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 to 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.     

Effects of Clostridium botulinum toxins on infant and adult mice.

Toxic supernatants of many Clostridium botulinum type B isolates from various sources, especially those of isolates obtained from cases of infant botulism, appeared to more toxic in infant mice than in adult mice. The significance of this finding in diagnostic situations is discussed.     

Some physical and chemical properties of trypsin-digested nucleoprotein

A DNA-peptide complex that is soluble in 0.2m-sodium chloride can be prepared by trypsin digestion of calf thymus nucleoprotein. The trypsin-digested nucleoprotein molecule contains about 70% of DNA and 30% of peptides by weight, and consists of one DNA molecule associated with arginine-rich peptides. A series of trypsin-digested nucleoprotein preparations differing only in molecular weight were prepared by blending. The intrinsic viscosity and average sedimentation coefficient were determined for each of these preparations. Then the DNA was isolated from each preparation and the hydrodynamic measurements were repeated on the DNA. From a comparison of these results it was concluded that the presence of the complex-forming peptides causes a large decrease in intrinsic viscosity of the DNA and an increase in sedimentation coefficient. In addition, the hydrodynamic data indicate that the DNA-peptide complex behaves like a coil in solution but is more compact than the same length of DNA. The ;melting' profiles, streptomycin precipitation curves and maximum viscosities obtained with ethidium bromide binding for the trypsin-digested nucleoprotein are similar to those of purified DNA, and markedly different from those of undigested nucleoprotein. These findings suggest that the peptides are not strongly associated with the DNA, and that secondary valency forces are involved in the binding.     

Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.     

Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27–34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16–26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.     

Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.