Activation of Clostridium botulinum type E toxin by trypsin.

Since 1936, when the type E strain of Clostridium botulinum was first established as a result of the work of Gunnison et al. (1936), 17 outbreaks of Type E botulism have been reported, involving 69 persons, of whom 21 died (Dolman and Chang, 1953; Dolman et al., 1955). Although the large proportion of fatalities suggests that this organism is highly toxigenic, published reports indicate that in the laboratory type E strains produce very low toxicities for mice, as compared with types A, B, C and D. The highest titer of type E toxin obtained by conventional methods was reported by Dolman, who obtained 4000 MLD/ml by growth of the VH strain in a peptone-beef infusion broth (Dolman et al., 1950). When this strain was grown by the cellophane bag method, titers that ranged from 10,000 to 50,000 mouse MLD's/ml were obtained (Barron and Reed, 1954). Investigations in this laboratory on the type E strain were directed toward development of a toxoid satisfactory for immunization. An inexpensive medium was required in which toxin production was consistent, and studies were carried out to determine the effect of various peptones, hydrolyzed caseins, amino acids, yeast extracts, and carbohydrates upon toxin production in bottle-grown cultures. Titers consistently greater than 5000 mouse intraperitoneal LD5o/ml were not obtained. Occasionally, a type E culture contaminated with unidentified organisms of the genus Bacilus produced higher titers of type E toxin than the pure culture. Devlin and Soderstrom (1954, personal communication), of the Parke, Davis and Company Research Laboratories, made similar observations and noted that when the toxin in these cultures was fractionated, yields greatly in excess of 100 per cent were frequently obtained. These observations suggested the presence in the cultures of an inactive toxin or protoxin similar to that described by Turner and Rodwell (1943a, b) for the epsilon toxin of Clostridium perfringens (C. welchii) type D, and by Ross et al. (1949) for the iota toxin of Clost*rium perfringens type E. These investigators reported that the toxicity of crude preparations of these toxins was greatly increased by the action of trypsin. We have found that the type E toxin of C. botulinum may be activated in a similar manner, and the present paper describes certain aspects of the activation phenomenon.