Theoretical and Technical Aspects of Diagnostic Techniques for Mammalian Tuberculosis
Tuberculosis (TB) infection, due to organisms of Mycobacterium tuberculosis and Mycobacterium bovis, causes significant disease in both humans and animals. Tuberculosis continues to persist in domestic livestock and captive cervid herds.17 Recent occurrences of TB at several zoos and in a privately owned elephant herd have prompted increased concern that zoo and wildlife species represent potential TB reservoirs (Mitch Essey, personal communication). The current test and depopulation method used to control tuberculosis in domestic cattle is unacceptable for endangered wildlife species.
A variety of techniques have been used to diagnose mammalian tuberculosis. Methods that directly detect bacteria from clinical specimens include acid fast and fluorescent smears, culture, and nucleic acid amplification such as polymerase chain reaction (PCR). Indirect methods include detection of antigen or antibody, and measures of cellular reactivity against mycobacterial antigen.
Direct Detection of Tuberculosis
Isolation of the mycobacterial organism by culture is currently the only definitive method to confirm tuberculosis infection in animals. Although acid fast smears of clinical specimens or histologic examination of tissues can detect mycobacteria, species identification requires either confirmation by culture or DNA probes. Acid fast smears have a lower limit of detection of about 1000 organisms/ml, whereas culture can detect approximately 100 organisms/ml.
Recent technological advances have seen the introduction of methods that detect mycobacteria by amplification of mycobacterial nucleic acids (DNA or RNA) followed by detection of the amplification product. Ideally, these methods can detect as few as 1–10 organisms. However, in clinical trials, these tests have shown approximately a 99% sensitivity at detecting acid fast positive, culture positive samples, but only a 60–80% sensitivity at detecting acid fast negative, culture positive samples. They are highly specific for TB complex organisms but cannot differentiate species. Since even dead or dying organisms can be detected, these methods cannot be used to monitor treatment.1,4,11
At present, two amplification methods have been FDA approved for detection of M. tuberculosis from sputum in humans: the Amplicor system (Roche Molecular Systems, Branchburg, NJ) and the AMTD system (Gen-Probe Inc., San Diego, CA). The Amplicor system has as its target the mycobacterial 16S ribosomal RNA gene. A small target sequence within this gene is amplified (billions of copies are made) using PCR. Because of small differences in the 16S ribosomal gene, the primers are specific to organisms only of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti) and only these species are detected. The AMTD system amplifies the ribosomal RNA through a method termed Transcription Mediated Amplification (TMA). Both amplification products are detected through fluorescent probes against the generated nucleic acids.1,11
Indirect Detection Methods
The standard tuberculin test is the simplest measure of cellular reactivity against mycobacterial antigens. A small aliquot of mycobacterial antigen is injected intradermally and the induration palpated or measured at 48 h (humans) or 72 h (humans, animals). Induration results from a local influx of lymphocytes because of cell-mediated immunity. Estimates of the sensitivity of skin testing range from 68–95% and specificity is estimated to be 96–99% in cattle.12 The efficacy of the intradermal test has not been established for the vast majority of wildlife species. Results vary between species and test sites and clinicopathological correlation with test results is often lacking.8-10,15 In addition, tuberculin testing practices vary widely between zoos.13 Differences include the tuberculin preparation used, antigen strength, injection site and interpretation of results.
The Blood TB Test (BTB) is currently based on two assays, a lymphocyte transformation assay and an ELISA to measure antibody formation against antigens (see below). Lymphocyte transformation is measured by incubating lymphocytes with mycobacterial antigens, typically 1 µg of PPD derived from M. bovis, in the presence of radiolabeled thymidine (a nucleic acid precursor). T-lymphocyte populations with prior memory of an antigen will “expand,” (i.e., begin to undergo cell division) and take up thymidine into newly synthesized DNA. The amount of radioactive uptake is then compared between PPD-stimulated and non-stimulated cells. The BTB test shows a sensitivity of 95% and a specificity of 92% in deer herds harboring mixed M. bovis and M. avium infections.7 Sensitivity and specificity in other species remain to be demonstrated.
ELISA measures antibody formation against specific antigens. The test is performed by adding serum to a well of a microtiter plate that contains the test antigen. Antibody against the antigen will bind to the antigen (protein, lipid, glycolipid, etc.) and remain after washing. The adherent antibody is then detected by a fluorescent tagged antibody against the animal’s antibody (for example, antibodies raised in goats against cattle IgG). ELISA shows a sensitivity 65.6% and a specificity of 56.4% in cattle.6 The test appears useful in detecting seriously infected deer but may fail to identify low-grade infections.5 Positive ELISA reactions were observed in llamas 8 wk following exposure to killed M. bovis.16
Two technical factors limit the application of ELISA. First, unless antibodies against the animals being tested are available, detection is severely limited (there are no commercially available anti-elephant antibodies) since cross-reactivity between species is limited. Secondly, the antigen against which antibodies are measured must be chosen carefully. For example, the BTB ELISA test measures antibodies against MPB70, a protein specific for M. bovis, and will not be able to detect the presence of antibodies against M. tuberculosis.
The gamma interferon test, accredited by the Standing Committee on Agriculture in Australia, measures the release of a cytokine gamma interferon following exposure of peripheral blood mononuclear cells to mycobacterial antigens, typically PPD-bovis.14,18 A recent modification has examined the utility of detecting gamma interferon and interleukin-2 messenger RNA induced in response to PPD-bovis exposure using reverse transcription quantitative competitive (RT-qc) PCR. These cytokines are elevated in TB-exposed humans who have mounted a protective immune response. Depressed levels of gamma interferon and interleukin-2 correlate with active lesions (Suzanne Kennedy-Stoskopf, personal communication).
Last, various mycobacterial antigens have been directly measured from the blood as an indirect marker of infection. These include the M. bovis-specific surface phenolic glycolipid (PGL) that has been shown to be able to detect experimental infection in cattle2 and the glycopeptide (GPL) specific to M. avium and M. leprae reported as being able to detect leprosy and M. avium infection in humans.3
Caveats for Use in Clinical Situations
Aside from the experience of skin testing, BTB test and ELISA measurements of antibody production in cattle and captive Cervidae, few data exist on the utility of these tests for other species of animals. Further, none of the other methods of detection have been validated for any animal species. Until any of the tests are accepted for use in non-domestic species, careful validation ideally based on results from histology and culture at the time of necropsy is mandated.
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