Mycobacterium tuberculosis Infection of Domesticated Asian Elephants, Thailand Taweepoke Angkawanish, Worawidh Wajjwalku, Anucha Sirimalaisuwan, Sittidet Mahasawangkul, Thattawan Kaewsakhorn, Kittikorn Boonsri, and Victor P.M.G. Rutten 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 in- fections, a combination of diagnostic assays is essential. D uring the past 2 decades, infections of captive Afri- can and Asian elephants with Mycobacterium bovis and M. tuberculosis have been diagnosed worldwide (1–4). Transmission of these infections to other mammals and vet- erinary 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 Sys- tems, 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. 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. 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 tuber- culous lesions in the respiratory tract, mediastinal lymph nodes, liver, kidney, and spleen. Histopathologic examina- tion 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 posi- tive 23 months later. Bacteria that were positive by ZN staining were cultured from a trunk wash sample. This el- ephant 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 nega- tive. The animal was severely ill and in lateral recumbency. Necropsy showed tuberculous lesions in the lungs, upper trachea, and mediastinal lymph nodes. Histopathologic ex- amination 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 mediasti- nal lymph nodes. Histopathologic examination showed ac- cumulation of macrophages and edema in the lung tissues. ZN staining did not show acid-fast bacilli. However, myco- bacteria 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 subse- quent sequencing was conducted by using an ABI 3070 system (Applied Biosystems, Foster City, CA, USA). Un- ambiguous sequences were compared with data available in GenBank (www.ncbi.nih.gov/BLAST) and analyzed by using ClustalW version 1.4 (www.ebi.ac.uk/Tools/ clustalw/). 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 Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 16, No. 12, December 2010 1949 Author affiliations: National Elephant Institute, Forest Industry Or- ganization, Lampang, Thailand (T. Angkawanish, S. Mahasawang- kul); Kasetsart University, Nakhonpathom, Thailand (W. Wajjwalku); Chiang Mai University, Chiang Mai, Thailand (A. Sirimalaisuwan, T. Kaewsakhorn, K. Boonsri); Utrecht University, Utrecht, the Nether- lands (T. Angkawanish, V.P.M.G. Rutten); and University of Preto- ria, Onderstepoort, South Africa (V.P.M.G. Rutten) DOI: 10.3201/eid1612.100862