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This study reports on an investigation of Mycobacterium tuberculosis cases in mostly captive wild animals using molecular typing tools [Variable Number of Tandem Repeat (VNTR) typing and Restriction Fragment Length Polymorphism typing]. The investigation included cases from (i) the National Zoological Gardens of South Africa ( NZG) recorded between 2002 and 2011; (ii) Johannesburg Zoo, where tuberculosis was first diagnosed in 2007 and has since been detected in three antelope species; (iii) a rehabilitation centre for vervet monkeys ( Chlorocebus pygerythrus) in which M. tuberculosis was diagnosed in 2008; and (iv) incidental cases in other facilities including a sable antelope ( Hippotragus niger), two unrelated cases in chacma baboons ( Papio ursinus) (one of which was from a free-ranging troop) and a colony of capuchin monkeys ( Cebus capucinus). Identical genetic profiles of the latter three isolates indicate the persistence of a single M. tuberculosis strain in this population since at least 2006. Results of the outbreak investigation in the captive vervet monkey colony indicate that it was caused by two unrelated strains, while all 13 M. tuberculosis isolates from 11 animal species in the NZG showed different VNTR patterns. A substantial increase in tuberculosis cases of 60% was recorded in the NZG, compared with the previous reporting period 1991-2001, and may indicate a countrywide trend of increasing spillover of human tuberculosis to wild animals. South Africa ranks among the countries with the highest-tuberculosis burden worldwide, complicated by an increasing rate of multidrug-resistant strains. Exposure and infection of captive wildlife in this high prevalence setting is therefore a growing concern for wildlife conservation but also for human health through potential spillback. [ABSTRACT FROM AUTHOR]