Evaluation of a fluorescence polarization assay using MAP whole-protein antigen for the detection of anti-Mycobacterium avium subsp. paratuberculosis antibodies in cattle and buffaloes

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis of ruminants that causes substantial economic losses worldwide due to reduced productivity. Rapid and reliable serological methods are important for disease surveillance and control. The present study evaluated the relative diagnostic performance of a fluorescence polarization assay (FPA) using fluorescein-labelled MAP whole-protein antigen for the detection of anti-MAP antibodies in cattle and buffalo sera. A total of 218 serum samples were collected from cattle and buffaloes with a history of chronic intermittent diarrhea from organized dairy farms in Punjab, India. Whole protein extracted from MAP strain K10 was used as the tracer antigen in the FPA. Receiver operating characteristic (ROC) curve analysis was performed, and the results were compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA). ELISA identified 20 seropositive samples, whereas FPA identified 35 seropositive samples. ROC analysis established an optimal cutoff value of >39.15 mP (operational threshold >39 mP), corresponding to a relative sensitivity of 85% and a relative specificity of 71.1%, with an area under the ROC curve (AUC) of 0.830 (P < 0.001), indicating good discrimination between ELISA-positive and ELISA-negative samples. These findings suggest that FPA using MAP whole-protein antigen is a simple and rapid serological assay that may serve as a useful complementary method to ELISA for the detection of anti-MAP antibodies in ruminants.