Fish husbandry Juvenile mono-sex Nile tilapia (15 ± 3 g) was obtained from a commercial fish farm in Jessore. The fish were habituated for two weeks in aerated seawater at 20°C to 25 °C in 500 l tanks prior to experiments. During the experiment, fishes were maintained with standard culture conditions and fed twice a day with commercial pelletized diet. Also, fishes were confirmed negative for S. iniae infection by culturing fish tissue in the brain heart infusion (BHI) agar (BD, USA).
Bacterial culture and inactivation of bacteria The S. iniae F-1 strain was cultured on tryptic soy agar (TS) with 1.5% NaCl (TSN) at 25 °C for 48 h. Colonies were subcultured onto brain heart infusion agar with 1.5% NaCl (BHIN) at 25 °C for 24 h. The cell concentration was then adjusted to an optical density of 3 (OD610 = 3; 1010 CFU/mL). The inactivated whole-cell vaccine was prepared as in (Tsai et al., 2013) [22] with slight modifications. The bacterial pellet was collected by centrifugation (5000 × g for 10 min at 4°C), washed twice in sterile PBS (pH 7.4) and resuspended in 30 ml of PBS. Following inactivation, the bacterial pellet was resuspended at a final concentration of 1010 CFU/mL in PBS containing 0.1 to 1.0 % of all chemicals. Formalin (0.2, 0.4 and 0.6%), ethanol (0.2, and 0.6%), chloroform (0.2, 0.4 and 0.6%), Na2SO4 (0.2 and 0.6%), CuSO4 (0.2, and 0.6%), KCl (0.2, 0.4 and 0.6%), phenol (0.2, and 0.6%) and EDTA (0.2, and 0.6%) were used as inactivating chemicals. To investigate the promoting effect of low amperage electric current (ECKC), inactivation was performed with 1, 3, 5, 7 and 9 mA for 5 min; heat 70ºC for 10 min and 100ºC for 30 min; Chemical with heat (0.4% phenol + 100 ºC for 30 min, 0.3% formalin + 100ºC for 10 min,0.6% formalin 100ºC for 50 min) after those treatment, the culture was incubated at 37ºC for 2 days to examine inactivation.
Virulence test (LD50) S. iniae was used in the experimental infection of tilapia to determine the LD50 dosage. Eight groups of 20 fish were tested. Through the serial dilution method, different concentrations of cells (10−1 to 10−7) or sterile PBS were intraperitoneally injected into tilapia. Ten (10) µl bacterial solutions from different concentrations were cultured onto TSN agar at 25 °C for 24–48 hours, notably, each cell concentration was run in duplicate. Mortalities were recorded daily for 14 days after challenge. S. iniae was re-isolated from the liver, spleen, kidney, and brain of moribund and dead tilapia after challenge.
Vaccination-Preparation of fish anti- S. iniae sera Fishes, maintained at 20°C to 25 °C were divided into two groups (n = 100) each in duplicate. One group was vaccinated intra-peritoneally (IP) with 100µl of 1 × 107 CFU ml−1 of S. iniae, while the other group (control) received the same quantity of PBS alone. The booster dose was injected after two week's interval. The mortality were recorded for a period of 6 weeks; the dead fish were examined to confirm the infection by the re-isolation of the pathogen from kidney, spleen, liver, brain, gill, skin on BHI agar. The blood sample was collected from live fishes once in two weeks at regular intervals, from both the vaccinated and control fishes. The blood was allowed to clot for 1 h at 25 °C and then centrifuged at 1000 × g for 10 min. The collected serum was stored at -70 °C until assayed for antibody titer and western blotting.
Preparation of rabbit anti- S. iniae sera and administration A rabbit was immunized with an intravenously administered ECKC-killed PBS-diluted suspension with S. iniae cells (1 × 107 CFU ml−1) twice a week with consecutive doses of 0.2, 0.4, 0.8, and 1.0 ml) [10]. One week after the last injection, the rabbit was bled from the ear vein. Two weeks later, the immunization procedure was repeated, now with 1.0 ml doses throughout. The antisera was collected and stored at -20 °C.
Agglutination assay Sixteen (16) days after challenge, surviving fish were randomly removed from each of the replicate tanks and blood samples collected following anesthetizing with MS-222. Approximately 100 ml serum/sample was collected following centrifugation at 1000g for 5 min then stored frozen at -80°C for subsequent determination of agglutinating antibody titers to S. iniae by modifying the method of Chen and Light (1994). S. iniae inactivated with formalin 0.4%, ethanol 0.6%, Na2SO4 0.6%, KCl 0.6%, ECKC 7mA for 5 min and heat 70°C for 10 min were grown in brain–heart infusion broth (BHI) for 24 h. The cells were centrifuged at 3000g. The resulting cell pellet was washed twice in 0.85% saline solution and suspended in saline solution to an optical density of 0.8 at 540 nm. Starting with a dilution of 1:10 (10-µl serum and 90 µl PBS), two-fold serial serum dilutions were made in 96-well round bottom microtiter plates by adding 50 Al of diluted serum into the remaining wells plated with 50 µl of PBS. Thereafter, 50 µl of bacterial cell suspension was added to each well; thus, the initial serum dilution was 1:20. The plates were covered with plastic film and incubated at room temperature for 16–18 h. The agglutination end point was established as the last serum dilution where cell agglutination was visible after incubation. Agglutination titers were reported as log10 of the reciprocal of the highest serum dilution showing visible agglutination as compared to the positive control. Prior to the bacterial challenge, four fish from each of the replicate tanks were bled to determine if they were negative for antibody to to S. iniae.