BACKGROUND: In the last two decades a variety of different molecular typing methods have been developed to differentiate strains of the genomically highly conserved Mycobacterium avium subsp. paratuberculosis (Map). The most successful techniques are based on insertion sequences, repetitive loci, comparative genomics or single nucleotide polymorphisms. In the latter class, AFLP, RAPD and PFGE have been applied with variable success. In this study a single Map gene was targeted that is coding for a member of the polymorphic PPE protein family for which roles in immune evasion, antigenic variation and virulence have been suggested. AIM: To examine whether polymorphic PPE proteins can serve as a means of differentiation of Map isolates.

METHODS: We have identified at least four Map PPE proteins to be surface exposed on the cell wall of Map by enzymatic trimming of viable Map bacteria in specific in vitro conditions. One of the corresponding genes, Map1506 was sequenced from a collection of 60 isolates from different sources, hosts and typing profiles, including 7 isolates of bovine (C) and 6 ovine (S and I) variants representing most predominant IS900-RFLP profiles. IS1311 PCR-REA was performed on all isolates to determine their bovine (C) or ovine (S) subtype. Sequence data for the entire Map1506 gene was collected and compared. These sequencing results were used to select polymorphic regions in the gene to amplify for analysis by denaturing gradient gel electrophoresis (DGGE).

RESULTS: Grouping based on the sequencing data corresponded in general to the C versus S subtyping by IS1311 PCR-REA. One of the Map1506 gene sequences differed substantially from the sequenced K10 strain with only 79% identity. Polymorphic regions of Map1506 were selected for analysis by denaturing gradient gel electrophoresis allowing visual discrimination of bovine and ovine M. avium subsp. paratuberculosis isolates as well as separation of ovine isolates into two subgroups (S and I).

CONCLUSION: The Map1506 gene encodes a surface exposed polymorphic PPE protein with putative roles that are relevant to Map pathogenicity. Sequence polymorphisms in this gene are readily detectable by DGGE, and allow distinction of isolates correlating with the known C, S and I variants.