Center of Veterinary Epidemiology and Animal Disease Surveillance Systems, College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA; †Present address: Environmental Medicine Consortium, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
Mycobacterium bovis is typically the agent of tuberculosis in hoofstock. In the past 20 years, M. bovis infection has occurred in ungulates in at least 20 U.S. zoos.14 The American Zoo and Aquarium Association and the American Association of Zoo Veterinarians have recommended testing for tuberculosis; however, prior to 2001, no standard testing protocols had been implemented. Federal regulations have only required testing prior to interstate shipment of federal eradication “program species,” including domestic cattle, bison, and cervids. With few exceptions, diagnostic tests have been validated only in these program species. The National Tuberculosis Working Group for Zoo and Wildlife Species was formed to address issues regarding tuberculosis in zoos, circuses, and other animal collections and this group has recently drafted guidelines for tuberculosis surveillance, including recommendations for testing.14
Intradermal (ID) Testing
In zoos, intradermal (ID) tests are typically the primary screening tools for detecting M. bovis infection. However, ID tests have rarely been validated in non-program hoofstock and some reports suggest low sensitivity and/or specificity in non-domestic species.3,9 It may be difficult to evaluate the response in some species because of skin thickness. Further data needs to be generated in order to make conclusions about the validity of ID testing in many non-domestic hoofstock species. Animals that respond to ID tests should be further evaluated by other techniques. ID tests are additionally problematic in that they require handling animals both for initial tuberculin injection and again for injection site palpation. Post-testing suppression of the immune response precludes retesting 10–60 days after ID testing (10–90 days in cervids).
The host’s response to tuberculin is primarily cell-mediated, depending on previous exposure to antigen(s) and resultant differentiation of lymphocytes. Responses become more pronounced as infections become more extensive. False-negative ID tests may occur for many reasons and anergy can be seen in severely infected animals that lose the cellular response. Poor immune responses can be caused by stress, concurrent infections, a lack of antigenic stimulation, variability in antigen recognition, or immune-modulating therapy.4 False-positives may occur in animals with exposure to non-tuberculous mycobacteria. The primary screening test in cattle and bison is the caudal fold test. While this test is an effective screening tool in cattle and bison, it is ineffective in cervids and has not been validated in most other hoofstock species.
The single cervical test (SCT) requires ID injection of bovine tuberculin in the neck. It is the official screening test in cervids and is recommended for many non-bovid hoofstock.14 A similar test can be applied in camelids, swine, and tapirs, however, in these species, non-cervical sites have been recommended.14 Extensive work has been conducted to validate both the SCT and the comparative cervical test (CCT) in cervids; however, most of this evaluation has been conducted on Cervus elaphus (red deer and elk) with few reports of validation in other species. In cervids, the SCT has had a high sensitivity (Se) of 86–97%, but a specificity (Sp) of only 70–78%.5,16 In contrast, the CCT has a lower Se (70–84%), but a higher Sp (80–99%) in cervids.5,10 This test is performed by injecting balanced bovine and avian tuberculins at separate sites; the two responses are then compared. The CCT’s low Se may lead to an increase in false-negative results, so it is currently recommended that it be used only as a secondary/confirmatory test for all hoofstock.
Whether testing for cellular or humoral immunity, blood testing for tuberculosis can be advantageous as it only requires handling the animal once. However, techniques for testing blood have had problems with sensitivity and specificity. Most blood techniques have not been validated in non-domestic hoofstock and some require special sample handling or preparation.
Lymphocyte transformation assays measure cellular immunity by quantitating the antigenic stimulation of T-helper lymphocytes.2 Lymphocytes are separated from the blood by differential centrifugation, challenged with antigens, and then incubated. Following incubation, cultures are pulsed with a radioisotope-labeled nucleotide. After further incubation, the cells are harvested and incorporation of the radioisotope is measured. Lymphocytes previously challenged with the antigen proliferate and incorporate elevated levels of the radioisotope. Blood samples must be carefully handled and typically need to be processed within 48 hours. In cervids, reported Se and Sp have been highly variable. Differences in results may depend on species tested, laboratory techniques, antigens, stress level of the animal, and/or statistic methods of evaluation.2,4,6
The gamma-interferon test is a whole blood assay that detects specific lymphokines produced when lymphocytes respond to specific antigens. It is used in attempt to differentiate nonspecific lymphocyte transformation from antigen-specific stimulation. Lymphocytes must remain viable and functional, so it is important that the sample be handled gently and processed quickly (within 8–12 hours). Once the lymphocytes have been stimulated, the plasma supernatant is drawn off and can be frozen and stored. Research in cattle has suggested that this assay has superior Se and Sp to the caudal fold test.17 However, the test uses specific monoclonal antibodies and it’s unclear how well gamma-interferon will be detected in other hoofstock species.
Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is a serologic test used to measure antigen or antibody. These tests only require serum, so sample collection, transport, and processing are simple and samples may be preserved indefinitely. Blood is collected and centrifuged. Serum is extracted and frozen (preferably at -70°C) until it is tested. An antigen is usually bound to a plate and the animal’s serum is placed in contact. Plates are washed and the ELISA measures the amount of bound antibody. The development of antibody detection methods that are not species-specific has facilitated the use of ELISA in non-domestic species.7,15 Many different antigens have been used for tuberculosis detection, including antigens from both M. tuberculosis and M. bovis. The multiple-antigen ELISA, using a combination of several antigens, appears to provide the best combination of Se and Sp. Animals may come into contact with non-tuberculous mycobacteria, such as M. avium or saprophytic mycobacteria, so antigens from these organisms have also been used in the multiple-antigen ELISA, in an effort to increase the test’s specificity.
Serologic assays have had considerable variation in Se and Sp. One multiple-antigen ELISA demonstrated Se of 85% and Sp of 100% in red deer,8 while an investigation in multiple cervid species revealed lower Se (66%) and Sp (56%)7. There is some evidence that serologic responses are species-specific and that different antigens, concentrations of antigens, and/or different “cut-off” levels are necessary for different species. When our laboratory evaluated infected and non-infected bongo (Tragelaphus eurycerus), an infected individual had a much stronger serologic response than its non-infected cohorts. However, the infected bongo’s response was much less than that typically seen in M. bovis infected cattle.
The primary host response to mycobacterial infection is cell-mediated and it is thought that humoral responses are often weak, generally occurring only in advanced disease. The utility of the ELISA for routine screening of non-domestic hoofstock is still unclear, however, even if only severely infected animals are detected, the ELISA will be helpful as severely infected animals are likely to be shedding large numbers of organisms and need to be detected rapidly. The ELISA may also be useful for detecting anergic animals that no longer have a cellular response but do have a humoral response. There is some evidence that the ELISA may be useful after ID testing, as exposure to ID antigens increases humoral antibody levels in infected animals.
Blood Test for Tuberculosis (BTB)
The results of LT and ELISA may be combined, along with plasma fibrinogen, to produce a composite assay termed the blood test for tuberculosis (BTB). Developed in New Zealand, this test has been reported to have excellent Se (>95%) and Sp (>92%) in red deer.8 Findings were not the same when the BTB was used on elk and other cervids in the United States, as it failed to identify infected animals on multiple occasions.16 It is not currently available in the United States.
Ag85 Dot Blot Immunoassay
Actively proliferating mycobacteria secrete Antigen 85 complex proteins.4 The presence of these products in an animal’s circulation may indicate active mycobacterial infection. The Ag85 assay is performed by blotting diluted serum samples and bovine reference samples to nitrocellulose.4 Ag85 is detected by dot immunobinding using monoclonal antibodies. Because this assay detects antigens, it should not depend on the immunocompetency of the animal, nor should host-specific assays be necessary. A report on Ag85 assay in non-domestic hoofstock (multiple species), described higher levels in infected groups than non-infected groups.11
Although culture is the current “gold-standard” for diagnosis of M. bovis, this test is often used only if there is a history or clinical suspicion of tuberculosis. Mycobacterial culture can detect as few as 100 organisms/ml,12 however, results may take up to 8 weeks. Recommended specimens have included lung, lymph nodes, and abscesses, but it may not be feasible to sample these tissues in live animals. For living non-domestic hoofstock, the tracheal wash is the currently recommended sample.14 Nasal washes or swabs may be collected but will likely be negative unless large numbers of mycobacteria are actively being shed. Other methods may include thoracoscopy, bronchoscopy, or thoracotomy. Infected animals may be culture-negative if samples are inappropriately collected, processed, or handled. Samples from non-domestic hoofstock should not be shipped using sodium borate as this may cause false-negative results. Sodium borate has been recommended for shipping tissues from cattle, to prevent overgrowth of non-tuberculous mycobacteria on the outer surface. Cattle samples are usually large enough that sodium borate doesn’t penetrate all of the tissue, but samples from non-domestic hoofstock are usually smaller; sodium borate may penetrate the entire sample, thereby killing all mycobacteria.
Histopathology and Acid-Fast Staining
Histopathologic analysis is routinely performed to help confirm a diagnosis of mycobacterial infection, particularly postmortem. Tissues are embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Sections with granulomas are stained by the Ziehl-Neelsen or Kinyon acid-fast technique.2 The presence of acid-fast bacteria is suggestive of M. bovis infection, although other non-tuberculous mycobacteria (as well as Nocardia spp.) can appear as acid-fast. Mycobacterial culture is typically necessary to confirm M. bovis, although a presumptive diagnosis may sometimes be made if there are histologic changes consistent with mycobacterial infection and a history of M. bovis in the herd.
Histopathology is not possible on many samples (tracheal washes, small tissue samples, etc.) that are taken from the live animal, and acid-fast smears may be performed instead. However, smears have a low sensitivity, as the lower limit of detection is about 1000 organisms/ml.12 Hence, false negatives are common with acid-fast smears. False positives may be caused by the presence of M. avium, saprophytic environmental mycobacteria, or Nocardia spp.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is typically used on samples that are taken for culture, histopathology, or acid-fast smear. The PCR is a nucleic acid amplification technique in which specific sequences of DNA/RNA are replicated, allowing for detection of target sequences that otherwise would not be present in high enough numbers. DNA/RNA probes are then used for detecting amplified mycobacterial sequences. PCR methods can detect as few as 20–50 mycobacterial organisms/ml1 and in cases where positive samples are contaminated with bacteria, recovery rates for the PCR appear to be better than those for culture. Clinical trials have shown PCR to be highly specific for M. tuberculosis-complex organisms and preliminary information on these tests are very promising, with high Se (93%) and Sp (100%).13 While PCR appears promising, this technology should still be utilized in conjunction with culture.
There are many diagnostic tests available and the tests used will vary by species, institution, and situation. No test is 100% sensitive or 100% specific. There appear to be species variations in response to tests of immunity and further information is needed for informed evaluation of these tests in non-domestic species. Many institutions that do not have documented M. bovis infection will continue to use ID testing for screening. A battery of diagnostic tests is more likely to be used in places that are addressing documented M. bovis infection. Clinicians are encouraged to consider submitting samples for tuberculosis testing when feasible, so that we may better understand the tests’ utility in managing the health of our non-domestic hoofstock populations.
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14. National Tuberculosis Working Group for Zoo and Wildlife Species. 2001. Tuberculosis Surveillance Plan for Non-Domestic Hoofstock. 30 pgs.
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