Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis

Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. We have made the hypothesis that it might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for bovine paratuberculosis. In a previous work, we isolated an amoeba from water sample from the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. A partial 18S rRNA gene allowed us to suggest that it was a Rosculus-like amoeba but at that time we were not able to cultivate it. In the present study, we succeeded in cultivating the amoeba at 20-25C. This amoeba is among the smallest (5-7 microm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the Rosculus genus, i.e., R. vilicus. Also, the mitochondrial genome is particularly large (184 954 bp). Finally, we showed that this amoeba was able to phagocyte Mycobacterium avium subsp. paratuberculosis and that the bacterium was still observed within amoebae after at least three days. In conclusion, we have characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. Therefore, R. vilicus is a potential candidate as an environmental reservoir for Mycobacterium avium subsp. paratuberculosis.