IDENTIFICATION OF A GENE UNIQUE TO MYCOBACTERIUM-AVIUM SUBSPECIES PARATUBERCULOSIS AND APPLICATION TO DIAGNOSIS OF PARATUBERCULOSIS

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the etiologic agent of paratuberculosis (Johne's disease), a chroni c 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 of M. paratuberculosis infection. ii M. paratuberculosis gene (hspX) was cloned, sequenced, and a 30 bp speci es-specific oligonucleotide was synthesized. As an internal control to identify mycobacterial strains, a 33 bp Mycobacterium genus-specific oligonucleotide was synthesized based on the conserved 5' terminus of the mycobacterial recA gene. Dioligonucleotide hybridization (dOH) ana lysis identified 28/28 (100%) mycobacterial strains and specifically i dentified 14/14 (100%) reference (ATCC 19698), bovine, ovine and human isolates of M. paratuberculosis. The M. paratuberculosis-specific oli gonucleotide distinguished M. paratuberculosis isolates from related m ycobacteria, including ail closely related members of the Mycobacteriu m avium complex (MAC) tested in this study. The members of MAC tested in this study included Mycobacterium avium subspecies avium (M. avium) , M. paratuberculosis, Mycobacterium avium subspecies silvaticum (M. s ilvaticum) and Mycobacterium intracellulare strains. Hybridization was not observed with DNA extracted from a selected group of other bacter ial pathogens. The experiments indicate that the dol-l analysis is a u seful diagnostic tool to detect mycobacterial infection, specifically M. paratuberculosis. The dOH method could be a good alternative to exi sting assays and will be adapted for specific identification of M. par atuberculosis from faecal samples, mixed bacteriologic cultures, tissu e specimens and whole blood. (C) 1998 Academic Press.