Molecular Epidemiology of Mycobacterium aviumsubsp. paratuberculosis: Evidence for Limited Strain Diversity, Strain Sharing, and Identification of Unique Targets for Diagnosis

ABSTRACTThe objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium aviumsubsp. paratuberculosisisolated in the United States and to identify M. aviumsubsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. aviumsubsp. paratuberculosisisolates recovered from animals (n= 203) and patients with Crohn's disease (n= 7) from diverse geographic localities. Six hundred bacterial cultures, including M. aviumsubsp. paratuberculosis(n= 303), non-M. aviumsubsp. paratuberculosismycobacteria (n= 129), and other nonmycobacterial species (n= 168), were analyzed to evaluate the specificity of two IS900integration loci and a newly described M. aviumsubsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. aviumsubsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. aviumsubsp. paratuberculosisisolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. aviumsubsp. paratuberculosisisolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. aviumsubsp. paratuberculosisstrains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. aviumsubsp. paratuberculosisisolates recovered from human and ovine sources.