Abstract
New molecular technologies provide a powerful means to diagnose and manage infectious disease agents in psittacine birds. The underlying theory and application to Psittacine Beak & Feather disease, avian polyomavirus, Pacheco’s disease virus, and Chlamydophila is discussed. Submission recommendations and test interpretations are provided.
Introduction
Historically, infectious diseases of avian species have been some of the most difficult to confirm by diagnostic testing and difficult to manage in an avian population. Advances in the field of molecular biology have allowed for the development of extremely sensitive and specific DNA-based testing to aid these diagnoses. The first commercial application to avian disease testing was in the diagnosis of Psittacine Beak & Feather Disease (PBFD) and Avian Polyomavirus (APV) infections (Research Associates Laboratory, Inc., 1992).1
DNA amplification techniques coupled with internal nucleic acid probes provide diagnostic tests of extreme accuracy. Practitioners should possess a good understanding of the principles involved in this new technology to best utilize its potential. Additionally, a thorough evaluation of the available laboratory services and tests being offered is essential to identify those best suited to provide valid test results.
Molecular Test Technology
Molecular-based testing comprises several distinct steps. The first is the isolation of nucleic acid (DNA or RNA) from the diagnostic sample. Samples may include blood, serum, tissue and environmental swabs, and feces. Strict attention to sample collection, storage, and transport is essential to avoid sample contamination and inaccurate (false positive) test results. Laboratories must also adhere to strict methods of sample handling. Improper sample handling can result in sample cross-contamination and contaminated work environments, leading to inaccurate test results.
The next step in the testing process involves the enzymatic amplification of the target DNA sequence. This is generally accomplished through a process known as the polymerase chain reaction (PCR). A thermal stable DNA polymerase is used to synthesize a complementary DNA strand from the target sequence. Target strand amplification occurs between two sets of nucleic acid primers. These are synthetic oligonucleotides, complementary to a particular sequence of DNA, found in the bacteria, virus or protozoa being tested. It is important to remember that the particular sequence of these primers confers specificity to the DNA amplification process. The design and location of these primers is usually determined by the individual laboratory and is crucial to the accuracy of the PCR test. If a laboratory designs primers in a variable or non-conserved region, the PCR test may not give a consistent positive result among various strains of the target organism. A (false) negative test result can occur in an actual positive test sample. The recent experience of practitioners screening birds for the Psittacine Beak and Feather Disease virus confirms this occurrence.
Throughout the PCR process, samples are ramped through a cycle of temperature settings in an instrument called a thermal cycler. Each cycle of the reaction consists of three distinct phases. A denaturing step in which the double stranded DNA template is denatured into two single strands, an annealing step where the primers bind to the complementary target DNA sequences, and an extension phase where DNA polymerase synthesizes a complementary copy of the target strand. The entire cycle is repeated for 25 to 35 times. Theoretically, the target DNA sequence is doubled in each cycle. During a routine testing process, target DNA sequences are amplified into millions of copies (2n, n=30–40). This ability to amplify small quantities of target DNA gives the technique its great sensitivity. PCR tests usually can detect less than 10 copies of target sequence. Cross-contamination can therefore be a major concern. When the same diagnostic test is performed week after week, the laboratory risks contamination with large amounts of amplified DNA (amplicons). The inadvertent introduction of small amounts of this material into a test sample before PCR amplification will result in a false-positive test result. Laboratories must have sufficient safeguards established to avoid this risk of sample contamination. While other potential sources of sample cross-contamination exist, amplicon contamination is by far the most important. It has been reported that very few laboratories use specific methods to destroy amplicons and thus prevent sample cross-contamination.2
The final step in the testing process involves the confirmation of amplified (positive) test samples. By determining that the amplified product has the correct genetic code, the specificity of the test process is greatly enhanced. This is generally accomplished by hybridizing a nucleic acid probe, to the internal sequence of the amplified DNA. The DNA probe has enzymes or fluorochromes covalently attached to it, which are used to visualize or detect the amplified product. If the hybridization test is positive, it confirms that the amplified product represents a true positive sample.
Several diagnostic laboratories in the United States offer molecular-based veterinary testing. The number of laboratories and available tests should increase as applied technologies become more widespread. While several laboratories may offer molecular testing for a particular disease, the methods and practices utilized are not identical. This can lead to variability in test results among those labs. It has even been reported that a number of laboratories doing PCR testing lack sufficient safeguards (controls) to ensure that the results are accurate.2 It is imperative that a practitioner keenly evaluate the available testing before selecting a laboratory to submit samples to. Validation of a laboratory’s testing should be supported by scientific presentation and publication. Performance of these tests in the field of use should also be reviewed. Only then will a practitioner be able to select laboratories that are best prepared to give valid results.
Molecular-Based Tests
Historically, the application of molecular-based testing in veterinary medicine has been most extensively applied to diseases of avian, and in particular, psittacine species. Currently, validated tests exist for Psittacine Beak and Feather Disease virus, avian Polyomavirus, Pacheco’s Disease virus, and Chlamydophila (psittacosis) infections. These tests can be utilized to provide a confirmed diagnosis of infection in both antemortem and postmortem samples. They also can detect subclinical infections in “carrier” birds and can be used to assess the level of environmental contamination in physical facilities. Culturette swabs of cut tissue sections and environmental surfaces are used for postmortem and environmental testing, respectively. Preferred sample submission and test interpretation for individual diseases are listed below.
Psittacine Beak and Feather Disease (PBFD)
Whole, unclotted blood and postmortem tissue swabs are the preferred samples to submit for testing birds for this infection. Currently, the majority of birds that test positive do not exhibit feather abnormalities or other outward signs of PBFD disease. Most of these are subclinically and transiently infected with the PBFD virus. Birds commonly come in contact with the virus through environmental contamination during shipment, in pet stores, at bird shows, etc. Although infected with the virus, these birds do not show clinical disease. 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.3 Chronic, low-grade clinical infections are evident particularly in lovebirds and lorikeet species. These birds serve as a source of infection to other birds and contaminate environmental areas. The ability to compartmentalize this virus has been theorized during which infected birds may test blood negative. Current research is investigating this theory, which has yet to be substantiated. The inability to detect these individuals may, however, be due to inadequacies in test design.
The genome of the PBFD virus has been shown to vary, as much as 12% among isolates from different species.4 Depending on design, DNA-based tests may not be able to identify this infection in all species of psittacine birds. The problem has been most evident in detecting infected lory and lorikeet species. Conserved regions in the genome, however, do exist and a properly designed test should be able to detect infection in various psittacine species.4 Interpretation of test results is listed in Table 1.
Table 1. PBFD DNA test interpretation
|
Age
|
Test result
|
PBFD clinical signs
|
Interpretation
|
|
Neonate
|
Positive
|
None
|
PBFD viral nucleic acid was identified in the sample; bird should be considered infected and may become clinical; isolate and retest the bird in 30–90 days.
|
|
Juvenile to adult
|
Positive
|
None
|
PBFD viral nucleic acid was identified in the sample; bird should be considered exposed and infected, but will most likely become immune and eliminate the virus; isolate and retest in 30–90 days.
|
|
Neonate, juvenile to adult
|
Positive
|
Yes
|
PBFD viral nucleic acid was identified in the sample; bird should be considered infected and clinical; isolate.
|
|
Neonate, juvenile to adult
|
Negative
|
None
|
PBFD viral nucleic acid was not identified in the sample; bird should be considered negative for PBFD infection
|
|
Neonate, juvenile to adult
|
Negative
|
Positive
|
Consider other disease etiologies; run swab test from affected feather pulp for PBFD and APV.
|
Avian Polyomavirus
Birds may be screened for infection with this virus using whole, unclotted blood or cloacal swabs. Avian polyomavirus is thought to shed intermittently with shedding being most evident in clinically ill birds. During a neonate outbreak, the ability to detect infected birds is similar between whole blood and cloacal swabs. Birds that become ill and recover from the disease often become nonclinical carriers. They can harbor and shed the virus well into their adult life.5 Blood testing is much more reliable in detecting these infected individuals. Swabs of liver, spleen, and bursa can be used to confirm the diagnosis in postmortem cases. Test sample interpretation is listed in Table 2.
Table 2. Avian polyomavirus blood test interpretation
|
Age
|
Test result
|
APV clinical signs
|
Interpretation
|
|
Neonate
|
Positive
|
None
|
APV viral nucleic acid was identified in the sample; Bird should be considered infected and a potential source of infection to other birds; bird may become clinical, generally within 2 wk; Isolate and retest the bird in 30–90 days.
|
|
Juvenile to adult
|
Positive
|
None
|
APV viral nucleic acid was identified in the sample; Bird should be considered exposed and infected, but will almost always become immune and eliminate the virus; Isolate and retest in 30–90 days.
|
|
Neonate
|
Positive
|
Yes
|
APV viral nucleic acid was identified in the sample; Bird should be considered infected and a source of APV infection to other birds; bird is clinical and may become a persistently infected “carrier” if it recovers. Isolate and retest at 30–90 days.
|
|
Juvenile to adult
|
Positive
|
Yes
|
APV viral nucleic acid was identified in the sample; bird may be exhibiting a rare case of Adult APV disease; An immunosuppressed state is considered and the bird should be tested for PBFD.
|
|
Neonate, juvenile to adult
|
Negative
|
None
|
APV viral nucleic acid was not identified in the sample; bird should be considered negative for APV infection.
|
|
Juvenile to adult
|
90 day positive retest
|
None
|
APV viral nucleic acid was identified in the sample; bird is persistently infected and may serve to spread APV to other birds; Bird will most likely remain non-clinical and will probably become negative after a variable period of time.
|
Pacheco’s Disease Virus
The cause of acute illness and death in adult psittacines, Pacheco’s disease is incompletely understood.6 Many strains of herpes virus belonging to at least five serologically distinct subtypes exist, but not all infections appear to cause disease in all birds. Conure and Amazon parrot species are frequently implicated as carriers of the virus; however, infections also appear to be far more common than previously thought. A DNA-based assay has been developed to identify birds infected with this virus.6 Cloacal swabs are the best sample to submit when screening birds, followed by fecal swabs, and whole, unclotted blood. One cloacal swab test will identify 75% of infected individuals; two tests, 30 days apart will detect 95% of those infected. Like herpes virus infections in other species, infected birds are considered to be infected for life.
Chlamydophila
Avian Chlamydophila is an important infection that has historically affected aviculture. Impact ranges directly from overt clinical disease and mortality to the often non-diagnosed effects on growth, health, and reproduction. The organism exhibits a pronounced variability in host susceptibility, pathogenicity, course of disease, and diagnostic parameters. While numerous diagnostic methods have become available over the years, test results are often equivocal, making a confirmed diagnosis in a live bird elusive.
A molecular-based test to identify birds infected with Chlamydia psittaci has been reported.7 The test appears ideally suited to detecting states where infectivity is low or where a rapid assay is desired. It provides a confirmed diagnosis of chlamydial infection in the clinically inapparent and/or persistently infected state. It also offers the advantage of providing a sensitive method for Chlamydia detection, which is not dependent upon a host immune response. Infectivity studies have shown that upon initial infection, oral/choanal swabs almost always immediately test positive. The organism becomes detectable in cloacal swabs around 10 days postinfection. By 15 days postinfection, both swabs and blood test positive for infection.7 It is recommended to submit a combined choanal and cloacal swab, and whole unclotted blood on each individual to be tested. Postmortem liver and splenic swabs will also confirm infected individuals. It should be noted that doxycycline can interfere with the PCR test process. Samples should be acquired before treatment is initiated.
Conclusion
Molecular-based testing offers a powerful means to diagnose and manage infectious disease in psittacine birds. It is important that practitioners understand the underlying technology and know how to properly submit samples for testing. Prudent sample collection and handling is essential to ensure accuracy of test results. They should also utilize only those laboratories offering tests with proven performance through scientific publication, presentation, and field use. These laboratories must have sufficient quality control and assurance procedures to report accurate results. When these criteria are met, practitioners will receive meaningful information to aid them in the control of these infectious diseases.
Literature Cited
1. Dahlhausen, R.D. and C.S. Radabaugh. 1993. Update on psittacine beak and feather disease and avian polyomavirus testing. Proceedings Annu. Conf. Avian Vet.: 5–7.
2. Sockett, D.D. 2001. Molecular Diagnostics: What the Practitioner Needs to Know. Midwest Veterinary Conference 2001, Columbus, Ohio. Session #216.
3. Dahlhausen, R.D. and C.S. Radabaugh. 1997. Molecular based diagnostics: New insights into PBFD and avian Polyomavirus. Proceedings Annu. Conf. Avian Vet.: 199–207.
4. Ypelaar, I., M.R. Bassami, G.E. Wilcox, and S.R. Raidal. 1999. A universal polymerase chain reaction for the detection of Psittacine Beak and Feather disease virus. Vet Microbiol. 68:141–148.
5. Dahlhausen, R.D. and C.S. Radabaugh. 1996. Improved detection and management of avian polyomavirus infection in psittacine birds. Proceedings Annu. Conf. Avian Vet.: 291–298.
6. Phalen, D.N., E.L. Tomaszewski, C.S. Radabaugh, and R.D. Dahlhausen. 2000. Psittacid Herpesviruses and Herpesvirus disease in psittacine birds. Proceedings Annu Conf Avian Vet.: 259–262.
7. Dahlhausen, R.D. and C.S. Radabaugh. 1997. Detection of Chlamydia psittaci infection in pet birds using a molecular based diagnostic assay. Proceedings Annu. Conf. Avian Vet.: 191–198.