Simple SummaryThe prevalence of Johne’s disease (JD) due toMycobacterium aviumssp.paratuberculosis(Map), responsible for considerable economic losses in the dairy industry worldwide, remains very high. In addition, recent studies have shown thatMapmay hamper the success of bovine tuberculosis (bTB) eradication programs due to frequent co-infection of hosts. Therefore, the risk ofMapcontamination by environmental sources must be studied given achieving improved control of this enzooty. Our previous work showed that environmental amoebae could function as reservoirs and vectors of these pathogenic mycobacteria. This study aims to describe amoeba-mycobacteria co-occurrence by sampling the water points of cattle herds monitored forMapandMycobacterium bovis(M. bovis) infection. Our study showed that a wide diversity of amoebae and non-tuberculous mycobacteria (NTM) species, includingMap, live closely together in water troughs of herds monitored for JD or bTB. The exact association between amoebae and mycobacteria calls for further investigation.AbstractOne of the obstacles to eradicatingparatuberculosisor Johne’s Disease (JD) seems to be the persistence ofMycobacterium aviumsubsp.paratuberculosis(Map) in the environment due to its ability to survive alone or vectorized. It has been shown thatMapis widely distributed in soils and water. Previously, we isolated amoebae associated withMapstrains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected withMaporMycobacterium bovis(M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized.Mapand other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae andMapmay be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination byMap.