Differentiating host-associated variants of Mycobacterium avium by PCR for detection of large sequence polymorphisms

Abstract

The Mycobacterium avium species consists of a group of organisms that are genetically related but phenotypically diverse, with certain variants presenting clear differences in terms of their host association and disease manifestations. The ability to distinguish between these subtypes is of relevance for accurate diagnosis and for control programs. Using a comparative genomics approach, we have uncovered large sequence polymorphisms that are, respectively, absent from bird-type M. avium isolates and from cattle types and sheep types of M. avium subsp. paratuberculosis. By evaluating the distribution of these genomic polymorphisms across a panel of strains, we were able to assign unique genomic signatures to these host-associated variants. We propose a simple PCR-based strategy based on these polymorphisms that can rapidly type M. avium isolates into these subgroups.

FIG. 1.

Schematic representation of large sequence polymorphisms. LSP^A 4-II and LSP^A 18 are specifically absent from M. avium subsp. paratuberculosis cattle type, LSP^A 20 is absent from M. avium subsp. paratuberculosis sheep type, and LSP^A 17 is absent from M. avium subsp. avium bird type. Coordinates on the genome are given as base pairs, starting from the first nucleotide of the start codon of dnaA in M. avium 104 and M. avium subsp. paratuberculosis K10, respectively. White boxes represent homologous sequences across M. avium 104, M. avium subsp. paratuberculosis sheep type, and M. avium subsp. paratuberculosis cattle type. (A) LSP^A 4-II is depicted by the black box, LSP^A 4-I is depicted by the striped box, and LSP^P 12 is indicated by the gray box. (B) The striped box represents a large sequence that is conserved but inverted in M. avium subsp. paratuberculosis cattle type, and the black box represents LSP^A 18. (C) The black box represents LSP^A 20. (D) The black box represents LSP^A 17. Thick arrows represent primers flanking the LSP (bridging primers); a PCR product is obtained if the region is missing. Thin arrows represent primers targeting a sequence that is within the LSP (internal primers); a PCR product is obtained if the region is present.

FIG. 2.

Detection of subspecies and subtypes of M. avium using PCR for large sequences polymorphic among strains of M. avium. On the top panel, 11 samples were tested for LSP^A 8 using a three-primer PCR. Lanes: L, 100-bp ladder; 1 to 4, M. avium subsp. paratuberculosis strains; 5 and 6, nonparatuberculosis strains of M. avium; 9, mixed sample (M. avium 104 and M. avium subsp. paratuberculosis K10); 10, M. intracellulare ATCC 13950 strain; 11, water. In the bottom panel, four samples were tested for LSP^A 20 and four samples are tested for LSP^A 17 using three-primer PCRs. Lanes: L, 100-bp ladder; 1 and 2, M. avium subsp. paratuberculosis C type; 3 and 4, M. avium subsp. paratuberculosis S type; 5, water; 6 and 7, M. avium subsp. hominissuis; 8 and 9, M. avium subsp. avium bird type; 10, water.

FIG. 3.

Diagnostic algorithm for PCR-based identification and typing of an M. avium isolate. *, testing for the presence or absence of LSP^A 4-II or LSP^A 18 are other alternatives for typing M. avium subsp. paratuberculosis isolates into cattle or sheep types.