Updates on disease testing in birds (Proceedings)

Bacterial diseases

Chlamydophila psittici

An excellent resource for information on the testing, treatment, zoonotic and legal implications of this disease can be found in the National Association of State Public Health Veterinarians (NASPHV) Psittacosis Compendium (www.nasphv.org)

Criteria for a confirmed case include the presence of one of the following:

     1) Isolation of organism from clinical specimen

     2) Identification of Chlamydial antigen by immunofluorescence of the bird's tissue.

     3) ≥ 4-fold change in serologic titer in 2 specimens from the bird obtained at least 2 weeks apart and assayed simultaneously at the same lab

     4) Identification of the organism within macrophages in smears of the tissue stained with Gimenez or Macchiavello stain.

Antibody tests for Chlamydophila indicate exposure to the organism with host immune response, not necessarily current infection.

Additional facts from this publication include:

     - Use of a combination of culture, antibody-detection and antigen-detection methods is recommended, particularly when only one bird is tested.

     - Although there is no epidemiologic evidence of increased disease risk to young, elderly, or immunocompromised humans, more rigorous testing should be considered for birds in contact with these individuals.

     - Consultation with an experienced avian veterinarian may help when selecting tests and interpreting results.

     - Because proper sample collection techniques and handling are critical to obtain accurate test results, clinical laboratories should be contacted for specifics on specimen submission.

Diagnostic methods for Chlamydophila include:

     1) Gross pathology – although cloudy air sacs and enlargement of the liver and spleen may be observed, no specific gross lesion is pathognomonic.

     2) Histopathology - Chromatic or immunologic staining of tissue or impression smears can be used to identify organisms in necropsy and biopsy specimens.

     3) Culture

          a. Live birds - with suggestive clinical signs of Chlamydiosis, a combined conjunctival, choanal and cloacal swab specimen or liver biopsy specimen is recommended

          b. Necropsy specimens - liver and spleen are the preferred sources for culture.

          c. If feces are chosen as a site for attempted detection of C. psittaci, serial fecal specimens should be collected for 3 to 5 consecutive days and pooled for submission as a single sample. NOTE: The diagnostic laboratory should be contacted for specific procedures required for collection and submission of specimens. The proper handling of specimens is critical for maintaining the viability of organisms for culture, and a special transport medium is required. Following collection, specimens should be refrigerated and sent to the laboratory packed in ice but not frozen.

Antibody tests

A positive serologic test result is evidence that the bird has been infected by Chlamydiaceae, but it might not indicate that the bird has an active infection. False-negative results can occur in birds that have acute infection when specimens are collected before seroconversion. Treatment with an antimicrobial agent can diminish the antibody response. However, IgG titers may persist following successful treatment. When specimens are obtained from a single bird, serologic testing is most useful when signs of disease and the history of the flock or aviary are considered and serologic results are compared with white blood cell counts and serum liver enzymes. A fourfold or greater increase in the titer of paired samples or a combination of a titer and antigen identification is needed to confirm a diagnosis of avian Chlamydiosis

          - Elementary-body agglutination (EBA) - The elementary body is the infectious form of C. psittaci. Elementary-body agglutination is commercially available and detects IgM antibodies, an indication of early infection. Titers greater than 10 in budgerigars, cockatiels, and lovebirds and titers greater than 20 in larger birds are frequently detected in cases of recent infection. However, increased titers can persist after treatment is completed.

          - Indirect Fluorescent Antibody Test (IFA) - Polyclonal secondary antibody is used to detect host antibodies (primarily IgG). Sensitivity and specificity varies with the immunoreactivity of the polyclonal antibody to various avian species. Low titers may occur because of non-specific reactivity.

          - Complement fixation (CF) - Direct CF is more sensitive than agglutination methods. False-negative results are possible in specimens from parakeets, young African gray parrots, and lovebirds. High titers can persist after treatment and complicate interpretation of subsequent tests. Modified direct CF is more sensitive than direct CF.

Antigen tests

Antigen Tests give rapid results and do not require live, viable organisms; however, false-positive results from cross-reacting antigens can occur. False-negative results can occur if there is insufficient antigen or if shedding is intermittent.

          - Enzyme-Linked Immunosorbent Assay (ELISA) The sensitivity and specificity of these tests for identifying suspected C. psittaci in birds is not documented. A positive ELISA result in a healthy bird should be suspect, as should a negative result in a suspect bird. (e.g., culture, serologic testing, or polymerase chain reaction [PCR] assay may need to be performed).

          - Fluorescent Antibody Test (FA) - are used to identify the organism in impression smears or other specimens. These tests have similar advantages and disadvantages to ELISA. This test is utilized by some state diagnostic laboratories.

          - DNA/PCR tests - these tests detect the presence of Chlamydophila DNA in submitted samples. There are no standardized PCR primers and laboratory techniques and sample handling will vary, affecting the specificity and accuracy of the test. Environmental contamination is also possible. Positive results should be interpreted in light of clinical signs of disease.

Mycobacteriosis

Several species may be involved in pet bird infections, including M. avium and subspecies, M. intracellulare and M. genevense. This disease is often difficult to diagnose antemortem. Methods of testing are listed below.

          - Acid-fast staining - this is often used on fecal samples. Limitations of this test include intermittent shedding of the organisms, the absence of GI affectation (i.e. the fact that some cases are limited to the liver or other location) and the quality of the stain and ability of the person evaluating the preparation.

          - Histopathology – requires biopsy of affected tissue when done antemortem. This can be impractical in debilitated birds.

          - Culture – Mycobacteria are notoriously difficult to culture and are very slow growers. Being a potential human pathogen, the number of laboratories offering this test is limited.

          - DNA probe/PCR (Polymerase chain reaction)- As with Chlamydia PCR, segments of the organisms DNA are what is isolated, so viability is not an issue. When feces are used, the bird must be shedding the organisms for the test to be positive, but a lower level of bacteria is needed to detect a positive then is needed with acid fast staining. Probes are available that will differentiate between species of Mycobacteria. This is important due to the variation between species in pathogenicity, zoonotic potential and prognosis.

Viral diseases

Bornavirus

Proventricular Dilatation Disease (PDD): Recent research has demonstrated compelling evidence of avian bornavirus (ABV) etiology for PDD. This disease syndrome, originally known as macaw wasting disease, has been identified in the US since 1978, and was originally seen in only macaws. It has since been diagnosed in most species of psittacine and in many non-psittacine birds.

Pathologists are able to recognize specific changes (lymphoplasmacytic ganglioneuritis) seen on histopathology in affected birds, but isolation of a causative agent has been elusive. The histopathologic changes can be seen on biopsy specimens or on necropsy. If aN antemortem diagnosis via biopsy is attempted, samples from the ingluvies are often preferred in debilitated clinically ill birds, since samples from the proventriculus are harder to obtain and healing is often compromised. If necropsy samples are submitted, preferred tissues include the proventriculus, crop, brain, possibly adrenal glands, heart, spine, and mesenteric plexus.

DNA Probe-Identifies Avian Borna Virus (ABV) DNA via amplification of bornavirus-specific DNA sequence to produce detectable levels. The specificity of this test, stability of samples and direct correlation between a positive bornavirus DNA test and disease are still under being determined.

Polyoma virus

Clinical disease occurs in young psittacines.

Serology is useful in documenting exposure; it does not indicate viral shedding. Antibody titers require serum or plasma for submission – check with the lab for specifics. Currently, virus neutralizing (VN) or ELISA serologic tests are available. Note that vaccination will not interfere with the VN test in neonates (no production of neutralizing antibody occurs). In adults however, a low neutralizing antibody titer may be present post-vaccination.

DNA probe identifies Polyoma virus DNA via PCR (Polymerase chain reaction). Sample sources include whole blood, cloacal and choanal swabs. Notes-Swab PCR may be more useful in detecting shedding than PCR of blood. Use of both PCR and serology concurrently is ideal.

Psittacine beak and feather disease (PBFD)

Avian circovirus includes two identified variants (PsCV1, PsCV2) that vary in pathogenicity. DNA Probe-Identifies avian circovirus DNA via PCR (Polymerase chain reaction). Sample required is whole blood. Environmental swab are also used to evaluate contamination of facilities. Note: Some labs use sequences common to both variants, others use tests that are specific for one variant. Birds positive on PCR but clinically normal should be isolated and retested in 3 months. Feather biopsies submitted for diagnosis require a blood feather with surrounding skin. Histopathology then identifies circovirus inclusion bodies.

Fungal

Aspergillosis

A. fumigatus and other species ubiquitous in the environment can cause infection. Multiple tests exist and definitive diagnosis may be difficult. Tests run as a panel increase the likelihood of diagnosis in infected patients. An Aspergillus panel is currently offered at the University of Miami

     1) Antibody titer: ELISA (Enzyme-linked immunosorbant assay) run on serum creates an enzyme-activated color change caused by the presence of AG-AB complexes. Many infected birds will have negative or weak positive titers. Positive titers currently correlate best with disease in raptors and much less consistently on psittacines.

     2) Antigen titers

          a. ELISA (Enzyme-linked immunosorbant assay), Antigen capture assays using serum. Note: Infected birds may have negative or weak positive titers. Strong positive antigen titers are often associated with negative antibody titers in infected birds.

          b. Galactomannan is an Aspergillus antigen also run on serum. Correlation with disease is still unknown, although empirically in psittacines, there is higher correlation than with the ELISA antigen test.

          c. Note that direct visualization of lesions with subsequent culture, cytology or biopsy, often via endoscopy, is often diagnostic for Aspergillosis.

Sarcocystosis and heavy metal testing will be discussed during the presentation.

References available upon request

**Thanks to Dr. Julie Yeager for portions of these Proceedings.