brain) and these birds will show head tremors. Microscopic examination of the tissues from these birds reveals virus inclusion bodies in cells of multiple organ systems, including the liver, spleen, kidney, feather follicles, skin, esophagus, brain, and heart.2,3,4,9,10,28,29

Not every budgerigar infected with APV will die. Some survivors will never become outwardly ill and will show no signs of infection. Other nestlings will fail to develop their primary and secondary wing feathers and/or their tail feathers.3,18,22 These birds have been referred to as runners or creepers and this form of the disease has been described as French molt. It is extremely important to note that another virus, the Psittacine Beak and Feather Disease Virus, can also cause similar signs. It is possible that one or more additional diseases, may also cause feather disease in nestling budgerigars.

Not all budgerigars appear to be equally susceptible to infection and disease. In one study in the United States, English budgerigars were rarely found to be infected with APV although they were housed with other birds shedding the virus.44

Nonbudgerigar parrots

Nonbudgerigar parrots are also susceptible to avian polyomavirus infection.5,17,20,32,33,58 Some are highly susceptible to disease, while others rarely if ever develop disease (Table 1).37 APV-disease in these birds occurs at different ages in different birds (Fig. 1). In conures, deaths typically occur in birds less than 6 weeks of age. Deaths in macaws and eclectus parrots occur in birds 14 weeks and younger. Most, possibly all, of the nestlings lost are being hand fed.5,17,20,39 Infected nestlings appear healthy, show very few premonitory signs, and then die suddenly. When signs do occur, they proceed death by only a few hours. Observant owners may notice delayed crop emptying, weakness, a generalized pallor, or bruising under the skin in the preceding hours before death. Yellow discoloration of the urates is another rare observation.5,17,20 Dr. Susan Clubb was able to predict which birds would die, up to 24 hrs before their death, by pulling out growing feathers. Birds developing disease would bleed extensively from the feather follicle.5

Necropsy findings commonly include generalized pallor with subcutaneous and subsersoal hemorrhage and enlargement of the spleen and liver. Less commonly, acites and pericardial effusion may be present. Microscopic examination of the tissues reveals extensive areas of necrosis (cell death) in the liver. Virus inclusion bodies are found in the spleen, mesangial cells of the kidney, and Kupffer cells of the liver. Necrosis of splenic cells is often massive. Less commonly, virus inclusions are found in other organ systems including the feather follicles.5,17,20 An immune complex glomerulopathy occurs in a significant percentage of the birds with this disease. These complexes contain antibody and viral proteins.36,43

Lovebirds

APV disease in lovebirds is distinct enough to merit special attention. Like the budgerigar, this disease occurs in nestling birds and inclusion bodies can be found in multiple organs. Unlike the budgerigar, birds up to 1 year of age can also be affected.32 This unusual age susceptibility has not been fully explained. However, in at least some of these older birds, concurrent infection with PBFDV is also occurring and may permit APV disease in a bird that would otherwise be resistant to it.34,41

INFECTION VERSES DISEASE

It has become evident that infection and disease are not synonymous. Many birds are infected by the virus but only a certain, and sometimes small, percentage of these birds will develop disease.6,7,30,44,48 Whether disease will develop is dependent on the species of bird infected, the age of the bird infected, whether that bird is also infected with the PBFDV, and other factors that remain unclear. Birds that are infected and do not develop disease still have virus replication within their bodies and shed virus in their droppings for a period of time.6,7,30,44,48 The length of time that virus shedding occurs, again, depends on the age the bird at the time of infection and the species of the bird.6,7,48

Infection in Budgerigars

As previously mentioned, in the United States, the English variety of budgerigar appears to have some resistence to APV infection.44 The most devastating outbreaks of disease occur in large commercial aviaries of the American variety of budgerigar where birds are bred in rooms containing tens or hundreds of free-flighted birds. Both nestling and adult budgerigars are susceptible to infection. Death, however, is confined to young birds between the ages of 10 and 25 days. The nestling mortality (death) rate is often high and may approach 100% when the virus is first introduced to an aviary. If there is no intervention, in subsequent breeding seasons mortality rates will decline but production will always remain depressed.34,37,38

Birds that survive infection may have abnormal feathering or appear completely healthy.18,32,38 Survivors shed virus in their droppings and probably their skin and feather dander for up to 6 months after infection.38 Virus shedding stops with the onset of sexual maturity or during the first breeding cycle. The infection cycle is then maintained through the shedding of virus by nestlings and young adult birds. Thus, birds are exposed to the virus immediately after hatch and have virus circulating in their blood by the time they are 7 to 10 days old.37 Fledglings and young adult birds are also important sources of virus exposure for other birds when they are taken to bird shows, bird marts, and sold to pet stores.

It has been suggested that egg transmission of APV occurs in the budgerigar.10,56 This conclusion is based on 2 observations. First, intra nuclear inclusion bodies where reported in day-old nestlings suggesting that these birds had virus growing in them before they hatched.2 Secondly, in a clinical trial, eggs were removed from a flock of budgerigars experiencing an outbreak of disease and placed under the hens of a clean flock. The young from these eggs subsequently developed disease. This author's experience, however, does not support this conclusion. I have not seen inclusion bodies in birds less than a week old. Also, there is another interpretation for the results of the clinical trial. If the transferred eggs were contaminated with virus, then the chicks could have been exposed at hatch. Additionally, budgerigar hens eat the egg shells. Thus they could have become infected and then passed the infection onto their young. In a paper I presented in Utrect, The Netherlands, I found very low concentrations of APV DNA in some embryos and very young nestling budgerigars.37 This data has also been used to suggest that egg transmission occurs.56 These birds never developed disease and subsequently, I found that one of the reagents used in this work was contaminated with viral DNA. Therefore, at this point in my understanding of APV disease in the budgerigar and other species, I feel that there is only very limited and circumstantial evidence that egg transmission occurs.

Dr. Branson Ritchie, citing my data, has stated that budgerigars are the only bird that is continuously infected with APV and remain sources of virus for life.56 In justifying this conclusion Dr. Ritchie cites one of my publications,38 but ignores another.42 In the first publication,38 I found that virus DNA could be detected in tissues of budgerigars at least to the age of 4 years. Virus concentrations were highest in 6 month old birds, but diminished in birds breeding for 4 months and were even lower in birds continuously breeding for 17 months. Although virus DNA was found in birds of all ages, it was not clear that the older experienced breeding birds were actually shedding virus. In my second study,42 I took older breeding birds that we new had been infected with virus and rested them from breeding for 7 months. These birds were then allowed to breed and their young were monitored for signs of infection and the development of antibody to the virus (an indication of infection). None of the 107 young birds produced by these previously infected budgerigars developed disease. Therefore, we must conclude that older experienced budgerigar breeders are not sources of virus infection and even if small concentrations of virus DNA can be found in their bodies, they do not actively shed the virus.

Infection in nonbudgerigar parrots

Susceptible birds infected with APV infection will die. Rarely, a susceptible bird will have transient signs and survive.5 In birds resistant to disease, infection is unapparent. In these birds, viral DNA can be first detected in blood after which it is detected in the cloaca.7,8,48 Cloacal samples may intermittently be negative, but generally the blood will remain positive.7,8,48 When the bird is about to stop shedding, the blood will become negative and within a week or two, cloacal swabs will also become negative.48 The length of time that birds are blood and cloacal positive is dependent on the species of bird and the age that it was infected. It appears, for the most part, that the older the bird is at the time of infection, the shorter the duration of shedding.7,8,48

Conures: Many, possibly most, conure nestlings exposed to APV at six weeks of age or younger will develop disease and die. In birds older than six weeks, APV causes an unapparent infection (Fig. 1). In conures, unapparent infections are best detected by examining the blood for virus DNA. Virus shedding can be expected for 4 to 8 weeks in most birds, but up to 16 weeks in the rare individual.7,8

Macaws: Macaws are susceptible to APV infection and disease up to approximately 14 weeks of age, after which infection is unapparent. Peak mortality in macaws occurs from 4 to 8 weeks of age (Fig. 1). Unapparently infected birds will become blood positive and cloaca positive. In a recently completed study, 2 blue and gold nestlings that survived infection shed virus 14 weeks. Two fledgling red-fronted macaws shed virus for 10 weeks. Adult blue and gold macaws and hyacinth macaws shed for 6 weeks or less. The nestling birds became blood negative first, then negative on the cloacal swab.48

Eclectus parrots: Infection of otherwise healthy nestling eclectus parrots will cause their death if they are less than 14 weeks old (Table 1). Specific studies on the length of virus shedding in these birds have not been done.39

Cockatoos: As a general statement, cockatoos of any age are highly susceptible to infection with APV, but are extremely resistant to disease. Healthy adult cockatoos are not expected to ever develop APV disease and the same is true for nestling cockatoos under most circumstances. In a recent study, it was found that citron-crested and umbrella cockatoo nestlings exposed to the virus at less than 3 weeks of age developed abnormal feathers. These birds showed transient signs of a systemic illness, then recovered with supportive care. Older birds and other cockatoo species remained healthy, although nearly all of them became infected.48 Virus shedding, as determined by cloacal swab, lasted 8 to 10 weeks. Virus could be detected in the blood consistently until just before shedding stopped. In this group of birds, cloacal swabs were not consistently positive and several birds that were originally cloacal positive became negative and then positive again.48

I have documented 2 cases of APV disease in nestling cockatoos that resulted in their deaths. Both birds were also infected with the PBFDV.47

APV INFECTION AND DISEASE IN ADULT PARROTS; THE PBFDV CONNECTION

APV readily infects adult parrots.30,36,44,48 Most infections, probably greater than 99.9% of then, are completely asymptomatic. These birds become infected, shed virus for a period of time, and never look or act ill. APV disease, however, has been documented in adult birds.24,25,46,50,58 So why do these few adult birds develop APV disease? The answer in most cases is that they are immunosuppressed with a concurrent infection of PBFDV.24,25,46 The author has documented an outbreak of APV disease in adult eclectus parrots.46 All birds had PBFDV. Disease has also been identified in adult cockatoos,24,25,34,58 again when these birds were tested for PBFDV, they have been found to be positive. I have previously mentioned that young adult lovebirds can die with APV disease. Again, concurrent infections with PBFDV may be the explanation for why. In the authors experience, on every occasion that APV outbreaks have occurred in lovebirds, PBFDV could also be found in the aviary.

PBFDV-infected birds are a common source for APV in an aviary.44 PBFDV-infected birds, like AIDS patients have a poorly functioning immune system. Therefore, if they become infected with APV they cannot clear the virus. Some of these birds will develop full blown APV disease and die. Most will become persistently infected. These persistently infected birds will then shed virus continuously from their skin and in their feather dust. This constant virus shedding contaminates the environment and makes it likely that it will be tracked into the nursery.

ARE CAIQUES MORE SUSCEPTIBLE TO APV INFECTION EVEN AS ADULTS, THAN OTHER PARROTS?

One of the first reports of APV disease in adult birds documented an outbreak where an eclectus, a painted conure, and 3 white-bellied caiques died.50 These birds clearly had APV-disease. They were not, however, tested for the PBFDV. So we do not know if this means that they were normal birds that have a predilection for APV disease, or were birds infected with PBFDV and were immunosuppressed. Since that time, the author has heard of a number of deaths in adult caiques. However, none of these birds were tested for PBFDV. Thus, the answer to this question remains elusive and requires further investigation. Because PBFDV may not cause histologic evidence of disease, the author feels that it is essential that when APV disease occurs in adult birds or in species where it is not normally a problem, that they be tested by DNA probes for the PBFDV.

TESTING

Currently there are 3 types of tests available for detecting APV infection in birds; serology, examination of blood for virus DNA, and examination of cloacal swabs for virus DNA.

Serology

Serology is the examination of the liquid portion of blood (plasma or serum) for antibodies that are made specifically against a virus, bacteria, or fungus. If a bird is infected with APV and survives, it will develop antibody to the virus.38,44,62 Antibody can be detected in the budgerigar by 9 days after infection, in most other birds antibody is not present in the blood until 2 to 3 weeks after infection.37 Antibody concentrations rise very quickly and by 4 to 6 weeks after infection reach maximal concentrations.48 Antibody to APV can be detected in the blood for months to many years after infection depending on the species.38,44 Budgerigars maintain an antibody titer for life.38 Cockatiels probably only maintain antibody titers for about 6 months.44 However, for most parrot species, antibody can be detected for at least 2 to 3 years following infection.44

So what does APV serology tell us? In the budgerigar, it tells us that the bird was infected with APV. If the bird is a young adult it is probably still shedding virus. If the bird is an older experienced breeder it is not shedding virus and most likely will not. A positive antibody titer in a cockatiel means that the cockatiel has been infected within the last 6 months and this bird may be shedding virus. In other parrots, it tells us very little. If the bird has antibody, then we know that it has been infected with virus, but we do not know whether the bird is shedding virus. If the bird was infected recently, then it probably is shedding virus. If the bird was infected over 16 weeks ago, then it is probably not shedding virus, unless it is also infected with PBFDV. Therefore, with the exception of the budgerigar, serology is generally not very helpful in detecting virus shedding birds. Unfortunately, this test has been inappropriately used in the past. The author is aware of people who have killed or given away their seropositive birds without understanding that they were not necessarily shedding virus.

The author is also concerned that not all serologic assays are the same. The test used by most investigators is a virus neutralization assay. This test measures both IgG and IgM and appears to be very accurate.14,30,38,48 A complement fixation assay has also been made available for testing parrot serum (Texas Veterinary Medical Diagnositic Laboratory, College Station, TX). In a comparison between the virus neutralization assay and the complement fixation assay, the complement fixation assay was only in agreement with the virus neutralization assay 60% of the time.41 At this writing the author strongly discourages veterinarians from using this complement fixation assay for APV serology.

PCR assay of cloacal swabs and blood.

The polymerase chain reaction, or PCR, is an assay that has become an incredibly important tool for the diagnosis and control of infectious diseases. This assay takes a low concentration of the APV DNA and amplifies it to a concentration that can be detected. Therefore, as few a 10 copies of the virus can be detected if the test is properly preformed.35 The sensitivity of this test is one of its greatest strengths as well as one of its greatest weaknesses. The potential problem with this assay is that even the smallest contamination of the sample, either at the collection site or in the laboratory will result in a negative sample becoming positive. Therefore, if one is testing multiple birds, it becomes very easy to get the sample from a negative bird contaminated with the feather dust or dried feces from a positive bird.48

Which PCR assay is better? The original discovery that APV could be detected in the live bird was made by Dr. Frank Niagro at the University of Georgia.30 He and his collaborators found that APV could be detected in cloacal swabs of unapparently infected birds. This technology was licensed to the Research Associate Laboratories (Drs. Dalhausen and Radabaugh) and has been offered by them for 5 years. During this time, these scientists have modified and improved this assay and have discovered that APV DNA can also be detected in the blood of birds recently infected with APV.6,7 The blood-based PCR assay has been heavily criticized by Dr. Ritchie and he has also questioned its scientific validity. His criticism is unfounded.

Both the blood and cloaca PCR -assays will pick up most birds shedding virus. So which one will you choose to screen birds? In a recently completed study, Drs. Dalhausen and Radabaugh and myself compared cloacal virus PCR, blood PCR and serology.48 Of all the birds (>50) that were examined with multiple tests, both tests picked up all but 1 of the birds that seroconverted. Not all birds were positive on both tests each time. In cocaktoos and conures, it was found that birds stayed consistently positive with the blood PCR, while a several were intermittently positive on the cloacal swab. As virus was cleared from the bird, the blood test became negative first and the cloacal swab became negative 2 to 4 weeks later. Therefore, for these species, I recommend that the blood PCR be used as a screening tool. If it is positive, the bird should be retested in 2 to 3 months, if negative, the bird should be quarantined for 4 additional weeks and then will be considered free of virus shedding. In macaws, we found that in most situations both tests were positive. Virus shedding and viremia stopped almost simultaneously. In the future, Research Associates may offer a PCR assay that screens both blood and cloacal samples from the same bird in the same reaction. This should be the most sensitive assay of all.8

It has been said that blood PCR testing of live birds following vaccination or swabs of tissue in recently vaccinated birds that die, will detect fragments of DNA from the vaccine. This assertion is totally invalid. Recent work by Drs. Dalhausen and Radabaugh has shown that viral DNA is never present in the blood of nestlings vaccinated for APV. The veterinarian must therefore conclude that if a bird is blood PCR positive, vaccinated or not, that it is infected with APV and is most likely shedding virus.

THE APV VACCINE; POSSIBLY A TOOL, NOT A PANACEA

In the past two years a vaccine developed by Dr. Ritchie and co-workers at the University of Georgia and the Biommune company has been on the market. The developers of this vaccine are advocating its use in essentially all parrots, and suggest that if adequate numbers of birds are vaccinated that we can essentially eliminate APV as a problem.1 This is a noble, but flawed, concept and has caused many a bird with no risk of APV disease to be vaccinated and given false hope to aviculturalists that they can protect their nestlings by the use of this vaccine alone.

In somewhat reverse order, consider the following 2 points. First, if the vaccine is effective, which birds can it be expected to protect from infection and disease.? Secondly, do we have significant and substantial data to suggest that the vaccine does work?

APV immunization to protect from disease

Adults. If our goal is to prevent APV disease by immunization, then it is essential to understand basic APV biology. As has been discussed, healthy adult parrots rarely if ever develop disease. Thus, vaccinating adult birds to protect them from APV disease is unnecessary.

Nestlings. It is the nestling that when infected with APV will die. Recall, however, that only certain nestling of certain species are susceptible to disease. To protect these nestling, according to the vaccination manufacturer, nestlings should be vaccinated at 5 weeks or older and then again 2 to 3 weeks later.1 They are said to be protected 4 weeks after the first immunization. Thus the vaccine has the potential to protect susceptible chicks from infection and disease in the window of 9 to 14 weeks. A review of Figure 1 demonstrates that we cannot immunized conures at an early enough age to protect them. The same is also true for most macaw and eclectus chicks. Therefore, APV immunization cannot protect most nestlings from infection and death if they are exposed to the virus before the age of 9 weeks. For macaws and eclectus parrots raised in a virus-free environment then moved to a high risk environment at 9 weeks of age, immunization may provide them with protection against infection.

The question then arises, can we immunize adult birds so that they will pass on antibody through the egg yolk and protect their young from infection? This is a valid and important question that does not have a complete answer. In the budgerigar, antibody positive parents still have young that develop disease. It has been shown, in this species of parrot, that antibody is transferred to the egg but does not reach the chicks circulation.40 We do not know if other parrots transfer antibody to their chicks through the egg. If they do, several points need to be considered. First, antibody concentrations in adult blood have to be high enough to result in a significant concentrations of antibody being incorporate into the yolk. Therefore, adult birds would have to be immunized close to the onset of breeding season every year. Any disruption of breeding birds at this time can be expected to have some negative consequences. A second point that needs to be considered is that we do not know if antibody alone will protect from infection. However, if we assume that it does and antibody is transferred to the chick through the egg, then passive transfer may conceivably protect chicks for approximately 5 weeks after hatch.

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