RAPID DETECTION OF MYCOBACTERIUM-PARATUBERCULOSIS IN CLINICAL-SAMPLESFROM RUMINANTS AND IN SPIKED ENVIRONMENTAL-SAMPLES BY MODIFIED BACTEC12B RADIOMETRIC CULTURE AND DIRECT CONFIRMATION BY IS900 PCR

The suitability of a radiometric culture medium consisting of BACTEC 1 2B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detec tion of Mycobacterium paratuberculosis in feces, mesenteric lymph node s, and intestinal walls from cattle, sheep, and goats, In addition, a simple method that would enable the rapid identification of Mycobacter ium paratuberculosis by IS900 PCR in the primary cultures was sought s o that subculture to secondary egg-free radiometric medium could be av oided, An ethanol extraction followed hy differential centrifugation w as used to separate M. paratuberculosis from PCR inhibitors in the pri mary culture, PCR was then undertaken with the pellet, after boiling t o lyse the mycobacteria; if this test was negative, the DNA in the lys ate was purified with guanidine thiocyanate and silica, Cultures of fe ces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats kn own to have or suspected of having Johne's disease yielded positive PC R results 1 to 7 weeks after inoculation. Similar results were obtaine d with soil and pasture samples that had been spiked with M. paratuber culosis. The results suggested that radiometric culture was more sensi tive than histopathology in detecting M. paratuberculosis infection in sheep and goats and more sensitive than culture on Herrold's egg yolk medium for the detection of the infection in cattle, Of 259 individua l PCR tests with samples from cultures with growth indices of greater than or equal to 10,237 (91.5%) were positive, with only 28 (11.8%) re quiring both ethanol and silica preparation to yield a positive result , Of the 22 negative PCR results for samples from cultures with growth indices of greater than or equal to 10, 18 were for samples from cult ures that had only just developed evidence of growth, PCR-positive cul tures tended to remain PCR positive over successive weeks, Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs fo r ruminants.