Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection of Mycobacterium avium subspecies paratuberculosis in bovine colostrum

Abstract

Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP; strain K-10) at increasing concentrations between 1×101 and 1×109 cells/ml were tested for recovery of MAP DNA using a nested ISMAP02 target polymerase chain reaction initially developed for detecting MAP DNA in fecal samples. The following detection rates were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1×107 and 1×109 cells/ml, 75% between 1×103 and 1×106 cells/ml, and 50% between 1×101and 1×102 cells/ml replicates. Detection rates achieved for the colostrum sample replicates inoculated with sonicated MAP cell suspension were 75% for 1×109 cells/ml, 100% between 1×107 and 1×108 cells/ml, 75% for 1×106 cells/ml, 0 for 1×104 cells/ml, and 25% between 1×101 and 1×103 cells/ml. When negative control colostrum samples were tested, 16 of 18 (89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the MAP DNA detection rates of the present assay improved with increasing concentrations of MAP in the colostrum sample replicates, although MAP DNA was also detected in 2 of 18 (11%) negative control samples, suggesting an undefined technical problem with the assay or, perhaps, sample contamination during preparation. Overall, the present findings suggest a potential role of the proposed polymerase chain reaction assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.