2.2. Challenge Test and Sampling

A detailed description of the challenge test has been previously reported [12]. In brief, after the growth trial, 60 fish were chosen from each group and inoculated intraperitoneally with a 1.0 mL nonlethal dosage of A. hydrophila (2.5 × 10⁶ CFU/mL). During the 14-day infection period, the same diets as in the feeding trial were fed to the grass carp. At the end of the challenge test, all grass carp in each group were anaesthetized using a benzocaine bath (50 mg/L) and sacrificed by a blow on the head, and the intestines were removed, divided into three parts (proximal intestine (PI), mid intestine (MI), and distal intestine (DI)), then immediately frozen in liquid nitrogen, followed by storage at −80 °C. The degree of intestinal red coloration, swelling, and hyperemia of the grass carp was recorded in our previous study [12].