Identification of a gene unique toMycobacterium aviumsubspeciesparatuberculosisand application to diagnosis of paratuberculosis

Mycobacterium aviumsubspeciesparatuberculosis(M. paratuberculosis) is the etiologic agent of paratuberculosis (Johne's disease), a chronic granulomatous enteritis in ruminants. Currently, there is a need for improved diagnostic tests because of the lack of methods for accurate, rapid and reliable detection ofM. paratuberculosisinfection. AM. paratuberculosisgene (hspX) was cloned, sequenced, and a 30 bp species-specific oligonucleotide was synthesized. As an internal control to identify mycobacterial strains, a 33 bpMycobacteriumgenus-specific oligonucleotide was synthesized based on the conserved 5 terminus of the mycobacterialrecAgene. Dioligonucleotide hybridization (dOH) analysis identified 28/28 (100%) mycobacterial strains and specifically identified 14/14 (100%) reference (ATCC 19698), bovine, ovine and human isolates ofM. paratuberculosis.TheM. paratuberculosis-specific oligonucleotide distinguishedM. paratuberculosisisolates from related mycobacteria, including all closely related members of theMycobacterium aviumcomplex (MAC) tested in this study. The members of MAC tested in this study includedMycobacterium aviumsubspeciesavium(M. avium),M. paratuberculosis,Mycobacterium aviumsubspeciessilvaticum(M. silvaticum) andMycobacterium intracellularestrains. Hybridization was not observed with DNA extracted from a selected group of other bacterial pathogens. The experiments indicate that the dOH analysis is a useful diagnostic tool to detect mycobacterial infection, specificallyM. paratuberculosis.The dOH method could be a good alternative to existing assays and will be adapted for specific identification ofM. paratuberculosisfrom faecal samples, mixed bacteriologic cultures, tissue specimens and whole blood.