The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Controlling the spread of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), in domestic livestock is challenging. Current diagnostic methods lack sufficient sensitivity to detect subclinically infected animals, and thus, better diagnostic methods are needed. This study was carried out to investigate the diagnostic potential of two novel peptide-mediated magnetic separation (PMS)-based tests-a PMS-phage assay and PMS-culture-both of which have been developed and optimized to detect viable MAP cells in bovine milk. Individual milk samples (50 ml) were obtained from 105 "non-infected" and 40 "MAP-infected" animals (classified as such on the basis of prior faecal culture and serum-ELISA results) in three dairy herds and tested in parallel by the PMS-phage assay and PMS-culture. Diagnostic sensitivity (DSe) and specificity (DSp) of the PMS-phage and PMS-culture methods were determined relative to the MAP infection status of the animal contributing the milk sample. The PMS-based tests applied individually showed moderate DSe (PMS-culture 0.250 and PMS-phage assay 0.325) and high DSp (0.962 and 1.000, respectively). When results of the two PMS-based tests were combined, DSe increased substantially to 0.525, and the DSp was calculated to be 0.962. It was concluded that combined application of the PMS-phage assay and PMS-culture provided the most complete picture regarding the presence of viable MAP in bovine milk samples. A comprehensive validation of the PMS-based assays relative to currently used diagnostic methods (faecal culture and serum-ELISA) would be the next step in assessment of the diagnostic potential of these novel PMS-based methods.