Background: Zoonotic tuberculosis (zTB) is the transmission of Mycobacterium tuberculosis complex (MTBC) subspecies from animals to humans. zTB is generally quantified by determining the proportion of human isolates that are Mycobacterium bovis . Although India has the world's largest number of human TB cases and the largest cattle population, where bovine TB is endemic, the burden of zTB is unknown. Methods: To obtain estimates of zTB in India, a PCR-based approach was applied to sub-speciate positive MGIT® cultures from 940 patients (548 pulmonary, 392 extrapulmonary disease) at a large referral hospital in India. Twenty-five isolates of interest were subject to whole genome sequencing (WGS) and compared with 715 publicly available MTBC sequences from South Asia. Findings: A conclusive identification was obtained for 939 samples; wildtype M. bovis was not identified (95% CI: 0 – 0.4%). There were 912 M. tuberculosis sensu stricto (97.0%, 95% CI: 95.7 – 98.0), 7 M. orygis (95% CI: 0.3 – 1.5%); 5 M. bovis BCG, and 15 non-tuberculous mycobacteria. WGS analysis of 715 MTBC sequences again identified no M. bovis (95% CI: 0 – 0.4%). Human and cattle MTBC isolates were interspersed within the M. orgyis and M. tuberculosis sensu stricto lineages. Interpretation: M. bovis prevalence in humans is an inadequate proxy of zTB in India. The recovery of M. orygis from humans, together with the finding of M. tuberculosis in cattle, underscores the need for One Health investigations to assess the burden of zTB in countries with endemic bovine TB. Funding: Bill & Melinda Gates Foundation, Canadian Institutes for Health Research

Zoonotic tuberculosis in India: looking beyond Mycobacterium bovis