Abstract:
Enterotoxaemia is an economically important disease of sheep, goats and calves. The disease
affects mainly young animals aged between four to six months. Enterotoxaemia is caused by
systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium
perfringens Type D. Due to almost certain death of affected animals, there is no form of
treatment. The only practical means of controlling the occurrence of enterotoxaemia is to
immunize animals through vaccination. The vaccine is prepared by toxoiding the bacterial
culture filtrate, and contains a range of proteins in addition to the epsilon toxin. Batches of the
vaccine are thus required to be tested for safety, efficacy and potency. The potency of the
vaccines is currently tested with the in vivo mouse neutralisation test (MNT). However, 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 ELISA (I-ELISA)
and the MNT.
To investigate, three groups of eight guinea pigs were immunized with one of three different
production batches of the enterotoxaemia vaccine. Guinea pig sera were collected prior to
vaccination and at five weeks post-vaccination, and the sera of four guinea pigs per group were
pooled to give six test sera. The levels of anti-epsilon toxin antibodies in the respective test
sera were subsequently determined using MNT, I-ELISA and I-CBA. The I-CBA assay
developed during the course of this study is based on coating of functional beads with purified
epsilon toxin that serves to capture anti-epsilon toxin antibodies from the test sera. Following
incubation with fluorescein isothiocyanate (FITC)-labelled anti-guinea pig IgG, the samples were
analyzed by flow cytometry and the anti-epsilon toxin antibody concentrations were extrapolated
from a standard curve using linear curve fitting. Although the intra- and inter-assay variability
was satisfactory, epsilon antitoxin levels of the guinea pig test sera determined by both the ICBA
and I-ELISA tests were higher than that of the MNT assay which is accepted as the current
“gold standard”. Moreover, in contrast to the MNT, all of the serum samples were identified as
having antitoxin levels above the required minimum (i.e. 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, cost-effective and efficient
method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus
for further optimization and standardization of the I-CBA assay described in this study.
