Sumiko Sakaguchi

Purification of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G cultured for 6 days at 30 degrees C in proteose peptone-yeast extract-glucose medium produced toxin of 1.3 x 10(4) LD50/ml. The toxin was precipitated at pH 4.0, extracted with 0.2 M phosphate buffer, pH 6.0, and activated with trypsin. Sonic treatment and trypsinization of the residual precipitate released additional toxin, the toxicity of which corresponded to that detected in whole culture. Activated toxin obtained from the first extract and that from the residual precipitate were combined and purified by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second gel filtration on Sephadex G-200. The yield of purified toxin from 10 liters of culture was 22.9 mg an 1.1 X 10(8) mouse ip LD50 with a specific toxicity of 3.0 X 10(7) mouse ip LD50/mg nitrogen. The molecular weight of the toxin was about 500,000, corresponding to that of L toxin of the other types. No M nor LL toxin was detected.

Primary multiplication of Clostridium botulinum type A in mustard-miso stuffing of ‘karashi-renkon’ (deep-fried mustard-stuffed lotus root)

Abstract In June, 1984, a large-scale outbreak of type A botulism occurred in 14 different prefectures in Japan, involving 36 cases and 11 deaths, due to consumption of vacuum-packed ‘karashi-renkon’ (deep-fried mustard-stuffed lotus root). The onset of symptoms ranged from June 9th through July 28th and many batches of ‘karashi-renkon’, having been manufactured by a factory during a period of three weeks from June 5th to June 25th, were found to be contaminated with type A spores. Type A spores (1.0 × 104 cfu/g) experimentally inoculated into the mustard-miso stuffing [water activity (aw) 0.92, pH 5.0, NaCl 5.7%] did not multiply. By adjusting the stuffing to aw 0.98 (pH 5.4) growth of type A was still inhibited. When the stuffing with elevated aw and pH was heat-treated for 60 min at 80°C or autoclaved, it supported growth of Clostridium botulinum type A as indicated by the achievement of toxin levels higher than 105 LD50/g. When mustard-miso was stuffed into the holes of lotus roots and refrigerated overnight or a longer period, both aw and pH of the stuffing increased to 0.98 and 5.3, respectively. The changes were apparently due to dialysis between the stuffing and the lotus root. This refrigeration process was actually performed at the plant, where the incriminated foodstuff was manufactured. The dialyzed mustard-miso stuffing, when heat-treated, supported the growth of type A organisms. Thus, we substantiated the possibility of primary multiplication of C. botulinum type A in mustard-miso stuffing subjected to refrigeration in lotus roots and then heat-treated.

Purification, Characterization, and Oral Toxicity of Botulinum Type G Progenitor Toxin

Clostridium botulinum type G (or C. argentinense) strain 2470 was grown overnight at 37°C in chopped meat glucose medium and transferred to a medium consisting of proteose peptone 4%, yeast extract 1%, glucose 1%, and L-cysteine 0.2%, pH 7.3, which was incubated for 6 days at 30°C. The 10-L culture with a potential toxicity of 1.3 x 108 mouse i.p. LD50 was brought to pH 4.0 with sulfuric acid and kept overnight at 4°C. The precipitate was packed by centrifugation and extracted twice with 0.2 M phosphate buffer, pH 6.0. The extract was treated with 0.5% trypsin (Difco 1:250) at 37°C for 60 min. The extract recovered 1.3 × 108 LD50. The residual cells were sonicated and treated with trypsin in a similar way, which recovered 1.1 × 108 LD50. The two extracts were combined, the toxicity of which was set as 100%. Ammonium sulfate was added to the combined extracts to a 0.5 saturation. The precipitate was dissolved in 150 ml of 0.2 M phosphate buffer, pH 6.0, which was clarified by centrifugation. This extract was dialyzed against 0.05 M acetate buffer, pH 4.0, which caused precipitation of the toxin. The precipitate was dissolved in 100 ml of 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. It was clarified by centrifugation, concentrated by salting out, followed by dialysis. The extract of the second salting out recovered 191 mg of protein and 3.3 × 108 LD50 (118%). The extract, divided into 20-ml portions, was subjected to gel filtration on Sephadex G-200 (2.5 × 90 cm). The effluent in the void volume contained 96.3 mg protein and 2.8 × 108 LD50 (100%). The fraction was dialyzed against 0.5 M NaC1-0.05 M acetate buffer, pH 4.0, and added were the same volume of 0.5 M NaCl-0.05 M citrate buffer, pH 4.5, and 0.01% protamine. This was clarified by centrifugation and percolated through SP-Sephadex C-50 (2.5 × 30 cm) equilibrated with 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. The percolate was diluted 2.5-fold with 0.05 M acetate buffer, pH 4.0, applied again to SP-Sephadex C-50 (1.6 × 12 cm) equilibrated with 0.2 M NaC1-0.05 M acetate buffer, pH 4.0, and eluted with NaC1 gradient from 0.2 to 0.7 M. The fractions eluted at 0.34 to 0.48 M NaC1 contained 48.0 mg of protein and 1.9 × 108 LD50 (68%). The toxin fraction was concentrated by salting out and subjected to the second gel filtration on Sephadex G-200 (2.5 × 90 cm) with the same buffer. A single protein peak eluted contained 22.9 mg of protein and 1.1 × 108 LD50 (39%). The specific toxicity was 3.0 × 107 LD50/mg N.

Purification and some properties ofClostridium botulinum type AB toxin

Abstract The progenitor toxin of Clostridium botulinum type AB was purified; both large-sized (L) and medium-sized (M) toxins were found. The toxicity of M toxin increased by about 10-fold upon trypsinization; the increase was due mostly to type B toxin and a little to type A toxin. M toxin appeared to consist of one molecule each of toxic and nontoxic components. The activated toxic component was made up of four fragments, A-H- and L-chains and B-H- and L-chains. AB toxin may be a mixture of A and B toxins.