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Journal of Clinical Microbiology, April 2004, p. 1694-1702, Vol. 42, No. 4
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.4.1694-1702.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Multilocus Short Sequence Repeat Sequencing Approach for Differentiating among Mycobacterium avium subsp. paratuberculosis Strains
Alongkorn Amonsin,1 Ling Ling Li,1 Qing Zhang,1 John P. Bannantine,2 Alifiya S. Motiwala,3 Srinand Sreevatsan,3 and Vivek Kapur1*
Department
of Microbiology and Biomedical Genomics Center, University of
Minnesota, St. Paul, Minnesota 55108,1
National Animal Disease
Center, U.S. Department of Agriculture, Ames, Iowa
50010,2
Food Animal Health Research
Program, Ohio Agricultural Research and Development Center,
and Department of Veterinary Preventive Medicine, The Ohio
State University, Wooster, Ohio
446913
Received 14 August 2003/
Returned for modification 23 October 2003/
Accepted 2 January 2004
We
describe a multilocus short sequence repeat (MLSSR) sequencing approach
for the genotyping of Mycobacterium avium subsp.
paratuberculosis (M. paratuberculosis) strains.
Preliminary analysis identified 185 mono-, di-, and trinucleotide
repeat sequences dispersed throughout the M. paratuberculosis
genome, of which 78 were perfect repeats. Comparative nucleotide
sequencing of the 78 loci of six M. paratuberculosis isolates
from different host species and geographic locations identified a
subset of 11 polymorphic short sequence repeats (SSRs), with an average
of 3.2 alleles per locus. Comparative sequencing of these 11 loci was
used to genotype a collection of 33 M. paratuberculosis
isolates representing different multiplex PCR for IS900 loci
(MPIL) or amplified fragment length polymorphism (AFLP) types. The
analysis differentiated the 33 M. paratuberculosis isolates
into 20 distinct MLSSR types, consistent with geographic and
epidemiologic correlates and with an index of discrimination of 0.96.
MLSSR analysis was also clearly able to distinguish between sheep and
cattle isolates of M. paratuberculosis and easily and
reproducibly differentiated strains representing the predominant MPIL
genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of
M. paratuberculosis described previously. Taken together, the
results of our studies suggest that MLSSR sequencing enables facile and
reproducible high-resolution subtyping of M. paratuberculosis
isolates for molecular epidemiologic and population genetic
analyses.
* Corresponding
author. Mailing address: University of Minnesota, 1500 Gortner Ave.,
St. Paul, MN 55108. Phone: (612) 625-7712. Fax: (612) 624-6264. E-mail:
vkapur{at}umn.edu.
Journal of Clinical Microbiology, April 2004, p. 1694-1702, Vol. 42, No. 4
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.4.1694-1702.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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Copyright © 2004 by the American Society for Microbiology. All rights reserved.