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Mycobacterium avium subsp. paratuberculosis (MAP) is an etiological agent of paratuberculosis (JD), causing chronic persistent diarrhoea in small and large ruminants. Now-a-days, it is increasingly considered as a problem which is adversely affecting animal health leading to significant economic losses to the livestock industry. Recent emerging evidences demonstrated a relationship between MAP and Inflammatory bowel disease (IBD), Crohn’s disease (CD) confirming its zoonotic significance hence the present study was carried out to record the MAP infection in buffaloes of Malwa region (Madhya Pradesh) by Dot ELISA. A total of 112 serum samples of buffaloes of either sex were examined for diagnosis of MAP infection which were slaughtered at Cantonment Board slaughter house, Mhow and Nagar Nigam Indore, belonging to different places of Malwa region aged between 1 to 10 years. Out of 112 serum samples, 92 (82.14%) samples were recorded positive for Anti-MAP antibodies or MAP infection in the serum samples of buffaloes by Dot-ELISA as a serological diagnostic test.