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An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum (strain NCMB571).Two fractions (GI and GII + III) obtained by Sephadex G-200 chromatography following DEAE-cellulose chromatography were lethal to rainbow trout and mice. Material separated from the GI fraction by Sepharose 4B affinity chromatography (GI-A fraction) was lethal to these animals.By sodium dodecylsulfate polyacrylamide gel electrophoresis, the GI and GI-A fractions were found to be composed of components with molecular weights of 44K and 34K, and 44K, respectively. The 44K protein band was associated with carbohydrate.Peripheral vascular disorder was observed in fish and mice that died after inoculation with GI or GI-A fraction. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100 C for 20 min and complete at 121 C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity.Homologous antitoxin completely neutralized the toxic activity.Vibriosis of fish is characterized by hemorrhagic septicemia and high morbidity and mortality among marine (16, 18) and freshwater (4, 14) fishes. The causative agents, Vibrio anguillarum and other Vibrio species isolated from fish, produce hemorrhagic and necrotic lesions in the skin, fins, and visceral organs as well as hypertrophy of the kidney, spleen, and liver (15). We found that the cell-free filtrate of V. anguillarum cultures contains substance(s) toxic to rainbow trout and mice, and also produce a cytotoxic effect on fish line cells (8, 12). Munn (13) observed that the hemolysin of V. anguillarum is lethal to eels. Inamura et al (6) and Toranzo et al (19) also reported a toxic substance of this bacterium. However, the relationship between these extracellular products and lethal toxicity of the organism in fish is still unclear. This is the first report on the purification and characterization of the extracellular toxic substance of V. anguillarum. The relationships of the toxic substance produced by V. anguillarum to hemolysin and protease and its role in the pathogenesis of vibriosis are discussed. MATERIALS AND METHODS Bacteria. A virulent strain of V. anguillarum (NCMB571 strain) which was 909 91 0 H. KODAMA ET AL

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