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Journal of Clinical Microbiology, May 2005, p. 2111-2117, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2111-2117.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Detection and Typing of Strains of Mycobacterium avium subsp. paratuberculosis from Broth Cultures

Alifiya S. Motiwala,1 Megan Strother,1 Natalie E. Theus,2 Roger W. Stich,2 Beverly Byrum,3 William P. Shulaw,2 Vivek Kapur,4 and Srinand Sreevatsan1*

Food Animal Health Research Program, Ohio Agricultural Research Development Center and Department of Veterinary Preventive Medicine, The Ohio State University, Wooster,1 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus,2 Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, Ohio,3 Biomedical Genomics Center and Department of Microbiology, University of Minnesota, St. Paul, Minnesota4

Received 17 September 2004/ Returned for modification 23 November 2004/ Accepted 27 December 2004

A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrold's Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johne's disease.

* Corresponding author. Present address: Veterinary Population Medicine, University of Minnesota, 136G Andrew Boss Bldg., 1354 Eckles Ave., St. Paul, MN 55108. Phone: (612) 625-3769. Fax: (612) 624-4906. E-mail: sreev001{at}umn.edu.

Journal of Clinical Microbiology, May 2005, p. 2111-2117, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2111-2117.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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