Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log10 50% lethal doses of 17 isolates were lower by greater-than-or-equal-to 3.5 log10 units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 50% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.

Eleven ornithine-positive strains of Aeromonas (9 A. veronii and 2 provisionally classified as Aeromonas species ornithine positive) were tested for ability to cause fluid accumulation in the rabbit ileal loop. All eight beta-hemolytic strains caused fluid accumulation. Gel diffusion analysis revealed that the A. veronii beta-hemolysin was serologically related to the A. hydrophila beta-hemolysin, a known enterotoxic molecule. The biological activity of the A. veronii hemolysin was neutralized by antiserum to A. hydrophila hemolysin. One of three strains that were not beta-hemolytic caused fluid accumulation but only when the ileal loops were inoculated with live cultures. These results suggest that A. veronii is a potential enteropathogen that can cause diarrhea by means of a cell-freed enterotoxin (beta-hemolysin) or by a second mechanism that requires the presence of whole cells.

Weakly virulent isolates of Vibrio vulnificus that were lethal only to simultaneously iron-overloaded and immunosuppressed mice were tested for ability to cause fluid accumulation in the permanently ligated rabbit ileal loop. Unlike the highly virulent isolates, which caused septicemia and death in rabbits, these isolates caused significant fluid accumulation in the rabbit loops. Fluid accumulation was also observed when culture filtrates were tested, indicating the existence of an enterotoxin. Enterotoxin activity did not correlate with the hemolysin or protease activities. Only one of three enterotoxigenic isolates caused diarrhea when administered to temporarily ligated rabbit ileal loops, suggesting involvement of some other pathogenic determinant(s) such as colonization.

Lethal doses of 11 clinical and environmental isolates of Vibrio vulnificus were determined in suckling mice after oral challenge. With one exception, isolates that were virulent to iron-overloaded adult mice after intraperitoneal inoculation were highly lethal to the infant mice (>50% lethality at 105 CFU/mouse). The virulent isolate that failed to kill infant mice at 105 CFU had lost its invasiveness. Conditionally virulent isolates that were virulent only to simultaneously iron-overloaded and immunosuppressed adult mice required > 109 CFU to kill the infant mice. Avirulent isolates failed to kill at >109 CFU/mouse. There were no significant differences in the lethalities of clinical and environmental isolates. These findings demonstrated a close correlation between virulence in the iron-overloaded adult mouse and infectivity by the oral route.

The lethality of Listeria isolates was determined with normal adult mice and mice that were immunocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of the pathogenic isolates were significantly lower (alpha = 0.05) in the immunocompromised mice than in the untreated mice, with an average reduction of 5.8 log10 units. In contrast, the mean LD50s of the nonpathogenic isolates were lower in the immunocompromised mice by an average of only 0.4 log10 unit, a difference that was not significant (alpha = 0.05). When immunocompromised mice were used, the LD50s of pathogenic Listeria monocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by greater than or equal to 6 log10 units and lower than those of nonpathogenic L. ivanovii isolates by greater than or equal to 4 log10 units. Pathogenic L. monocytogenes isolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in immunocompromised mice in 3 days at a dose of approximately 10(4) CFU per mouse. An alternative procedure using iron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.

Klebsiella pneumoniae isolates were recovered in increased numbers from oysters harvested during the warm months of the year from approved and classified waters. A total of 76 oyster isolates was examined using in vivo and in vitro assays. The nonpathogenic responses of the strains studied suggest that environmental strains are not a public health risk.

Thirteen Clostridium perfringens isolates classified as nonenterotoxigenic by radioimmunoassay (RIA) were tested for biological activity in rabbit ileal loops to determine whether these organisms produced enterotoxins serologically unrelated to the classical C. perfringens enterotoxin. None of these strains was active in the ileal loop assays. The large number of RIA-negative isolates obtained from food-poisoning outbreaks is more likely due to the failure to isolate causative strains rather than to the existence of novel enterotoxins.

Twelve strains of Vibrio parahaemolyticus from four epidemiologically distinct groups were examined for their ability to hemagglutinate human, bovine, chicken, guinea pig, and rabbit erythrocytes and to adhere to human buccal mucosal epithelial cells in the presence and absence of mannose. Four of six Kanagawa-positive but none of six Kanagawa-negative strains showed mannose-sensitive hemagglutination with erythrocytes of rabbits and of one or more additional species. Mannose-resistant hemagglutination was shown by one strain in each group with no apparent relationship to strain source or hemolytic capability. All strains adhered to human buccal mucosal cells, with but a single strain showing significant difference in adherence at the alpha = 0.05 level. The adherence pattern had no relationship to the four epidemiological groups. Although adhesive processes may well be involved in disease caused by V. parahaemolyticus, our results do not support a role for adherence as a predictor of pathogenicity.

Three of 36 raw milk isolates of Yersinia enterocolitica produced enterotoxin in milk at 25 degrees C, but not at 4 degrees C. No strain tested could survive pasteurization.

Groups of 16 rabbits per strain were injected with broth culture dilutions of three Kanagawa-positive Vibrio parahaemolyticus strains. The effective dose required to produce ileal loop dilatation in 50% of rabbits for pure cultures of strains 10136-76 and 553-72 from patients stools and NY 477 from incriminated food was 1.1 x 10(6), 2.6 x 10(5), and 7.7 x 10(6) organisms, respectively. When each of these cultures was admixed with greater than or equal to 10(9) Vibrio alginolyticus cells, the 50% effective dose was 1.2 x 10(6), 1.1 x 10(7), and 1.3 x 10(8) cells, respectively. Although concomitant injection of large numbers of competitive nonvirulent cells did not affect the 50% effective dose for strain 10136-76, that for the remaining two was increased 20- to 40-fold. The initiation of ileal loop response as estimated from sigmoidal plots of proportion of positive loops versus cell concentrations was given by as few as 10(2) cells of strain 553-72. Strains NY 477 and 10136-76 required approximately 10(5) cells. Half of the maximal response from these plots corresponded well with the 50% effective dose for the strains. These results suggest that pathogenicity of Kanagawa-positive V. parahaemolyticus strains may involve the participation of some virulence mechanisms in addition to the Kanagawa hemolysin.

The enteropathogenicity of Vibrio parahaemolyticus was investigated by contrasting the effects of whole cells, cell fragments, cell-free preparations, and media constituents injected into rabbit ileal loops. Three of 20 cultures utilized were Kanagawa-negative strains from seawater and sea fish. The remaining 17 cultures included both Kanagawa-positive and -negative strains from Japanese victims of gastroenteritis. Broth culture filtrates concentrated 10-fold by dialysis against 30% Carbowax were unreactive, whereas lyophilized filtrates, regardless of Kanagawa type, as well as all sterile broth preparations containing greater than or equal to 5% NaCl gave positive reactions in the rabbit gut. In contrast, crude lysates derived from broth cultures of Kanagawa-positive strains caused loop dilatation; lysate supernatants were unreactive. Lysates of cells washed from brain heart infusion agar were more reactive than lysates from Trypticase soy agar-grown cells. When agar-grown cell lysates prepared by disruption in saline were dialyzed against distilled water, they were devoid of gut reactivity. Reactivity was restored in dialysands resuspended in saline and in dialysates concentrated 10-fold. The agar-grown cell lysates exhibited Kanagawa-type hemolysis. Our data support the conclusion that the rabbit loop reactivity observed with lyophilized, cell-free culture filtrates may result from excessively elevated NaCl concentrations, and that a toxic factor associated with large-cell particles may be dialyzable, depends on saline for expression, and resembles the Kanagawa hemolysin.

The antigenic relationships of 79 strains of Vibrio parahaemolyticus representing 46 assigned K-types were studied by tube agglutination. Homologous titers of 46 anti-K sera ranged from 80 to 2,560. All but three sera exhibited from one to six heterologous reactions, the majority of which gave titers of ≤20, but 19 sera showed cross-reactions whose titers exceeded 40. Nine reciprocal and 16 unilateral relationships were described. Some of the cross-reactions involved heat-extractable K-antigens, as determined by absorption with nonheated and heated heterologous antigens, whereas others did not involve K-antigens since absorption with the heterologous antigens had no effect on the homologous systems. On the basis of the reactions of selected antisera to the O-antigens of some of the K-strains, the cross-reactions could not be explained solely on the basis of O-specificities.

The hemolytic activities of 91 strains of Vibrio parahaemolyticus isolated from human diarrheal stools, sea fish, and sea water; 21 suspected V. parahaemolyticus cultures isolated from wound infections; 14 nonpathogenic marine vibrios; and 21 V. parahaemolyticus isolated from moribund blue crabs Callinectes sapidus were compared. Potentially pathogenic V. parahaemolyticus strains could be differentiated from the related nonpathogenic marine vibrios, because the former hemolyzed hamster, sheep, and human blood, whereas the latter were nonhemolytic. In addition, V. parahaemolyticus isolated from tissue infections could be differentiated from those of the first group isolated from sea fish or human stools, because the former exhibited primarily an α-hemolytic reaction on chicken blood; the latter exhibited mostly β. It is suggested that V. parahaemolyticus isolated from blue crabs may be differentiated from the first group on the basis of their hemolysis of human blood. A useful schema of the differential hemolytic reactions, exhibited by V. parahaemolyticus, tissue infection vibrios, and nonpathogens on hamster, sheep, chicken, goose, and human blood is given. The patterns of hemolytic activity of these groups on special human blood-agar plates (Kanagawa hemolysis) resembled that seen on ordinary human blood-agar.

Morphological, cultural, biochemical, and serological characteristics of 79 strains of Vibrio parahemolyticus isolated from patients suffering from gastroenteric disease in Japan were compared with 17 suspected V. parahemolyticus cultures isolated from wound infections and 14 nonpathogenic vibrios isolated from an estuarine environment in the United States. These groups were differentiated on the basis of several key reactions which included: the range of growth temperature and salt tolerance; the production of catalase and acetoin; the hydrolysis of starch; the fermentation and utilization as single carbon source of sucrose, cellobiose, and arabinose; and the ability to swarm on 1% agar. The separation of the groups on the basis of cultural and biochemical analyses was confirmed by means of slide agglutinations with specific anti-K antisera. The results of this study strongly suggest that the wound infection isolates are V. parahemolyticus species which are easily distinguished from the nonpathogenic estuarine vibrios.