Sample handling substantially affects Johne's ELISA

Detection methods for Mycobacterium avium subsp. paratuberculosis (MAP) are imperfect, yet crucial for diagnosis of Johne's disease. Our purpose was to test for significant and biologically relevant changes in Johne's ELISA results associated with how field-collected blood samples were transported to the laboratory, prepared and stored prior to testing, while removing potential confounding by test kit and laboratory variables. Blood samples were collected from 21 cows that previously had MAP ELISA scores ranging from negative to highly positive. Samples for immediate laboratory processing were subjected to different transportation temperatures (on ice, 26 degrees C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), but were tested using the same ELISA kit in the same laboratory. Samples for laboratory processing after one week of storage were subjected to different storage temperatures (4 degrees C, -20 degrees C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), and again were tested using the same ELISA kit in the same laboratory. Finally, samples were evaluated by time to processing (one day, one week) and storage temperature (4 degrees C, -20 degrees C). Data were checked for normality and analyzed with repeated measures ANOVAs. Significantly (P=0.027) higher MAP ELISA scores were recorded for whole blood and hemolyzed samples transported at 26 degrees C than serum separated samples. Sample storage for one week at -20 degrees C resulted in significantly (P<0.001) lower MAP ELISA scores, regardless of handling method, compared to samples stored at 4 degrees C for one week. Method of sample preparation, as well as transportation temperature and medium-term storage temperature, affects MAP ELISA results. Such discrepancies will inevitably result in improper classification of MAP-infected cattle, impeding both biosecurity measures on uninfected farms and MAP control programs.