Purification of Mycobacterium avium subsp. Paratuberculosis from intestinal mucosa of buffaloes showing lesions of natural paratuberculosis

Two methods, namely density gradient centrifugation and xylene extraction, and a modified enzyme-digestion centrifugation method developed in this study were compared for purification of Mycobacterium avium subsp. paratuberculosis from the intestinal tissues of buffaloes showing lesions of natural paratuberculosis. Out of 12 animals examined by direct impression smears, only 4 animals showed acid-fast bacilli compatible to M.a.paratuberculosis, whereas smears from bacterial pellets purified from large amount of intestinal tissues by above three methods showed AFB in all 12 cases examined. Results of acid-fast smears and IS900 PCR confirmed the specificity of M.a.paratuberculosis in the bacterial pellets. On comparison of the efficacy, the first two methods resulted in lesser bacterial recovery with more cellular debris and contaminating bacteria (average wet weight of bacterial pellets ranged 50–90 mg/3 g of mucosal homogenate). The enzyme digestion-centrifugation (EDC) method yielded better results in terms of bacterial recovery (30–60 mg) and purity. It was concluded that enzyme digestion-centrifugation method expedited the disease diagnosis and molecular confirmation of isolates in less than 6 hours with simple procedures using cheaper and commonly available chemicals. It also increased the test sensitivity of smear examination and genome based diagnostic tests.