Mycobacterium paratuberculosis detection in cow’s milk in Argentina by immunomagnetic separation-PCR

Abstract

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium aviumsubsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected anduninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with thoseobtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were usedto coat magnetic beads. All of the 118 samples from the Map uninfected herds were negativefor the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture,and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement betweenthe different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCRmethod detected Map in milk of the cows that were not positive in other techniques. Thisis the first report dealing with the application of IMS-IS1 PCR in the detection of Map in rawmilk samples under field conditions in Argentina.