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Journal of Clinical Microbiology, August 2004, p. 3600-3606, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3600-3606.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Amplified Fragment Length Polymorphism Reveals Genomic Variability among Mycobacterium avium subsp. paratuberculosis Isolates

B. O'Shea,1 S. Khare,1 K. Bliss,2 P. Klein,3 T. A. Ficht,1 L. G. Adams,1 and A. C. Rice-Ficht2*

Department of Veterinary Pathobiology,1 Department of Biochemistry and Biophysics, Texas A&M University,3 Department of Medical Biochemistry and Genetics, The Texas A&M University Health Science Center, College Station, Texas2

Received 1 October 2003/ Returned for modification 20 November 2003/ Accepted 12 May 2004

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.

* Corresponding author. Mailing address: Medical Biochemistry and Genetics, 440 Reynolds Medical Building, Texas A&M University, College Station, TX 77843-1114. Phone: (979) 845-3466. Fax: (979) 847-9481. E-mail: aficht{at}neo.tamu.edu.

Journal of Clinical Microbiology, August 2004, p. 3600-3606, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3600-3606.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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