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 in 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 separation media for differential and density gradient centrifugation, yields of approximately 90 mg dry weight M. avium subsp. paratuberculosis per 5 g intestinal mucosa were obtained. The preparations of purified M. avium subsp. paratuberculosis were visually free of non-acid fast bacteria and contained 10(2)-10(3) aerobic/ facultatively anaerobic organisms per gram wet weight. DNA extracted from purified M. avium subsp. paratuberculosis was examined by hybridisation with an IS900 probe after digestion with BstEII and RFLP patterns distinct from isolates from cattle were obtained. The RFLP pattern of purified M. avium subsp. paratuberculosis from five sheep matched that obtained previously from organisms cultured from sheep in studies in New Zealand, indicating that the purification and RFLP method is robust.