BRIEF REPORT Folia Microbiol. 37 (2), 157-158 (1992)
Effect of pH on the Stability of Type-C Toxin of Clostridium botulinum J. HALOUZKA a n d Z . HUBALEK
Institute of Systematic and Ecological Biology, Czechoslovak Academy of Sciences, 603 65 Brno, Czechoslovakia Received March 25, 1991 ABSTRACT. Stability of type-C botulinum toxin at pH 1.8-12.0 and during exposure to 5 and 28 ~ for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7-10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.
Botulism, an intoxication of the organism by the type-C toxin of Clostridium botulinum, is often responsible for a mass killing of wild and domestic water fowl. Our knowledge of the epizootiology of this poisoning and of the sensitivity of the toxin to environmental conditions are rather incomplete. In previous work we investigated the resistance of the toxin to temperature (Hubfilek and Halouzka 1988). In this study, the effect of pH on the stability of botulinum toxin was examined (cf. Segner et aL 1971; Smart and Rush 1987; Sagmeister and Willinger 1989). The type-C toxin of C. bolTllinttm was obtained by homogenization of about 40 toxic larvae of the flies Lucilia sericata and Calliphora vomitoria collected on carcasses of swans (Cygnus olor) during an epizootic in southern Moravia in autumn 1988. The type of the toxin was identified (antisera A, B, C, D, E - hnuna, ~arigsk6 Micharany, Czechoslovakia) by using the toxin neutralization mouse test (Hubfilek and Halouzka 1991). The suspension in saline solution was filtered (Synpor, 230-nm pores) and gentamicin added to 200 mg/L. The toxin titer in the filtrate was 790 LD50/mL. To prepare samples with a defined pH value, a Mcllvain's buffer (citric acid 0.1 mol/L and Na2HPO4 0.2 mol/L) containing 50 ppm of phenol red was used. The limit values were obtained by using HCI (1 mol/L) or NaOH (1 tool/L). In this way a range of nine pH values (pH 1.8-12.0) was prepared. A volume of 0.3 mL of the toxin suspension was added to 1.2 mL of the buffer having a defined pH. By measuring pH (Radelkis OP-208 pH-meter), the change of the actual pH was checked not to exceed _+ 0.05 unit. Then the samples were incubated at 5 (20 h) or 28 ~ (16 h). No opalescence or precipitation were observed during the incubation as a result of the pH change. After incubation, the samples were immediately neutralized to reach pH 7 and the toxin titrated on adult SPF mice (mass, 20-25 g) by using intraperitoneal (i.p.) inoculation (0.4 mL of sample per mouse). In addition, samples of the original (exposed) suspension diluted 5-, 25-, and 125 times were also tested. The mice were observed for 3 d. The results showed a significant resistance of type C-botulinum toxin over a broad Table I. Number of micea that died of botulism after inocularange of pH at both low and high incubation tion with toxin samples exposed (dilution 5-, 25-fold) to various temperature (Table I). Only at extreme pH pH at 5 or 28 ~ was the toxin inactivated or its activity decreased below the value detectable by the 5~ 28 ~ method used. Exposed at 5 or 28 ~ a 100 % pH killing was observed within 2 d with the 5 525-foldb 525-fold times diluted toxin suspension at pH 2.7-10.2 and with the 25 times diluted one at pH 1.8 0 0 0 0 2.77.3. 2.7-7.3 e 2 2 2 2 8.4 2 1 2 2 The results obtained support the hypothe10.2 2 0 2 0 sis of persistence of type C-botulinum toxin in 12.0 0 0 0 0 the environment even at extreme pH. Sagmeister and Willinger (1987) also reported ~rl'wo mice tested in each variant. that the toxin remained active over a broad bNo killing observed at a 125-fold dilution. range of pH (3.0-9.0) after a 24-h exposure CpH values tested: 2.7, 3.9, 5.3, 6.2, 7.3. at 20 ~ Our data show that this range is broader (2.7-10.2), despite the higher temperature of 28 ~
158
J. HALOUZKA and Z. HUBALEK
Vol. 37
This resistance could explain the persistence of the toxin in developmental stages of Diptera contaminated by feeding on bodies of animals that had died of botulism. A very low pH (2.8-3.3 Greenberg 1973) inside the bodies of larvae and pupae of sarcophagous Diptera need not lead to inactivation of botulinum toxin. The intoxicated developmental stages of these flies can, therefore, play a role in the outbreak of a botulism epizootic. Another fact must also be taken into account, that, in contrast to the bacterium, the toxin is resistant to the effect of extreme values of pH even at temperatures normally attainable in the mild climatic zone in summer. The above findings could help in understanding the mechanism of circulation of botulinum toxin in nature.
REFERENCES GREENBERGB." Flies and Disease, Vol. II. Princeton University Press, Princeton 1973. HUa.~LEKZ., HALOUZKAJ.: Thermal sensitivity of Clostridium bolulinum type C toxin. Epidem.lnf. 101, 321-325 (1988). HUaALEKZ., HALOUZlO.J.: Persistence of CIoslridium botulinum type C toxin in blow fly (Calliphoridae) larvae as a possible cause of avian botulism in spring. J.WildI.Dis. 21, 81-85 (1991). SAGMEISTERH., W|LLINGERl-l.: Beitrag zur Epidemiologie des massenhaften Wasservogelsterben durch Clostridium botulinum Typ C. ~en.tier~rtztI.Mschr. 76, 205- 209 (1989). SF~SER W.P., SCnMIDT C.F., BOLTZJ.K.: Minimal growth temperature, sodium chloride tolerance, pH sensitivity and toxin production of marine and terrestrial strains of CIosrridium bomlinum type C..4ppI.Microbiol. 22, 1025-1029 (1971). SMART J.L, RUSH P.A.: In-vitro heat denaturation of Clostridium botulinum toxin A, B and C. Im.J.Food $r 22, 293- 298 (1987).
Effect of pH on the Stability of Type-C Toxin of Clostridium botulinum J. HALOUZKA a n d Z . HUBALEK
Institute of Systematic and Ecological Biology, Czechoslovak Academy of Sciences, 603 65 Brno, Czechoslovakia Received March 25, 1991 ABSTRACT. Stability of type-C botulinum toxin at pH 1.8-12.0 and during exposure to 5 and 28 ~ for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7-10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.
Botulism, an intoxication of the organism by the type-C toxin of Clostridium botulinum, is often responsible for a mass killing of wild and domestic water fowl. Our knowledge of the epizootiology of this poisoning and of the sensitivity of the toxin to environmental conditions are rather incomplete. In previous work we investigated the resistance of the toxin to temperature (Hubfilek and Halouzka 1988). In this study, the effect of pH on the stability of botulinum toxin was examined (cf. Segner et aL 1971; Smart and Rush 1987; Sagmeister and Willinger 1989). The type-C toxin of C. bolTllinttm was obtained by homogenization of about 40 toxic larvae of the flies Lucilia sericata and Calliphora vomitoria collected on carcasses of swans (Cygnus olor) during an epizootic in southern Moravia in autumn 1988. The type of the toxin was identified (antisera A, B, C, D, E - hnuna, ~arigsk6 Micharany, Czechoslovakia) by using the toxin neutralization mouse test (Hubfilek and Halouzka 1991). The suspension in saline solution was filtered (Synpor, 230-nm pores) and gentamicin added to 200 mg/L. The toxin titer in the filtrate was 790 LD50/mL. To prepare samples with a defined pH value, a Mcllvain's buffer (citric acid 0.1 mol/L and Na2HPO4 0.2 mol/L) containing 50 ppm of phenol red was used. The limit values were obtained by using HCI (1 mol/L) or NaOH (1 tool/L). In this way a range of nine pH values (pH 1.8-12.0) was prepared. A volume of 0.3 mL of the toxin suspension was added to 1.2 mL of the buffer having a defined pH. By measuring pH (Radelkis OP-208 pH-meter), the change of the actual pH was checked not to exceed _+ 0.05 unit. Then the samples were incubated at 5 (20 h) or 28 ~ (16 h). No opalescence or precipitation were observed during the incubation as a result of the pH change. After incubation, the samples were immediately neutralized to reach pH 7 and the toxin titrated on adult SPF mice (mass, 20-25 g) by using intraperitoneal (i.p.) inoculation (0.4 mL of sample per mouse). In addition, samples of the original (exposed) suspension diluted 5-, 25-, and 125 times were also tested. The mice were observed for 3 d. The results showed a significant resistance of type C-botulinum toxin over a broad Table I. Number of micea that died of botulism after inocularange of pH at both low and high incubation tion with toxin samples exposed (dilution 5-, 25-fold) to various temperature (Table I). Only at extreme pH pH at 5 or 28 ~ was the toxin inactivated or its activity decreased below the value detectable by the 5~ 28 ~ method used. Exposed at 5 or 28 ~ a 100 % pH killing was observed within 2 d with the 5 525-foldb 525-fold times diluted toxin suspension at pH 2.7-10.2 and with the 25 times diluted one at pH 1.8 0 0 0 0 2.77.3. 2.7-7.3 e 2 2 2 2 8.4 2 1 2 2 The results obtained support the hypothe10.2 2 0 2 0 sis of persistence of type C-botulinum toxin in 12.0 0 0 0 0 the environment even at extreme pH. Sagmeister and Willinger (1987) also reported ~rl'wo mice tested in each variant. that the toxin remained active over a broad bNo killing observed at a 125-fold dilution. range of pH (3.0-9.0) after a 24-h exposure CpH values tested: 2.7, 3.9, 5.3, 6.2, 7.3. at 20 ~ Our data show that this range is broader (2.7-10.2), despite the higher temperature of 28 ~
158
J. HALOUZKA and Z. HUBALEK
Vol. 37
This resistance could explain the persistence of the toxin in developmental stages of Diptera contaminated by feeding on bodies of animals that had died of botulism. A very low pH (2.8-3.3 Greenberg 1973) inside the bodies of larvae and pupae of sarcophagous Diptera need not lead to inactivation of botulinum toxin. The intoxicated developmental stages of these flies can, therefore, play a role in the outbreak of a botulism epizootic. Another fact must also be taken into account, that, in contrast to the bacterium, the toxin is resistant to the effect of extreme values of pH even at temperatures normally attainable in the mild climatic zone in summer. The above findings could help in understanding the mechanism of circulation of botulinum toxin in nature.
REFERENCES GREENBERGB." Flies and Disease, Vol. II. Princeton University Press, Princeton 1973. HUa.~LEKZ., HALOUZKAJ.: Thermal sensitivity of Clostridium bolulinum type C toxin. Epidem.lnf. 101, 321-325 (1988). HUaALEKZ., HALOUZlO.J.: Persistence of CIoslridium botulinum type C toxin in blow fly (Calliphoridae) larvae as a possible cause of avian botulism in spring. J.WildI.Dis. 21, 81-85 (1991). SAGMEISTERH., W|LLINGERl-l.: Beitrag zur Epidemiologie des massenhaften Wasservogelsterben durch Clostridium botulinum Typ C. ~en.tier~rtztI.Mschr. 76, 205- 209 (1989). SF~SER W.P., SCnMIDT C.F., BOLTZJ.K.: Minimal growth temperature, sodium chloride tolerance, pH sensitivity and toxin production of marine and terrestrial strains of CIosrridium bomlinum type C..4ppI.Microbiol. 22, 1025-1029 (1971). SMART J.L, RUSH P.A.: In-vitro heat denaturation of Clostridium botulinum toxin A, B and C. Im.J.Food $r 22, 293- 298 (1987).
