Molecular tools for the characterization of mycobacterium avium subspecies paratuberculosis

Abstract

Several strain typing techniques are available to categorize Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) isolates into cattle, sheep, bison, and Intermediate groups. The majority of isolates studied were identified as members of the cattle associated group, regardless of sample host origin, suggesting that the cattle group of M. paratuberculosis isolates are very successful. This may be because host specificity is not critical for this group or the small differences required to demonstrate host specificity have not yet been found. A major limitation to the epidemiological study of M. paratuberculosis has been the difficulty associated with laboratory cultivation of this micro-organism. The new typing techniques described in this thesis do not require viable M. paratuberculosis bacteria and therefore open a door to novel typing practices.

The new molecular techniques, single stranded conformation polymorphism (SSCP) analysis and satellite typing, were applied to M. paratuberculosis isolates (n=75) from a broad range of ruminant hosts and geographic locations. SSCP analysis and satellite typing were compared to currently accepted techniques (PCR-REA, RFLP, PFGE) for their ability to rapidly and reliably differentiate among M. paratuberculosis isolates. PCR-REA segregated isolates (n=75) into cattle (n=72), sheep (n=1) or bison (n=2) associated strain types. Two isolates from cattle in Canada were typed as RFLP-BstEII C5 by RFLP analysis. PFGE grouped a subset (n=8) of M. paratuberculosis isolates into 4 different PFGE types. Satellite typing resulted in 4 different satellite types (A, B, C, D). SSCP analysis identified 2 regions (IS900-2 and HSP70) where sequence polymorphisms could be targeted to display differences among M. paratuberculosis isolates.