Aims: The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture. Methods and Results: The new method couples Map-specific peptide-mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3x10(2)-6x10(8)phageml(-1). When low numbers of Map were present (10(2)CFUml(-1)), the burst size of a single host Map cell was maximal (10(3) phage per cell) resulting in a highly sensitive screening assay. Conclusion: A rapid, sensitive immuno-based screening method suitable for the detection of viable Map in milk and faeces was developed. Significance and Impact of the Study: The novel PMS-phage-ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48h, representing a substantial decrease in time to detection compared with current culture methods for Map.