Characterization of IS900 loci in Mycobacterium avium subsp paratuberculosis and development of multiplex PCR typing

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chron ic inflammation of the intestine in many animals, including primates, and i s implicated in Crohn's disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserv ed loci in its genome. In the present study, genomic DNA flanking 14 of the se insertions was characterized and homologues in the Mycobacterium tubercu losis and M. avium subsp. avium genomes were identified. These included reg ions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at loc us 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, lo cus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RES of a gene sub stituting an RES encoded by IS900 sited two bases closer to the initiation codon, IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp, paratuberculosis isolates lac ked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. aviu m, from one M, avium subsp. silvaticum and from six other mycobacterial spe cies. A multiplex PCR of IS900 loci (MPIL) typing method was developed whic h was able to discriminate 10 different types of M. avium subsp. paratuberc ulosis from the panel of 81 isolates with consistent differences between th ose of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/BstEII RFLP type, suggesting that this method may be applicable to typ ing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstE II RFLP types. Further resolution of these may come from sequencing the rem aining four uncharacterized IS900 loci.