DNA PCR Technology:
Its Importance In Avian and Exotic Medicine

When it comes to performing diagnostic tests on avian and exotic animal patients, deciding which tests to perform under differing circumstances can be very confusing and challenging. Now, labs offer different tests for the same disease; for example, if you want to test a bird for chlamydiosis (psittacosis, Parrot Fever), you have the choice of performing chlamydial isolation (the gold standard, but may get false negatives), EBA titer (measures IGM, best for early infection), IFA titer (measures IGG, better for chronic infections and follow-up testing), DNA PCR testing (of a pooled choanal/cloacal swab, which detects the organism in the secretions), and DNA PCR testing of the blood (which detects the organism in the bloodstream). Choosing the correct test is very important in obtaining an accurate diagnosis, since there are many variables that can affect the outcome.

While most veterinary laboratories offer DNA PCR testing, there is often some confusion as to when this technology should be used in place of other testing methods. To better understand when and why to use DNA PCR tests, let's go over the basics of how this test works. PCR stands for polymerase chain reaction. PCR testing can be used to amplify the production of highly specific DNA fragments of Chlamydial, Mycoplasmal or viral genomes. This makes PCR testing both highly sensitive and highly specific. PCR testing can also be used to identify the sex of a bird, as well, by amplifying a portion of the W or Z chromosome, however the primers for sexing are generally species-specific. (The most widely used DNA method for sexing is called restricted fragment length polymorphism, RFPL, which is also extremely accurate).

By amplifying specific portions of the genome of a disease in combination with an avian probe, it is possible to identify specific disease strains. The major disadvantage to this technology is its extreme sensitivity. It is possible to contaminate a blood sample with organism particles from the skin or a toenail. For this reason, it is vital that the blood sample be procured aseptically and from a clean venipuncture stick. A toenail clip should never be used for the blood sample, since it is impossible to adequately disinfect the toe and nail to prevent contamination of the specimen, which may result in false-positive results. Tubes used for sample submission should be stored to prevent potential contamination. Often, bullet tubes are used, and these are supplied with the caps open. It is possible for environmental dust from a clinic to contaminate these tubes, which may also result in false positive test results. Make sure to store these tubes in sealed plastic bags, and close the caps for storage, as well.

DNA PCR technology may be applied to many different samples. It can be used to screen the blood for the presence of the organism. Whole blood is the usual sample for testing, so an anticoagulant should be used to prevent it from clotting. Heparin is often preferred, but before you pull the blood sample, it is always best to check with the lab to ascertain which anticoagulant is recommended. This technology only requires a small sample of blood, so drawing a large volume of blood is not necessary. This is very beneficial when blood is being drawn for a variety of tests, as saving a drop or two for a specific DNA PCR test is usually not a problem. DNA PCR testing for Chlamydiosis performed on the blood will show if the sample contained the Chlamydia psittaci organism.

There are DNA PCR tests for several viruses, as well. This is the blood test of choice for Psittacine Beak and Feather Disease (PBFD). However, the presence of the virus in the bloodstream must be interpreted in light of the bird's age and clinical signs. Research has shown that a high percentage of young birds that test positive for PBFD, and have no beak or feather lesions, will develop a natural immunity to the virus and will actually clear the virus and become naturally vaccinated. The Bursa of Fabricius (a lymphoid organ found in baby and juvenile birds, just inside the cloaca) is very important in determining whether or not a bird can acquire the virus. It is much more difficult for an adult bird (in which the Bursa has involuted) to acquire PBFD, unless there is actual blood contact with the virus. Any clinically normal bird (of any age) that tests positive for PBFD should be retested in 90 days. Most young birds will clear the virus and be negative by the retest. A clinically normal adult bird that tests positive may be transiently viremic, and may also test negative in 90 days. A clinically normal adult bird that remains positive may be latently infected or continually exposed to viral particles in the environment. A bird with beak and/or feather lesions that tests positive may be considered infected with PBFD, but it is still prudent to retest in 90 days, in case the feather lesions are the result of other causes (certain medications such as fenbendazole or metronidazole can also cause feather abnormalities). In addition to PBFD DNA PCR testing, biopsies of follicles containing abnormal feathers can be tested for the presence of the virus, or can be examined histopathologically for evidence of the disease.

There is also a DNA PCR test for polyoma virus. Blood can be tested for the presence of virus, or a cloacal swab can also be tested. A positive blood test shows the presence of the virus in the bloodstream. A positive cloacal swab shows that the virus is present in the sample. This can indicate that the bird is actively shedding the polyoma virus, or in some cases, it may mean that the bird has swallowed some of the virus particles, which are just passing through the digestive tract without causing disease. For this reason, the positive test must be evaluated in conjunction with the bird's clinical signs and other test results. In most cases, it is most important to know if a bird is shedding polyoma, as that bird can expose other birds to the virus.

DNA PCR tests can also be performed on swabs taken from the choanal slit, cloaca, conjunctiva or nasal discharge. Sterile saline (with or without preservatives) can be used to moisten a sterile cotton-tipped applicator prior to swabbing the patient. It is not necessary to use a sterile culturette (and actually, if one is used, it should not be pushed into the transport medium). It is not necessary for the swab to remain moist during transport to the lab, as the DNA of the organism will still be present. The test swab can be placed in a clean plastic bag for transport to the lab. Swabbing the choanal slit and cloaca of a bird is useful to submit for Chlamydial DNA PCR testing, to determine if a bird is shedding at the time of submission, since Chlamydia is shed in respiratory and fecal secretions. Of course, a negative test result just tells us that the bird was not shedding at the time of submission.

Testing a bird or tortoise for mycoplasmosis used to be quite difficult. Culturing an animal for mycoplasma was likely to result in a false negative test result as this fastidious organism is difficult to grow in the lab. Now, it is possible to swab the conjunctiva or choanal slit of a suspect cockatiel, or nasal or ocular secretions from a tortoise that might be harboring mycoplasma, and submit the swab for DNA PCR testing. If the organism is present in the sample, the test will be positive, facilitating a diagnosis. Mycoplasmosis is a serious respiratory problem in many Florida gopher tortoises and Western Desert tortoises, and DNA testing will provide an accurate method of screening tortoises prior to introducing new tortoises.

One very exciting application of DNA PCR technology involves being able to apply a probe to a histopathological sample to determine if a specific pathogen is present. For example, if a liver specimen from a bird shows suspicious inclusion bodies on histopath, it is possible to use PCR testing to determine if there is a specific virus in the sample. In this way, it is possible to rule in or rule out certain pathogens. This is very exciting technology that can shorten the diagnostic process by combining steps.

There are times when choosing DNA PCR testing is not the best way to accurately diagnose a disease. For example, once a bird has been started on antibiotic therapy, most birds will stop shedding the chlamydial organism within 3-5 days. So, if a patient is already on antibiotics, swabbing a bird for chlamydia is likely to result in a false-negative result, and the organism may be unpredictably picked up in the blood stream, as well. One researcher also has indicated that the presence of doxycycline in the bird's system may interfere with DNA PCR testing, itself. So, keep in mind that antibiotic therapy could possibly cause a false-negative test result for one of several reasons.

DNA PCR tests have been developed for chlamydia, mycoplasma, Lyme disease, polyoma virus, Psittacine Beak and Feather Disease, Pasteurella multocida (for rabbits) and for certain herpes diseases. I'm sure there are other PCR tests being developed all the time, and I apologize if I have omitted any. They definitely have their place in avian and exotic practice and when utilized correctly, they can aid in diagnosing some previously difficult-to-detect diseases.

Cadeusus
Copyright 2006 Margaret A. Wissman, D.V.M., D.A.B.V.P.
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