Comparison of Two Immunomagnetic Separation Methods for Detection of Mycobacterium avium subsp Paratuberculosis in Bovine Feces

Abstract

Detection of Mycobacterium paratubeculosis in feces is complicated by the slowdivision and the lipid composition of the cell wall of the bacterium. In this study, we compared a rapid and sensitive method for direct detection of M. paratuberculosis with anti-paratuberculosis chicken IgY-conjugated magnetic nanoparticles versus commercially available micro-sized paramagnetic beads. Tenfold serial dilutions of a standard M. paratuberculosis strain and clinical isolation strains were spiked in pooled stool and detected by using antibody-conjugated magnetic nanoparticles or paramagnetic beads, and confirmed by polymerase chain reaction. The binding ratio of antibody-nanoparticles was 96.44 +/- 0.77%, and the detection limit was 20 M. paratuberculosis cells per gram of feces. Also, magnetic nanoparticles showed significantly intensive results than paramagnetic beads in dose-effects evaluation. The magnetic nanoparticles were also cost-effective. Antibody-conjugated magnetic nanoparticles did not react with other bacteria, thus providing specific immunomagnetic separation. We conclude that the antibody-conjugated magnetic nanoparticles are more effective than paramagnetic beads for low-cost, direct detection of M. paratuberculosis in cattle feces; in addition, confirmation time was reduced from 12-16 weeks to 2-3 days. This antibody-based immunocapture polymerase chain reaction method is expected to provide a platform of rapid screening of M. paratuberculosis in fecal samples in one step.