A triplex real-time (TRT-PCR) assay was developed to ensure a rapid and reliable detection of Mycobacterium avium subsp. paratuberculosis (Map) in faecal samples and to allow routine detection of Map in farmed livestock and wildlife species. The TRT-PCR assay was designed using IS900, ISMAP02 and f57 molecular targets. Specificity of TRT-PCR was first confirmed on a panel of control mycobacterial Map and non-Map strains and on faecal samples from Map-negative cows (n = 35) and from Map-positive cows (n = 20). The TRTPCR assay was compared to direct examination after Ziehl-Neelsen (ZN) staining and to culture on 197 faecal samples collected serially from five calves experimentally exposed to Map over a 3-year period during the sub-clinical phase of the disease. The data showed a good agreement between culture and TRT-PCR (kappa score = 0.63), with the TRT-PCR limit of detection of 2.5 102 microorganisms/g of faeces spiked with Map. ZN agreement with TRT-PCR was not good (kappa = 0.02). Sequence analysis of IS900 amplicons from three single IS900 positive samples confirmed the true Map positivity of the samples. Highly specific IS900 amplification suggests therefore that each single IS900 positive
sample from experimentally exposed animals was a true Map-positive specimen. In this
controlled experimental setting, the TRT-PCT was rapid, specific and displayed a very high
sensitivity for Map detection in faecal samples compared to conventional methods.