Cultivation of Mycobacterium paratuberculosis from bovine fecal samples by using elements of the Roche MB Check system

Abstract

Components of a commercially available, nonradiometric, biphasic (liquid medium then solid medium) system for the detection of Mycobacterial species, Roche MB Check, were adapted for the isolation of Mycobacterium paratuberculosis from bovine fecal specimens. A two-stage culture procedure was developed in which processed fecal samples were incubated in modified commercial liquid medium and then subcultured onto Herrold's egg yolk medium with mycobactin. By using known culture-positive samples and/or samples from animals clinically affected with paratuberculosis, it was found that visible colonial growth on solid media could be obtained after 4 weeks of incubation in liquid medium containing egg yolk and mycobactin followed by 8 weeks of incubation on Herrold's egg yolk medium. In the second part of the study, conventional fecal culture (sample sedimentation in hexadecylcetylpyridinium chloride followed by incubation on Herrold's egg yolk medium) was compared with the two-stage system using a two-step centrifugation technique for sample preparation. One hundred fecal samples from clinically normal but absorbed-enzyme immunoassay-positive cattle were used for the comparison. Conventional culture yielded a sensitivity of 16.5%, whereas the sensitivity of the two-stage system was 29.4%. When used in parallel, the tests detected 36.5% of the samples. There was no significant difference between the two methods in the time taken to obtain visible colonies. These results indicate that the two-stage method is a sensitive method for isolation of M. paratuberculosis from fecal samples obtained from cattle with clinical paratuberculosis. In addition, the two-stage system is more sensitive than conventional culture for the isolation of M. paratuberculosis from subclinically infected cattle.