Monoclonal antibodies (MAbs) obtained from a mouse immunised with Clostridium botulinum type D toxoid were developed into a sandwich ELISA (sELISA) format that was able to detect type D toxin and types C and D toxin complexes. The sELISA was examined for its potential to replace the mouse bioassay as an alternative in vitro assay for the diagnosis of cattle botulism. Its application directly to intestinal samples collected from suspect cattle botulism cases and prepared for testing for the standard mouse bioassay showed poor correlation and sensitivity with the mouse bioassay results. However, anaerobic pre-enrichment of the samples after heat treatment at 80 degrees C for 10 min to activate any residual C. botulinum spores greatly improved the sELISA detection rate of the toxin by increasing the sample toxin levels. All of the mouse bioassay positive cattle cases tested were detected by the sELISA from the heated and pre-enriched samples tested after 24h incubation. Toxin was detected by sELISA and subsequently confirmed by mouse bioassay in samples from an additional 3 cases that had been originally mouse bioassay negative. The results indicate that the application of this procedure for screening intestinal samples for C. botulinum strains that produce types C and D toxins from suspect cattle botulism cases would improve the diagnostic rate as well as significantly reduce the number of mice involved in diagnosis.