Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/17/2009
Publication Date: 3/1/2010
Citation: Pithua, P., Wells, S.J., Godden, S.M., Sreevatsan, S., Stabel, J.R. 2010. Experimental Validation of a Nested Polymerase Chain Reaction Targeting the Genetic Element ISMAP02 for Detection of Mycobacterium avium subspecies paratuberculosis in Bovine Colostrum. Journal of Veterinary Diagnostic Investigation. 22(2):253-256. Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced production by these animals through reduced milk production, they also present a potential infective threat to the rest of the herd. Johne’s disease is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is dependent upon understanding the immune responses of the host animal during infection. Use of the polymerase chain reaction (PCR) assay for pathogen detection in milk and colostrum of dairy cows is one such diagnostic tool. In the present study, the sensitivity and specificity of a colostrum detection test was evaluated. Results suggest that this test would be useful for the detection of infection within a herd. This information may offer alternative methods for a diagnostic tool for control of Johne's disease in the US.
Technical Abstract: Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP) (strain K-10) at increasing concentrations between 1×10**1 and 1× 10**9 cells/mL were tested for recovery of MAP DNA using a modified nested ISMAP02 target PCR initially developed for detecting MAP DNA in fecal samples. The following detection limits were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1 × 10**7 and 1×10**9 cells/mL, 75% between 1 ×10**3and 1×10**6 cells/mL, and 50% between 1×10**1 and 1×10**2 cells/mL replicates, respectively. Detection limits achieved for the colostrum sample replicates inoculated with sonicated MAP cells suspension were: 75% for 1 ×10**9 cells/mL, 100% between 1 × 10**7and 1× 10**8 cells/mL, 75% for 1×10**6 cells/mL, 0 for 1 ×10**4 cells/mL, and 25% between 1 × 10**1 and 1×10**3 cells/mL, respectively. When the known negative colostrum samples were tested, 16/18(89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the analytic sensitivity of the present assay improved with increasing concentration of MAP in the colostrum sample replicates but specificity was compromised possibly due to contamination during sample preparations. Overall the findings presented here suggest that the nested PCR protocol evaluated in this study was useful for the detection of the presence of MAP DNA in experimentally infected bovine colostrum.