2.7. Challenge experiment
Apparently healthy Nile tilapia juveniles, O. niloticus(~28 g), were obtained from a commercial tilapia hatchery in northern Vietnam for challenge experiments. The fish were acclimatized to the experimental conditions for one week before conducting experiments. Ten fish were randomly checked for E. ictaluri free status by Gram staining, inspection of the spleen and head kidney for the presence of bacteria, and by streaking these tissue samples on BHIA plates before beginning the experiments. Four representative bacterial isolates (Ed.HB-02, Ed.HD-09, Ed.YB-08, and Ed.TB-07) were selected for experimental infection. Each isolate was grown in BHIB medium at 28 °C for 36 h. The viable bacterial density of the stock suspensions was determined using the plate count method. The bacterial density of the stock suspension was then adjusted to approximately 1 × 108 colony forming units (CFUs)/mL by adding an equivalent volume of phosphate-buffered saline. Ten-fold serial dilutions of bacteria from 102–108 CFU/mL were prepared for the virulence test. With each bacterial isolate, fish were divided into eight 100 L groups (15 fish/tank, two replicates). Fish from seven groups were intraperitoneally injected with 0.1 mL of the serial bacterial suspensions to reach the respective bacterial concentrations of 101–107 CFU/fish. In the control group, fish were injected with normal saline solution without bacteria. Mortality was observed daily for 2 weeks. Representative moribund and freshly dead fish (n = 3) from each challenge group and apparently healthy fish from the control groups (at the end of the experiment, n = 3) were subjected to bacterial re-isolation and histopathological analysis.