Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species

Abstract

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.

FIG. 1.

Dendrogram showing the distribution of MPIL fingerprints. The assigned fingerprints, isolate identities, and host species are shown. The proportion distance of each cluster is also indicated. MPIL analysis had a Simpson's diversity index of 0.533, indicative of a limited degree of strain discrimination capability. Shannon's diversity index was 1.067. MAP, M. paratuberculosis; MAIC, non-M. paratuberculosis M. avium-M. intracellulare complex; Outside MAIC, mycobacteria outside the M. avium-M. intracellulare complex.

FIG. 2.

Phylogenetic tree showing the distribution of strains by the numbers of G-residue repeats. The numbers of G-residue repeats, isolate identities, host species, and major fingerprints obtained by MPIL analysis are shown. Also shown at the clade origins are the bootstrap values generated by 1,000 replications in a maximum-parsimony model. SSR analysis had a Simpson's diversity index of 0.751, indicative of a high degree of strain discrimination capability. Shannon's diversity index was 1.593. MAP, M. paratuberculosis; MAIC, non-M. paratuberculosis M. avium-M. intracellulare complex; Outside MAIC, mycobacteria outside the M. avium-M. intracellulare complex.