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Journal of Clinical Microbiology, March 2008, p. 972-981, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01801-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High Genetic Diversity among Mycobacterium avium subsp. paratuberculosis Strains from German Cattle Herds Shown by Combination of IS900 Restriction Fragment Length Polymorphism Analysis and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem-Repeat Typing{triangledown}

Petra Möbius,1* Gabriele Luyven,2 Helmut Hotzel,3 and Heike Köhler1

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany,1 Consortium Chemical and Veterinary Laboratories Agency (Rhine-Ruhr-Wupper), Deutscher Ring 100, 47798 Krefeld, Germany,2 Institute for Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany3

Received 10 September 2007/ Returned for modification 25 November 2007/ Accepted 23 December 2007

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.

* Corresponding author. Mailing address: Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany. Phone: (49) 3641 804280. Fax: (49) 3641 804228. E-mail: petra.moebius{at}fli.bund.de

{triangledown} Published ahead of print on 3 January 2008.

Journal of Clinical Microbiology, March 2008, p. 972-981, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01801-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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