Toxin Isolation from a Kanagawa‐Phenomenon Negative Strain of Vibrio parahaemolyticus

Abstract

Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawaphenomenon negative strain) and consistently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.Prominent among symptoms recorded for victims of Vibrio parahaemolyticusassociated food poisoning are diarrhae, abdominal pain and vomiting, with chills, fever, headache and bloody stools less frequently reported (2,3,7,8,10,19,33,(36)(37)(38). V. parahaemolyticus illnesses have frequently been diagnosed as dysentery and non-typhoidal salmonellosis (1-3). Bacterial-induced diarrhea is currently thought to be mediated by at least three mechanisms : (i) cell-free protein enterotoxins, liberated by nonpenetrating organisms and initiating fluid accumulation in the gut, viz., Vibrio cholerae (12), Escherichia coli (29) and Clostridium perfringens (10) , (ii) invasion by organisms, such as Shigella (15) and E. coli (11), manifesting cytotoxic properties which interfere with host cell functions and cause diarrhea; and (iii) a combination of these. Because of the variety of symptoms reported by V. parahaemolyticus gastroenteritis victims and the conflicting pathology data, it is not clear whether the disease is a manifestation of effects of enterotoxin on the host, invasion of the causative agent via the gut, or to toxic components produced only by certain serotypes of V. parahaemolyticus. Another member of the genus Vibrio, V. cholerae, is known to produce an exo-enterotoxin, associated with symptoms elicited by the organism (12). Because of the strong similarity of V. parahaemolyticus-associated disease with cholera, there is reason to suspect that V. parahaemolyticus may produce an enterotoxin (6, 40).

Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin

“…Vibrio parahaemolyticus is a marine bacterium that can cause gastroenteritis, the major clinical symptoms being diarrhea, abdominal cramps, nausea, and vomiting (1, 17,19). Although the pathogenic mechanisms of this organism are not well understood, proposed virulence factors or properties include thermostable direct hemolysin (TDH), other extracellular or cell-associated toxins or enzymes (3,14,16,17,19,21,34,36,38,39), adherence (13,17,19,20), and invasiveness (4,19). Of these potential virulence factors, TDH has been considered very important because of a striking epidemiological correlation with the Kanagawa phenomenon (KP), which is beta-hemolysis induced by TDH in a special blood agar medium (Wagatsuma agar).…”

Experimental pathogenicity of Vibrio parahaemolyticus for the schistosome-bearing snail Biomphalaria glabrata

“…We studied a Kanagawa phenomenon negative strain of Vibrio parahaernolyticus, which has been shown to elaborate a water-soluble, proteinaceous exotoxin (Sochard and Colwell 1977). In our experiments the cell-free culture filtrates did not kill any snails, suggesting that the exotoxin was not active against B. glabrata (Table 1).…”

“…This strain is nonhemolytic, nonenteritis associated. and Kanagawa phenomenon negative (Sochard and Colwell 1976).…”

Analysis of Vibrio parahaemolyticus soluble antigens by employing passive hemagglutination

“…(iii) Toxic fractions. A culture filtrate, ranging from 50 to 80% protein, extracted from V. parahaemolyticus FC 1011, isolated from a normal Chesapeake Bay blue crab, was found to be toxic on forced feeding, producing diarrhea and death in BALB/c mice of 15 to 22 g. Intraperitoneal injection produced similar results (21). Briefly, this fraction was prepared as follows: V. parahaemolyticus FC 1011 was cultured overnight at 37°C in 2-liter volumes of seawater-yeast extract broth without agitation.…”

“…HA titer against V. parahaemolyticus FC 1011 (1.0 mg) showed no reaction, most likely representing inhibition by excess antigen, although other possibilities exist. This antigen was found to possess diarrhea-provoking properties in mice when force-fed to the mice or injected intraperitoneally (21). Antigen preparations of strain 11590 and Sak-3 revealed little or no toxic effect in animals, perhaps accounting for the relatively weak response in the HA test.…”