Prospects for the refinement and reduction of animal usage in the potency testing of Cl. chauvoei vaccines.

Protection has been elicited by both cellular and supernatant antigens from Cl. chauvoei. The cellular antigen has been solubilised by various treatments described for removing flagella, and has been detected by an ELISA method. The level of protection elicited by these preparations was poor, and immunoassays using these antigen preparations did not correlate with in vivo results. The supernatant antigen has been fractionated by ammonium sulphate precipitation and gel filtration. Using the latter technique, the protective antigen has been partially purified. Immunoassays using this soluble antigen did not correlate with in vivo protection levels. It is concluded that in vitro assays based on the partially characterized antigens are not yet suitable for the measurement of protective antibody.

In vitro tests for the measurement of veterinary clostridial toxins, toxoids and antisera. I. Titration of Clostridium septicum toxins and antitoxins in cell culture.

The assay of Clostridium septicum antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in vivo counterparts, permitting detection of substantially lower titres than is possible using in vivo indicators. It is suggested that cell culture indicators may prove useful for the titration of Cl septicum antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations.

In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture.

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.