Inter- and Intra-subtype genotypic differences that differentiate <it>Mycobacterium avium</it> subspecies <it>paratuberculosis</it> strains

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium</it> subspecies <it>paratuberculosis</it> (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the <it>Mycobacterium avium</it> 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 <it>gyr</it> genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS<it>900</it> (IS<it>900</it>-RFLP) analysis.</p> <p>Results</p> <p>The presence of LSP^A4 and absence of LSP^A20 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 IS<it>900</it>-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS<it>900</it>-RFLP segregated the S-type strains into types I and III and the results concurred with those of the <it>gyr</it> SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of <it>Mycobacterium avium</it> Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III.</p> <p>Conclusion</p> <p>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 (IS<it>900</it>-RFLP, PFGE and SNP analysis of the <it>gyr</it> 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.</p>