Introduction.

The aim of this study was to establish usefulness of the Immunomagnetic separation (IMS) for the selective isolation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from milk samples for the IS900 PCR.

Material and methods.

The milk samples from 38 seropositive cows and milk samples spiked with known number of M. paratuberculosis cells (ATCC 43015, 1200, 600, 120, 60 and 12 bacterial cells per ml of milk) were tested. IMS was performed using Dynabeads-Protein A (Dynal, Norway) and M. paratuberculosis-specific goat hyperimmune serum, according to the manufacturer's instructions, followed by DNA extraction with High Pure PCR Template Preparation Kit (Boehringer-Mannheim, Germany) and IS900 PCR. The PCR detection level of the target bacterium in milk was determined also with the standard method (sample concentration and decontamination with 0.75% HPC).

Results and discussion.

The procedure incorporating IMS was found to be capable of detecting less than 12 M. paratuberculosis bacterial cells per ml of milk and was significantly more sensitive than the classical PCR procedure without IMS (detection limit: 600 M. paratuberculosis bacterial cells per ml). Among 38 milk samples of seropositive animals 6 (15.79%) samples were positive in IMS-PCR. 3 of them were also culture positive. Our results suggest that IMS considerably improves the sensitivity of PCR IS900 for the detection of M. paratuberculosis in milk samples and should consequently increase the sensitivity of other direct methods in the diagnostics of paratuberculosis.