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

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 LSP(A)4 and absence of LSP(A)20 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.

Figure 1

Detection of types and subtypes of strains based on of the absence or presence of large sequences LSPA4 (A) and LSPA20 (B) investigated by PCR.

Figure 2

UPGMA Dendrogram showing the profiles of Map strain obtained by PFGE using SnaB1 (A) or (B) Spe1. The numbering codes of the profiles obtained for each enzyme were assigned according to the nomenclature available at http://www.moredun.org.uk/PFGE-mycobacteria. The colored squares indicate the animal origin of strains: cattle (sky blue), sheep (orange), goat (dark blue) and deer (purple).

Figure 3

UPGMA dendrogram based on IS900 RFLP typing, using Bst EII on a panel of strains of S-type and C-types. 28 strains were isolated from cattle (sky blue), 22 from sheep (orange), 5 from goat (dark blue), 3 from deer (purple) and one from pig (light green).

Figure 4

Minimum spanning tree based on MIRU-VNTR genotypes among Mycobacterium avium subsp. paratuberculosis of types S and C, Mycobacterium avium subsp. avium, Mycobacterium avium subsp. hominissuis, and Mycobacterium avium subsp. silvaticum. 135 strains were isolated from cattle (sky blue), 23 strains from sheep (orange), 17 strains from goat (dark blue), 63 strains from pigs (light green), 17 strains from birds (yellow), 17 strains from humans (white), 6 strains from deer (purple), 5 strains from other sources (red), 4 strains from wood pigeons (brown), and 2 different vaccine strains (316 F from France and United Kingdom) (light blue). Each genotype is displayed as a pie chart, the size of which is proportional to the number of strains, with color-coded distribution of the strain origins. The number of loci differing between the genotypes is indicated by the style of the connecting lines: thick and short, 1 difference; intermediate, 2 differences; thin and long: 3 differences.