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The morphology and the drug sensitivity of the strain GYX2014-1 isolated from the hepatic pancreatic tissue of moribund Litopenaeus vannamei were evaluated by conventional culture characteristics, physical and chemical characteristics, and molecular biology methods. Detection of extracellulase and hemolysin activity shows that the isolated GYX2014-1 has protease, lipase, gelatinase activity, but none of amylase, or lecithinase activity. The 16S rRNA gene (GenBank accession number: KT781675) was analyzed, and a phylogenetic tree analysis showed that the isolated pathogen was most closely related to V. vulnificus (GenBank accession number: NR 118570)—a match of more than 99%. The phenotypic traits and molecular biology of isolated bacteria, determined their identity as Vibrio vulnificus (V. vulnificus). In addition, artificially infected L. vannamei with Vibrio vulnificus appeared with the same disease symptoms as those of naturally infected shrimp. Drug sensitivity tests showed that V. vulnificus is highly sensitive to fosfomycin, cefradine and sinomin, and was resistant to penicillin, amikacin and kanamycin. This experiment is the first to separate V. vulnificus from L. vannamei, and the findings of this study can be used as a reference for disease control and health management. [ABSTRACT FROM AUTHOR]