Although semen is not a common way for spread of paratuberculosis, isolation of MAP from semen has been reported. However, as the risk of spread by artificial insemination is not fully investigated, there is a demand for a semen test intended for use in paratuberculosis free herds. The rapidity, sensitivity and specificity of real-time PCR would greatly benefit the detection of MAP. We have applied the following method on artificially infected bovine semen. Samples of diluted MAP free semen were spiked with MAP, counted in bürker-chamber since CFU-count usually underestimates the number of detectable genomes. The MAP suspension used was also washed from free DNA to avoid overestimating the sensitivity of the test. Incubation with lysisbuffer and proteinase K followed by mechanical disruption by beadbeating released the DNA, which was recovered with phenol/chloroform extraction. Real-time PCR with a fluorescent probe targeting the insertion element IS900 could detect as few as 10 MAP per sample of 100 µl semen. PCR-inhibition was monitored by the addition of an internal control to the samples and it was shown that the applied method was sufficient for removing inhibiting components.

Sponsorship

Attendance to this Congress was sponsored by the EU-funded project SSPE-CT-2004-501903