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The dissemination and abundances of Vibrio species in aquatic environments are of interest, as some species cause emerging diseases in humans and in aquatic organisms like fish. It is suggested that Vibrio cholerae non-O1 infections of Plecoglossus altivelis ('ayu' were spread to various parts of Japan through the annual transplantation of juvenile fish. To investigate this, we used genome-aided tracing of 17 V. cholerae strains isolated from ayu between the 1970s and 1990s in different Japanese freshwater systems. The strains formed a