Development of antitoxin with each of two complementary fragments of Clostridium botulinum type B derivative toxin.

Two fragments with molecular weights of 111,000 (fragment I) and 59,000 (fragment II) were separated from each other by gel filtration of dithiothreitol and urea-treated, trypsinized derivative toxin (molecular weight, 170,000) of the proteolytic Okra strain of Clostridium botulinum type B on a column of Sephadex G-200 (superfine) with a buffer containing dithiothreitol and urea. Upon removal of dithiothreitol and urea by dialysis, the two fragments reassembled to reconstruct the derivative toxin molecule. Both fragments were immunogenic, and both anti-fragments neutralized type B toxin.

Intestinal absorption of botulinum toxins of different molecular sizes in rats.

During a period of 10 to 12 h after injection of type B 16S (L) toxin into the ligated duodenum of rats, 0.01 to 0.1% of the total toxicity administered was found in the lymph drawn by cannulation of the thoracic duct.

Clostridium botulinum type D toxin: purification, molecular structure, and some immunological properties.

Clostridium botulinum type D progenitor toxin was purified. The addition of ribonucleic acid to the whole culture helped initial acid precipitation of the toxin. As with type B, both L (16S) and M toxins (12S) obtained from a hemagglutinin-positive strain, whereas M toxin only was produced by a hemagglutinin-negative strain.

Response of type B and E Botulinum toxins to purified sulfhydryl-dependent protease produced by Clostridium botulinum type F.

A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C.

Activation of botulinum toxins in the absence of nicking.

The derivative toxins purified from cultures of proteolytic strains of Clostridium botulinum types A and F were found to have been only partially nicked but were fully activated. Trypsinization of C. botulinum type B derivative toxin at pH 6.

Correlation between oral toxicity and in vitro stability of Clostridium botulinum type A and B toxins of different molecular sizes.

The in vitro sensitivity to acid and pepsin differed markedly among Clostridium botulinum type A and B toxins of different molecular sizes. The larger the molecular size of the toxin, the higher the resistance to these agents. Tye B derivative toxin was rapidly inactivated, but the progenitor toxins resisted in vitro exposure to rat intestinal juice.

Oral toxicities of Clostridium botulinum toxins in response to molecular size.

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.

Comparison of progenitor toxins of nonproteolytic with those of proteolytic Clostridium botulinum Type B.

A nonproteolytic strain of Clostridium botulinum type B produces two toxins of different molecular weight (16S and 12S) that are indistinguishable from the corresponding toxins of a proteolytic strain in molecular weight and construction but differ in potential toxicity, activation ratio, and hemagglutinability. Successful hybridization between the toxic and nontoxic components (both7S) of 12S toxins of biologically heterologous type B strains confirmed the physico-chemical similarity between the toxic as well as the nontoxic components.

Molecular construction of Clostridium botulinum type A toxins.

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 10(8) to 3.

Responses of Clostridium botulinum type B and E progenitor toxins to some clostridial sulfhydryl-dependent proteases.

Sulfhydryl-dependent proteases produced by Clostridium botulinum types A, B, and F, Clostridium histolyticum, Clostridium sporogenes and Clostridium perfringens activate preferentially type E over type B progenitor toxin but less efficiently than trypsin. The results explain why activable toxin is demonstrable in culture of a strongly proteolytic type B strain.

Experimental diarrhea in cynomolgus monkeys by oral administration with Clostridium perfringens type A viable cells or enterotoxin.

Purified C. perfringens type A enterotoxin fed orally in an amount of 5 mg caused both vomiting and diarrhea in the monkey only when the gastric juice had been neutralized. Exposure of enterotoxin to pH 4.

Antigenicites of fragments of Clostridium botulinum type B derivative toxin.

Two fragments with molecular weights of 110,000 and 60,000 were separated in a preparatory scale by gel filtration of the reduced Clostridium botulinum type B trypsinized derivative toxin on Sephadex G-200 with 0.05 M tris(hydroxymethyl)aminomethane-0.38 M glycine buffer, pH 8.

Molecular construction of Clostridium botulinum type F progenitor toxin.

Molecular dissociation of purified type F progenitor toxin with an S20,W of 10.3 and a molecular weight of 235,000 into two components, toxic and atoxic, was demonstrated by ultracentrifugation, gel filtration, and diethylaminoethyl-Sephadex chromatography at pH 7.5.

Purification of Clostridium botuliunum type F progenitor toxin.

Clostridium botulinum type F progenitor toxin was purified to a homogeneous state as judged by gel filtration on Sephadex G-200, ultracentrifugation, and disc electrophoresis. The sedimentation constant, corrected to water at 20 C, of type F progenitor toxin was determined to be 10.3 and the molecular weight to be 235,000 by ultracentrifugation at pH 6.

Purification and some properties of progenitor toxins of Clostridium botulinum type B.

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.