Fish and experimental conditions
The study was carried out at Fish Hatchery Unit in Laboratory of Marine Biotechnology (MARSLAB), Institute of Bio-Science (IBS), Universiti Putra Malaysia, Malaysia. A total of 605 apparently healthy hybrid red tilapia (Oreochromis mossambicus × O. niloticus) with an average weight of 61.23 ± 4.95 g, were purchased from a local fish farm (Kam Sing Fish farm, Selangor, Malaysia). The collected red tilapia were randomly distributed into 18 tanks with 400 litres capacity. The fish were acclimatized for 14 days before vaccination and fed with an available commercial diet (Star Feed, Star Feed Mills SDN. BHD, Malaysia) with 32% protein containing feed at 3% body weight per day. The fish faces and waste materials were siphoned out 3 h after feeding. Prior to commence the experiment, thirty fish were randomly dissecting for screening pathogenic bacteria and checking the antibody level to confirm that they were free from Streptococcus sp. and Aeromonas sp. One day prior to vaccination or challenge fish were taken off feed. The water quality of the rearing tanks like temperature, pH, dissolved oxygen, ammonia and nitrites were observed throughout the study period. Anesthetics were applied on the experimental fish using 120 mg/L of tricaine methanesulfonate, MS-222 (Aldrich, USA) prior to collect blood samples and bacterial challenge protocols.
Formalin-killed bacteria preparation
The pathogenic strains of S. iniae and A. hydrophila were isolated from diseased hybrid red tilapia and obtained from the previous study [57, 58]. The formalin killed bacterin were prepared as stated in the previous studies [25, 28]. Briefly, the bacterial strains of S. iniae and A. hydrophila were cultured individually on 5% blood agar (Oxoid, UK) and further grown in separate flasks of 500 ml containing Brain Heart Infusion Broth (BHIB, Oxoid, UK) at 30oC in a shaker incubator at 150 rpm for overnight. The following incubation, the bacterial concentrations were calculated by applying the establish plate count. The individually cultured bacteria cells were then inactivated by treating with neutral-buffered formalin to the concentration of 0.5% formalin in PBS (phosphate buffered saline) and kept at 4°C for 24 h. After that, the bacterial cells were washed four times with the sterile PBS by centrifugation at 6000 x g for 15 min to remove the medium and formalin residue from the culture. Afterwards, the inactivated bacteria were again suspended in sterile PBS to keep the final concentration of 6.7 × 109 CFU/ml. The bacterial suspension was again streaked onto BHIA and incubated at 37°C for overnight to confirm that all S. iniae or A. hydrophila cells were inactivated. For formulations of bivalent vaccine, formalin killed whole cells (FKCs) of two vaccine strains were combined at a ratio of 1:1 (v/v) and kept at 4°C. Subsequently, for improving the vaccine antigenicity, palm oil (Vesawit, Malaysia) as an adjuvant was mixed to a final concentration of 10% before it was thoroughly sprayed on commercial pellet or formulate feed to obtain a final concentration of 6.7 x 109 cells/g of feed [25, 28].
Feed-based vaccine preparation
Bivalent spray vaccine
The feed-based vaccine was formulated according to the method described earlier Ismail  with some modifications. Briefly, the formalin-killed bactrin (FKB) of S. iniae (6.7 x 109 CFU/ml) and A. hydrophila (6.7 x 109 CFU/ml) with 10% palm oil were mixed and re-suspended properly in PBS for preparing bivalent vaccine. Next, the bivalent FKB solution was directly sprayed onto the commercial floating pellet feed (Star feed, containing 32% protein) to obtain the individual FKB a final concentration of 6.7 x 109 cells/g of feed. A homogenizer or mixer (Golden Bull B10-A Universal Mixer, Malaysia) was used to distribute and impregnate the bivalent FKB vaccine properly into the pellet feed. Finally, the vaccine added pellet was dried up at 30°C for overnight in the oven prior to the experiment.
Preparation of formulated vaccine
A commercially available pellet feed (Star feed, containing 32% protein) was blended with a blender machine to form a very fine mesh powder. To incorporate the vaccine, the formalin-killed bacterin (FKB) of monovalent or bivalent vaccine with 10% palm oil was re-suspended in PBS to a final concentration of 6.7 x 109 CFU/ml. Afterwards, the FKB solution of monovalent or bivalent vaccine was sprayed properly onto the fish feed powder to obtain the individual FKB a final concentration of 6.7 x 109 cells/g of feed. A homogenizer was used to distribute and impregnate the monovalent or bivalent FKB vaccine properly onto the fish feed powder. In unvaccinated group, only 10% palm oil was added in fish feed as a control group. Finally, the vaccine added feed paste was loaded into the auto mini pellet machine (Golden Avill, China) to make the pellet size of 4 x 4 mm and kept at 30°C for overnight in the oven prior to the feed-based immunization.
A total of 495 hybrid red tilapia (Oreochromis mossambicus × O. niloticus) were randomly distributed into 15 glass aquaria with 400 litres capacity. The experiment was conducted with five different experimental major groups, and each group consisted of 99 fish for the 3 replicates; each replicate containing 33 fish in 400 L glass aquaria. Group-1 (unvaccinated) was fed non-vaccine containing commercial pellet feed (incorporated only 10% palm oil); group-2 (Bivalent Spray, BS) was vaccinated by bivalent mixture of S. iniae and A. hydrophila vaccine directly sprayed on commercial pellet feed; group-3 (Bivalent Formulate, BF) was vaccinated by bivalent vaccine incorporated in feed; group-4 (Monovalent S. iniae, MS) was vaccinated by only monovalent S. iniae vaccine incorporated in feed and group-5 (Monovalent A. hydrophila, MA) was vaccinated by only monovalent A. hydrophila vaccine incorporated in feed. At the start of the vaccination, the feed-based vaccine was orally applied in all vaccination groups only on day 0 at 5% body weight four times daily up to 5 consecutive days. Except for unvaccinated control group, all other groups were double boosted with the same immunization on 14 and 42 days after first dose vaccination (Fig. 3). The water was dechlorinated and aerated
continuously throughout the trials. The water quality of the experimental glass aquaria were maintained at temperature of (27.73±2.45°C), dissolved oxygen (6.97±2.43 mg/l), pH (7.65±1.45) and ammonia (0.01±0.00 mg/l) ranged on acceptable levels overall the experimental periods.
The challenge tests were performed on 70 days post-vaccination with single bacterial infections of S. iniae (3.4×108 CFU/ml), A. hydrophila (6.8 ×109 CFU/ml) and the co-infection of both pathogenic bacteria (S. iniae, 3.4×108 CFU/ml and A. hydrophila, 6.8 ×109 CFU/ml) with a composition ratio of 1:1. The fish were challenged via intraperitoneal (i.p.) route at a dose of 0.5 ml. Besides the four vaccinated groups, four sub-control groups were set up for challenge test, including control (negative)-1 (unvaccinated, without challenge), control-2 (unvaccinated, but challenged with S. iniae) and control-3 (unvaccinated, but challenged with A. hydrophila) and control-4 (unvaccinated, but challenged with co-infection, S. iniae and A. hydrophila). For each bivalent vaccinated group, fish were separated into three sub-groups: The sub-groups were challenged separately with S. iniae, A. hydrophila and another with co-infection. In every vaccinated group or sub-group was two replicates and each replicate had consisted of 15 fish. All of the unvaccinated or non-challenged each fish was also injected with 0.5 ml PBS. Fish mortalities in each group was subsequently recorded daily for 14 days after the challenges. Following 14 days post-challenges, vaccine efficacies were estimated by comparing the average cumulative mortalities (%) and the relative percentage of survival (RPS: [1- (% mortality in vaccinated fish/ % mortality in unvaccinated fish) x 100]). All remaining fishes at the end of the challenge trials and experiment were euthanized in overdose concentration of 400 mg/L of tricaine methanesulfonate, MS-222 (Aldrich, USA) for at least 10 min and soaked in 25% sodium hypochlorite for 30 minutes prior disposed as clinical waste.
Sampling of fish blood and spleen organ were done from five groups on 7, 21 and 49 days post-vaccination (dpv) and also 96 hours post-infection. The blood sample was collected through the caudal veins from 9 fish of each group and the collected blood were kept in two different type tubes, in one EDTA-containing tubes while another without EDTA-containing tubes. The EDTA-containing blood samples were immediately sent to the laboratory for haematological parameters analysis but without EDTA-containing samples were used to assess immune responses. The spleen samples were collected to determine the phagocytic activity.
The anti-coagulate blood samples were used to determine the erythrocyte, thrombocytes, leukocyte, haemoglobin, lymphocytes, haematocrit, MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), monocytes and granulocytes count using an automatic Mythic 18 Vet Haematology analyser machine (Woodley Veterinary Diagnostics, England and Wales).
Serum lysozyme activity
The lysozyme assay was done according to the methods of Anderson and Swicki . The pH of the PBS was adjusted to 6.2 at 25oC using 1M HCl and 1M KOH. This (0.01 M PBS) was used to prepare 0.4 mg/ml of Micrococcus lysodeikticus. Firstly, 100 μl blood serum and then prepared 100 μl M. lysodeikticus was put into the microplate. Afterwards, the resulting absorbance was read at 450 nm (optical density, OD) using microplate reader (Multiskan™ GO Microplate Spectrophotometer, USA) at the time interval of 30 sec and after 30 min. The lysozyme activity was calculated based on a decrease in OD of 0.001/minute. The following formula was used to estimate the units of enzyme activity per one ml of the serum. (see Formula 1 in the Supplementary Files)
Phagocytosis by spleen performed using the method of Anderson and Swicki . Briefly, in this method; yeast cells were the particulate cells, where equal volumes of spleen cell suspension and yeast cells (0.1 ml) were mixed well with a pipette and incubated for 20 min at 25oC. Five microliters of the incubated solution was placed on a glass slide (pre-coated with 10 % Poly L-Lysine (PLL) solution and dried) and made a smear, air dried and fixed with 95 % methanol for 1 min, transferred into May-Grunwald (MG) solution for 5 min. Finally, the cells were stained with 7% Giemsa stain for 20 min, air-dried. The cells were set under oil immersion (100 x magnification) and one hundred cells were counted from different portions of the slide and finally, the percentage of phagocytic cells were determined.
Enzyme-linked immunosorbent assay (ELISA)
The samples of serum were subjected to indirect ELISA to determine the antibody titre against S. iniae and A. hydrophila using the method described by Ismail  with minor modification. Coating antigen was prepared by culturing S. iniae and A. hydrophila into BHIB and incubated for overnight in shaker incubator for 150 rpm at 30°C. The concentration of the cultured bacteria was calculated with the following of the standard plate count method prior to harvest through centrifugation and washed with PBS. After that, the bacterial pellets were suspended in carbonate-bicarbonate buffer (pH 9.6). The prepared bacteria was inactivated through boiling in a water bath at 90°C for 20 min and incubated at room temperature before to use as coating antigen (2.5 x 105 CFU/ml). Then, 100 μl coating antigen was coated into the microtitre plates and kept at 4°C for 24 h before washed two times with PBST (PBS + 0.05 % Tween 20). This was followed by adding 200 μl of 1% BSA to block unspecific binding sites and kept at 37°C for one hour. Thereafter, 100 μl of diluted serum (1:1000) was added into the reaction and incubated. Afterwards, goat anti-tilapia hyperimmune serum (Aquatic Diagnostics Ltd, Scotland) was diluted at the ratio of 1:10000, added 100 μl into the reaction and incubated at 37°C for one hour again. Then, 100 μl of conjugated rabbit anti-goat IgM horseradish peroxidase (Nordic, Netherland), diluted 1:10000 was added and incubated. The following microtitre plates was added 100 μl of TMB (Promega, USA) after washed for thrice with PBST and finally added100 μl of TMB (Promega, USA) before 0.2 mol/l sulphuric acid. The absorbance was calculated by setting microplate reader (Multiskan™ GO Microplate Spectrophotometer, Finland) at 450 nm wavelength.
Data were analysed using SPSS-16 software (SPSS Inc., Chicago IL). Differences in haemato-immunological parameters and RPS between unvaccinated and vaccinated groups were examined using one-way ANOVA with Duncan post hoc tests. Statistical significance was considered at p values < 0.05.