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Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. While a partial 18S rRNA gene has allowed us to suggest that this amoeba was Rosculus-like, at that time we were not able to subcultivate it. In the present study, we succeeded in cultivating this strain at 20–25°C. This amoeba is among the smallest (5–7  μm) 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. Of note, 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 3  days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. As a result, R. vilicus is a potential candidate as environmental reservoir for Mycobacterium avium subsp. paratuberculosis but further experiments are needed to test this hypothesis. [ABSTRACT FROM AUTHOR]

A Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), is the main etiologic agent of bovine tuberculosis (TB). In France and other countries in Europe, this ancient zoonotic [...]

Abstract: The Bovigam® gamma interferon (IFNγ) assay was used to complement official skin-test screening in a low bovine tuberculosis (bTB) prevalence region in France. The aim of our work was to determine decisional cut-off values for protein purified derivatives (PPD) and ESAT6-CFP10 antigens (R) in order to optimize the efficacy of the modified Bovigam® test, in this low-prevalence area, for optimal classification of infected or non-infected herds following positive skin tests. The sensitivity of the IFNγ assay relative to post-mortem bTB-positive animals (Ser) was studied in 60 cattle from 20 bTB-infected herds. Its absolute specificity (Sp) was studied in 492 cattle from 25 bTB-free herds from a bTB-free zone. Its operational specificity (relative to the positive skin test) (Spr) was also studied in 547 skin-test positive cattle from 172 bTB-free herds from an infected zone. Using normalized interpretations for individual (PPD or R) results, the cut-off values at 0.02 for PPD and 0.01 for R were obtained with a view to employ them in low prevalence areas with no previously observed non-specific reactions to SITT. Concerning its use after positive skin tests, cut-off values were set at 0.05 for PPD and at 0.03 for R. The choice of an interpretation method considering positive results with PPD and/or R (PPDUR), justified in a high risk context, provided a test Ser of 93% [84–98] and Spr of 71.8% [67.9–75.6]. Analysis of positive results with PPD and R (PPD∩R), ideal for low-risk contexts, provided a test Spr of 94.3% [92.0–96.1] and Ser of 77% [64–87]. Thus, adapting the criteria to the region''s infection status and to the conditions for its application is essential for the appropriate use of the IFNγ assay. [Copyright &y& Elsevier]

Free-living amoebae (FLA) are described as environmental reservoirs for some bacteria able to resist their phagocytosis. In the environment, the fate of Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) responsible for bovine tuberculosis and paratuberculosis, respectively, remains poorly understood and is considered potentially problematic in the eradication and control of these diseases. We hypothesize that FLA may play a role in the persistence of Mbo and Map in the environment. In this study, 90 samples were collected from herds affected by one or both diseases to investigate the diversity of amoeba and their associated bacteria. Metabarcoding analyses revealed that Acanthamoeba, Copromyxa, Naegleria, and Vermamoeba were the most represented genera of FLA, with Pseudomonadota being the bacteria most commonly found associated with FLA. Although no Mbo and Map DNA were identified by sequencing, traces were detected by ddPCR (digital droplet PCR), specifically targeting these bacteria. In conclusion, we described a wide diversity of FLA and associated bacteria in this environment. It also suggests that Map and Mbo could be associated, even weakly, with FLA in the environment. However, this needs to be confirmed by detecting a highest amount of DNA and, if possible, cultivable Map and/or Mbo associated with these environmental FLA., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)