Partial purification of extracellular substance of Vibrio anguillarum toxigenic for rainbow trout and mouse.

Abstract

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

A Comparison of Pathological Changes Caused by Vibrio anguillarum and Its Extracellular Products in Rainbow Trout(Oncorhynchus mykiss).

“…Clinical symptoms shown by infected fish were similar in every case, the most common being skin ulcers, hemorrhages at the fin bases, hem orrhagic septicaemia and ocular changes. It is thought that the extracellular products (ECPs) are implicated in the virulence of V. anguillarum, as ECPs alone is lethal to fish (Umbreit and Tripp, 1975;Inamura et al, 1984;Kodama et al, 1985;Santos, 1991;Santos et al, 1991). In addition, it has been reported that the ECPs of some strains contain hemolysin, proteases and are cytotoxic to different fish cell lines (Munn, 1980;Toranzo et al, 1983;Kodama et al, 1984Kodama et al, , 1985Inamura et al, 1985;Kanemori et al, 1987;Krovacek et al, 1987).…”

“…It is thought that the extracellular products (ECPs) are implicated in the virulence of V. anguillarum, as ECPs alone is lethal to fish (Umbreit and Tripp, 1975;Inamura et al, 1984;Kodama et al, 1985;Santos, 1991;Santos et al, 1991). In addition, it has been reported that the ECPs of some strains contain hemolysin, proteases and are cytotoxic to different fish cell lines (Munn, 1980;Toranzo et al, 1983;Kodama et al, 1984Kodama et al, , 1985Inamura et al, 1985;Kanemori et al, 1987;Krovacek et al, 1987). His tological studies have shown that the bacterium causes hemorrhagic lesions in most internal organs (Chart and Munn, 1980;Richards, 1980;Ransom et al, 1984;Ming-Chen et al, 1985;Lamas, 1991).…”

Use of prawn blood agar hemolysis to screen for bacteria pathogenic to cultured tiger prawns Penaeus monodon

“…However, both pathogenic and non-pathogenic bacteria are often found in the prawn hepatopancreas (Ruangpan & Kitao 1991, Chang et al 1996a) and it has been suggested that their hemolytic activity may be correlated to their pathogenicity, since factors such as adhesion and invasion (Ruangpan et al 1994) have been excluded. Although there is still much disagreement about the relationship between hemolysis and pathogenicity (Inamura et al 1984, Chung & Kou 1985, Kodama et al 1985, hemocytolytic assay is still commonly used to differentiate among suspected pathogens (Ullah & Arai 1983, Lee et al 1995.…”

Metalloprotease Produced by Listonella anguillarum Shows Similar Activity to Plasma Activated Protein C in Rainbow Trout Coagulation Cascade.

“…Among them,the well-characterized virulence prop erty of L.anguillarum is the ability to sequester iron from high-affinity iron-binding proteins of a host via a plasmid-encoded mechanism (Crosa, 1980;Actis et al, 1986).Extracellular products such as toxin, hemolysin, and proteolytic enzymes are also thought to be involved in the virulence. Tissue damage from vibriosis is caused by extracellular substances of the organism (Kodama et al, 1984;Kodama et al,1985;Krovacek et al,1987;Santos et al, 1991). Metallo protease is known to be one of these extracellular products (Inamura et al,1985;Norqvist et al,1990;Farrel andCrosa, 1991, Milton et al,1992;Stensvag et al,1993).…”