Wild raccoons () as a potential reservoir of cytolethal distending toxin-producing strains in Japan.

In view of increasing reports of infections due to virulent species including cytolethal distending toxin () gene-positive strains, it is important to identify the reservoirs and transmission routes of such pathogenic strains. Raccoons considered to be a source of zoonotic pathogens were monitored for the presence of species in Japan and analyzed for genes. Of 384 wild raccoon rectal swabs analyzed, 60% were positive for species, of which 20% carried -genes. Among seven species isolated ( and ), genes were distributed in (63%), (16%), and novel in (21%). Complete gene clusters were identified in and strains, whereas had intact but truncated and genes. Phylogenetic analyses showed divergent pulsotypes among the gene-positive strains. Cytotoxicity assay revealed that and produced CDT more toxic to eukaryotic cells compared to human clinical strains, which were neutralized by anti-PaCdtB serum. As expected, the strains with truncated genes had no biological activity. Molecular analysis revealed that all the genes were located on plasmids as determined by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization assay. Intriguingly, the gene in strains was detected on dual plasmids. Notably, all the gene-positive strains were found to carry plasmid-mediated T3SS-related genes. These results suggest that wild raccoons are possible reservoir of virulent strains in Japan.IMPORTANCE species considered normal flora are occasionally associated with gastroenteritis in healthy humans. Cytolethal distending toxin (CDT), a bacterial virulence factor found in various Gram-negative bacteria and associated with gastroenteritis and extra-intestinal infection has also been reported in at least two species ( and ). Determination of the transmission routes of such virulent is crucial for the implementation of evidence-based control programs. In this study, we identified raccoons as the probable reservoir of the gene-positive strains in Japan. Interestingly, CDTs produced by raccoon-derived strains exerted more toxic effects on the eukaryotic cells compared to the clinical strains. In addition, the identification of a novel gene cluster in another species isolated from raccoons suggests that may be categorized as an emerging zoonotic pathogen.