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Open Access Highly Accessed Research article

Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains

Franck Biet1*, Iker A Sevilla2, Thierry Cochard1, Louise H Lefrançois1, Joseba M Garrido2, Ian Heron3, Ramón A Juste2, Joyce McLuckie3, Virginie C Thibault1, Philip Supply4567, Desmond M Collins8, Marcel A Behr9 and Karen Stevenson3

Author Affiliations

1 INRA, UMR1282, Infectiologie Santé Publique (ISP-311), Nouzilly, F-37380, France

2 Neiker-tecnalia, Dpto. de Producción y Sanidad Animal, Berreaga 1, Derio, Bizkaia, 48160, Spain

3 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland, EH26 0PZ, UK

4 INSERM, Lille, U1019, France

5 CNRS UMR, Lille, 8204, France

6 Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France

7 Univ Lille Nord de France, Lille, France

8 AgResearch, Wallaceville, Upper Hutt, P.O. Box 40063, New Zealand

9 McGill University, Montreal, Quebec, H3G 1A4, Canada

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BMC Microbiology 2012, 12:264  doi:10.1186/1471-2180-12-264

Published: 19 November 2012

Abstract

Background

Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis.

Results

The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III.

Conclusion

This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.