Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 16, Number 12—December 2010

Mycobacterium tuberculosis Infection of Domesticated Asian Elephants, Thailand

Taweepoke Angkawanish, Worawidh WajjwalkuComments to Author , Anucha Sirimalaisuwan, Thattawan Kaewsakhorn, Kittikorn Boonsri, Victor P.M.G. Rutten, and SittidetMahasawangkul
Author affiliations: Author affiliations: National Elephant Institute, Forest Industry Organization, Lampang, Thailand (T. Angkawanish, S. Mahasawangkul); Kasetsart University, Nakhonpathom, Thailand (W. Wajjwalku); Chiang Mai University, Chiang Mai, Thailand (A. Sirimalaisuwan, T. Kaewsakhorn, K. Boonsri); Utrecht University, Utrecht, the Netherlands (T. Angkawanish, V.P.M.G. Rutten); University of Pretoria, Onderstepoort, South Africa (V.P.M.G. Rutten)

Cite This Article


Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.

During the past 2 decades, infections of captive African and Asian elephants with Mycobacterium bovis and M. tuberculosis have been diagnosed worldwide (14). Transmission of these infections to other mammals and veterinary personnel has also been observed (5). To date, M. tuberculosis infection has not been reported in elephants in Thailand. Four elephants referred to the National Elephant Institute (NEI) Hospital during 2005–2008, three of which showed signs of weakness and chronic weight loss, and 1 showed serous nasal discharge. Tuberculosis was confirmed by using conventional and molecular diagnostic assays.

The Study

The ElephantTB Stat-Pak (Chembio Diagnostic Systems, Inc, Medford, NY, USA), which detects antibodies specific to M. tuberculosis in elephants, was performed. Trunk wash sampling of elephants 1, 2, and 4, according to the Guidelines for the Control of Tuberculosis in Elephants, 2008 (6), was followed by culture for bacteria (Technical Appendix Table 1). Necropsy of elephants 1, 3, and 4 was performed at 21 months, 7 days, and 33 months after admission, respectively, and lesion tissues were collected for bacterial culture, Ziehl-Neelsen (ZN) staining, and histopathologic examination (Technical Appendix Table 2 ).

A serum sample from elephant 1 was negative for M. tuberculosis at admission, but a sample obtained 10 months later was positive. Bacteria could not be grown from trunk wash samples. Necropsy showed that elephant 1 had tuberculous lesions in the respiratory tract, mediastinal lymph nodes, liver, kidney, and spleen. Histopathologic examination showed caseous necrosis; infiltration of lymphocytes; and accumulation of macrophages and giant cells in lung tissue, lymph nodes, and liver. ZN staining identified acid-fast bacilli. Mycobacteria were cultured from lesion tissue.

A serum specimen from elephant 2 was negative for mycobacteria at admission, but a second sample was positive 23 months later. Bacteria that were positive by ZN staining were cultured from a trunk wash sample. This elephant is still alive and being kept in a restricted area.

Serum samples from elephant 3 were negative at days 1 and 7 after admission, and the elephant died a few hours after the second sample was tested. A stored serum sample from elephant 3, obtained 4 months earlier was also negative. The animal was severely ill and in lateral recumbency. Necropsy showed tuberculous lesions in the lungs, upper trachea, and mediastinal lymph nodes. Histopathologic examination showed caseous necrosis and accumulation of macrophages and giant cells in the lung and lymph nodes. ZN staining showed acid-fast bacilli. Mycobacteria were cultured from lesion tissues.

A serum specimen from elephant 4 was positive at admission. Initially, M. avium bacteria were grown from cultures of trunk wash samples. At necropsy, tuberculous lesions were found in the respiratory organs and mediastinal lymph nodes. Histopathologic examination showed accumulation of macrophages and edema in the lung tissues. ZN staining did not show acid-fast bacilli. However, mycobacteria were cultured from lesion tissues.

Bacteria cultured from trunk wash and tissue samples were further identified by PCR reactions by using 16S rRNA, 16S–23S-rDNA internal transcribed spacers (ITS) (7,8), and gyrase B (gyrB) primers (Table 1). The subsequent sequencing was conducted by using an ABI 3070 system (Applied Biosystems, Foster City, CA, USA). Unambiguous sequences were compared with data available in GenBank ( and analyzed by using ClustalW version 1.4 ( The 16S rRNA and ITS sequencing confirmed that bacteria from lesion tissues of elephants 1, 3, and 4 and from a trunk wash sample of elephant 2 belong to the M. tuberculosis complex. The gyrB sequences of isolates from elephants 2, 3, and 4 were identical to those of M. tuberculosis strain American Type Culture Collection (ATCC) 27294 and others (Table 2); the gyrB sequence of the isolate from elephant 1 differed at position 482, which is similar to the M. tuberculosis strain KPM KY679, the ancient TbD1-positive strain (911). The mycobacterial interspersed, repetitive-unit variable number tandem repeat typing of the exact tandem repeat-A (ETR-A) locus was performed according to protocols of Fleche et al. (12). The sequence of the ETR-A locus showed that different types of M. tuberculosis were present in elephants 2, 3, and 4 because the sequence had 3, 2, and 4 repeats of the typical 75-bp sequence, respectively.


We report M. tuberculosis infection in elephants in Thailand. Clinical signs shown by these 4 elephants varied considerably. Elephant 2 showed nasal discharge only; in contrast, elephant 3, showed severe clinical signs and lateral recumbency. Elephant 3 had no antibodies, which may indicate an anergic status of the mycobacteria-specific immune response (13). Histopathologic examination showed that this elephant was severely affected by the infection. Elephant 2 is still alive; cultures of trunk wash samples contain mycobacteria. The elephant was seropositive for M. tuberculosis antigens as defined by the StatPak assay. The other 2 elephants (1 and 4) showed anorexia, chronic weight loss, and comparable lesions at necropsy, but diagnostic assays showed variable results. Trunk wash culture, considered to be the standard for confirmation of M. tuberculosis complex infection in elephants, has its limitations, as described elsewhere (14). This study, which included 3 elephants positive for mycobacteria in tissue culture at necropsy, showed that bacterial cultures of only 2 of 60 trunk wash samples were positive for mycobacteria. The study indicates that serologic tests or other diagnostic procedures could not unequivocally identify infected animals, perhaps because of differences in specific immune responsiveness among species and length of time after infection (13). However, the combination of the different diagnostic observations after infection holds promise for improving the likelihood of confirmed M. tuberculosis infection.

Sequence analysis of 16S and ITS indicated M. tuberculosis complex bacteria in each elephant. Nucleotide sequence polymorphism in the gyrB gene of the mycobacteria isolates (911) confirmed the identity of M. tuberculosis for all 4 elephants. M. tuberculosis may be classified into ancestral and modern strains based on M. tuberculosis–specific deletion (TbD1) (15). M. tuberculosis isolated from elephant 1 had a gyrB gene sequence identical to strains of the ancient TbD1-positive strain (Table 2). The other 3 elephants were infected with strains identical to M. tuberculosis ATCC 27294, the modern type, potentially related to major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters (15).

On the basis of these molecular studies, we believe that M. tuberculosis was probably transmitted to these 4 elephants from humans. In addition, mycobacterial interspersed, repetitive-unit variable-number tandem-repeat typing of the ETR-A gene M. tuberculosis strains in elephants 2, 3, and 4 showed different numbers of the typical 75-bp repeat. Therefore, we conclude that the sources of infection were of different origins. Annual health checks of mahouts and veterinarians who were in contact with the infected animals for >4 years at the NEI did not identify any persons with positive results by chest radiograph when tested as part of the tuberculosis control program in Thailand. To control M. tuberculosis complex transmission from humans and other species to wild animals, including elephants, or from wild animals to humans, assays that enable early diagnosis of infection are necessary. Because no assay unequivocally defines the infectious status, a combination of diagnostic approaches is essential.

Further investigation of tuberculosis transmission and surveillance and monitoring of this disease in Thailand will enhance the understanding of its epidemiology. Increased epidemiologic knowledge is essential to control and prevent tuberculosis in elephants.

Mr Angkawanish is a PhD candidate in the Department of Infectious Disease and Immunology, Faculty of Veterinary Medicine, at Utrecht University, the Netherlands. He is also employed by the National Elephant Institute, Forest Industry Organization, Lampang, Thailand. His research interests include the prevalence of elephant tuberculosis, diagnostic methods, and epidemiology of disease.



We thank the staff of the Department of Livestock Development and the National Elephant Institute, Thailand, and Bjarne Clausen for his suggestions to the manuscript.

This work was supported by the EU-Asia Link project, TH/Asia-Link/012(141-055).



  1. Mikota  SK, Peddie  L, Peddie  J, Isaza  R, Dunker  F, West  G, Epidemiology and diagnosis of Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus). J Zoo Wildl Med. 2001;32:116.PubMedGoogle Scholar
  2. Payeur  JB, Jarnagin  JL, Marquardt  JG, Whipple  DL. Mycobacterium isolation in captive elephants in USA. Ann N Y Acad Sci. 2002;969:2568. DOIPubMedGoogle Scholar
  3. Michalak  K, Austin  C, Diesel  S, Bacon  JM, Zimmerman  P, Maslow  JN. Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg Infect Dis. 1998;2:2837. DOIPubMedGoogle Scholar
  4. Lewerin  SS, Olsson  SL, Eld  K, Röken  B, Ghebremichael  S, Koivula  T, Outbreak of Mycobacterium tuberculosis infection among captive Asian elephants in a Swedish zoo. Vet Rec. 2005;156:1715.PubMedGoogle Scholar
  5. Une  Y, Mori  T. Tuberculosis as a zoonosis from a veterinary perspective. Comp Immunol Microbiol Infect Dis. 2007;30:41525. DOIPubMedGoogle Scholar
  6. The National Tuberculosis Working Group for Zoo and Wildlife Species. Guidelines for the control of tuberculosis in elephants, 2008 [cited 2010 Oct 2].
  7. Weisburg  WG, Barns  SM, Pelletier  DA, Lane  DJ. 16S Ribosomal DNA amplification of phylogenetic study. J Bacteriol. 1991;173:697703.PubMedGoogle Scholar
  8. Jones  SE, Shade  AL, McMahon  KD, Kent  AD. Comparison of primer set for use in automated ribosomal intergenic space analysis of aquatic bacterial communities: an ecological perspective. Appl Environ Microbiol. 2007;73:65962. DOIPubMedGoogle Scholar
  9. Kasai  H, Ezaki  T, Harayama  S. Differentiation of phylogenetically related slowly growing Mycobacteria by their gyrB sequences. J Clin Microbiol. 2000;38:3018.PubMedGoogle Scholar
  10. Gutierrez  MC, Brisse  S, Brosch  R, Fabre  M, Omais  B, Marmiesse  M, Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog. 2005;1:e5. DOIPubMedGoogle Scholar
  11. Niemann  S, Harmsen  D, Rusch-Gerdes  S, Richter  E. Differentiation of clinical Mycobacterium tuberculosis complex isolated by gyrB DNA sequence polymorphism Analysis. J Clin Microbiol. 2000;38:32314.PubMedGoogle Scholar
  12. Fleche  PL, Fabre  M, Denoueud  F, Koeck  JL, Vergnaud  G. High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol. 2002;2:1471–2180. 2180–2-37.DOIGoogle Scholar
  13. Van Rhijn  I, Godfroid  J, Michel  A, Rutten  VP. Bovine tuberculosis as a model for human tuberculosis: advantages over small animal models. Microbes Infect. 2008;10:7115. DOIPubMedGoogle Scholar
  14. Lyashchenko  KP, Greenwald  R, Esfandiari  J, Olsen  JH, Ball  R, Dumonceaux  G, Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment. Clin Vaccine Immunol. 2006;13:72232 .DOIPubMedGoogle Scholar
  15. Brocsh  R, Gordon  SV, Marmiesse  M, Brodin  P, Buchrieser  C, Eiglmeier  K, A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A. 2002;6:36849.




Cite This Article

DOI: 10.3201/eid1612.100862

Table of Contents – Volume 16, Number 12—December 2010

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Worawidh Wajjwalku, Faculty of Veterinary Medicine, Kasetsart University, Nakhonpathom, Thailand

Send To

10000 character(s) remaining.


Page created: August 29, 2011
Page updated: August 29, 2011
Page reviewed: August 29, 2011
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.