Abstract Zoonotic tuberculosis (TB) caused mainly by Mycobacterium bovis is a neglected public health problem in areas with intensive livestock farming. The present study was conducted to determine the prevalence and molecular characteristics of Mycobacterium tuberculosis complex (MTBC) isolates from humans and livestock in Hamedan Province, Iran, using region-of-difference (RD) typing. Sputum samples were collected from 21 PPD-positive (purified protein derivative or tuberculin skin test) individuals suspected of TB after screening 1503 individuals. Tissue and lymph node samples were collected from 17 tuberculin-positive dairy cattle after screening 31,202 animals. Samples were cultured on Lowenstein–Jensen medium with and without pyruvate. Acid-fast bacilli (AFB) were confirmed by Kinyoun staining, and species identification was performed by Polymerase Chain Reaction (PCR) for the 16S rRNA gene, IS6110, and RD typing (RD1, RD4, RD9, RD12). Culture-positive results were obtained in 16 out of 21 (76.2%) of human samples and 7 out of 17 (41.2%) of animal samples. All isolates were AFB-positive and confirmed as members of the MTBC by PCR. RD typing revealed that all human isolates exhibited the M. tuberculosis profile, whereas all animal isolates showed the M. bovis profile (absence of RD1 and RD4, presence of RD9 and RD12). No mixed infections, atypical banding patterns, or cross species transmission were detected. The study provides molecular evidence for the circulation of M. tuberculosis in humans and M. bovis in cattle in Hamedan, an endemic region in western Iran. No M. bovis was isolated from human samples in this cohort; given the pulmonary-only sampling frame, this finding indicates no detectable zoonotic infection among the enrolled patients rather than absence of zoonotic TB in the province.
