Pennsylvania Animal Diagnostic Laboratory System
PADLS Conferences Spring 2001 6. IS900-PCR assay
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A total of 180 cows from 4 herds with known history of Johne's disease were examined for M. paratuberculosis. Pooled quarter milk (50 ml), serum (1 ml) and fecal samples (10 g) were collected for analysis. Bulk tank samples (200 ml) were also collected and split into 4 samples (50 ml). Quarter milk and bulk tank milk samples were tested by IS 900-PCR assay and cultured also for M. paratuberculosis on Herrold's egg yolk medium slants supplemented with mycobactin. Serum samples were evaluated for M. paratuberculosis by a Kinetics based ELISA (KELA). Fecal samples were cultured for M. paratuberculosis on Herrold's egg yolk medium slants according to the Cornell method. The IS900-PCR assay was optimized to detect M. paratuberculosis directly from milk samples. It was observed that 68 of 180 (36%) pooled quarter milk samples were positive for M. paratuberculosis by the IS900-PCR assay, while fecal culture, KELA and milk culture detected M. paratuberculosis in 27/180(14%), 33/180(17%) and 9/180(5%) samples, respectively. Fifteen cows (8%) were positive by both ELISA and fecal culture, and 16 (8%) were positive by both IS 900-PCR from milk and fecal culture. IS 900-PCR assay allowed detection of M. paratuberculosis from 10/16 (63%) samples of bulk tank milk, while milk culture detected M. paratuberculosis in 1 of 16 (6%) samples. Preliminary results suggest that IS 900-PCR assay can be applied to screen herds using bulk tank milk samples and quarter milk samples to detect cows with Johne's disease. The IS900-PCR assay needs to be further evaluated on both Johne's positive and negative herds.

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