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Journal of Clinical Microbiology, August 2007, p. 2404-2410, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00476-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

New Variable-Number Tandem-Repeat Markers for Typing Mycobacterium avium subsp. paratuberculosis and M. avium Strains: Comparison with IS900 and IS1245 Restriction Fragment Length Polymorphism Typing{triangledown} ,{dagger}

Virginie C. Thibault,1 Maggy Grayon,1 Maria Laura Boschiroli,2 Christine Hubbans,3 Pieter Overduin,4 Karen Stevenson,5 Maria Cristina Gutierrez,6 Philip Supply,3,{ddagger} and Franck Biet1*,{ddagger}

UR1282, Infectiologie Animale, Santé Publique (IASP-311), INRA Centre de Tours, F-37380 Nouzilly, France,1 Unité Zoonoses Bactériennes, Agence Française de Sécurité Sanitaire des Aliments, 23 av. du Général de Gaulle, F-94706 Maisons-Alfort Cedex, France,2 U629 INSERM Institut de Biologie de Lille, 1 rue du Professeur Calmette BP 447, F-59021 Lille Cedex, France,3 Virology Department, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3721 MA Bilthoven, The Netherlands,4 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland, United Kingdom,5 Unité Biodiversité des Bactéries Pathogènes Emergentes, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France6

Received 2 March 2007/ Returned for modification 17 April 2007/ Accepted 18 May 2007

Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

* Corresponding author. Mailing address: Infectiologie Animale, Santé Publique (IASP-311), INRA Centre de Tours, F-37380 Nouzilly, France. Phone: (33) 2 47 42 78 69. Fax: (33) 2 47 42 77 79. E-mail: Franck.biet{at}tours.inra.fr

{triangledown} Published ahead of print on 30 May 2007.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{ddagger} P.S. and F.B. contributed equally to this work.

Journal of Clinical Microbiology, August 2007, p. 2404-2410, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00476-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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