Comparison of Immunomagnetic Bead Separation-Immunosensor Detection and Nested-PCR Methods for Detecting Mycobacterium avium Subspecies paratuberculosis in Cattle Feces

Background: Johne's disease, also known as paratuberculosis, is a chronic granulomatous enteritis disease that affects ruminants worldwide. Objective: The objective of this study was to assess the effectiveness of the immunomagnetic bead separation-immunosensor (IMB-IS) detection method compared to Nested-PCR for identifying Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle feces samples. Methods: Ninety rectal fecal samples were collected from selected cattle, comprising 59 serum-positive and 31 serum-negative cases based on serum ELISA. Following DNA extraction, nested-PCR was conducted using the IS900 primer sequence targeting the MAP-specific gene. Immunomagnetic bead (IMB) nanoparticles were synthesized by purifying hyperimmune donkey IgG through affinity chromatography and then conjugating it to Fe nanoparticles. Rhodamine-B hydrazone immunosensor (IS) was synthesized and conjugated to hyperimmune rabbit IgG. The synthesized IMB and IS were used to identify MAP in cattle fecal samples. Results: The results of this study revealed that of the 90 stool samples tested using the nested-PCR method, 62 samples (68.88%) were positive, while 28 samples (31.12%) were negative. In the IMB-IS test based on optical density (OD), 64 samples were positive (71.1%), while 26 samples were negative (28.8%). This test exhibited a sensitivity of 100%, specificity of 92.85%, and an overall test accuracy of 97.77%. Conclusion: Given the considerations of cost, time, positive and negative predictive values, and acceptable accuracy of the IMB-IS test, it is recommended for evaluation in screening and epidemiological studies.