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Vibrioses are the predominant bacterial infections in marine shrimp farms.Vibrio nigripulchritudois an emerging pathogen of the cultured shrimpLitopenaeus stylirostrisin New Caledonia and other regions in the Indo-Pacific. The molecular determinants ofV. nigripulchritudopathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenicV. nigripulchritudoisolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulentV. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence-linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence ofV. nigripulchritudoin several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non-pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage ofV. nigripulchritudo.