Objectives

determine the potential of nested PCR on blood, milk, semen and placental fluid to detect clinical and subclinical cattle infected with Mycobacterium avium subsp. paratuberculosis.

Procedures

A nested PCR (nPCR) probing for IS900 was developed and its efficiency in detecting Map DNA in peripheral blood and milk samples was compared with that of ELISA and AGID serology in a cohort of 11 clinical and 46 subclincal lactating Holstein cows from a herd with confirmed paratuberculosis. Placental fluid of 8 pregnant animals from the same herd and of 3 pregnant cows from a beef herd with confirmed paratuberculosis was analyzed for evidence of Map DNA via nPCR. A Holstein bull with clinical signs of Johne's disease from the dairy herd had the semen examined for evidence of Map DNA via nPCR.

Results

The detection rate for the nPCR in peripheral blood and milk samples of clinical animals was 100% (11 animals), but also 100% on ELISA and 55% ( 6 animals) on AGID in this group. The detection rate of the nPCR was 52% (24 animals) on blood and milk samples of the subclincal animal group as compared to 39% positive or suspicious (18 animals) on ELISA and none on AGID. In 5 of 12 animals (42%) the nPCR identified subclincal animals when these were negative on ELISA and AGID. More animals were detected by nPCR on milk samples than on blood samples. Some cows had both positive blood and milk samples. The clinical bull was positive on blood and semen samples. Of the 11 pregnant cows 18% (2 animals ) tested positive on the allantochorionic placental fluid. [Positve fetal tissue samples in 36% (4 feti )]

Conclusion

Nested PCR testing of peripheral blood, milk, semen and placental fluid samples has potential as an ante mortem test to detect clinical and subclincal animals infected with Map by taking advantage of a hematological phase of circulating infected monocytes in paratuberculosis. Positive signals obtained in subgroups of pregnant animals make it an ante mortem test to determine in-utero infection with Map in late gestation animals. Likewise, semen can be analyzed for the presence of Map DNA. Positive signals obtained in subgroups of animals when the ELISA and AGID readings were negative, make the assay a candidate for detection of early, silent infection with Map.