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Journal of Clinical Microbiology, May 2003, p. 2015-2026, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2015-2026.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster,¹ Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg,⁴ Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, Ohio,² National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa,⁶ Department of Molecular Microbiology, University of Central Florida, Orlando, Florida,⁵ Biomedical Genomics Center and Departments of Microbiology and Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota³

Received 8 November 2002/ Returned for modification 8 January 2003/ Accepted 31 January 2003

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates 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. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.

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