, 2016. Isolation and molecular confirmation of Mycobacterium avium subspecies paratuberculosis in cattle and buffaloes from three states of India. Bulg. J. Vet. Med., 19, No 3,[189][190][191][192][193][194][195][196][197][198][199] In a random survey for identification of Mycobacterium avium subsp paratuberculosis (MAP) in cattle and buffaloes from states of Telangana, Maharashtra and Gujarat in India, a single time point sampling of 1,008 animals was undertaken. The samples: milk (n=605), faeces (n=381) and necropsy/post-mortem tissues (n=22) were initially processed for isolation of MAP in the MGIT BACTEC 960 system, which detected 22 out of 1,008 as MAP. All 22 samples were sub-cultured in Middlebrook 7H10 agar, Herrold's Egg Yolk medium (HYEM) and 7H9 broth supplemented with Mycobactin J. The three media showed different sensitivities in supporting the growth of field MAP strains. Only 9 out of 22 BACTEC 960 positive cultures grew on Middlebrook 7H10 agar, and 7H9 broth supplemented with Mycobactin J, while 8 out of 22 cultures grew on HYEM. Seven out of 9 positive cultures originated from milk samples from cattle, while the remaining 2 positive cultures were from necropsy and post-mortem tissues of cattle and buffalo respectively. MAP could not be isolated from faecal samples. All cultures grown on 7H10 agar, HYEM, 7H9 broth supplemented with Mycobactin J revealed acid fast bacilli on staining. In a recently developed in-house Real Time PCR targeting the MAP-specific insertion element ISMav2, only 8 out of the 9 field isolates were identified as MAP.