Isolates Clostridium botulinum from foodborne and infant botulism cases in the United States were compared on the basis of toxigenicity, cultural and biochemical characteristics, metabolic products, and susceptibility to antimicrobial agents. Seventy-eight strains, including 42 from foodborne and 36 from infant botulism sources, were examined. Cultures on anaerobic blood agar exhibited circular, spindle, and rhizoid (medusa head) colonies. Overall, the characteristics of isolates from foodborne and infant botulism cases were quite similar. We concluded that it was not possible to differentiate C. botulinum isolates associated with foodborne botulism from those recovered from infant botulism cases. All of the 78 strains produced an unidentified indole derivative(s), detected with paradimethylaminocinnamaldehyde reagent, and hydrocinnamic acid, detected by gas-liquid chromatography; all exhibited a high degree of resistance to cycloserine, sulfamethoxazole, and trimethoprim. These characteristics should prove to be useful in the isolation and identification of C. botulinum from mixed microbial populations.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 ,ug/ml), sulfamethoxazole (76 ytg/ml), and trimethoprim (4 gg/ ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.

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Immunological tolerance is a state of unresponsiveness to foreign substances (antigens) which can develop in human and animal species as the result of continued exposure to antigens early in life. We utilized this principle for the preparation of antibodies against Clostridium botulinum type A toxin. By selective suppression of the immunological response of rabbits to unwanted antigens and subsequent immunization with a toxoid, we were able to produce a specific type A antitoxin without the need to purify the toxin. Despite cross-reactivity with C. botulinum type B, our type A antitoxin was otherwise specific since it did not react with culture filtrates of nontoxigenic variants of type B, any other C. botulinum type (C, D, E, F, and G), nor with 18 other Clostridium species, including Clostridium sporogenes. Using this antitoxin, we developed a sensitive enzyme-linked immunosorbent assay for detection of C. botulinum type A toxin.

An enzyme-linked immunosorbent assay (ELISA) for Clostridium botulinum type A and type B toxins was assessed for diagpiostic accuracy in cases of infant botulism. This test was positive in all 22 cases confirmed by the conventional tests, which included the mouse lethality assay and stool culture. Stool specimens from five cases were positive by culture, but the mouse lethality bioassay was either negative or toxicity was judged nonspecific since it could not be neutralized by specific antitoxin. The positive ELISA results in these specimens suggeste4 that this assay may be more reliable, in some cases, than the mouse bioassay. Of the 21 fecal specimens from suspected foodborne cases, 2 contained botulinal toxin demonstrable by the mouse assay and the ELISA. With regard to specificity, 35 fecal specimens from infants and 19 from suspected foodborne cases which were negative in the bioassay for botulinal toxins A and B were also negative in the ELISA. Only two fecal specimens with negative bioassay gave positive ELISA readings, providing a specificity rate of 96%. These results suggest that the ELISA may serve as a useful screening test to detect C. botu(inum toxin in clinical specimens.