Investigations have been made on the purification of Clostridium botulinum type E toxin and an attempt made to elucidate the phenomenon of trypsin activation. Type E botulinus toxin produced in meat infusion medium in dialysis sacs was partially purified by Seitz filtration and ethanol precipitation. Treatment of this toxic preparation with trypsin produced a 20 - 50 fold increase in potency. Both non-activated and trypsin-activated toxins were further purified by elution through cellulose ion-exchange columns and dried by lyophilization. Refractionation of these dried toxins effected even further purity. These highly purified preparations of both non-activated and trypsin-activated type E toxin were then analysed in the ultracentrifuge. The former was found to have a sedimentation constant of 5.6 Svedburg units whereas activated toxin did not form a boundary under identical conditions. Together with other considerations, this evidence indicates that the activation mechanism involves a fragmentation process whereby more toxic sites become exposed on the toxin molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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The aflatoxins, carcinogenic and toxic mold metabolites of Aspergillus flavus, were isolated and determined in cottonseed meal and other feedstuffs. This new quantitative method uses an acetone soxhlet extraction to remove the toxins from the defatted meal. The residual triglycerides, phospholipids and pigments were removed from the acetone by filtration of the cooled solution. The aflatoxin was further isolated by evaporating the acetone and redissolving the residue in hot methanol. After cooling, the insolubles were discarded and the methanol-soluble materials taken up in chloroform. The chloroform solution was spotted on silicic acid thin layer chromatographic plates along with suitable standards. After development with 9:1 (v/v) chloroform:acetone, the chromatographic plates were examined under ultraviolet light. Complete recovery of aflatoxin, within limits of visual discrimination, was obtained by this isolation procedure as indicated by extraction efficiency and internal standard data. Twenty-five cottonseed meals were analyzed by this method and nine meals contained aflatoxin B₁ at levels from 17 to 190 parts per billion. Fluorodensitometry, a new instrumental technique, was used to compare standards and samples containing aflatoxin directly from the thin-layer chromatographic plate. This procedure eliminates the errors inherent in the visual comparison method and permits greater sensitivity and accuracy. Amounts of aflatoxin B₁ as low as 8.0 x 10⁻⁵ micrograms can be detected by this technique. The results obtained by this procedure were substantiated by duckling assay and trout feeding trials. / Graduation date: 1966

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