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.