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.