Abstract

Tuberculosis (TB) remains a serious re-emerging disease in wildlife and zoo animals. Mycobacterium tuberculosis has been isolated from 30 captive Asian elephant (Elephas maximus) within 14 herds in the United States (1994–2004) and Mycobacterium bovis has been isolated from one African elephant (Loxodonta africana) (Mikota pers. comm.).³ There are several challenges with elephant TB diagnosis. Culture of trunk wash has relatively poor sensitivity and is subject to contamination. Skin test is not validated in elephants and there is little reliability in these results.⁴ Serologic tests are appealing because samples can be stored for future analysis, archived samples can be analyzed, various assay platforms can be directly compared, and these assays are amenable to serial analysis (e.g., to monitor therapy). There is currently a multiple-antigen ELISA test available for experimental use in elephants.¹

To improve tuberculosis control, new diagnostic tools should be rapid, accurate, and host species independent. Two novel serologic methods, multi-antigen print immunoassay (MAPIA) and lateral flow technology (rapid test), have been adapted for use in white-tailed deer, European badger, cattle, and Asian and African elephants for the detection of TB-specific antibody. Serologic markers of diagnostic importance have been identified for each host tested so far. With MAPIA, a machine prints specific antigens horizontally on a nitrocellulose membrane which can be cut into strips and used in western blot.² Strips are incubated with test serum samples, then an anti-Ig conjugate and color developer. Using this assay, an antibody response to multiple mycobacterial antigens has been observed in sera from M. tb-infected elephants. No antibody response was detected to any antigens in noninfected elephant sera. Additionally, the kinetics of antibody responses by elephants undergoing antibiotic therapy indicates that the MAPIA could be used for monitoring treatment and to determine recrudescence of infection.

Using selected antigens, a lateral flow test was developed for rapid antibody detection that can be used in multiple species. The rapid test can use serum, plasma, or whole blood and provides results within 15 minutes. These tests are similar to in-clinic tests for FIV/FeLV detection (snap test, IDDEX). If a band is present in the test strip, it indicates a positive reaction (antibody present). A panel of sera from healthy and TB infected elephants showed good correlation between the MAPIA and the rapid test (Table 1).

Table 1. Comparison of serodiagnostic results for tuberculosis in elephants

In summary, it appears that TB-infected elephants produce a robust antibody response that can be detected in serologic assays. Of special significance is the kinetics of the response, which may permit earlier detection of infection than current diagnostic methods. While initial results are promising, additional studies are required to validate these two assays. A relatively small set of serum samples from documented infected and noninfected elephants was used, and more samples are needed to further validate the tests. MAPIA has been used to optimize antigen selection in order to make the most sensitive and specific rapid test. This strategy may also allow for identification of “treatment-sensitive” antigens that could be used in the MAPIA format to monitor TB therapy. While elephants will be used as an initial “proof of concept” species for test development, additional samples from other species will also be evaluated to determine applicability to other species (i.e., a host species independent test), thus benefiting other groups such as primates, rhinos, cervids, etc.

Acknowledgments

The authors thank the zoos and individuals that have provided samples and assistance with this research, including Ray Ball, Carol Buckley, Jenifer Chatfield, Genny Dumonceaux, Javan Esfandiary, Rena Greenwald, Scott Larsen, Susan Mikota, Torsten Moller, Dick Montali, Mike Richards, Heidi Riddle, Mo Salman, Scott Terrell, and many others. This research was supported by Chembio Diagnostics, Inc.

Literature Cited

1.  Larsen, R.S., M.D. Salman, S.K. Mikota, R. Isaza, R.J. Montali, and J. Triantis. 2000. Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis infection in captive elephants. J Zoo Wildl Med. 31:291–302.

2.  Lyashchenko, K., et al. 2000. A multiantigen print immunoassay for the serological diagnosis of infectious diseases. J Immunol Methods. 242:91–100.

3.  Mikota, S.K., and J. Maslow. 2002. Epidemiology and treatment of tuberculosis in elephants: 2002. In: Proc Am Assoc Zoo Vet Annu Meet. 384–387.

4.  Mikota, S.K., L. Peddie, J. Peddie, et al. 2001. Epidemiology and diagnosis of Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus). J Zoo Wildl Med. 32:1–16.