(Reprinted with permission from Research Associates Laboratory, Inc. Milford, Ohio 45150.)
Viral diseases of pet birds have some of the most severe emotional and economic impact upon aviculture of any disease group. They also have been historically the most difficult diseases to confirm on diagnosis and to manage in our avian population. Traditionally diagnostic methods have inherent problems in detection (sensitivity & specificity) and interpretation of results. They also often fail to accurately identify the actively infected individual, especially in the sub-clinical "carrier" state.
Over the last 15 years, advances in the field of molecular biology have allowed for the development of extremely sensitive and specific nucleic acid (DNA, RNA) detection methods. Diagnostic testing and improvements in test methodologies have allowed for a greater understanding of the epidemiology and management of avian disease.
DNA Based Diagnostics
The first applied use of viral specific DNA technology in avian disease diagnosis, was marked by the development of tests for the Psittacine Beak and Feather Disease (PBFD) and avian polyoma (APV) viruses (Psittacine Research Group, University of Geaorgia).(1) Research Associates Laboratory (RAL) has commercially offered these tests since 1992 and is both the oldest and largest molecular biology based laboratory serving the veterinary community.
RAL's DNA based diagnostics, use viral specific nucleic acid probes, to identify the unique DNA sequence making up the desired viral genome. These sequences are detected in DNA extracts from submitted blood and tissue swab samples. DNA amplification techniques coupled with internal sequence probes allow for diagnostic tests of extreme specificity and sensitivity.
The performance of RAL's diagnostic tests has been evaluated both in-house and by an independent biotechnology laboratory. Test sensitivity, which is how accurate the test is in reporting a positive infected bird, as positive, is 99.7% for PBFD and 98.2% for APV.(2) The accuracy of the tests in reporting a negative infected bird as negative was 100% for both tests.
Aviculturalists and veterinarians should be keenly aware of the intense capabilities of these diagnostic methods. The presence of contaminating virus in areas of high avian traffic (hospitals, stores, aviaries etc ... ) should not be underestimated. Prudent sample collection and handling is necessary to prevent environmental contaminants from producing a positive test result. The levels of test specificity demonstrated above show that the incidence of "false-positive" test results is virtually non-existent in these tests.
Aviculturalists should also keep in mind that infection does not always equal disease. The majority of birds exposed to these viruses will remain clinically normal and mount an effective immune response which eliminates the virus. These birds are in essence "naturally vaccinated" and test positive only throughout the time active virus is present. Recent advances in RAL test technology allow for a quantitative (numerical) value to be assigned to the DNA test results. This valuation of test data helps to differentiate an active viral infection from non-progressive viral exposures. Results of this nature will prove useful in the management of test positive individuals in the near future.
PSITTACINE BEAK AND FEATHER DISEASE
The characteristics of PBFD have been well described.(1) The acute form of this disease is most commonly observed in young or fledgling birds during their first feather formation after replacement of the neonatal down. Chicks as young as 2 months of age have been described with classic PBFD feather lesions.(1) These infections may be characterized by necrosis, fracture, bending, hemorrhage, or premature shedding of developing feathers. Chicks that develop clinical lesions while the majority of feathers are still in the developmental stage exhibit the most severe feather pathology. The clinical progression of disease is less dramatic in young birds that develop clinical signs after body contour feathers are mature. In these birds, feather changes may be limited to the still developing primary flight and tail feathers.
In some peracute cases in young birds, PBFD may manifest itself as depression, anorexia, crop stasis, & diarrhea followed by death in 1 - 2 weeks. Since these birds are covered only with neonatal down, no feather abnormalities will be observed. This clinical picture appears to be particularly common in young cockatoos, African greys, and lovebirds.
A more chronic form of the disease is observed in older birds in which dystrophic feathers, that stop growing shortly after emerging from the follicle, appear during each successive molt. The powdery down feathers located over the flank region are typically the first to show signs. The disease then progresses to involve the contour feathers in most feather tracts, followed by dystrophic changes in the primary and secondary feathers of the wings, tail, and crest.
PBFD is generally considered fatal, with most infected birds surviving 6 months to 2 years after the onset of clinical signs. The PBFD virus is immunosuppressive and death usually occurs from complications due to secondary bacterial, viral, or fungal infections, or from terminal disease changes which necessitate euthanasia.
We have previously reported on the analysis of approximately 10,000 PBFD tests at this laboratory. (2) Approximately 5% of birds tested positive for PBFD. The majority of these birds were not exhibiting feather abnormalities or other outward signs of PBFD disease. Most of these birds were sub-clinically and transiently infected with the PBFD virus. With a mature, functioning immune system, most birds are capable of mounting an effective and protective immune response, which results in elimination of the PBFD virus. Retesting of these individuals 90 days later is recommended, at which time most will show a negative test result. These transiently infected birds represent a large portion of positively identified birds at our laboratory. Birds that continue to remain test positive should be considered latently infected and may break with clinical disease at a future date. All test positive individuals should be isolated from other birds until they test negative.
Old World psittaciformes show the highest incidence of positive tests. Eclectus species had an overall positive rate of 10% followed by 8.7% for cockatoos and 8% for African Grey parrots. New World psittaciformes had shown a much lower positive test incidence. The rate for macaw and amazon parrot species was approximately 4%. Of interest is the apparently high rate of positive tests in lovebird species, which exceeds 30%. This positive test rate most probably results from selectively using the test for diagnostic purposes (suspected clinical cases) as compared to testing as a screening tool to document a bird's health status (as is common with the larger pet bird species).
The current positive test rate for PBFD at this laboratory is approximately 3.5% - 4%. Essentially the rate has decreased only slightly over the past 5 years. Many avian practitioners however, report a dramatic decrease in the incidence of observed clinical PBFD disease. This is probably due to the fact that by testing and isolating positive individuals, we have reduced the exposure of susceptible individuals (neonates), which are more likely to develop the clinical disease in response to PBFD viral infection.
Situations have arisen where multiple clutches of baby birds have tested positive and shown clinical disease, in light of negative blood tests on parent birds. Environmental contamination, as determined by testing environmental swabs, is the major source of infective virus in these situations. The duration of time that the PBFD virus remains viable in the environment has not been determined. It is generally accepted however that PBFD is viable for a prolonged period of time. Thorough cleaning of the nursery premises has eliminated the problem of pediatric infection in most of these cases. Contaminated nursery environments should be considered a major source of PBFD (and APV) viral transmission.
Observations of clinical PBFD in baby birds demonstrates that the time of viral exposure in relation to the maturity of their immune system is a determining factor in the progression of clinical disease. In one aviary situation, baby scarlet macaws were pulled from the aviary at 3-4 weeks of age. In the nursery, these birds developed transient feather abnormalities compatible with PBFD disease. Abnormalities were evident in new feather growth that occurred over a 2-3 week period of time. Blood and feather pulp tests during this time were positive for the PBFD virus. After several weeks, abnormal feather growth had ceased and all subsequent new feather growth was normal. Future blood tests on these birds were negative. Existing abnormal feather pulp at this time was still test positive. Eventually, no abnormal feathers could be identified and all tests were negative. Environmental and parent bird testing revealed that these babies were infected from exposure in the contaminated nursery envirorunent. They successfully mounted an effective immune response, which resulted in elimination of the virus.
Another recent case involved a 4-week-old African Grey parrot that was tested negative for PBFD immediately prior to sale to a store. The bird was sold from the store at 8 weeks of age at, which time it tested, positive. Evaluation of the pet store revealed a contaminated nursery environment with several other hand-feeding birds PBFD positive. The store requested euthanasia but the 8-week-old parrot was not showing any evidence of clinical PBFD disease. RAL's new quantitative viral testing indicated a transient rather than progressive viral infection. The baby was isolated and retested 4 weeks later. Quantitative testing revealed that while still positive levels of circulating virus were significantly lower. Repeat testing at 60 and 90 days later were negative and the bird remains negative and clinically normal to date.
DNA probe testing has had a tremendous impact on the incidence of observed clinical PBFD disease. It has proven to be a useful means by which to control and reduce this deadly disease. Observations derived from clinical testing and consulting with the avian community across the country summarize new points in our understanding of PBFD disease:
1. Most birds with adequate immune system function, when exposed and infected with the PBFD virus, mount an effective immune response, which results in elimination of the virus. These birds are in essence "naturally vaccinated."
2. New World Psittacines appear to be inherently more resistant to PBFD infection and disease when compared to Old World species.
3.The positive test rate incidence in birds has shown little change over the past 5 years.
4.The incidence of chronic clinical disease is declining in the U.S. avian population.
5.Most observed infections are now transient in nature.
6.Contaminated avian environments remain a major source of PBFD viral transmission.AVIAN POLYOMAVIRUS
Although the APV virus that infects budgerigars and other psittacine species appears to be the same, the clinical disease, distribution of lesions, and epidemiology of infection differ. (1,7,8,14 -17) APV, also described as budgerigar fledgling disease, causes variable morbidity and mortality, and abnormal feather development (French Molt) in this species. The feather abnormalities caused by APV resemble those of PBFD. The difference is that normal feathers will return with the next molt if the initial abnormalities were due to APV. With PBFD, abnormalities become progressively worse with successive molts.
The true impact of APV disease in budgerigars has been subject to some misrepresentation. It has often been stated, albeit incorrectly, that 1) all budgies are infected with APV and that 2) once infected, budgies remain infected for life. Individuals and breeding colonies have been identified that have no history of APV disease. These birds have negative APV antibody titers and are negative on APV DNA testing. The obvious conclusion is that there is no evidence that these budgerigars are infected with the APV virus. APV negative budgerigars do exist. In regards to point #2, positive infected budgerigars that have converted to negative status and also produced negative offspring have been documented (Dr.D.N. Phalen, pers. communication). Once APV infected, all budgerigars are not infected for life.
In non-budgerigar psittacine birds, APV typically affects young birds, 2 weeks to 14 weeks of age. It generally causes death within 48 hours after the onset of acute depression, crop stasis, biliverdinuria, diarrhea, and hemorrhage. (1,5,7,8,17) While APV mortality in susceptible young birds ranges from 20% to 80%, (1,5,7) infection does not always result in death or disease. Some young birds infected with APV recover after a brief illness, still others do not show any evidence of clinical disease. Others may also become persistently infected with APV. These "carrier" birds have been considered a source of APV maintenance and spread within aviary populations. (18,19)
Diagnosis of polomavirus infection in the live bird was previously based upon observation of the characteristic clinical disease. Other infectious agents however, may produce similar disease signs. Serological assays, which measure a bird's antibody levels, can help determine a history of APV exposure. While antibody assays may not accurately predict current infection, one assay does show excellent correlation with blood based DNA testing (Dr. D.N. Phalen, collab. research). (19-22) The molecular DNA based assay, developed by the Psittacine Research Group at the University of Georgia, has been offered by this laboratory since 1992. (2,4) The test uses virus-specific DNA probes to confirm the presence of APV nucleic acid in cloacal, fecal, tissue, and environmental swab samples. This test detects birds that are infected with and shedding polyomavirus in their droppings.(23) The fact that APV infected birds shed the virus intermittently however, makes this test unreliable in predicting the infected status of birds with negative swab test results.
While the identification of non-clinical, persistently infected "carrier" birds has been difficult, circulating APV has been demonstrated in blood and serum samples by DNA based diagnostics. (1,17) While this method was thought to have value as a screening tool, it did not appear reliable for individual bird testing. (1,11) New improved technologies for APV detection, were designed and researched at RAL. This new molecular based assay has been shown to consistently and reliably detect the presence of APV in blood samples from infected birds, independent of clinical disease or virus shedding. (23)
It is unfortunate that some veterinarians still erroneously recommend the swab test over the blood APV assay for screening birds infected with APV. They support this recommendation by stating that the swab test provides the aviculturalist with more pertinent clinical information because it detects birds that are actively shedding the virus. This is a disadvantage however, not a benefit. The swab test only detects birds that are shedding. The blood APV assay detects birds that are shedding the virus and those that are infected but not shedding. It has been documented in one APV study that the swab test only detected ~14% of the positive APV infected birds. This means that 86% of infected birds were diagnosed as negative when indeed they were infected with the virus. Would you feel comfortable adding one of these birds to your aviary collection? While other attempts to develop a blood APV assay have been made, RAL researchers were the first to develop and validate the blood DNA, APV assay. This assay is currently the best test to perform when screening a bird for APV infection.
The practical applications of this new test technology, to the study of the epidemiology and management of APV disease, were previously reported. (23) Individual birds in an aviary with a confirmed APV epornitic, were tested for APV infection. The aviary population of 143 birds consisted of cockatiels (n=21), lovebirds (n=11), conures (n=88), Quaker parakeets (n=5), Senegal parrots (n=3), Amazon parrot (n=1), macaws (n=13), and cockatoo (n=1) species. All birds were initially tested for APV infection on blood and cloacal swab samples. Test positive birds were isolated in a physically separate facility and retested at monthly intervals. Negative retest individuals were isolated to minimize reinfection exposure and eventually returned to the aviary. Cleaning efforts were also conducted within the aviary to eliminate environmental contamination as a source of future APV infection.
Of the 143 birds in this aviary collection, 22 birds (15%) tested positive for APV. All positive test birds showed positive test results on whole blood samples whereas only three (14%) were positive for APV shedding on the cloacal swab assay. None of the birds tested showed a swab positive, blood negative test result and no adult birds were positive for APV viral shedding on the cloacal swab assay. The blood test was superior in detecting birds infected with the APV virus, when compared to the cloacal swab assay.
The original source of the virus was traced to two conures that were recently added to the aviary collection. Within two weeks of the onset of testing and isolation procedures, no new cases of APV disease were observed. Eventually all birds were returned back to aviary with the exception of the two conures. Over the last two years, this aviary has produced in excess of 300 babies and has not experienced any recurrence of APV disease. The blood APV assay has proven useful in the control and elimination of APV infection in this aviary.
However, aviculture has been misled about the benefits of this useful technology. It has been reported by some individuals, albeit incorrectly, that "fragmented DNA", the product of cellular viral processing, results in a positive diagnostic test. This infers that the blood APV test identifies many birds as positive for infection when active virus is not actually present. Analysis of extracted DNA, in actual diagnostic samples submitted to our laboratory, does not reveal evidence that DNA fragmentation is a significant problem. Isn't it odd that the criticism of "fragemented" DNA is directed only at blood APV testing when testing for PBFD, which uses the exact same sample and test format, is the "saving grace" for eliminating this deadly disease? These same molecular biology methods are considered "gold standard" in human medicine where fragmented DNA is not a confounding issue.
There exists a purposeful attempt to perplex both avian veterinarians and aviculturists in regards to this DNA technology. Lay individuals and veterinarians, with no background in the field of molecular biology, have been supported in their supposed factual interpretations of blood polyomavirus testing. These articles are wrought with factual error and distort the true scientific facts. Such misinformation serves only to confuse and confound the industry. Aviculture must demand a higher level of honesty and integrity from these "leaders."
Researchers at RAL were the first scientists to develop and prove the benefits of blood polyomavirus testing. Our interpretations have been dervied from scientific study and compare favorably with the findings of other prominent researches in the field. Additionally, RAL consults with aviculturalists and veterinarians across the country on a daily basis where these interpretations continue to prove valid.
Age related infection and disease
The age of a bird at the time of infection and more specifically, the competence of it's immune system, appear to affect the outcome of APV infection. (17,19) While some young birds exhibit mortality characteristically associated with APV infection, others appear to recover, while still others show no clinical illness. It has also been suggested that resistance to APV disease results from a modified host response to virus infection and not by an increased resistance to infection. (17) Young Blue and Gold macaws (Ara ararauna), experimentally infected with APV, developed high virus neutralization antibody titers indicating infection, but failed to show clinical disease.(1) Viral induced cytopathic changes, were observed in experimentally infected Budgerigar nestlings, although the birds never showed signs of APV disease.(3) No birds over two months of age, which tested positive in the RAL aviary study, showed evidence of clinical disease. Additionally, most positive testing birds, including new aviary additions, became consistently blood negative in a short time. These observations suggest that most immune competent psittacine birds, when infected with APV, mount an effective response, sufficient to prevent disease and eliminate the virus.
Persistently infected "carrier" birds
It has been theorized that birds infected before they are inununocompetent, may become tolerant of APV and remain persistently infected. (1,20) A clinically ill and recovered Goldcap conure in the aviary study, continued to test APV positive until 10 months of age. It is interesting to note that this bird had both polyoma virus and high levels of APV antibodies in its blood. Antibody titers do not successfully eliminate the viral infection in these "carrier" birds. Many researchers believe that active cellular immunity (killer T lymphocytes) not antibody, are needed to eliminate the viral infection. We have also confirmed circulating APV in the blood of an adult Sun conure (Aratinga solstitialis) that was diagnosed as a "carrier" for a period of four years (Dr. D. Phalen pers.com). It appears that birds can remain asymptomatically infected with APV for extended periods of time. Intermittent viral shedding from these "carriers" can serve to maintain and spread APV throughout an avian population.
Polyomavirus disease in adult birds
While it has been reported that polyomaviral disease can occur in adult psittacines, the incidence of this is extremely rare. Contrary to what the avicultural community has been led to believe, almost all infections in adult birds are asymptomatic. (11,27) Combined infections of APV and PBFD have been shown to occur. (11,13,23) It appears that APV disease in adult birds requires immunosuppresion, such as that from PBFD infection, for clinical APV disease to occur. (11,16) We recently tested DNA samples from three adult Ecelectus parrots and two cockatoos which died from confirmed APV disease. All these birds also tested positive for concurrent PBFD infection. APV disease in finches has also been suggested to result from immune suppresion. (28) Of all 143 birds tested in the aviary study, no adult birds were observed to exhibit clinical APV disease. It has also been suggested that adult birds recently infected with APV, serve as an amplification source for virus infection within the aviary. However, none of the adult study birds which tested blood positive, were shown to be actively shedding APV virus on cloacal swab testing.
APV and AVIARY MANAGEMENT
The aviary we studied had a 14 month history of morbidity and mortality from confirmed APV disease. With the implementation of blood testing and strict preventive practices, the last clinically observed case occurred at the end of 1995. Through the 1996 and 1997 breeding seasons, the aviary has produced over 300 neonates, with no incidence of APV disease. Additionally, random testing of young birds has confirmed the negative APV status in this aviary. Blood APV testing has proven to be a useful tool for the management and elimination of APV in closed aviary populations. It is the best and most economical method to screen new birds and prevent the introduction of this virus into your aviary population. Absolutely, no bird should be added to your aviary unless it has tested negative on blood APV testing.
The following statements derived from blood DNA and serology APV tests along with clinical disease observations, summarize new points in our understanding of APV disease:
1. Most birds with adequate immune system function, when exposed and infected with the APV virus, mount an effective immune response, which results in elimination of the virus. These birds are in essence "naturally vaccinated".
2. Infection in adult birds is inapparent, as is infection in many juvenile birds.
3. APV disease most commonly occurs in New -World species. Disease is most often observed in lovebirds, budgies, ringneck parakeets, conures, macaws, caiques, and eclectus parrots.
4. Clinically diseased birds most often show signs within 2 weeks of virus infection. They are viremic at this time and have begun to develop circulating antibody.
5. Birds that are infected but do not die, still develop a viremia and also develop high antibody titers. Viremia persists in these birds even in the face of high circulating antibody. These birds often become persistently infected for extended periods of time and can serve to spread the virus in a population.
6. Blood APV testing, combined with effective management procedures, can reduce and eliminate APV disease in a closed aviary population.
Much controversy exists surrounding our interpretation of APV disease. There is a strong disagreement and difference of opinion among prominent researchers in regards to the epidemiology, pathogenesis, and management of APV disease. Much of this controversy however is "man-made" and due to a rampant distortion of the true scientific facts. The prudent aviculturist should use common sense and relay on the honest interpretation of scientific observation and data. Likewise, it is unfair that aviculturists should be demeaned into thinking they are inferior or sub-standard for not complying with a particular technology, especially when such disagreement exists. Remember many statements and opinions are made but they are often meaningless without the firm support of sound scientific data.
There is no perfect solution to controlling APV disease. It is unlikely that we will ever achieve 100% compliance with any control method and ever totally eliminate a particular viral disease threat. We can however achieve control and reduce the likelihood of these diseases in our avian populations. If you have not had a problem with PBFD or APV you can insure that you will not in the future by not bringing the virus into your collection. The DNA-based tests offered by Research Associates Laboratory provide the avian community with the best method currently available for insuring that a new bird is negative for these disease agents.
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