Repression of toxin production by tryptophan in Clostridium botulinum type E

Of the seven amino acids required by Clostridium botulinum type E, tryptophan is the most essential and may provide the cell with nitrogen. The addition of excess tryptophan (10–20 mM) or other nitrogenous nutrients to minimal growth medium markedly decreased toxin formation but did not affect growth in C. botulinum type E. On the other hand, the addition of an enzymatic digest of casein (NZ Case) stimulated toxin formation and overcame repression by tryptophan. Immunoblots of proteins in culture fluids using antibodies to type E toxin indicated that tryptophan-repressed cultures produced less neurotoxin protein. Inhibitors of neurotoxin did not accumulate in cultures grown in minimal medium supplemented with high tryptophan. The results suggest that tryptophan availability in foods or in the intestine may be important for toxin formation by C. botulinum type E.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Thermal inactivation of type E botulinum toxin

The theoretical required cooking times for inactivation of type E Clostridium botulinum toxin (5,000 ld(50) mouse units per 0.5 ml) in haddock fillets of various sizes were calculated by graphical integration of the toxin inactivation rate and heat penetration data. The results indicated that normal cooking procedures should suffice to inactivate this amount of toxin. This conclusion was substantiated by the following additional experimental observations which revealed that the original experiments had been conducted under conservative conditions. First, maximal heat stability of the toxin was found to occur at about pH 5.5, with decreasing resistance upon increasing pH. The theoretical cooking times were based on destruction of the toxin at pH 6.7. The pH of radio-pasteurized inoculated haddock, when toxin production had occurred, was on the alkaline side, at which condition the toxin is heat-labile. Second, when spoilage was discernible in radio-pasteurized inoculated haddock, the toxin titer was low, about 50 ld(50) mouse units per 0.5 ml. Third, the toxin was adequately inactivated in toxic fillets after deep-fat frying for 3 min at 375 F (190.6 C) or after pan frying for 5 min per side at 400 F (204.4 C). Fourth, in this study, residual toxin activity was assayed by intraperitoneal injection of mice. It was shown that the oral toxic dose was 50 to 100 times greater than the intraperitoneal toxic dose.     

Immunochemical study of the toxin of C1. botulinum type E]

Toxins extracted from the cells and the culture fluid of C1. butulinum type E had a similar mol wt and antigenic composition. Trypsin activation led to the appearance of an additional antigen. Coefficient of the cell toxin activation was much greater than the coefficient of activation of the toxin contained in the culture fluid.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.