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Journal of Clinical Microbiology, April 2004, p. 1703-1712, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1703-1712.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis Isolates Recovered from Wild Animal Species

Alifiya S. Motiwala,1 Alongkorn Amonsin,2 Megan Strother,1 Elizabeth J. B. Manning,3 Vivek Kapur,2 and Srinand Sreevatsan1*

Food Animal Health Research Program, Ohio Agricultural Research and Development Center, and Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio,1 Biomedical Genomics Center and Department of Microbiology, University of Minnesota, St. Paul, Minnesota,2 Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin3

Received 15 August 2003/ Returned for modification 8 October 2003/ Accepted 30 October 2003

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.

* Corresponding author. Mailing address: FAHRP-OARDC, The Ohio State University, 1680 Madison Ave., Room 170, Wooster, OH 44691. Phone: (330) 263-3744. Fax: (330) 263-3677. E-mail: sreevatsan.1{at}osu.edu.

Journal of Clinical Microbiology, April 2004, p. 1703-1712, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1703-1712.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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