Botulism in horses in the USA is attributed to Clostridium botulinum types A, B 
or C. In this study, a duplex quantitative real-time PCR (qPCR) for detection of 
the neurotoxin genes of C. botulinum types A and B, and a singleplex qPCR for 
detection of the neurotoxin gene of C. botulinum type C, were optimized and 
validated for equine gastrointestinal, faecal and feed samples. The performance 
of these assays was evaluated and compared to the standard mouse bioassay (MBA) 
using 148 well-characterized samples, most of which were acquired from a 
repository of veterinary diagnostic samples from cases of botulism: 106 samples 
positive for C. botulinum (25 type A, 27 type B, 28 type C, 1 type D and 25 type 
E) and 42 negative samples. The sensitivities of the qPCR assays were 89%, 86% 
and 96% for C. botulinum types A, B and C, respectively. The overall sensitivity 
of the mouse bioassay for types A, B and C was 81%. The specificities of the 
qPCR assays were 99-100% and the specificity of the mouse bioassay was 95%.