Evaluation of the efficacy of a bivalent inactivated whole-cell vaccine against Edwardsiella tarda and Vibrio anguillarum in Tilapia (Oreochromis niloticus)

MARINE REPORTS 4(2) (2025) : 116-129 DOI: 10.5281/zenodo.18001946 [email protected] MARINE REPORTS e-ISSN: 2822-5155 Journal homepage: https://scopesscience.com/index.php/marep/ Received: 21 November 2025; Received in revised form: 20 December 2025 Accepted: 20 December 2025; Available online: 21 December 2025 RESEARCH PAPER Citation: Liu, Z., Endo, M., & Futami, K. (2025). Evaluation of the efficacy of a bivalent inactivated whole-cell vaccine against Edwardsiella tarda and Vibrio anguillarum in Tilapia (Oreochromis niloticus). Marine Reports, 4(2), 116-129. https://doi.org/10.5281/zenodo.18001946 EVALUATION OF THE EFFICACY OF A BIVALENT INACTIVATED WHOLE-CELL VACCINE AGAINST Edwardsiella tarda AND Vibrio anguillarum IN TILAPIA (Oreochromis niloticus) Zheyu LIU1, Masato ENDO2, Kunihiko FUTAMI3* 1 Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 2 Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 3* Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Zheyu LIU: [email protected], https://orcid.org/0000-0002-9429-8658 Masato ENDO: [email protected], https://orcid.org/0000-0001-7325-101X Kunihiko FUTAMI: [email protected], https://orcid.org/0009-0001-9003-4161 *Corresponding author: Kunihiko FUTAMI, [email protected], +81-3-5463-4268 Abstract Bacterial co-infections constrain tilapia aquaculture. We evaluated the efficacy of an adjuvantfree, formalin-inactivated bivalent whole-cell vaccine combining Edwardsiella tarda and Vibrio anguillarum in Nile tilapia. Fish received the bivalent formulation, matched monovalent vaccines, or phosphate-buffered saline. Systemic and skin-mucus immunoglobulin M levels were quantified by indirect enzyme-linked immunosorbent assay, and serum and mucus bactericidal activity were assessed at 1, 7, 14, and 21-days post-vaccination. At 21 days postvaccination, fish were challenged intraperitoneally with E. tarda or V. anguillarum, or both. By 14–21 days post-vaccination, the bivalent vaccine induced strong systemic and mucosal immunoglobulin M responses and increased bactericidal activity; across readouts, responses were significantly greater than (p < 0.05) or equal to (p > 0.05) the corresponding monovalent vaccine. After challenge with E. tarda, 21-day post-challenge survival was higher in the bivalent (54.17%, Relative Percent Survival = 38.9%) than in the monovalent (29.17%) or control (25.00%) group (both p < 0.05). After V. anguillarum challenge, survival was similar in the bivalent (87.50%, Relative Percent Survival = 78.6%) and monovalent (75.00%) groups (p > 0.05), both of which showed greater survival than the control (41.67%) (p < 0.05). After mixed E. tarda + V. anguillarum challenge, survival was significantly higher (50.00%, Relative Percent Survival = 47.8%) in the bivalent group than in the control group (4.17%) (p < 0.05). No acute adverse reactions or behavioral abnormalities were observed. These findings indicate Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 117 that an adjuvant-free bivalent vaccine elicits robust midto late-phase humoral responses and provides broad protection without evidence of antigenic interference, supporting a practical polyvalent strategy where E. tarda and V. anguillarum co-circulate. Keywords: Bactericidal activity, Edwardsiella tarda, indirect ELISA, Tilapia, Vibrio anguillarum, whole-cell vaccine Introduction Bacterial pathogens remain a persistent constraint to tilapia aquaculture, where Edwardsiella tarda and Vibrio anguillarum are among the most consistently reported agents and may co-circulate under intensive farming stressors (e.g., deteriorating water quality or high stocking density) (Haenen et al., 2023; Frans et al., 2011). E. tarda is a Gram-negative, facultative intracellular bacterium that is capable of surviving within phagocytes and evading killing by the complement system (Li et al., 2022), whereas V. anguillarum is classically extracellular and rapidly colonizes tissues via adhesins, motility, iron acquisition and secreted toxins (Frans et al., 2011). Outbreaks of either pathogen, and especially their concurrence, may escalate mortality and undermine productivity in warm-water aquaculture systems (Haenen et al., 2023). Although chemotherapeutics have historically been used to treat outbreaks, concerns about antimicrobial resistance and residues have catalyzed a shift toward vaccination as a preventive, pathogen-specific measure (Schar et al., 2020). In practice, fish in intensive systems face multi-pathogen pressure; as a result, polyvalent vaccines are attractive for their broader coverage and reduction in handling events. However, the combination of multiple antigens can also introduce immunological interference (antigenic competition), whereby the immune response to one component is reduced relative to when the antigen is delivered alone, a phenomenon that has been documented in experimental polyvalent fish vaccines (Nikoskelainen et al., 2007). In tilapia and related species, bivalent formulations delivered either orally via feed or by injection have been shown to achieve elevated IgM responses and relatively high percentage survival in experimental and field settings, supporting their operational feasibility (Mohd Ali et al., 2023; Rivas et al., 2023; Shoemaker et al., 2012; Sun et al., 2011). Conceptually, combining antigens from the intracellular pathogen E. tarda and the extracellular pathogen V. anguillarum may broaden effector engagement across systemic and mucosal compartments and mitigate the risk of antigenic gaps. From a vaccine-development perspective, this potential benefit must be balanced against the risk of antigenic competition in multivalent vaccines, highlighting the importance of evaluating combined formulations alongside matched monovalent vaccines (Kuang et al., 2022) However, the use of adjuvant-free, inactivated whole-cell bivalent approaches targeting these two bacteria in tilapia, and their performance across serological, mucosal, and survival endpoints including mixed-pathogen challenge remain to be explored. Here, we evaluated a formalin-inactivated, adjuvant-free bivalent vaccine comprising E. tarda and V. anguillarum antigens. We hypothesized that the bivalent formulation would elicit stronger functional immunity relative to monovalent vaccines without evidence of antigenic competition, thereby offering a practical option for controlling co-circulating E. tarda and V. anguillarum in tilapia aquaculture. We quantified systemic and mucosal IgM responses alongside serum and mucus bactericidal activity at multiple timepoints, and assessed the protective efficacy of the vaccine against homologous and mixed challenges. Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 118 Materials and methods Fish and maintenance Tilapia (Oreochromis niloticus) weighing approximately 30–50 g were obtained from the Laboratory of Aquaculture Science, Tokyo University of Marine Science and Technology (TUMSAT). Before the experiments, the fish were acclimated for a minimum of 2 weeks in a 60-L freshwater tank maintained at 25°C (±1°C) under a 12-h/12-h light/dark cycle. The fish were fed daily with commercial pellets at 1% of their body weight. One-third of the tank volume was replaced daily to maintain water quality. Dissolved oxygen, pH, and other water parameters were monitored regularly to ensure optimal conditions. All husbandry and experimental procedures involving live fish were conducted in accordance with the Animal Experiment Handling Regulations of TUMSAT. The Edwardsiella strain was maintained and used under its original designation, as reported in previous studies. Bacterial strains and maintenance E. tarda FPC498 and V. anguillarum were obtained from the Laboratory of Fish Health and Management, TUMSAT. E. tarda FPC498 was originally isolated from the ascitic fluid of a naturally infected Japanese flounder (Paralichthys olivaceus) (Yamasaki et al., 2015) and previously used by Cao et al. (2023). V. anguillarum was isolated from rainbow trout Oncorhynchus mykiss (Gallage et al., 2016). Both strains are non-reference field isolates that have been maintained and propagated in the source laboratory as previously described. The identity of each strain was confirmed by standard biochemical profiling and colony characteristics on selective agar; for V. anguillarum, species-specific PCR targeting the amiB gene was performed as previously described (Hong et al., 2007). All bacterial strains were stored at −80 °C until use. For E. tarda, frozen stocks were streaked onto tryptic soy agar (TSA); for V. anguillarum, the TSA was supplemented with 3% (w/v) NaCl. After incubation at 25–30 °C for 24–48 h, single colonies were inoculated into tryptic soy broth (TSB) for E. tarda and TSB containing 3% (w/v) NaCl for V. anguillarum. Cultures were incubated at 30 °C with shaking at 150 rpm for 24 h; bacterial cells were harvested by centrifugation, washed three times with phosphate-buffered saline (PBS; pH 7.2), and resuspended in PBS. Vaccine preparation and experimental groups Vaccines were prepared as a formalin-inactivated whole-cell suspension (FKC). In brief, logphase cultures of E. tarda and V. anguillarum were resuspended in PBS (pH 7.2) to approximately 1 × 10⁹ CFU/mL. Formalin (0.3% v/v) was added, and the suspensions were incubated at 4 °C with gentle agitation for 24 h. Complete inactivation was verified by a lack of colonies after plating 100 µL of the treated suspension on TSA (E. tarda) or NaCl–TSA (V. anguillarum), followed by incubation at 28 °C for 72 h. Cells were washed three times with PBS to remove residual formalin and resuspended to the target concentrations. Four groups were established and injected intraperitoneally (100 µL/fish) with adjuvant-free suspensions as follows: PBS control (n = 96; PBS only), mono-E. tarda (n = 48; 1×10⁸ CFU/fish FKC), mono-V. anguillarum (n = 48; 1×10⁷ CFU/fish FKC), and bivalent (n = 96; both antigens at the same per-fish doses). Sample sizes were chosen to provide adequate numbers for the immunological assays and challenge tests described below. The overall experimental design, including group allocation, dosing, sampling timepoints, and the challenge scheme, is summarized in Figure 1. Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 119 Figure 1. Experimental design. After a 14-day acclimatization, tilapia allocated to the bivalent (n = 96), Figure 3 monovalent (E. tarda, n = 48; V. anguillarum, n = 48), or PBS control (n = 96) group were intraperitoneally injected (100 µL/fish) with formalin-killed bacterial suspensions prepared without adjuvant (bivalent, E. tarda 1×10⁸ + V. anguillarum 1×10⁷ CFU/fish; monovalent matched) or PBS. Serum and skin-mucus samples were collected at 1, 7, 14, and 21-days post-vaccination (dpv) (n = 6/timepoint/group) for IgM ELISA and serum/mucus bactericidal activity. At 21 dpv, the remaining fish were challenged with E. tarda or V. anguillarum, or both (3 tanks × 8 fish; n = 24 fish/arm) and observed for 21 days post-challenge (dpc); the co-infection arms comprised only the bivalent and PBS groups. Sample collection and timepoints Serum and skin mucus samples were collected at 1, 7, 14, and 21 dpv. At each timepoint, six fish per group were randomly netted and anaesthetised by immersion in 2-phenoxyethanol (300 µL/L; Wako, Japan) until respiratory and opercular movements slowed and the righting reflex was lost. For serum collection, blood was withdrawn from the caudal vein using a plain, nonheparinized 1-mL syringe and transferred into a microtube without anticoagulant. Samples were allowed to clot for 60 min at room temperature before centrifugation at 3,000 × g for 10 min at 4 °C. The clarified serum was aliquoted into single-use vials and stored at –80 °C to avoid repeated freeze–thaw cycles. The sampling timeline is depicted in Figure 1. While still under anaesthesia, each fish was placed in a sterile polyethylene bag containing 0.5 mL of PBS (pH 7.2) supplemented with EDTA-free protease inhibitor cocktail (1 tablet/10 mL; Roche, Germany). The body surface was gently massaged for approximately 60 s to release mucus into the buffer. The PBS–mucus mixture was transferred to a 1.5-mL microtube and centrifuged at 3,000 × g for 10 min at 4 °C. Supernatants were aliquoted and stored at –80 °C for subsequent immunological assays. All sampling procedures were completed within 3 min per fish to minimize handling stress. Measurement of specific antibody titers Serum and mucus levels of IgM specific for E. tarda and V. anguillarum were measured by indirect ELISA. In brief, ImmulonTM Immunoassay plates (Thermo Fisher Scientific, USA) Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 120 were coated with 100 µL of FKC of each pathogen (1 × 10⁹ CFU/mL) suspended in PBS and incubated overnight at 4 °C. After washing with PBS-Tween, the plates were incubated in 5% skim milk in PBS-Tween to prevent non-specific binding. Next, 100 µL of appropriately diluted serum or mucus sample was added to each well, and the plates were and incubated for 1 h at room temperature. After a washing step, 100 µL of mouse monoclonal anti-tilapia IgM antibody (Aquatic Diagnostics Ltd., UK; product code F04) was added at a dilution of 1:33 in blocking buffer, according to the manufacturer’s recommendation, and the plates were incubated for 1 h. After a second washing step, 100 µL of HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, USA) diluted 1:1000 was added, and the plates were incubated for 1 h. Subsequently, 100 µL of 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate substrate (Sigma-Aldrich, USA) was added to each well. After 10 min in the dark, the reaction was stopped by adding 50 µL of 2 M H₂SO₄. Absorbance at 450 nm (OD₄₅₀) was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). Antibody levels were expressed as absorbance values and statistically compared among treatment groups and sampling timepoints. Bactericidal activity assay E. tarda and V. anguillarum were cultured to the logarithmic growth phase, harvested, washed twice with PBS, and resuspended to approximately 1 × 10⁵ CFU/mL (for V. anguillarum, PBS and NaCl–TSA were used as appropriate). For each assay, 50 µL of serum or mucus was combined with 10 µL of bacterial suspension in a tissue culture treated 96-well test plate (TPP, Switzerland) and incubated at 30 °C for 2 h. The mixtures were then serially diluted in PBS and plated onto NaCl–TSA. Plates were incubated at 30 °C for 24 h, and colonies were enumerated as CFU. Bactericidal activity was determined by the method of Gallage et al. (2016) and expressed as the percentage of growth inhibition relative to the PBS control: Inhibition (%) = （1 − !"#!"#$!#% !"#&'($%&'()&* ）# × #100 where CFUtreated denotes the colony count from serumor mucus-treated wells, and CFUPBS control the count from bacteria incubated with PBS alone. Challenge tests At 21 dpv, three separate challenge models were conducted: E. tarda, V. anguillarum, and co-infection (see Figure 1). All challenges were performed by intraperitoneal injection using a final volume of 100 µL per fish. The fish remaining in each vaccination group were allocated to challenge arms in three replicate tanks (8 fish/tank; n = 24 fish/arm). Fish were observed twice daily for 21 dpc; moribund or dead individuals were recorded and promptly removed. The challenge organism was confirmed by re-isolation on TSA for E. tarda and NaCl–TSA for V. anguillarum. For the E. tarda model, the mono-E. tarda, bivalent, and PBS control groups were injected with 100 µL of E. tarda suspension at 1×108 CFU/fish. For the V. anguillarum model, the mono-V. anguillarum, bivalent, and PBS control groups were injected with 100 µL of V. anguillarum suspension at 1×107 CFU/ fish. For the co-infection model, the bivalent and PBS control groups received a mixed inoculum prepared immediately before use: 50 µL of E. tarda (1×108 CFU/fish) plus 50 µL of V. anguillarum (1×107 CFU/fish). The co-infection challenge in the Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 121 present study did not include monovalent comparator groups. Accordingly, this model was not intended to determine whether the bivalent formulation outperforms monovalent vaccines under mixed-pathogen exposure, but rather to confirm that protection is maintained when both pathogens are encountered simultaneously. Future studies incorporating matched monovalent vaccine groups in co-infection models will be necessary to directly compare relative efficacy and to determine whether bivalent vaccination confers additive or synergistic protection. Cumulative survival was summarized at 21 dpc for each arm. Relative percent survival (RPS) was calculated as: RPS = [1 − +&)(,*-(_/,%%-',(01 +&)(,*-(_234$%&'()&*]#× #100, where higher values indicate greater vaccine-induced protection. Statistical analysis All data are presented as mean ± standard deviation (SD). Antibody titers, bactericidal activities, and survival rates at 21 dpc were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test to determine statistical significance among groups. The significance level was set at p <0.05. All statistical analyses were performed using IBM SPSS Statistics for macOS, version 30.0 (IBM Corp., Armonk, NY, USA). Results IgM antibody responses in serum and mucus First, we measured specific IgM antibody responses in fish serum and mucus for 21 dpv. From 7 to 21 dpv, vaccination elicited antigen-specific IgM responses in both serum and skin mucus, with vaccinated groups generally exceeding the PBS control group. For E. tarda-specific responses (Figure 2A–B), in serum at 21 dpv the bivalent group (OD450 = 0.22) was numerically higher than the monovalent group (OD450 = 0.20) but not significantly different; both vaccinated groups were significantly higher than the PBS group (OD450 = 0.08; p <0.05). In mucus, at 21 dpv the bivalent group (OD450 = 0.43) did not differ significantly from the monovalent group (OD450 = 0.38), while both groups were significantly higher than the PBS group (OD450 = 0.09; p <0.05). For V. anguillarum-specific responses (Figure 2C–D), in serum at 14 dpv the bivalent group (OD450 = 0.27) was not significantly different from the monovalent group (OD450 = 0.18), but was significantly higher than the PBS control (OD450 = 0.08; p <0.05). At 21 dpv, the bivalent group was significantly higher than both the monovalent and PBS groups; and the monovalent group was significantly higher than the PBS group (p <0.05). In mucus, at 14–21 dpv the bivalent group (1.38–1.44) was significantly higher than the monovalent (0.35–0.34) and PBS (0.08–0.09) groups at corresponding timepoints (p <0.05). Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 122 Figure 2. Serum and mucosal IgM antibody responses against E. tarda and V. anguillarum in tilapia immunized with monovalent vaccines, a bivalent vaccine, or PBS control. (A) Serum IgM responses against E. tarda. (B) Mucosal IgM responses against E. tarda. (C) Serum IgM responses against V. anguillarum. (D) Mucosal IgM responses against V. anguillarum. All responses were measured at 1, 7, 14, and 21 dpv. Data are mean ± SD (n = 6 fish/group), and the error bars represent standard deviation. At each timepoint, different lowercase letters denote significant differences among groups (ANOVA with Tukey’s test, p <0.05), and uppercase letters indicate significant differences over time within the same group. Collectively, midto late-phase IgM responses were robust across systemic and mucosal compartments, with the bivalent vaccine performing better than or equal to the monovalent vaccine in most comparisons and showing a consistent advantage in mucosal IgM and V. anguillarum serum IgM at 14 dpv. Bactericidal activity in serum and mucus We also measured the bactericidal activity of serum and mucus collected post-vaccination. Overall, vaccination increased bactericidal activity across compartments compared with PBS controls from 7 to 21 dpv. For E. tarda-specific bactericidal activity (Figure 3A–B), the bivalent group showed significantly higher serum activity than the monovalent group at 7 and 21 dpv (p < 0.05), while the difference at 14 dpv was not significant (p > 0.05). Serum activity in the PBS group remained <7% at each timepoint, and values within the bivalent group did not differ significantly between 14 and 21 dpv (p > 0.05). In mucus, all pairwise comparisons among the three groups were significant at 14 dpv (p < 0.05). At 21 dpv, activity in the bivalent group (40.53%) was significantly higher than that in the PBS group (1.40%; p < 0.05), but not significantly different from that in the monovalent group (23.10%; p > 0.05). For V. anguillarum-specific bactericidal activity (Figure 3C–D), serum activity in the bivalent group reached 76.00% and 80.12% at 14 and 21 dpv, respectively; these values were significantly higher than those in the monovalent group (60.50% and 66.70%) and PBS controls (<7%; p < 0.05). In mucus, activity at 21 dpv was 48.60% in the bivalent group and 44.80% in Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 123 the monovalent group; although these values did not differ significantly (p > 0.05), both were significantly higher than the inhibition observed in the PBS group (1.50%; p < 0.05). Figure 3. Serum and mucosal bactericidal activity against E. tarda and V. anguillarum in tilapia immunized with monovalent vaccines, a bivalent vaccine, or PBS control. (A) Serum bactericidal activity against E. tarda. (B) Mucosal bactericidal activity against E. tarda. (C) Serum bactericidal activity against V. anguillarum. (D) Mucosal bactericidal activity against V. anguillarum. All responses were measured at 1, 7, 14, and 21 dpv. Data are mean ± SD (n = 6 fish/group), and the error bars represent standard deviation. At each timepoint, different lowercase letters denote significant differences among groups (ANOVA with Tukey’s test, p < 0.05), and uppercase letters indicate significant differences over time within the same group. Overall, the observed bactericidal responses aligned with the IgM kinetics. The bivalent vaccine showed clear advantages over the monvalent vaccine in serum bactericidal activity and comparable activity to the monovalent vaccine against V. anguillarum at later timepoints. Protective efficacy against single and co-infections Lastly, we monitored survival of the fish for 21 dpc. After E. tarda challenge (Figure 4A), survival was significantly higher in the bivalent group (54.17%) than in the monovalent (29.17%) or PBS (25.00%) group (p <0.05). After V. anguillarum challenge (Figure 4B), survival was 87.50% in the bivalent group and 75.00% in the monovalent group; the difference between the vaccinated groups was not significant (p > 0.05), but survival in both groups was significantly higher than that in the PBS group (41.67%; p <0.05). Under mixed (E. tarda + V. anguillarum) challenge (Figure 4C), survival in the bivalent group (50.00%) was significantly higher than that in the PBS group (4.17%; p <0.05). Taken altogether, these results demonstrate that the bivalent vaccine was superior or comparable to monovalent formulations in single-pathogen models and conferred a clear benefit under co-infection, consistent with the humoral and bactericidal data. Liu et al. Bivalent vaccine for tilapia Marine Reports 4(2) (2025) 116-129 124 Figure 4. Cumulative survival of tilapia following experimental challenge with E. tarda, V. anguillarum, or a combination of both after immunization with monovalent vaccines, a bivalent vaccine, or PBS control. (A) Cumulative survival after E. tarda challenge. (B) Cumulative survival after V. anguillarum challenge. (C) Cumulative survival after mixed (E. tarda + V. anguillarum) challenge. Data are the cumulative survival rate (%) over 21 dpc. Each treatment had three replicate tanks (8 fish/tank; n = 24 fish/group). Different letters indicate significant differences among groups at 21 dpc (ANOVA with Tukey’s test, p < 0.05).