C. perfringens 3
Chickens (12 birds per group) were randomly assigned to uninfected sham control (NV) and Clostridium perfringens-infected groups. Briefly., the 1- to 14-day-old birds post-hatch were provided with an antibiotic-free starter diet with 20% protein. From 15-day, the feed was changed to a high-protein diet (1:1 mixture with 100% fish meal and diet of 20% protein). At day 20, the chickens were orally infected with 2.0 ml of C. perfringens netB+tpeL+ starin culture (2.0~4  108 colony-forming units per chicken), twice daily for five consecutive days. Uninfected sham control birds were mock-inoculated with an equal volume of BYC culture medium by oral gavage. At day 5 post-first infection, all the birds were weighed and sacrificed, and an around 2.5 cm-long section of mid jejunum (with intestinal contents) was collected and placed into liquid RNALater according to the manufacturers instructions (Sigma-Aldrich, St Louis, MO, USA). The RNA was extracted with a RNeasy PowerMicrobiome Kit (Qiagen Inc, Gaithersburg, MD, USA). Fresh RNAs were extracted from three randomly selected and pooled samples each group and sent for RNA sequencing.