Title : TOXIGENIC AND NON - TOXIGENIC STRAINS OF CLOSTRIDIUM BOTULINUM TYPE E A . DIFFERENTIATION BY IMMUNODIFFUSION B . ATTEMPTS TO INDUCE TOXIGENICITY

approved: K. S. Pilcher In this study of toxigenic and non -toxigenic strains of Clostridium botulinum type E, attempts were made to induce toxin production in non -toxigenic cultures by exposure to mutagenic agents and to differentiate the organisms by immunodiffusion. First, cultures of non -toxic Cl. botulinum type E were exposed to various mutagenic agents in an attempt to produce toxigenic mutants. Next, an attempt was made to separate toxic and non -toxic Cl. botulinum type E colonies on the basis of their microscopic appearance by reflected and transmitted light. Finally, the use of micro immunodiffusion tests for the identification of Cl. botulinum type E toxin and consequent differentiation of toxic and non -toxic cultures was studied. Nine non -toxic Cl. botulinum type E strains were exposed to a variety of mutagenic agents. These included 2aminopurine, azaserine, acriflavin, 5bromouracil and gamma ray irradiation. The degree of exposure was sufficient to kill 90% or more of the cells. After exposure, the survivors were transferred to fresh medium and the resulting cultures tested for toxigenicity. No toxin production was demonstrated in any of these cultures. An irradiated culture of a non -toxic E -like strain was cultured on brain heart agar with sodium thioglycollate. The colonies which developed were examined microscopically by reflected and transmitted light. Of approximately 10, 000 colonies observed, seven were picked because of their resemblance to the toxic colonies described by Dolman (1957b) and tested for toxicity. None proved to be toxic. Two micro immunodiffusion methods were studied. Culture filtrates of 14 toxic and nine non -toxic strains of Cl. botulinum type E were concentrated approximately ten fold by dialysis against polyethylene glycol 4000. They were tested against type E antitoxin using the agar well micro immunodiffusion method. All toxic strains reacted with this antiserum giving a single line precipitin reaction visible in a strong beam of light. These strains did not react with type B antitoxin and gave a faint, fading reaction with type A antitoxin. The non -toxic filtrates did not react with types B or E antitoxin and gave a faint, fading reaction with type A antiserum. Concentrated culture filtrates of nine other clostridial species were tested against types A, B and E antitoxin. None reacted with type E antitoxin. However, some did react with type A and B antitoxin, indicating the presence of antigens common to Cl. botulinum type A or B and some other clostridial species. Using the template micro immunodiffusion method, concentrated culture filtrates of Cl. botulinum types A, B and E were tested against the same antitoxins. Again type E antitoxin proved specific and reacted with toxic type E cultures only; these cultures again failed to cross react with type A or B antitoxin. Because of the specificity of the reactions between type E culture filtrates and antitoxin, micro immunodiffusion methods appear to be very promising for differentiating pure cultures of toxic and non -toxic type E strains under laboratory conditions without the use of animals. Further work is needed to explore possible application to mixed cultures from fish and other specimens. The sensitivity of the two micro immunodiffusion methods was compared by first determining the toxin concentration in a concentrated filtrate of a type E culture in terms of mouse LD50 /ml and then determining the minimum concentration of this toxin detectable by the two in vitro procedures. Visible precipitate lines developed only with the template micro method for this particular concentrate. Thus, the template micro method appeared more sensitive. It was able to detect a minimum toxin concentration of 123 LD50 /ml or about 3. 1 LD50 per 0. 025 ml, the approximate volume used in the test. Toxigenic and Non -Toxigenic Strains of Clostridium Botulinum Type E A. Differentiation by Immunodiffusion B. Attempts to Induce Toxigenicity by Mutagenic Agents