Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink.

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay.

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Immunogenicity of Clostridium septicum in guinea pigs.

Five strains of Clostridium septicum were used to prepare bacterins, bacterin-toxoids, toxoid, and combinations of bacterins or bacterin-toxoids. These preparations were tested for immunogenicity in guinea pigs vaccinated subcutaneously with 1.0 ml of product. Usually, a second vaccination was given 21 to 24 days later. The immunity of groups of vaccinated guinea pigs was challenged with as many as 22 strains of C septicum. When challenge exposed with homologous strains at 21 to 24 days after one vaccination or 10 t0 18 days after a second vaccination, 60% to 100% of the guinea pigs in each group survived. Demonstrable cross-protection among strains of C septicum varied from none to 100% protection in vaccinated guinea pigs. A combination of bacterin-toxoid prepared from four selected strains protected 70% to 100% of the vaccinated guinea pigs challenge exposed with 21 strains. Duration-of-immunity studies demonstrated a twofold to fourfold decrease in protection when the vaccination-to-challenge interval was extended an additional 3 weeks. Strains of C septicum do not have an effective common immunogen and the stimulated immunity appears to be of short duration. Antitoxin was demonstrated to be less important than other factors in protecting against C septicum infection.

Potency testing of Clostridium septicum bacterins in sheep and laboratory animals.

The immunogenic potency of bacterins containing Clostridium septicum was evaluated in mice, hamsters, guinea pigs, rabbits and sheep to establish potency requirements for these bacterins. Two subcutaneous injections of Cl. septicum bacterins were shown to provide resistance against intramuscular spore challenge. Protection in mice, hamsters and guinea pigs was generally at low levels, in the range of 1 to 5 LD50. Efficacy estimates, at these levels of challenge, indicated that a degree of relationship existed between the response of rabbits and the response of sheep. This relationship was confirmed at higher levels of spore challenge, at greater than 5 to 500 LD50. Undiluted bacterins were classed as good or poor based on percent survival responses in sheep. Bacterins providing 90 to 100% survival were classed as good; those providing 30 to 57% survival were classed as poor. In rabbits, this classification was less evident with the undiluted bacterins. Dilution of bacterins to 1:32 provided a discriminatory level with 7 of 8 bacterins examined and represented correlation of the sheep and rabbits responses. A similar relationship was demonstrated between protection in sheep against spore challenge and the antitoxin response in rabbits to undiluted bacterins. Antitoxin responses, when obtained, were generally higher in rabbits than those obtained in sheep. Further evaluation of a good and a poor bacterin in rabbits indicated that no direct relationship existed between antitoxin titer in rabbits and resistance to spore challenge, when compared at the same dilutions of bacterins. Evaluation of serum agglutination responses to types I and II somatic antigens indicated a poor relationship between agglutination response and resistance to spore challenge in both sheep and rabbits. Cl. septicum toxoids were shown to provide resistance to spore challenge in rabbits.