IS1311 PCR-RFLP molecular epidemiological approach for genotyping of Mycobacterium avium subspecies paratuberculosis in sheep and goat milk.

Background: Johne's disease, a chronic wasting diarrheal illness, affecting ruminants, poses diagnostic challenges due to the prolonged incubation period of the agent.

Aims: The present study aimed to assess the efficacy of the IS900 PCR protocol, for detecting the causative agent and identifying predominant Mycobacterium avium paratuberculosis (MAP) genotypes circulating among small ruminants in the Punjab (India) through the application of IS1311 PCR-RFLP using milk samples.

Methods: A total of 205 milk samples, comprising 102 from goats and 103 from sheep, were tested for MAP DNA through IS900 PCR. Samples positive in IS900 PCR assay were subjected to IS1311 PCR assay, using M56 and M119 primers, followed by digestion of the products using Hinf I and Mse I restriction endonuclease enzymes.

Results: Of the 205 samples, 37 (16 sheep and 21 goats) tested positive. The IS1311 RFLP analysis of all positive samples exhibited a restriction pattern (67, 218, and 323 bp) corresponding to the Bison type MAP Strain.

Conclusion: Identification of a single predominant RFLP type (Bison) in both sheep and goats underscores the potential interspecies transmission and endemic co-circulation of Bison type MAP. These findings emphasize the need for further studies at regional and national levels, offering valuable insights for the development of comprehensive, monitoring and surveillance programs.