Appendix A METHODS FOR DIAGNOSING AVIAN CHLAMYDIOSIS

Histopathologic Findings

In birds that have avian chlamydiosis (AC), cloudy air sacs and an enlarged liver and spleen usually are observed, but no specific gross lesion is pathognomonic. The chromatic or immunologic staining of tissue-impression smears can be used to identify organisms.

Culture Technique

Isolation of the etiologic agent, Chlamydia psittaci, from the bird's spleen, liver, air sacs, pericardium, heart, or intestines is the optimal means for verifying the diagnosis. Chlamydia organisms are obligate intracellular bacteria that must be isolated in tissue culture, mice, or chick embryos. Specialized laboratory facilities and training are necessary both for reliable identification of chlamydial isolates and for adequate protection of microbiologists. Consequently, few laboratories perform chlamydial cultures.

In live birds, depending on which clinical signs they exhibit, combined cloacal and choanal-swab specimens should be collected, refrigerated, and sent to the laboratory packed in ice, but not frozen. The proper handling of samples is critical for maintaining the viability of organisms for culture, and a special transport medium is required. Veterinarians should contact their specific diagnostic laboratory for procedures required for submission of specimens for isolation.

Live birds being screened for C. psittaci might not shed the microorganism daily. Therefore, to reduce laboratory costs, serial specimens should be collected for 3-5 consecutive days and pooled before being cultured. Tissue samples from the bird's liver and spleen are the preferred necropsy specimens for isolation of C. psittaci. When legal actions may result from chlamydiosis cases, use of culture is recommended to avoid limitations associated with other tests.

Tests for Antibody

A major problem with serologic testing is the interpretation of results. A positive serologic test result is evidence that the bird was infected by C. psittaci in the past, but it does not prove that the bird currently has active disease. False-negative results may occur for birds that have acute infection when they are sampled before seroconversion. Treatment with an antimicrobial agent may diminish the antibody response.

A single testing method may not be adequate because of the diversity of reactions with immunoglobulins from the various avian species. Therefore, the use of a combination of antibody- and antigen-detection methods for the diagnosis of chlamydiosis is recommended, particularly when only one bird is tested. When specimens are obtained from a single bird, serologic testing is most useful when a) signs of disease and the history of the flock or aviary are considered and b) serologic results are compared with the white blood cell counts and liver-enzyme activities. A greater than fourfold increase in titer of paired samples or a combination of a titer and antigen identification is needed to confirm a diagnosis of chlamydiosis. Some of the advantages and disadvantages of two serologic tests for antibodies are described in the following paragraphs.

Direct Complement-Fixation (CF) Test

Direct CF is more sensitive to antibody activity than are agglutination methods. No commercial antigen is available. False-negative results are possible in specimens from small psittacine birds (e.g., budgerigars, young African grey parrots, and lovebirds). High titers may persist after treatment and complicate interpretation of subsequent tests. Modified direct CF is more sensitive than direct CF.

Elementary-Body Agglutination (EBA)

EBA is commercially available and can detect early infection. Titers greater than or equal to 10 in budgerigars, cockatiels, and lovebirds and titers greater than or equal to 20 in larger birds indicate current infection. However, positive titers may persist after treatment is completed, and EBA is performed only by one U.S. laboratory.

Tests for Antigen Immunofluorescent-Staining Tests

Monoclonal or polyclonal antibodies, fluorescein-staining techniques, and fluorescent microscopy are used to identify infectious agents in impression smears from dead birds. When used with cloacal or fecal smears, the sensitivity and specificity of the test are questioned by some authorities. The test is most useful if the bird is shedding antigen. Its advantages are that it gives rapid results and does not require live, viable organisms. Laboratory experience is important for accurate interpretation of immunofluorescent stains.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA tests (i.e., IDEIA ) now used to identify C. psittaci were originally developed for identification of the lipopolysaccharide antigen on Chlamydia trachomatis, which is a human pathogen. The exact sensitivity and specificity of these tests for identifying C. psittaci are not known. Although the test is most useful in clinically ill birds, the sensitivity may be low in asymptomatic birds because of intermittent shedding. Moreover, some tests may be falsely positive because of cross-reaction with other bacteria. The test results must be evaluated in conjunction with other clinical findings. If a bird has a positive ELISA result but is clinically healthy, the veterinarian should attempt to verify that the bird is shedding antigen through isolation of the organism. When a clinically ill bird has a negative ELISA result, a diagnosis of AC cannot be excluded without further testing (e.g., isolation, serologic testing, or fluorescent antibody).

Additional Tests

Additional diagnostic techniques are in use or under development, including polymerase chain reaction tests. Readers are encouraged to research peer-reviewed reports on such tests before use.

Laboratories that Test for C. psittaci

The National Association of State Public Health Veterinarians (NASPHV) can provide a list of laboratories that offer testing for C. psittaci. Address requests to NASPHV, RSA Tower, Suite 1310, P.O. Box 303017, Montgomery, AL 36130-3017.

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