Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused
by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium
perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia
is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from
the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse
neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in
vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA)
was developed for use in vaccine potency testing and the results were compared with those
obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT.
Sera were collected from guinea pigs immunised with three different production batches
of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined.
Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels
determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests
were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples
were identified as having antitoxin levels above the required minimum (not less than 5 U/mL).
These results indicate that the respective in vitro tests in their current formats are not yet
suitable alternatives to the in vivo MNT. The growing demand for a more humane, costeffective
and efficient method for testing the potency of enterotoxaemia vaccines, however,
provides a strong impetus for further optimisation and standardisation of the I-CBA assay but
further analytical research is required.