Fifty-four commercial 1-day-old male white Plymouth Rock broiler chickens (Bonnie's Chick Hatchery, Elmira, ON, Canada) were randomly divided into 4 experimental groups (n = 13-14 per group). The chickens were fed an antibiotic-free chicken starter containing 20% protein for 11 days followed by turkey starter containing 28% protein (Arkell Research Station, University of Guelph). For the experimental infection (challenge) of birds, wild type CP1, CP1DeltapCP1netB and CP1DeltapCP1netB(netB +) were grown in cooked meat medium (Difco) (CMM) for 24 h at 37  C under anaerobic conditions. FTG medium (Difco) was then inoculated 3% (v/v) with an overnight CMM culture and incubated aerobically at 37  C for 15 h. The growth at 15 h was approximately log10 8.45 +- 0.14 C. perfringens colony-forming units (CFUs) per ml. The FTG-grown culture was then mixed with feed at a ratio of 2:1 (v/w). The chickens were starved for 24 h before the C. perfringens contaminated feed was administered on day 13. Inoculated feed was prepared fresh twice daily and fed to chickens for 5 days.
Chickens were euthanized with carbon dioxide on the day following a 5-day challenge, and at necropsy the small intestine was examined for grossly visible lesions. Necrotic enteritis lesions were scored blindly using the system described by Keyburn et al..
To determine if there were differences in the number of birds with lesions between two challenge groups, a two-tailed Fisher's exact test was used to assess the null hypothesis that the proportion of birds with lesions among the two groups was the same. The null hypothesis was rejected at p <= 0.05.