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Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.

Mycobacterium tuberculosis is a primary cause of tuberculosis (TB) in elephants (1). Culture of trunk wash samples is regarded as the standard method for the diagnosis of TB in elephants; however, this method has many limitations (2). We previously reported TB in 3 elephants in Nepal that was caused by M. tuberculosis of IndoOceanic lineage (3). Here, we report on mixed M. tuberculosis lineage infection in 2 captive elephants from Chitwan National Park (CNP) in Nepal.

Elephant A was a female elephant [approximately equal to]65-70 years old. She had been in retirement for 3 years before she died in February 2013. We observed TB-like lesions in the lungs postmortem (Appendix Figure 1, EID/article/2 5/5/18-1898-App1.pdf). Elephant B was a 32-year-old male. His body condition had substantially deteriorated for several months before he died. We found extensive TB-like lesions in the lungs at postmortem.

We performed the DPP VetTB Assay (Chembio Inc.,, a serologic test, on the postmortem lung fluid (an off-label use) of elephant A and the serum of elephant B; results were reactive in both cases, indicating the presence of antibodies to TB. We processed the suspected lung lesions according to standard guidelines (4) and performed culture by using Lowenstein-Jensen media.

We performed genetic analyses on the 2 M. tuberculosis isolates by using spoligotyping and large-sequence polymorphism (LSP) as described previously (5). We amplified the direct-repeat region with a primer pair and hybridized the PCR products to a set of 43 oligonucleotide probes corresponding to each spacer covalently bound to the membrane. We identified the spoligo-international type by comparing spoligotypes with the international spoligotyping database (SpolDB4) (6). We performed LSP on the isolates by using specific primers for respective lineages, as described previously (7).

We identified the elephant isolates as a mixture of 2 strains based on uneven spoligotyping color development (suggesting mixture) and LSP detection PCR results (2 bands were observed). The spoligotyping results showed that the elephant A isolate had a new spoligotype that was not found in the international spoligotyping database. The elephant B isolate belonged to the Indo-Oceanic lineage (East African-Indian 5 spoligo-international type 1365) (Table). The prevalence of the Indo-Oceanic lineage among human TB patients in Nepal is only 11.5% (8). The drug resistance-associated region sequences rpoB, katG, inhA promoter region, and gyrA were all wild types in both isolates. Similarly, LSP results showed that elephant A was infected by the Indo-Oceanic and East AfricanIndian lineages (CAS-Delhi) (Appendix Figure 2), whereas elephant B was infected with the East Asian type (Beijing type) (Appendix Figure 3). The prevalence of CAS-Delhi and Beijing type lineages in Nepal in human TB patients is 40.6% and 32.2%, respectively (8). In the gyrA sequence, both of the samples showed a mixed peak of T231C, suggesting that the East African-Indian type is a Nepal-specific lineage.

Our study shows that the first elephant was infected with the Indo-Oceanic and East African-Indian (CAS-Delhi) M. tuberculosis lineages, whereas the second elephant was infected with the Indo-Oceanic and East Asian (Beijing) lineages. We previously identified the Indo-Oceanic lineage in 3 elephants from Nepal (3). We suspect that this lineage might be well adapted in elephants in Nepal.

We diagnosed the mixed lineage infection postmortem in both elephants. However, a successful antemortem diagnosis of mixed infection in a single elephant would enable a precise TB diagnosis and selection of an appropriate antiTB treatment, which could eventually lead to the control of this disease at the herd level.

The source of these mixed infections is unknown and could be from humans or elephants infected with these lineages. Infected elephant handlers who have daily close contact would be a likely human source. Genotyping of additional isolates from elephants and their handlers will help to determine the source of infection. We recommend regular TB screening of elephant handlers to safeguard human health and help prevent transmission of TB from humans to elephants.


We thank Arjun Pandit, Chitra B. Khadka, and Kiran Rijal for helping with the collection of elephant samples; technicians at the German Nepal Tuberculosis Project in Kathmandu for performing the laboratory work; and Yukari Fukushima and Haruka Suzuki for technical support at the Hokkaido University Research Centre for Zoonosis Control.

The National Trust for Nature Conservation Nepal and the Department of National Parks and Wildlife Conservation of Nepal's Ministry for Forests and Soil Conservation supported this work.

Mr. Paudel is an assistant professor in the Department of Cell Physiology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. His research interests include the mechanism of TB bacteria entry into cells, TB in elephants, and development of TB diagnostic tools for elephants.


(1.) Mikota SK, Lyashchenko KP, Lowenstine L, Agnew D, Maslow JN. Mycobacterial infections in elephants. In: Mukundan H, Chambers MA, Waters WR, Larsen MH, editors. Tuberculosis, leprosy and other mycobacterial diseases of man and animals: the many hosts of mycobacteria. Wallingford (UK): CABI Publishing House; 2015. p. 259-76.

(2.) Lyashchenko KP, Greenwald R, Esfandiari J, Olsen JH, Ball R, Dumonceaux G, et al. Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment. Clin Vaccine Immunol. 2006;13:722-32. CVI.00133-06

(3.) Paudel S, Mikota SK, Nakajima C, Gairhe KP, Maharjan B, Thapa J, et al. Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal. Tuberculosis (Edinb). 2014;94:287-92.

(4.) Groothuis DG, Yates MD. Decontamination, microscopy and isolation. In: Groothius DG, Yates MD, editors. Diagnostic and public health mycobacteriology. 2nd ed. London: Bureau of Hygiene and Tropical Diseases, European Society for Mycobacteriology; 1991. p. 63.

(5.) Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997;35:907-14.

(6.) Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, et al. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol. 2006;6:23.

(7.) Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, Narayanan S, et al. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 2006;103:2869-73.

(8.) Malla B, Stucki D, Borrell S, Feldmann J, Maharjan B, Shrestha B, et al. First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal. PLoS One. 2012;7:e52297.

Address for correspondence: Toshio Tsubota, Hokkaido University, Laboratory of Wildlife Biology and Medicine, Graduate School of Veterinary Medicine, Kita 18 Nishi 9, Sapporo, Hokkaido 060-0818, Japan; email:

Author affiliations: Hokkaido University, Sapporo, Japan (S. Paudel, C. Nakajima, M. Sashika, M. Shimozuru, Y. Suzuki, T Tsubota); Elephant Care International, Hohenwald, Tennessee, USA (S.K. Mikota); Department of National Parks and Wildlife Conservation, Kathmandu, Nepal (K.P. Gairhe); German Nepal Tuberculosis Project, Kathmandu (B. Maharjan); National Trust for Nature Conservation, Lalitpur, Nepal (S. Subedi); Chitwan Medical College, Chitwan, Nepal (A. Poudel)


Sarad Paudel, [1] Chie Nakajima, [1] Susan K. Mikota, Kamal P. Gairhe, Bhagwan Maharjan, Suraj Subedi, Ajay Poudel, Mariko Sashika, Michito Shimozuru, Yasuhiko Suzuki, Toshio Tsubota

[1] These first authors contributed equally to this article.
Table. Genotypic characteristics of Mycobacterium
tuberculosis isolates from 2 elephants, Nepal *

Source         Spoligotypef binary code               SIT

Elephant A     1110000111111111111111001111     New ([section])
Elephant B     00000000000000000000000011110         1365

Source              Clade        gyr A ([double dagger])

Elephant A     New ([section])            T231C

Elephant B          EAI5                  T231C

* EAI5, East African-Indian 5; SIT, spoligo-international type.

([dagger]) Spoligotype was determined as previously described by
Brudey et al. (6).

([double dagger]) Mutation in a partial sequence of gyrA. The gyrA
sequence of both elephant isolates had a synonymous single
nucleotide polymorphism from T to C at position 231.

([section]) Not found in the international spoligotyping database
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Author:Paudel, Sarad; Nakajima, Chie; Mikota, Susan K.; Gairhe, Kamal P.; Maharjan, Bhagwan; Subedi, Suraj;
Publication:Emerging Infectious Diseases
Geographic Code:9JAPA
Date:May 1, 2019
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