Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test

Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium. Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C. perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia development is peracute in most cases. Animals die suddenly while grazing on the pasture, without any prior signs of disease. Therefore, treatment is possible only in very rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time. The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for veterinary use. After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified. For this purpose, 10 rabbits are immunised twice with the product to be tested. Their blood is taken 14 days after the last immunisation and the serum is pooled. The pooled serum is then tested for its protective effect. This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum. The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin. The exact efficacy of the test serum is given in International Units (IU). The tested serum must show a minimum content of 5 IU. This in vivo method requires a very high number of experimental animals. Approximately 400 mice (or 50 guinea pigs) are used per vaccine batch. The monograph for C. perfringens vaccines, which has recently been revised, expressly indicates that a validated serological method may be used for batch testing. In addition, a reference serum known as clostridium multicomponent serum has been available since 2000. The objective is to test vaccine batches against this reference and by means of a competitive ELISA developed in the precursor project, using a monoclonal antibody for direct determination of specific antitoxins in rabbit sera. This ELISA method was subjected to an international validation to verify whether the protocol and the precision can be transferred within and between the participating laboratories.