The present report concerns the toxin yield in the filtrates observed on the basis of cytopathic effect on HeLa cells in cultures, and immunogenic activities of cholera experimental vaccines consisting of both the toxic filtrates combined with the dead bacteria by mouse protection test.
Cell free culture filtrates of Vibrio cholerae serotype Ogawa 41 were prepared by filtering through a 0.22U membrane filter peptone water which had 0.5^5% peptone, pH6.5 or 7. 8, and was cultivated at 29C for 17^-72 hours. Then, 17^72 hour filtrates were inoculated on HeLa cells in monolayer cultures respectively and cytopathic effect was checked in 4 hours.
Heated filtrates (56C, 45 minutes) were also tested in this same way. In this way two kinds of toxins in the filtrates were found: heat labile toxin and heat stable toxin. These toxic filtrates were tested for mouse lethal activity, agglutinating, vibriccidal, and neutralizing antibody.
Finally, experimental vaccines were prepared with the filtrates only and the filtrates plus dead cells in accordance with the conventional procedure for the preparation of cholera vaccine, and were tested for the protective activity against vibrio challenge by mouse protection test, compared with conventional vaccine.
The results obtained are summarized as follows:
1) Heat Iabile toxin and heat stable toxin were found easily in cell-free culture filtrates of Vibrio cholerae on the basis of cytopathic effect on HeLa cells in culture. These toxins, which were also different in antigenicity, were neutralized specifically with their respective antiserum. Heat labile toxin was produced on culture at 29C in peptone water containing 0. 5, or 1.0% peptone with nearly absence of heat stable toxin in 17 hour culture and the toxin appeared in the early stage of culture, the logarithmic phase. In contrast, heat stable toxin yield was greater on culture at 37C in peptone water containing above 3% peptone and the toxin appeared in the later stage of culture,, the stationary phase. Activity of heat labile toxin was suppressed temporarily by the rabbit normal serum but heat stable toxin was not suppressed.
2) Toxicity of heat labile and stable toxic filtrates to mice increased in direct proportion to
the peptone concentration in peptone water, incubation time and temperature, and heat stable toxic filtrate had a tendency to be more toxic than heat labile toxin., This tendency was similar to toxicity in vitro, based on cytopathic effects.
3) Agglutinins and vibriocidins were induced in rabbits at low and moderate level by both heat labile toxic filtrates and heat labile plus stable toxic filtrates respectively. The titers of agglutinins and vibriocidins were higher in heat labile toxic filtrates than heat stable toxic filtrates.
4) Experemental cholera vaccines consisting of dead cholera vibrio-containing toxic filtrates had more mouse protective activity than the conventional cholera vaccines but the cholera culture filtrates only, had similar protective activity to the cholera conventional vaccines diluted 100 times.
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