Free-living amoebae (FLA) are ubiquitous in environment and feed on bacteria by phagocytosis. Depending on the mycobacteria species, they can be digested, survive or grow within FLA. Studies have shown that Mycobacterium avium subsp. paratuberculosis (Map) can grow in FLA in vitro but too few have studied this interaction in an environmental context. Understanding its survival and its persistence in environment is important to better understand its transmission cycle. Our hypothesis is that FLA can be an environmental reservoir and vector of Map. To test this hypothesis, water and soil samples from infected farm were used to culture FLA, which were lysed to extract total DNA and intracellular bacteria. Total DNA were used to detect Map by a nested qPCR. Lysates were used to culture Map. For all qPCR-positive samples, FLA were identified and Map genotyped. Environmental FLA were finally used to characterise the interaction with Map by infection assays. For all samples, we isolated FLA. The nested qPCR method developed showed a higher sensitivity than the classical qPCR, allowing us to detect trace of Map DNA in the total DNA. Several samples were positive for qPCR-positive for Map. This underlined that at least Map DNA could be found in association with FLA. The identification of Map and FLA strains are in progress and will be presented. The permissiveness of FLA to Map is investigated by infection assays using a FLA isolated from cattle environment, Rosculus like, and with newly isolated FLA. Our work led to isolate many environmental FLA from infected farms, improve the sensitivity of Map detection by nested qPCR, confirm the presence of Map DNA in interaction with environmental FLA and follow the permissiveness of these FLA to Map infection. These results highlight the possible role of FLA as reservoirs of Map in the environment.