Iwao Ohishi

ANTIBODIES TO Clostridium botulinum TOXINS IN FREE-LIVING BIRDS AND MAMMALS

Naturally-occuring antibodies against Clostridium botulinum toxins were found in Cathartes aura (turkey vultures), Canis latrans (coyotes) and Corvus brachyrhynchos (crows) by the passive hemagglutination (PHA) test and verified by the serum neutralization (SN) test. The prevalence of IHA antibodies was 18 of 20 vultures (90%), 5 of 12 crows (42%) and 25 of 110 coyotes (23%). Vultures and coyotes were seropositive by the PHA test against A, B, C, D and F toxins. The highest antibody titer 1:8192 was in vulture serum against type C. In descending order, the highest antibody levels were against type C, D, F, E, A and B toxins.

Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components, designated components I and II. The toxin, a mixture of untrypsinized component I and trypsinized component II, induced marked morphological changes of tissue-cultured cynomolgus monkey kidney cells; the characteristic response of the cells to the toxin was rounding, which increased proportionally to log dose of the toxin. The components alone and a combination of untrypsinized components I and II showed little activity. The rounding of the cultured cells was not accompanied by inhibition of protein and nucleic acid syntheses of the cells, although the rounded cells ultimately lost viability. Immunofluorescence studies showed that component II, either trypsinized or untrypsinized, bound to the cell surface, whereas component I bound to the cells only in the presence of trypsinized component II. The present results support the previously proposed idea concerning the mode of action of C2T, that components I and II of C2T act together as a molecule with dual functions; component II as the recognizer of the receptor site on the cell surface membranes and component I as the effector in the cytoplasm by preferential inactivation of cytoskeletal actin, which results in alteration of cell morphology, and subsequently in cellular damage.

Molecular Structure and Biological Activities of Clostridium Botulinum C 2 Toxin

Clostridium botulinum is known to produce an extremely potent neurotoxin. In 1922, Bengtson isolated a nonproteolytic C. botulinum strain from Lucilia caeser larvae.1 The toxin produced by this strain was not neutralized with anti-botulinum type A or type B toxin serum. Types A and B were the only C. botulinum types known at the time. For this reason, the new strain was designated C. botulinum type C. Since then, a number of C. botulinum type C and D strains have been isolated. However, the immunological cross - reactions of toxins produced by type C and D strains have not been clearly elucided. In 1935, Mason and Robinson reported that type C strains produced three toxins, C1 and C2 and a small amount of D, and that type D strains produced a small amount of C toxin in addition to a large amount of D toxin.4 Jansen also reported that a type C2 strain produced C1, C2 and D toxins, that a type Cβ strain produced only C2 toxin, and that a type D strain produced C1 and D toxins.3 It was also reported that non- toxigenic type C and D strains, either cured of their prophages or isolated from environmental sources, produced C2 toxin.2,6 Although it had been recognized that certain strains of C. botulinum types C and D produced C2 toxin in addition to C and D toxins, little was known about the molecular structure and the biological activity of C2 toxin until the toxin was purified and characterized.

Inhibition of phagocytosis by rainbow trout (Oncorhynchus mykiss) macrophages by botulinum C2 toxin and its trypsinized component II

Botulinum C2 toxin (C2T) is composed of two nonlinked protein components, components I and II. The toxin reconstituted with component I and trypsinized component II inhibited phagocytosis of rainbow trout (Oncorhynchus mykiss) and mouse macrophages. Inhibition in both cell types was observed over a range of toxin concentrations that were not toxic to the cells. Because cytoplasmic action of rainbow trout macrophages is ADP-ribosylated by component I, the inhibition of phagocytosis in trout cells by C2T is probably due to inactivation of cytoplasmic actin. Moreover, phagocytosis by trout cells was also inhibited in a dose-dependent manner by trypsinized component II alone, and did not cause cell death. The present results show that the macrophages of aquatic vertebrates are susceptible to C2T, and that trypsinized component II elicits a novel biological activity by binding to the cell membrane of the macrophages.

Growth-Factor-Like Substance in Amniotic Fluid in the Rat: Effect on the Development of Fetal Colonic Goblet Cells

Extra- and intrauterine fetuses were studied to explore the effect of amniotic fluid on the development of colonic goblet cells. Significant differences in the density of goblet cells in developing colon were observed during fetal days 19-22 between these two groups, suggesting that amniotic fluid affects the development of the colonic goblet cells, especially during the period between days 19 and 21 when rat fetuses began to swallow amniotic fluid, as demonstrated by injecting India ink into the amniotic cavity. By immunological dot blot analysis, the amniotic fluid and gastric juice from intrauterine fetuses were positive for epidermal growth factor (EGF), whereas maternal abdominal serous fluid and gastric juice from extrauterine fetus were negative for EGF. The present results indicate that the amniotic fluid during late gestation greatly affects the development of the colonic goblet cells in rat fetuses and that the trophic factors for the cells seems to be EGF or an EGF-like substance in the amniotic fluid.