A method for purification and characterisation of Mycobacterium avium subsp. paratuberculosis from the intestinal mucosa of sheep with Johne's disease

Mycobacterium avium subsp. paratuberculosis, the cause of Johne's disease i n ruminants, cannot be cultured in large quantities from affected sheep in Australia. A method is described for the purification of the organism from the intestinal mucosa of sheep with multibacillary Johne's disease in order to undertake restriction fragment length polymorphism (RFLP) analysis for epidemiological purposes. Using sucrose and potassium chloride as separatio n media for differential and density gradient centrifugation, yields of app roximately 90 mg dry weight M. avium subsp. paratuberculosis per 5 g intest inal mucosa were obtained. The preparations of purified M. avium subsp. par atuberculosis were visually free of non-acid fast bacteria and contained 10 (2)-10(3) aerobic/facultatively anaerobic organisms per gram wet weight. DN A extracted from purified M. avium subsp. paratuberculosis was examined by hybridisation with an IS900 probe after digestion with BstEII and RFLP patt erns distinct from isolates from cattle were obtained. The RFLP pattern of purified M. avium subsp. paratuberculosis from five sheep matched that obta ined previously from organisms cultured from sheep in studies in New Zealan d, indicating that the purification and RFLP method is robust. (C) 1998 Els evier Science B.V. All rights reserved.