Comparative insights into molecular detection and IS1311-based genotyping of Mycobacterium avium subsp. paratuberculosis in cattle from Indian Gaushalas

Background Paratuberculosis (Johne’s disease) is a chronic livestock infection caused by Mycobacterium avium subsp. paratuberculosis , resulting in major economic losses. Molecular assays targeting the IS900 , ISMav02 , and F57 gene are widely used for detection, but differences in analytical sensitivity and specificity influence diagnostic agreement and prevalence estimation. Methods Fecal DNA from 213 livestock samples were analyzed using molecular PCR assays and culture. The data were compared in pairs for diagnostic agreement. MAP-positive samples were subjected to IS1311 PCR-REA and IS1311 locus-2 PCR-REA for genotyping and sub-genotyping, respectively. The concordance between assays was examined using Cohen’s kappa and Fisher’s exact test. Results Traditional IS900 PCR and ISMav02 PCR comparison showed 54 TP (25.4%) and 157 TN (73.7%) with two FP (0.9%) and no FN. Traditional IS900 PCR vs. F57 PCR, 56 TP (26.3%) and 152 TN (71.4%) were recorded, no FP and five FN (2.3%). Compared to F57 PCR, ISMav02 PCR revealed 54 TP (25.3%) and 152 TN (71.4%) with no FP and 7 FN (3.2%). The greatest apparent prevalence values were obtained using F57 PCR (28.6%), followed by Traditional IS900 PCR (26.3%) and ISMav02 PCR (25.4%). IS1311 PCR-REA showed the most frequent genotype in genuine MAP positives cattle was ‘Indian Bison type’. Conclusion The three molecular tests were highly concordant with F57 PCR identifying a higher MAP prevalence indicating greater analytical sensitivity. The observation of false negatives with the IS900 and ISMav02 tests indicates that the use of these assays alone might result in an underestimating of the true prevalence. The combination of F57 with IS900 and ISMav02 gene based PCRs improves the diagnostic accuracy and boosts the paratuberculosis monitoring in domestic animals.