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. Dahlhausen 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. Dahlhausen 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. Dahlhausen 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.