Abstract

Antemortem diagnosis of tuberculosis (TB) in wildlife has always proved challenging. Confirmation of TB as the disease process causing the clinical signs often occurs after a lengthy period of mycobacterial culture of diagnostic samples collected antemortem (e.g., tracheal wash) or postmortem (e.g., lesions in tissue). New diagnostic tools are needed to improve the ability to diagnose TB more quickly. Two novel serologic methods, Multi-Antigen Print ImmunoAssay (MAPIA) and lateral-flow technology (Rapid Test or RT) (Chembio Diagnostic Systems, Inc., Medford, New York 11763 USA) have been adapted for use in several species for the detection of Mycobacteria-specific antibody.^1,2 With MAPIA, specific antigens are printed as horizontal stripes on a nitrocellulose membrane. Strips can be cut from this print-out and incubated with test serum samples as a Western blot with an anti-immunoglobulin conjugate and color developer. Using this assay, an antibody response to mycobacterial antigens has been observed in sera from Mycobacteria-infected animals. Using selected mycobacterial antigens, RT was developed for rapid antibody detection that can use serum, plasma, or whole blood and provides results within 15 min for validated species. This technology is similar to in-clinic tests for FIV/FeLV detection (Snap FIV/FeLV Test, IDEXX Laboratories, Inc., Westbrook, Maine 04092 USA).²

Elephants were the “proof of concept” species for the development of this test methodology.² Although this methodology shows promise for use in other species, validation of each test is required with sera from individuals confirmed culture positive for mycobacterial infections. This report presents two species: addra gazelle (Gazella dama ruficollis) and jaguar (Panthera onca onca), previously not tested with MAPIA and RT and demonstrates their usefulness with clinical cases.

Case Studies

Addra Gazelle

A 15-year-old female addra gazelle was euthanatized August 2002 due to severe weight loss and persistent hyperglobulinemia. Gross necropsy revealed a severe, diffuse granulomatous pneumonia and bronchial lymphadenopathy. Histopathology demonstrated extensive multifocal acid-fast positive granulomatous pneumonia, moderate multifocal erosive rumenitis, and moderate renal fibrosis and interstitial nephritis. Mycobacterial culture of the lung tissues grew Mycobacterium tuberculosis. Retrospective serum samples from several dates were submitted for RT and MAPIA.

While the globulins were elevated in this animal as early as May 1997, no specific indicators of mycobacterial disease were observed during that time on either test. However, RT and MAPIA appeared to detect mycobacterial reactivity (ESAT-6 and polyprotein ESAT-6/CFP10) as early as February 2002 with the serum sample dated 24 August 2002 demonstrating even more noticeable bands consistent with M. tuberculosis (ESAT6, CFP10, rMPB83, nMPB83, CFP10/ESAT6, F10 and Acrl/MPB83). These results support the presence of tuberculosis in the gazelle at least 6 months before euthanasia and show an increasing immune response to the mycobacteria over time.

This herd of gazelle has historically had health issues with parasites, high prevalence to Johne’s disease, rumenitis and renal failure. Any of these conditions would allow an opportunistic organism such as M. tuberculosis to become an established and active infection. The remaining members of the herd were examined diagnostically with thoracic radiographs, bronchial lavage, intradermal skin testing, and ancillary testing on serum with RT and MAPIA. No clinical or serologic evidence of tuberculosis was found.

Jaguar

A 12-year-old female jaguar was imported from Venezuela in July 1998. In October 2000, this animal presented with lethargy, inappetence and abdominal distension. One week following exploratory abdominal surgery for excisional biopsy of an infiltrative mass in the greater omentum, the animal died in November 2000. Gross necropsy showed multiple, infiltrative granulomas of the omentum and lymph nodes and hepatic abscessation. Histopathologic findings included multifocal necrogranulomatous peritonitis, hepatitis and lymphadenitis. The granulomatous lesions were acid fast positive and cultured M. bovis.

Serum samples (dated 15 October 1999 and 3 November 2000) were submitted for retrospective analysis by MAPIA and RT. Sera from four other jaguars in the same collection were submitted for assessment; two of these cats were imported with and exposed to the TB-positive jaguar. Also, sera from three jaguars under consideration for acquisition from Guyana were assessed. All jaguar samples were submitted without disclosure of history.

Both tests demonstrated reactivity to mycobacterial antigens in the serum samples from the M. bovis-positive cat (MPB83, nMPB83 and 16/83 zones) with a band pattern noted as suggestive of M. bovis infection. Both samples submitted from the TB-positive cat were similar in band pattern, although the first sample was collected during a normal physical examination. The serum samples from the other cats showed a complete lack of any band pattern suggesting no antibody formation and presumably a lack of exposure to mycobacteria.

Discussion

The addition of these two species to the demonstrated ability of RT and MAPIA to diagnose TB broadens the potential application of these testing methodologies. It is encouraging that these diagnostics did identify as positive the two mycobacteria culture-positive individuals. These tests will only have predictive value if truly no mycobacterial disease is present in the animals which test negative on the MAPIA and RT. Continuation of testing within these and other species using all available mycobacterial diagnostics will be important in validating these tests.

The band pattern of the MAPIA appears to correlate with particular Mycobacteria species and may have predictive ability as to the species of infecting organism. This information could allow exploration and investigation of the epidemiology of the disease in a more timely fashion than occurs with confirmatory culture results. Additionally, the gradual development of the intensity of the band pattern over time shows the individual’s growing immunologic response to the mycobacterial infection so the course of the disease process can be documented.

The imported jaguar likely arrived at the institution with the mycobacterial infection from ingestion of a mycobacteria-infected carcass at the originating institution where whole carcass feeding was common practice. Multiple diagnostics (e.g., hepatic biopsy for histopathology and mycobacterial culture, mycobacterial blood culture, and tuberculin skin testing) on the two other remaining Venezuelan-imported cats have failed to confirm any underlying mycobacterial disease. The hepatic biopsies in the male have shown granulomatous changes but acid-fast organisms have neither been demonstrated nor Mycobacteria species cultured. Negative results from the MAPIA and RT are encouraging, but due to the insidious nature of the disease, these mycobacteria-exposed cats will continue to be monitored with the available diagnostics. This will allow for further validation of these new testing methodologies.

The ability to utilize the MAPIA and RT as non-invasive and relatively rapid turnaround tests will be of tremendous assistance in such clinical cases. Additionally, when other jaguars from Guyana were being considered for importation, they were assessed for tuberculosis prior to acquisition due to the problems experienced from the other imported jaguar. Based on the possibility of acquiring animals with pre-existing mycobacterial infections, especially when imported from tuberculosis-risk countries, it is encouraged that TB diagnostics be performed. RT and MAPIA can assist in expedient testing in such circumstances, as more species are validated.

Conclusion

The RT and MAPIA appear as very promising tools in the diagnosis of tuberculosis. As this methodology develops and the sensitivity and specificity of the tests are confirmed in other species, earlier detection of TB will be possible and result in earlier initiation of treatment or response. Additionally, positive MAPIA and RT results can help support that other diagnostics should be performed to identify the presence and location of mycobacterial disease in an individual.

Acknowledgments

This research was supported by Chembio Diagnostic Systems, Inc.

Literature Cited

1.  Lyashchenko, K., M. Singh, R. Colangeli, and M. Gennaro. 2000. A multiantigen print immunoassay for the serological diagnosis of infectious diseases. J. Immunol. Methods 242: 91–100.

2.  Lyashchenko, K., M. Miller, and W.R. Waters. 2005. Application of multiple antigen print immunoassay and rapid lateral flow technology for tuberculosis testing of elephants. Proc. Am. Assoc. Zoo Vet. Conf., Omaha, NE. Pp. 64–65.