A high prevalence of Clostridium botulinum type E in Finnish freshwater and Baltic Sea sediment samples

The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined. A total of 110 samples were tested with a neurotoxin-specific PCR assay. Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. Spore counts were quantified by the most probable number method, Cl. botulinum type E kg(-1) averaging 940 in sea and 370 in freshwater samples. The overall prevalence and spore counts of Cl. botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature. These findings indicate the possibility of Cl. botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.     

Proteolytic mutants obtained from Clostridium botulinum type E.

Proteolytic mutants were isolated from toxigenic strains of Clostridium botulinum type E after several transfers. When these cultures were plated on blood agar, almost all of the colonies obtained were proteolytic, and there were fewer toxigenic colonies than nontoxigenic colonies. The proteolytic mutants and nonproteolytic original strains were different in their biological properties.     

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.     

Clostridium botulinum type E in fish from the Great Lakes

Bott, Thomas L. (University of Wisconsin, Madison), Janet S. Deffner, Elizabeth McCoy, and E. M. Foster. Clostridium botulinum type E in fish from the Great Lakes. J. Bacteriol. 91:919-924. 1966.-The intestinal contents of more than 3,000 fish from Lakes Erie, Superior, Huron, and Michigan were examined for Clostridium botulinum type E. Demonstration of the organism was accomplished by identifying its toxin in liquid cultures inoculated with material from the alimentary tract. Incidence figures, expressed as per cent of the fish tested, were: Lake Erie, 1%; Lake Superior, 1%; Lake Huron, 4%; the main body of Lake Michigan, 9%; and Green Bay (on Lake Michigan), 57%. Thus, C. botulinum type E appears to be widely but unevenly distributed in the Great Lakes, and fish from all areas are potential carriers of it.     

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.