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A hemolysin produced by a strain of Aeromonas hydrophila isolated from a patient with diarrhea was purified by acid precipitation and quarternary aminoethyl-Sephadex chromatography. The molecular weight of the hemolysin was estimated at 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 48,000 by Sephadex G-100 gel filtration. In polyacrylamide gel electrophoresis at pH 4.0 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the hemolysin migrated as a single band, whereas electrophoresis at pH 9.4 and thin-layer isoelectric focusing demonstrated multiple bands. The results may indicate charge isomers of the hemolysin. The purified hemolysin had a hemolytic activity of 134 hemolytic units per microgram of protein on rabbit erythrocytes. It caused fluid accumulation in infant mouse intestines and rabbit ligated ileal loops. Purified hemolysin also elicited cytotoxicity to Vero cells and lethal toxicity to mice. All these biological activities were lost on heating for 5 min at 56 degrees C. These findings support the notion that A. hydrophila hemolysin is a cytotoxic enterotoxin.

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Purification of progenitor toxin of Clostridium botulinum type B strain Okra was undertaken by sequential steps of acid precipitation, extraction, ammonium sulfate precipitation, ribonuclease digestion, acid precipitation, protamine treatment, sulphopropyl-Sephadex chromatography, and Sephadex G-200 gel filtration. Two different molecular-sized toxins, named large (L) and medium (M) toxins, were obtained. L toxin was centrifugally homogeneous but electrophoretically heterogeneous. It contained 2.5 x 108 to 3.0 x 108 mean lethal doses per mg of nitrogen, and its sedimentation constant was 16S. M toxin was centrif'ugally and electrophoretically homogeneous. It contained 5.5 x 108 to 6.0 x 108 mean lethal doses per mg of nitrogen, and its sedimentation constant was 12S. The presence of both L and M toxins in spent culture was demonstrated. It seems justified, therefore, to call both progenitor toxins. Both consisted of toxic and nontoxic components. The toxic components of L and M toxins appeared to be identical with each other. The nontoxic component of L toxin was 12S and

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Clostridium botulinum type A, B, and F toxins of different molecular sizes were fed to mice to compare the oral toxicities. The progenitor toxin, a complex of a toxic and nontoxic component, of any type was higher in oral toxicity to mice than the dissociated toxic component or the derivative toxin. The former may no doubt play a more important role in the pathogenesis of food-borne botulism. The higher oral toxicity possessed by the progenitor toxin, including the exceptionally high one found with type B-L toxin, can be explained solely by the protection afforded by the nontoxic component attached to the toxic component. The possibility of the highest oral toxicity of type B-L toxin to humans is discussed.

Two Clostridium botulinum type A toxic fractions, named large (L) and medium (M) toxins, were eluted from Sephadex G-200. Sucrose density gradient centrifugation resolved L toxin (2.5 x 10W to 3.0 x 108 mean lethal doses per mg of N) into two fractions, 19S and 16S. The same procedure performed at pH 8 resolved it into three fractions; the heavier two were both nontoxic and hemagglutinin positive, and the lightest one (7S) was toxic. M toxin (12S) (4.5 x 10W to 5.0 x 108 mean lethal doses per mg of N) was homogeneous in electrophoresis and centrifugation at pH 6. The latter procedure performed at pH 8 demonstrated that it dissociated into uniform 7S components. The nontoxic component of M toxin was free from hemagglutinin. M toxin alone was demonstrated in culture by sucrose density gradient centrifugation at pH 6. Dialysis of the culture supernatant resulted in partial formation of 16S toxin. Centrifugation of the crystalline toxin in 1 M NaCl demonstrated 16S toxin only. The toxic components of L, M, and crystalline toxins were antigenically identical. The nontoxic components of the crystalline and L toxins, consisting of two distinct antigens, were antigenically identical; that of M toxin was identical with one of these two antigens.