|Abstract or Summary
- 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 2-aminopurine, aza-serine,
acriflavin, 5-bromouracil 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