CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy

CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy Mianlong Huang, Xiufang Li, Taian Pan, Donghai Wu, Gonghe Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9209433/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant E. coli DH5α-ORF4-GFP (targeting GBS ScpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays. Results The optimal regimen was single immersion at 1.5×10⁴ CFU/mL, with a maximum RPS of 73.12% and stable 65.26% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and enhanced globulin synthesis, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not change intestinal microbial richness but optimized its structure, enriching beneficial taxa like Alphaproteobacteria via the microbiota-immunity axis, enhancing host’s defense capacity via the "microbiota-immunity" axis. Conclusions In conclusion, this study successfully developed a highly effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines. CRISPR-Cas9 Streptococcus agalactiae tilapia vaccine immune protection Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Streptococcus agalactiae (Group B Streptococcus, GBS) is a major bacterial pathogen in tilapia aquaculture. Widely distributed in natural environments, it can colonize the human urogenital and gastrointestinal tracts, making it an important zoonotic pathogen[1, 2]. GBS primarily infects fish via skin lesion or the fecal–oral route, readily causing outbreaks in healthy fish populations. China is the world’s largest producer and exporter of farmed tilapia, accounting for over 40% of global production. However, large-scale streptococcosis outbreaks have occurred in major tilapia-farming regions of China over the past decade. with > 90% of clinical isolates have been identified as S. agalactiae . Cumulative mortality rates ranging from 30% to 80%, resulting in substantial economic losses to the aquaculture industry[3, 4]. Vaccination is a core strategy for preventing and controlling infectious diseases in aquaculture. Various vaccine types targeting GBS in tilapia have been developed, including inactivated vaccines, DNA vaccines, and live attenuated vaccines[5–7]。Zhang et al.[8] constructed an attenuated S. agalactiae strain (△cps), which achieved an RPS of 90.47% following intraperitoneal immunization. Li et al.[9] reported RPS values of 96.11% and 74.80% for an attenuated strain (△2) via intraperitoneal injection and oral administration, respectively. Despite their strong protective efficacy, live attenuated vaccines require, intraperitoneal administration involving fish anesthesia and handling, which is labor-intensive, time-consuming, and may induce stress-related immunosuppression[10]. Significant progress has also been made in genetically engineered subunit vaccines. Zhu et al. [11] developed an oral subunit vaccine (SIP-MSN@HP55) by encapsulating the surface immunogenic protein (SIP) in mesoporous silica nanoparticles(MSN) and hydroxypropyl methylcelluose phthalate(HP55), achieving a maximum RPS of 76.31%. Cao et al.[12] constructed a nanocarrier vaccine (BNC-rSip) by conjugating the antigenic protein Sip to biodegradable bacterial nanocellulose (BNC), providing up to 78.95% immune protection via immersion immunization. Lu et al.[13] incorporated chitooligosaccharides (COS) as an adjuvant into an inactivated streptococcal vaccine and compared four administration routes: intraperitoneal injection (Ip), immersion plus intraperitoneal injection (Im + Ip), immersion plus oral administration (Im + Or), and oral administration (Or). The Im + Ip group exhibited the highest RPS (78.6%). Nevertheless, oral vaccines are inherently limited by antigen degradation in the harsh acidic gastrointestinal environment, leading to weaker immune responses than conventional vaccines[14]. In contrast, immersion immunization is simple, efficient, and minimally invasive, but generally provides shorter immune protection duration than injection-based vaccination[12]. The emergence of CRISPR/Cas9 technology has revolutionized genome editing due to its high efficiency, simplicity, and specificity. This technology has been widely applied in bacterial genetic manipulation, facilitating both fundamental research and applied development[15–17]. C5a peptidase is a highly conserved surface-exposed serine protease of S. agalactiae . designated ScpB (streptococcal C5a peptidase from group B Streptococcus ), which is encoded by the scpB gene and secreted to the bacterial surface. ScpB is the largest surface protein identified in GBS and is present in all serotypes[18]. It inhibits neutrophil chemotaxis by cleaving complement component C5a[18], thereby facilitating immune evasion and enhancing bacterial adhesion and invasion[19]. Owing to its surface exposure and functional properties, ScpB is efficiently recognized by the host immune system and exhibits strong intrinsic immunogenicity. Previous studies have demonstrated that ScpB-targeted subunit vaccines confer significant immune protection in tilapia, particularly when combined with advanced delivery systems such as poly(lactic-co-glycolic acid) (PLGA) microspheres [20]. Sequence analysis of S. agalactiae strain HN016 (GenBank accession no. CP011325.1) revealed that the C5a peptidase is encoded by the scpB gene with a full-length open reading frame (ORF) of 3703 bp, consisting of two encoded regions: ORF1 (1275 bp) and ORF4 (2403 bp). Among them, the ORF4 open reading frame encodes the large subunit of the C5a peptidase, which plays a central role in antibody recognition and immune protection. Meanwhile, as it does not represent the full-length C5a peptidase, its toxicity is attenuated[21]. In the present study, ORF4-specific primers were designed to amplify the ORF4 fragment from S. agalactiae genomic DNA. Using CRISPR/Cas9-mediated genome editing, the ORF4 fragment was inserted into Escherichia coli DH5α along with a green fluorescent protein (GFP) reporter, resulting in the successful construction of the recombinant strain Escherichia coli DH5α-ORF4-GFP. Immunoprotection assays demonstrated that this strain conferred effective protection against GBS infection in tilapia, providing a solid theoretical and technical foundation for its potential application in aquaculture vaccines. Results Construction of the recombinant strain To construct the plasmid pSC101-PBAD-sgRNA-ORF4-GFP, genomic DNA from E. coli DH5α and S. agalactiae strain HN016, together with plasmids pBAV1K-T5-sfGFP and pSC101-PBAD-sgRNA-Donor26D, were used as templates. Using primers listed in Supplementary Material S1, the corresponding gene fragments were successfully amplified (Fig. S1 ). All fragments were assembled using the pEASY®-Uni Seamless Cloning and Assembly Kit and transformed into E. coli DH5α. Recombinant plasmids were extracted using a Plasmid Mini Kit II (OMEGA, USA) and designated as pSC101-PBAD-sgRNA-ORF4-GFP (Fig. 1 A).Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-ORF4-GFP were subsequently co-transformed into E. coli DH5α. Following induction, gene-edited colonies were obtained and named DH5α-ORF4-GFP (Fig. 1 B)(CCTCC M 20252368). PCR amplification using primers HA-F/ORF4-R and sequence analysis confirmed successful genome editing (Fig. 1 C; sequencing data are shown in Supplementary Material 1 ). Western blot analysis demonstrated that the DH5α-ORF4-GFP strain expressed the ORF4-GFP fusion protein at the expected molecular weight of approximately 116.3 kDa (Fig. 1 D). Growth curve analysis showed no significant difference in growth rate between DH5α-ORF4-GFP and the parental E. coli DH5α strain (Fig. 1 E). Immunoprotection against Streptococcus agalactiae infection Immunogenicity of the recombinant strain DH5α-ORF4-GFP was evaluated through dose optimization, immunization frequency analysis, and validation experiments to assess protection against S. agalactiae HN016 infection in tilapia. In the dose-selection experiment, fish were immunized once by immersion with DH5α-ORF4-GFP at concentrations of 1.5 × 10², 1.5 × 10³, and 1.5 × 10⁴ CFU/mL. Unimmunized fish served as the positive control, and fish immunized with E. coli DH5α served as the bacterial control. At 35 days post-immunization, fish were challenged by intraperitoneal injection with 3 × 10⁸ CFU/fish of GBS. Results showed that all DH5α-ORF4-GFP –immunized groups exhibited significant protective effects compared with controls (P < 0.05; Table 1 ), with protection increasing in a dose-dependent manner. The high-dose group achieved an RPS of 73.12%, whereas the DH5α control group showed an RPS of 0, confirming the absence of anti-GBS activity in the control strain. Based on the optimal dose, the effect of immunization frequency was evaluated. Fish received 1, 2, or 3 immersion immunizations at 14-d intervals. The corresponding RPS values were 60.00%, 53.30%, and 48.00%, respectively (Table 2 ). Although a declining trend in protection was observed with increasing immunization frequency, statistical analysis indicated no significant differences among groups (P > 0.05). In the validation experiment, a single immersion immunization at 1.5 × 10⁴ CFU/mL resulted in an RPS of 65.30% (Table 3 ), which was consistent with the dose- and frequency-optimization results and showed no significant differences (P > 0.05). Collectively, these results demonstrate that DH5α-ORF4-GFP confers effective protection against GBS infection in tilapia, with a single immersion immunization at 1.5 × 10⁴ CFU/mL representing the optimal and reliable regimen. Table 1 Protective efficacy of different immersion doses of DH5α-ORF4-GFP live vaccine against Streptococcus agalactiae in tilapia Treatment Viable vaccine concentration (CFU/mL) Immersion time (h) Immunization times (times) Challenge dose/fish (CFU) Challenge time No. dead/ No. totala Mean Mortality/% RPS/% PC 3×10 8 14 18/30 60 NC 0 14 30/30 0 DH5α 1.5×10 3 1 1 3×10 8 14 20/30 66.67 0 DH5α-ORF4-GFP 1.5×10 2 1 1 3×10 8 14 8/31 25.81 56.98 DH5α-ORF4-GFP 1.5×10 3 1 1 3×10 8 14 6/31 19.35 67.75 DH5α-ORF4-GFP 1.5×10 4 1 1 3×10 8 14 5/31 16.13 73.12 a: Fish were challenged 35-days post-immunization with 3×10 8 CFU/fish of HN016 strain and monitored for 14 days post challenge. RPS: Relative percent survival. PC:Positive Control Group. NC:Negative Control Group,fish were intraperitoneally injected with 0.2 mL of sterile PBS. Table 2 Protective efficacy of DH5α-ORF4-GFP live vaccine against Streptococcus agalactiae in tilapia with different immersion immunization frequencies Treatment Viable vaccine concentration (CFU/mL) Immersion time (h) Immunization times (times) Challenge dose/fish (CFU) Challenge time No. dead/ No. totala Mean Mortality/% RPS/% PC 3×10 8 14 25/36 69.44 NC 0 14 30/30 0 DH5α 1.5×10 4 1 1 3×10 8 14 22/33 66.67 4.00 DH5α-ORF4-GFP 1.5×10 4 1 1 3×10 8 14 10/36 27.78 60.00 DH5α-ORF4-GFP 1.5×10 4 1 2 3×10 8 14 12/37 32.43 53.30 DH5α-ORF4-GFP 1.5×10 4 1 3 3×10 8 14 13/36 36.11 48.00 a: Fish were challenged 7 d post-final immunization with 3×10 8 CFU/fish of HN016 strain and monitored for 14 d post challenge. RPS: Relative percent survival. PC:Positive Control Group. NC:Negative Control Group,fish were intraperitoneally injected with 0.2 mL of sterile PBS. Table 3 Protective efficacy of DH5α-ORF4-GFP live vaccine at 1.5×10⁴ CFU/mL with single immersion against Streptococcus agalactiae in tilapia Treatment Viable vaccine concentration (CFU/mL) Immersion time (h) Immunization times (times) Challenge dose/fish (CFU) Challenge time No. dead/ No. totala Mean Mortality/% RPS/% PC 3×10 8 14 19/33 57.58 DH5α-ORF4-GFP 1.5×10 4 1 1 3×10 8 14 7/35 20.00 65.26 a: Fish were challenged 15 d post-immunization with 3×10 8 CFU/fish of HN016 strain andmonitored for 14 days post challenge. RPS: Relative percent survival. PC:Positive Control Group. Hematological and serum biochemical characteristics Hematological and serum biochemical analyses were performed to compare physiological responses between the immunized group (MY) and the control group (CK). As shown in Table 4 , significant differences were observed in platelet-related parameters (P < 0.05). Platelet counts (PLT) in the MY group increased by 161.40% compared with the CK group, accompanied by increases in monocyte percentage (+ 56.01%) and basophil percentage (+ 88.74%), indicating activation of immune responses. In contrast, erythrocyte-related parameters showed only minor fluctuations, with hemoglobin (HGB) and red blood cell counts (RBC) decreasing by 10.14% and 5.92%, respectively. Serum biochemical parameters displayed coordinated functional changes (Table 5 ). In the MY group, globulin (GLO) levels increased by 25.90%, and alanine aminotransferase (ALT) activity increased by 31.62%, suggesting enhanced immune protein synthesis and hepatic metabolic activity. Meanwhile, cholesterol (CHOL) levels decreased by 16.54%, and glucose (GLU) exhibited only minor variation, indicating a metabolic shift toward supporting immune demands. Although some parameters, such as total protein (TP) and albumin (ALB), showed slight decreases, the overall pattern of immune- and metabolism-associated indicators suggests that immunization activated immune defenses while maintaining general physiological stability in tilapia. Table 4 Hematological parameters(n = 6) Parameter CK (mean ± SEM) MY (mean ± SEM) WBC(10 9 /L) 4.92 ± 1.30 4.38 ± 2.17 LYM%(%) 43.24 ± 16.07 42.55 ± 12.27 MON%(%) 12.04 ± 6.94 18.78 ± 15.13 NEU%(%) 42.77 ± 22.54 36.41 ± 21.40 EOS%(%) 1.61 ± 0.73 1.62 ± 0.60 BASO%(%) 0.34 ± 0.19 0.64 ± 0.46 LYM#(10 9 /L) 2.04 ± 0.86 1.91 ± 1.31 MON#(10 9 /L) 0.5662 ± 0.3873 0.614 ± 0.2583 NEU#(10 9 /L) 2.22 ± 1.61 1.75 ± 1.10 EOS#(10 9 /L) 0.07 ± 0.02 0.08 ± 0.05 BASO#(10 9 /L) 0.02 ± 0.01 0.03 ± 0.02 RBC(10 12 /L) 2.34 ± 0.20 2.20 ± 0.27 HGB(g/L) 106.83 ± 8.89 96.00 ± 11.37 HCT(%) 28.42 ± 1.43 26.18 ± 2.55 MCV(fL) 122.12 ± 5.16 119.77 ± 5.94 MCH(pg) 45.70 ± 1.99 43.68 ± 2.08 MCHC(g/L) 375.00 ± 16.12 365.83 ± 12.83 RDW-CV(%) 8.43 ± 0.61 8.42 ± 0.35 PLT(10 9 /L) 9.50 ± 3.83 a 24.83 ± 5.81 b MPV(fL) 11.12 ± 1.84 12.87 ± 1.25 Note: CK group : Blank Control Group; MY group: Single immersion immunization with DH5α-ORF4-GFP ( 1.5 × 10⁴ CFU/mL). Table 5 Serum biochemical parameters(n = 6) Parameters CK (mean ± SEM) MY (mean ± SEM) TP(g/L) 29.78 ± 2.78 30.70 ± 3.77 ALB(g/L) 19.10 ± 1.11 17.25 ± 3.90 GLO(g/L) 10.68 ± 3.60 13.45 ± 5.26 A/G 2.02 ± 0.82 1.43 ± 0.56 TBIL(umol/L) 5.06 ± 1.28 4.70 ± 0.86 ALT(U/L) 39.00 ± 18.41 51.33 ± 27.23 ALP(U/L) 21.50 ± 9.14 23.83 ± 4.67 BUN(umol/L) 0.02 ± 0.06 0.00 ± 0.00 CRE(umol/L) 39.50 ± 17.31 38.83 ± 12.29 CK(U/L) 2201.17 ± 1040.46 2432.83 ± 884.28 AMY(U/L) 8.00 ± 11.33 15.50 ± 12.32 GLU(umol/L) 6.06 ± 1.62 6.19 ± 2.00 CHOL(umol/L) 3.36 ± 1.13 2.81 ± 1.18 Ca(umol/L) 2.94 ± 0.24 3.09 ± 0.18 P(umol/L) 1.74 ± 0.58 2.07 ± 0.21 Note: CK group : Blank Control Group; MY group: Single immersion immunization with DH5α-ORF4-GFP ( 1.5 × 10⁴ CFU/mL). Measurement of antioxidant indices The superoxide dismutase (SOD) activities in the PC group and MY+Challenge group were the highest, and were significantly higher than those in the CK group and MY group (P < 0.05). The malondialdehyde (MDA) content in the positive control group was significantly higher than that in the CK group and MY group (P < 0.05), while the MDA content in the MY+Challenge group was lower than that in the PC group (Fig. 2 ). PC: Positive Control Group; CK: Blank Control Group; MY: Single immersion immunization group treated with DH5α-ORF4-GFP (1.5 × 10⁴ CFU/mL); MY+Challenge: Single immersion immunization group immunized with DH5α-ORF4-GFP (1.5×10⁴ CFU/mL) and challenged with HN016. (n = 6). Histopathological analysis Histological examination revealed no marked differences in tissue integrity between the MY and CK groups. In both groups, hepatic sinusoidal structures were clearly defined, and no obvious pathological alterations were observed in renal glomeruli or tubules (Fig. 3 ). Consistent with these observations, serum biochemical indices related to liver and kidney function (e.g., ALT, creatinine[ CRE]) showed no significant abnormalities. These results indicate that vaccination did not induce detectable liver or kidney damage, demonstrating favorable biosafety. Scale bar = 20 µm. (HE, ×400) Effects of vaccination on intestinal microbiota Given that vaccination can modulate intestinal microbial communities and influence host health[ 22 , 23 ], 16S rRNA gene sequencing was performed to examine the effects of DH5α-ORF4-GFP vaccination on the gut microbiota of tilapia. These sequence data have been submitted to the GSA databases under accession number CRA036623. As shown in Fig. 4 A and 4 B, no significant differences were observed in Chao or Shannon indices between the MY and CK groups, indicating comparable microbial richness and diversity. However, principal coordinate analysis (PCoA) revealed a clear separation between the two groups, suggesting significant differences in community structure (Fig. 4 C). Microbial composition was further analyzed at the phylum and genus levels (Fig. 4 D, 4 E). At the phylum level, the CK group was dominated by Fusobacteriota (45.8%) and Bacillota (27.5%). Compared with the CK group, the MY group showed a reduced abundance of Fusobacteriota (27.3%) and an increased abundance of Pseudomonadota (23.9%). At the genus level, the relative abundance of Cetobacterium decreased from 45.8% (CK group) to 27.3% ( MY group). Linear discriminant analysis effect size (LEfSe) identified differentially enriched taxa between groups (Fig. 4 F). In the CK group, o_Enterobacterales, f_Enterobacteriaceae, and g_Ligilactobacillus were significantly enriched, whereas in the MY group, c_Alphaproteobacteria, p_Actinomycetota, and c_Actinobacteria were significantly enriched. Discussion In the present study, a CRISPR/Cas9-based strategy was first employed to generate a recombinant E. coli DH5α strain expressing the ScpB-derived ORF4 antigen, and the successful expression and biological stability of the recombinant strain were confirmed at both the molecular and phenotypic levels. ScpB is a highly conserved virulence determinant among all Streptococcus agalactiae serotypes, and the encoded C5a peptidase facilitates bacterial invasion by degrading complement C5a and thereby suppressing host immune responses, making it an attractive target for vaccine development[18, 24].In this study, ORF4—encoding the major subunit of the C5a peptidase—was predicted to be surface-exposed and immunogenic. Importantly, ORF4 is a truncated form rather than the full-length C5a peptidase, which may reduce potential virulence-associated risks[21]. The functional ORF4 fragment was amplified using specific ORF4-F/R primers and expressed in E.coli DH5α via CRISPR/Cas9-mediated genome editing. Compared with conventional homologous recombination or plasmid-based transformation, the high targeting precision of CRISPR/Cas9 reduces unintended events, while GFP labeling enables rapid identification of positive clones. Consequently, the construction cycle was shortened to 5 days, outperforming the 7–10 days typically required by traditional methods [25], and providing a practical basis for scalable vaccine preparation. Consistent with the immunization and challenge results, the DH5α-ORF4-GFP vaccine conferred moderate but stable protection via a single immersion immunization, highlighting the feasibility of non-invasive delivery for GBS control in tilapia. Evaluation of protective efficacy is central to vaccine development. Relative to existing strategies, the DH5α-ORF4-GFP vaccine exhibited several advantages. Although inactivated vaccines are generally considered safe, their RPS values are often limited to ~ 50–65%[26, 27] and typically rely on injection, which is labor-intensive and may induce stress responses in fish. Live attenuated vaccines can achieve RPS values > 75% [28], yet concerns regarding reversion to virulence constrain their use, particularly under high-density farming conditions. In contrast, DH5α-ORF4-GFP delivered by immersion conferred a maximal RPS of 73.12%, and the validation assay yielded a stable RPS of 65.26% (P > 0.05), without reversion risk, while remaining operationally simple and cost-manageable for large-scale aquaculture. Notably, the protection level was lower than that reported for the attenuated vaccine developed by Zhang et al. (RPS = 90.47%) [8]. Future work may explore adjuvant strategies (e.g., fucoidan or IL-11) to enhance antigen presentation and further improve protective performance [27, 29, 30]. An observation warranting further discussion is that during immunization frequency optimization, a “single-dose outperforming multiple-dose” trend was noted (RPS: 1 dose, 60.00%; 2 doses, 53.30%; 3 doses, 48.00%), although differences were not statistically significant. This pattern may be linked to mucosal immunity characteristics. Immersion immunization primarily stimulates mucosa-associated lymphoid tissues (MALT). Repeated antigen exposure at short intervals may promote CD103 + dendritic cells to secrete TGF-β and retinoic acid, thereby driving regulatory T cell (Treg) expansion [31, 32]. Such responses can dampen effector T-cell activation, resulting in a gradual decline in protective immunity. These findings suggest that mucosal vaccines in aquaculture should avoid a potential “immune tolerance trap” and that increasing booster frequency does not necessarily improve protection. In future studies, antibody titers and memory lymphocyte proportions at 7, 14, and 30 days post-immunization should be monitored to define an optimal immunization window, thereby enabling evidence-based vaccination schedules for farming practice. Hematological, biochemical, antioxidant indices and histopathological analysis further confirmed that the DH5α-ORF4-GFP vaccine was biosafe under the optimized immunization protocol. In teleost immunity, thrombocytes contribute not only to hemostasis but also to innate defense via pathogen adhesion and the release of antimicrobial peptides and chemotactic mediators [33]. In the present study, only platelet counts (PLT) were significantly elevated in the MY group ( 161.40% increase vs. CK group; P < 0.05), while other parameters showed no significantly differences.In the present study, the SOD activity and MDA content in the PC group were significantly increased compared with the CK group, which was consistent with the typical changes of oxidative stress in tilapia infected with Streptococcus agalactiae[34]. In addition, the MDA content in the MY+Challenge group was lower than that in the PC group, while the SOD activity remained at a high level comparable to the PC group, suggesting that vaccination could enhance the stress resistance of tilapia by regulating oxidative balance.Meanwhile, there were no significant differences in MDA content and SOD activity between the MY group and the CK group, indicating that the recombinant vaccine itself had no adverse effects on the oxidative balance of tilapia and did not induce additional oxidative stress. Together with histological observations of no overt pathological lesions in the liver and kidney, these data support the vaccine’s biosafety and suggest no apparent adverse impact on tilapia health. Although the DH5α-ORF4-GFP vaccination did not significantly alter overall microbial richness or diversity, the observed shift in community structure suggests a potential indirect interaction between vaccination and intestinal microbial composition.Accumulating evidence indicates that the gut microbiota is closely associated with fish immune function, and enrichment of beneficial taxa can indirectly enhance host resistance via competitive exclusion, reinforcement of mucosal barriers, and modulation of local immune responses [35–37]. In this study, vaccination did not significantly affect alpha diversity (richness/diversity) but significantly reshaped community composition (distinct PCoA separation). The MY group showed increased relative abundance of taxa such as c_Alphaproteobacteria and p_Actinomycetota, implying that DH5α-ORF4-GFP may not only elicit antigen-specific immunity but also positively modulate the intestinal micro-ecosystem via a “microbiota–immunity” axis, thereby providing an additional protective barrier [37, 38]. Despite demonstrating the potential of a CRISPR/Cas9-edited vaccine strategy, several limitations should be acknowledged. First, the observation period for protective efficacy was relatively short; the durability of vaccine-induced immune memory should be evaluated in long-term farming trials (≥ 3–6 months), with challenge tests and antibody monitoring at multiple time points to determine long-lasting protection. Second, mechanistic investigations did not include immune-related gene expression (e.g., IL-1β, TNF-α, IgM ), limiting molecular-level interpretation of immune regulation. Future studies integrating transcriptomics and proteomics could clarify the signaling pathways by which this vaccine activates innate and adaptive immunity in tilapia. Third, laboratory conditions differ from the complex environments of production ponds (water quality fluctuations, climate, and diverse microbial communities); therefore, field-scale performance should be validated under commercial settings. Fourth, protection was assessed only against S. agalactiae strain HN016, and cross-protection against other serotypes remains unknown. Given the high conservation of ScpB among serotypes [18], broader protection is plausible; nevertheless, challenges with multiple serotypes are required to define the breadth of cross-protection and to enhance practical value. Conclusions Using CRISPR/Cas9-mediated genome editing, we successfully constructed recombinant E. coli DH5α-ORF4-GFP expressing the Streptococcus agalactiae ScpB-derived ORF4 antigen, with the optimal immunization regimen identified as a single immersion at 1.5 × 10⁴ CFU/mL. This regimen elicited effective protection against GBS infection in tilapia. Hematological, biochemical and gut microbiota analyses collectively confirmed the feasibility and application potential of this recombinant strain as a vaccine candidate against S. agalactiae in tilapia aquaculture, providing an alternative technical strategy for the immunoprophylaxis of streptococcosis. Methods Experimental materials Streptococcus agalactiae strain HN016 (GenBank accession no. CP011325.1), Escherichia coli DH5α (GenBank accession no. CP026085), recombinant ORF4 protein, and mouse-derived polyclonal antibodies against ORF4 were provided by the Clinical Laboratory of the College of Animal Science and Technology, Guangxi University. Plasmid pBAV1K-T5-sfGFP was purchased from HonorGene (HG-VYH1347). Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-Donor26D were obtained from Shandong Qibang Biotechnology Co., Ltd. Nile tilapia (“Hainan No. 1”; average body weight ≈ 20 g) were purchased from a commercial hatchery in Nanning, Guangxi, China. Fish were confirmed to be free of bacterial infection by bacteriological examination of brain and kidney tissues and were acclimated in disinfected tanks (7.0 m × 1.5 m × 1.2 m) with continuous aeration and daily feeding of a commercial diet. Plasmid construction Recombinant plasmids were generated using the pEASY®-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). DNA polymerases and PCR reagents were purchased from Takara Bio (Beijing, China). The sgRNA was designed using the sgRNAcas9 v3.0 software ( https://www.biootools.com/ ). Primer were designed using Primer Premier 5.0, and primer sequences are listed in Supplementary Material Table S1 . Primer synthesis, gene synthesis, and DNA sequencing were conducted by Tsingke Bio (Beijing, China) and BGI Genomics (Guangdong, China). The pSC101-PBAD-sgRNA-ORF4-GFP vector was constructed according to the method described by Huang et al.[ 39 ]. genomic DNA from S. agalactiae HN016 was used as a template to amplify a 408 bp promoter fragment ( primers QDZ-F/R) and a 2432 bp ORF4 fragment ( primers ORF4-F/R). Genomic DNA from E. coli DH5α was used to amplify a 512 bp left homology arm (LHA) and a 539 bp right homology arm (RHA) with primers LHA-F/R and RHA-F/R, respectively. The GFP gene (757 bp) was amplified from plasmid pBAV1K-T5-sfGFP using primers GFP-F/R. Five additional fragments (2127, 367, 301, 1118, and 341 bp) were amplified from pSC101-PBAD-sgRNA-Donor26D using primers yw1-5. All ten fragments were purified and assembled by isothermal assembly to generate the final plasmid pSC101-PBAD-sgRNA-ORF4-GFP. Plasmid sequences are provided in Supplementary Material 1. Construction of recombinant strains The construction of recombinant strains followed the procedure illustrated in Fig. 1 A[ 40 ]. Competent E. coli DH5α cells were prepared according to the protocol described by Rachel et al.[ 41 ]. Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-ORF4-GFP were co-transformed into competent cells via heat shock at 42°C. Following recovery in LB medium at 30°C for 1 h, transformed cells were plated onto LB agar containing ampicillin (100 µg/mL) and kanamycin (50 µg/mL) and incubated at 30°C for approximately 48 h. Induction and screening of positive clones were performed with slight modifications to the method described by Huang et al.[ 39 ]. Single colonies were cultured in LB medium, induced with IPTG and L-arabinose, and subsequently screened on kanamycin-containing plates. Colonies exhibiting green fluorescence were selected for PCR verification. The successfully edited strain was designated DH5α-ORF4-GFP . Growth curve assay The target strains were cultured in LB broth at 37°C with shaking (180 rpm) to the exponential phase, inoculated (1%, v/v) into fresh LB, and incubated under the same conditions. Samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, and 24 h. Optical density at 600 nm (OD 600 ) was measured using uninoculated LB as a blank. Three independent replicates were performed for each time point, and mean values were used for analysis. Protein extraction and Western blotting Bacterial cells were harvested by centrifugation (6000 ×g, 10 min, 4°C), washed, and resuspended in 1× PBS. Cells were lysed by ultrasonication and clarified by centrifugation (10,000 ×g, 10 min, 4°C). Total protein concentration was determined using the Bradford method. Samples were mixed with 6× loading buffer and denatured at 95°C for 10 min, separated by SDS–PAGE (4% stacking gel; 80 V, 2 h), and transferred onto nitrocellulose (NC) membranes using a semi-dry transfer system. Membranes were blocked with 5% (w/v) non-fat milk and incubated with mouse polyclonal anti-ORF4 antibody, followed by HRP-conjugated goat anti-mouse IgG (H + L) (ABclonal, China). Signals were visualized using BeyoECL Star (Beyotime, China). Immunization and challenge experiments Based on the preliminary experimental results, no significant difference was observed in the protective efficacy among intraperitoneal injection, oral gavage, and immersion administration routes (P > 0.05) (Table S2 ). Meanwhile, bacterial virulence tests revealed that immunization with the vaccine at a concentration of 1.5×10⁵ CFU/mL resulted in mortality in a small number of tilapia (Table S3 ). Therefore, the immersion administration route was adopted for all subsequent experiments. All animal experiments were conducted in strict compliance with standardized procedures and obtained approval from the Animal Ethics Review Committee of Guangxi University (Approval No.: GXU-2025-282). 1. Dose optimization Tilapia were acclimated for 5 d in a recirculating system (28 ± 1°C; dissolved oxygen ≥ 5 mg/L; pH 7.0–7.5; ammonia nitrogen ≤ 0.05 mg/L) and fasted for 24 h prior to immunization. Fish were assigned to six groups (≥ 30 fish/group): three DH5α-ORF4-GFP immersion-immunized groups (1 h) at concentrations of 1.5 × 10², 1.5 × 10³, or 1.5 × 10⁴ CFU/mL; one E. coli DH5α immersion-immunized group (1 h) at 1.5 × 10³ CFU/mL (empty vector control);a positive control (unimmunized, challenged); and a blank control (unimmunized, unchallenged). At 35 d post-immunization, all groups except the blank control were challenged by intraperitoneal injection of GBS (1.5 × 10⁹ CFU/mL; 0.2 mL/fish). Mortality was recorded for 14 d, and cumulative mortality and relative percent survival were calculated (RPS = [1 − (mortality of immunized group/mortality of positive control)] × 100%). 2. Immunization frequency Under the same rearing conditions and immersion procedure, fish were allocated to six groups (≥ 30 fish/group):three DH5α-ORF4-GFP immunized groups receiving 1, 2, or 3 immersion vaccinations at 14-d intervals (1.5 × 10⁴ CFU/mL; 1 h); one DH5α immunized group receiving a single immersion vaccination (1.5 × 10 4 CFU/mL; 1 h); and corresponding controls (positive and blank). At 7 d after the final immunization, fish (except the blank control) were challenged as described above, and mortality was monitored for 14 d to calculate RPS. 3. Confirmation of the optimal regimen Fish were divided into an immunized group (single immersion at 1.5 × 10⁴ CFU/mL) and a blank control (≥ 30 fish/group). After 14 d of rearing at 28 ± 1°C, six fish per group were sampled. The remaining fish were then challenged as described above, and mortality was recorded for 14 d to calculate RPS. Hematology and serum biochemistry For the groups in Confirmation of the optimal regimen, fish were anesthetized by immersion in MS-222 (Tricaine Methanesulfonate, 100 mg/L) prior to blood collection from the caudal vein as previously described[ 42 ]. For hematology, whole blood was mixed with EDTA-K2 anticoagulant and analyzed within 2 h using an automated five-part differential hematology analyzer. For serum biochemistry, 200 µL serum was transferred to sterile tubes and analyzed using an automated biochemical analyzer (Celercare® M5). Measurement of antioxidant indices Serum superoxide dismutase (SOD) and malondialdehyde (MDA) in tilapia were measured using commercial kits, which were purchased from Beijing Solarbio Science & Technology Co., Ltd. Serum samples were collected as described in the previous section (6 samples per group). All detection procedures were performed strictly in accordance with the manufacturer’s instructions. Histology Histological procedures followed Liu et al.[ 43 ]. Briefly, liver and kidney tissues (three fish per group from the optimal regimen group) were collected after euthanasia by overdose immersion in MS-222 (200 mg/L) until opercular movement ceased, then fixed in 4% paraformaldehyde, paraffin-embedded, sectioned at 6 µm, and stained with hematoxylin and eosin (H&E). Images were acquired using a digital slide scanner (LG-S80, Servicebio, China). Histological evaluation was performed using a semi-quantitative scoring system adapted from Bernet et al.[ 44 ] and Rey et al.[ 45 ]. Liver and kidney sections were examined for the presence and severity of pathological alterations, including hepatocellular vacuolation, necrosis, inflammatory infiltration, renal tubular degeneration, and glomerular abnormalities. Each lesion was scored on a scale of 0 to 3 (0 = absent, 1 = mild, 2 = moderate, 3 = severe) as described previously in tilapia studies[ 46 ]. Gut microbiota analysis Intestinal contents were collected from six fish per group (from the optimal regimen group) following euthanasia by overdose immersion in MS-222 (200 mg/L). Total genomic DNA was extracted using the E.Z.N.A.® Soil DNA Kit (OMEGA). The V3–V4 region of bacterial 16S rRNA genes was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplicons were quantified using a Quant-iT PicoGreen dsDNA assay (Microplate reader; BioTek FLx800) and sequenced on an Illumina NovaSeq platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.) for absolute abundance profiling. Paired-end reads were processed using QIIME 2. Downstream analyses, including alpha diversity, principal coordinate analysis (PCoA), and linear discriminant analysis (LDA), were conducted using the Majorbio Cloud( https://www.majorbio.com ). Statistical analysis Data are presented as mean ± SEM. Statistical analyses were performed in GraphPad Prism 8.0 (GraphPad Software, USA). Differences among groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test. Values with different lowercase letters in the same row indicate significant differences among treatments (P < 0.05). Abbreviations Abbreviation Full name GBS Streptococcus agalactiae SOD superoxide dismutase MDA malondialdehyde MS-222 Tricaine Methanesulfonate A/G Albumin globulin ratio ALB Albumin ALP Alkaline phosphatase ALT Alanine amiotransferase AMY Serum amylase Bas# Absolute value of basophils Bas% Basophil proportion BUN Blood urea nitrogen BUN Blood urea nitrogen Ca Calcium CHOL Cholesterol CK Creatine kinase CRE Creatinine Eos# Absolute value of eosinophils Eos% Eosinophil ratio GLO Globulin GLU Glucose HCT Hematocrit HGB Hemoglobin Lym# Absolute value of lymphocytes MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentratiom MCV Mean corpuscular volume Mon# Monocyte absolute value Mon% Monocyte ratio MPV Mean platelet volume Neu# Neutrophil Neu% Neutrophil ratio P Posphate PCT Plateletocrit PDW Platelet distribution width PLT Platelet count RBC Red blood cell count RDW-CV Erythrocyte variation coefficient RDW-SD Standard deviation of distribution width TBIL Total bilirubin TP Total protein WBC White blood cell count Declarations Ethics approval and consent to participate All animal experiments were conducted in strict compliance with standardized procedures and obtained approval from the Animal Ethics Review Committee of Guangxi University (Approval No.: GXU-2025-282). All the methods and procedures of this study comply with the requirements of the ARRIVE Guidelines. Consent for publication The manuscript has been given publication permission by all authors.Not applicable. Competing interests The authors declared no potential conflicts of interest with respect to the research, authorship and publication of this article. Funding The work was supported by grants from the Fangchenggang Science and Technology Program (AB22013007) and Guangxi Science and Technology Program (AA17204081-1). Author Contribution **MH** : Conceptualization, Methodology, Investigation, Writing – original draft. **XL** : Resources, Validation. **TP** : Formal analysis, Data curation. **DW** : Formal analysis, Data curation. **GL** : Investigation, Visualization. **WW** : Supervision, Funding acquisition, Writing – review & editing. Acknowledgement We thank the staff of the College of Animal Science and Technology, Guangxi University, for their technical support. Data Availability The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA036623) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA036623. References Xu DH, Shoemaker CA, Klesius PH. Evaluation of the link between gyrodactylosis and streptococcosis of Nile tilapia, Oreochromis niloticus (L). J FISH DIS. 2007;30(4):233–8. Nguyen HT, Kanai K, Yoshikoshi K. Ecological investigation of Streptococcus iniae in cultured Japanese flounder (Paralichthys olivaceus) using selective isolation procedures. Aquaculture. 2002;205(1):7–17. Ye X, Li J, Lu M, Deng G, Jiang X, Tian Y, Quan Y, Jian Q. Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China. 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Supplementary Files TableS123.xlsx SupplementaryMaterial1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviews received at journal 15 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviewers invited by journal 03 Apr, 2026 Editor assigned by journal 03 Apr, 2026 Editor invited by journal 31 Mar, 2026 Submission checks completed at journal 30 Mar, 2026 First submitted to journal 30 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9209433","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618516414,"identity":"6a9fe258-4ff5-4781-9f64-2bc95a001c63","order_by":0,"name":"Mianlong Huang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Mianlong","middleName":"","lastName":"Huang","suffix":""},{"id":618516415,"identity":"ed511347-10f5-4f11-b00f-4403bdf3fd05","order_by":1,"name":"Xiufang Li","email":"","orcid":"","institution":"Guangxi Vocational University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Xiufang","middleName":"","lastName":"Li","suffix":""},{"id":618516417,"identity":"dded3388-a42a-4305-95a2-93da4e4bbfcc","order_by":2,"name":"Taian Pan","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Taian","middleName":"","lastName":"Pan","suffix":""},{"id":618516425,"identity":"9deed1a2-b660-40f1-80de-da1f8d801c20","order_by":3,"name":"Donghai Wu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Donghai","middleName":"","lastName":"Wu","suffix":""},{"id":618516427,"identity":"a1aa7a74-d7a8-4230-8fdb-e04e6b24468d","order_by":4,"name":"Gonghe Li","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Gonghe","middleName":"","lastName":"Li","suffix":""},{"id":618516428,"identity":"d2c22f69-e03a-4262-9169-319f56894768","order_by":5,"name":"Wende Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYHACgwMJFWxybOztB4jWYvjgwRk+Yz6eMwlEazE2fNgmlzhPwsGASFfdSN4mkdhmlt4mwZDA8KNiGzFa0sokEs6l5bZJNx5g7Dlzm7AWs9s5ZhIJZcdy22QOJDAzthGthe1/OptEggHRWowNEtrYEojXYn//WeGDhDNshm3AQD5IlF8kew5vOPijgk1evr394IMfFURoQQEHSFQ/CkbBKBgFowAXAAA3Az9+Zb0+ewAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi University","correspondingAuthor":true,"prefix":"","firstName":"Wende","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-03-24 09:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9209433/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9209433/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106725103,"identity":"f2e4f821-064b-4542-9973-c0b8181df4d0","added_by":"auto","created_at":"2026-04-12 18:31:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":525270,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of the gene-recombinant strain.(A) Schematic diagram of \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e strain construction.(B) Fluorescence visualization assay of \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e DH5α strains.(C) PCR identification of gene-edited \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e strains using HA-F/ORF4-R primers. 1: \u003cem\u003eE. coli\u003c/em\u003e DH5α strain; 2–3: \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e strains.(D) Detection of ORF4 protein expression in \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003estrain. 1: \u003cem\u003eE. coli\u003c/em\u003e DH5α strain; 2: \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e strain; 3: Recombinant ORF4 protein.(E) Growth curve determination of \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003estrain. All cultures were performed in triplicate (\u003cem\u003en\u003c/em\u003e=3)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/acd77dfb9ff2634264a572f4.png"},{"id":106725269,"identity":"58b485cc-e539-4ca9-ae03-676528b45501","added_by":"auto","created_at":"2026-04-12 18:32:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51297,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination Results of Antioxidant Indexes. (A): Superoxide Dismutase; (B): malonaldehyde.\u003c/p\u003e\n\u003cp\u003ePC: Positive Control Group; CK: Blank Control Group; MY: Single immersion immunization group treated with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e (1.5 × 10⁴ CFU/mL); MY+Challenge: Single immersion immunization group immunized with DH5α-ORF4-GFP (1.5×10⁴ CFU/mL) and challenged with HN016. (n=6).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/0bb6899af7bc3a2ea7d4cd6c.png"},{"id":106725105,"identity":"95b501d8-f47a-4414-8c8f-9ba508be677d","added_by":"auto","created_at":"2026-04-12 18:31:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2012675,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of the CK and MY groups.(A) Liver tissue; (B) Kidney tissue . CK group = blank control group, MY group = single immersion immunization group treated with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e ( 1.5 × 10⁴ CFU/mL).\u003c/p\u003e\n\u003cp\u003eScale bar =20 μm. (HE, ×400)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/073a08f8453a18a079825d3a.png"},{"id":106555935,"identity":"23ae1789-16bb-43fe-b05a-9f4ec7df0067","added_by":"auto","created_at":"2026-04-09 19:59:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":219471,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e on the intestinal microbiota composition of tilapia.(A) Chao index; (B) Shannon index; (C) Principal Component Analysis (PCA); (D) Microbiota composition of each sample at the phylum level; (E) Microbiota composition of each sample at the genus level; (F) Linear discriminant analysis (LDA) value distribution histogram. CK Group: Blank Control ; MY group : Single immersion immunization group with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e ( 1.5 × 10⁴ CFU/mL).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/b8893f8961268a24446ce25d.png"},{"id":106959577,"identity":"2fb0d211-a7ae-4b70-b5ee-e7f12267b73a","added_by":"auto","created_at":"2026-04-15 09:11:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4312009,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/b118bbdf-debe-412d-b7d5-cad993e0a3a5.pdf"},{"id":106555931,"identity":"88baa47d-20b1-4b79-b153-1564efed8418","added_by":"auto","created_at":"2026-04-09 19:59:27","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15918,"visible":true,"origin":"","legend":"","description":"","filename":"TableS123.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/a9f40bbdee3cdc606fa02c71.xlsx"},{"id":106555933,"identity":"174afbbc-a044-4558-995c-829c06741af7","added_by":"auto","created_at":"2026-04-09 19:59:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":94660,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9209433/v1/9bec0807fdf964f68c1c725f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eStreptococcus agalactiae\u003c/em\u003e (Group B Streptococcus, GBS) is a major bacterial pathogen in tilapia aquaculture. Widely distributed in natural environments, it can colonize the human urogenital and gastrointestinal tracts, making it an important zoonotic pathogen[1, 2]. GBS primarily infects fish via skin lesion or the fecal\u0026ndash;oral route, readily causing outbreaks in healthy fish populations. China is the world\u0026rsquo;s largest producer and exporter of farmed tilapia, accounting for over 40% of global production. However, large-scale streptococcosis outbreaks have occurred in major tilapia-farming regions of China over the past decade. with \u0026gt;\u0026thinsp;90% of clinical isolates have been identified as \u003cem\u003eS. agalactiae\u003c/em\u003e. Cumulative mortality rates ranging from 30% to 80%, resulting in substantial economic losses to the aquaculture industry[3, 4]. Vaccination is a core strategy for preventing and controlling infectious diseases in aquaculture. Various vaccine types targeting GBS in tilapia have been developed, including inactivated vaccines, DNA vaccines, and live attenuated vaccines[5\u0026ndash;7]。Zhang et al.[8] constructed an attenuated \u003cem\u003eS. agalactiae\u003c/em\u003e strain (△cps), which achieved an RPS of 90.47% following intraperitoneal immunization. Li et al.[9] reported RPS values of 96.11% and 74.80% for an attenuated strain (△2) via intraperitoneal injection and oral administration, respectively. Despite their strong protective efficacy, live attenuated vaccines require, intraperitoneal administration involving fish anesthesia and handling, which is labor-intensive, time-consuming, and may induce stress-related immunosuppression[10]. Significant progress has also been made in genetically engineered subunit vaccines. Zhu et al. [11] developed an oral subunit vaccine (SIP-MSN@HP55) by encapsulating the surface immunogenic protein (SIP) in mesoporous silica nanoparticles(MSN) and hydroxypropyl methylcelluose phthalate(HP55), achieving a maximum RPS of 76.31%. Cao et al.[12] constructed a nanocarrier vaccine (BNC-rSip) by conjugating the antigenic protein Sip to biodegradable bacterial nanocellulose (BNC), providing up to 78.95% immune protection via immersion immunization. Lu et al.[13] incorporated chitooligosaccharides (COS) as an adjuvant into an inactivated streptococcal vaccine and compared four administration routes: intraperitoneal injection (Ip), immersion plus intraperitoneal injection (Im\u0026thinsp;+\u0026thinsp;Ip), immersion plus oral administration (Im\u0026thinsp;+\u0026thinsp;Or), and oral administration (Or). The Im\u0026thinsp;+\u0026thinsp;Ip group exhibited the highest RPS (78.6%). Nevertheless, oral vaccines are inherently limited by antigen degradation in the harsh acidic gastrointestinal environment, leading to weaker immune responses than conventional vaccines[14]. In contrast, immersion immunization is simple, efficient, and minimally invasive, but generally provides shorter immune protection duration than injection-based vaccination[12].\u003c/p\u003e\n\u003cp\u003eThe emergence of CRISPR/Cas9 technology has revolutionized genome editing due to its high efficiency, simplicity, and specificity. This technology has been widely applied in bacterial genetic manipulation, facilitating both fundamental research and applied development[15\u0026ndash;17].\u003c/p\u003e\n\u003cp\u003eC5a peptidase is a highly conserved surface-exposed serine protease of \u003cem\u003eS. agalactiae\u003c/em\u003e. designated ScpB (streptococcal C5a peptidase from group B \u003cem\u003eStreptococcus\u003c/em\u003e), which is encoded by the \u003cem\u003escpB\u003c/em\u003e gene and secreted to the bacterial surface. ScpB is the largest surface protein identified in GBS and is present in all serotypes[18]. It inhibits neutrophil chemotaxis by cleaving complement component C5a[18], thereby facilitating immune evasion and enhancing bacterial adhesion and invasion[19]. Owing to its surface exposure and functional properties, ScpB is efficiently recognized by the host immune system and exhibits strong intrinsic immunogenicity. Previous studies have demonstrated that ScpB-targeted subunit vaccines confer significant immune protection in tilapia, particularly when combined with advanced delivery systems such as poly(lactic-co-glycolic acid) (PLGA) microspheres [20].\u003c/p\u003e\n\u003cp\u003eSequence analysis of \u003cem\u003eS. agalactiae\u003c/em\u003e strain HN016 (GenBank accession no. CP011325.1) revealed that the C5a peptidase is encoded by the \u003cem\u003escpB\u003c/em\u003e gene with a full-length open reading frame (ORF) of 3703 bp, consisting of two encoded regions: ORF1 (1275 bp) and ORF4 (2403 bp). Among them, the ORF4 open reading frame encodes the large subunit of the C5a peptidase, which plays a central role in antibody recognition and immune protection. Meanwhile, as it does not represent the full-length C5a peptidase, its toxicity is attenuated[21]. In the present study, ORF4-specific primers were designed to amplify the ORF4 fragment from \u003cem\u003eS. agalactiae\u003c/em\u003e genomic DNA. Using CRISPR/Cas9-mediated genome editing, the ORF4 fragment was inserted into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5\u0026alpha; along with a green fluorescent protein (GFP) reporter, resulting in the successful construction of the recombinant strain \u003cem\u003eEscherichia coli DH5\u0026alpha;-ORF4-GFP.\u003c/em\u003e Immunoprotection assays demonstrated that this strain conferred effective protection against GBS infection in tilapia, providing a solid theoretical and technical foundation for its potential application in aquaculture vaccines.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the recombinant strain\u003c/h2\u003e \u003cp\u003eTo construct the plasmid pSC101-PBAD-sgRNA-ORF4-GFP, genomic DNA from \u003cem\u003eE. coli\u003c/em\u003e DH5α and \u003cem\u003eS. agalactiae\u003c/em\u003e strain HN016, together with plasmids pBAV1K-T5-sfGFP and pSC101-PBAD-sgRNA-Donor26D, were used as templates. Using primers listed in Supplementary Material S1, the corresponding gene fragments were successfully amplified (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All fragments were assembled using the pEASY\u0026reg;-Uni Seamless Cloning and Assembly Kit and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α. Recombinant plasmids were extracted using a Plasmid Mini Kit II (OMEGA, USA) and designated as pSC101-PBAD-sgRNA-ORF4-GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-ORF4-GFP were subsequently co-transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α. Following induction, gene-edited colonies were obtained and named \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)(CCTCC M 20252368). PCR amplification using primers HA-F/ORF4-R and sequence analysis confirmed successful genome editing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; sequencing data are shown in Supplementary Material 1 ). Western blot analysis demonstrated that the \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e strain expressed the ORF4-GFP fusion protein at the expected molecular weight of approximately 116.3 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Growth curve analysis showed no significant difference in growth rate between \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e and the parental \u003cem\u003eE. coli\u003c/em\u003e DH5α strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprotection against\u003c/b\u003e \u003cb\u003eStreptococcus agalactiae\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eImmunogenicity of the recombinant strain \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e was evaluated through dose optimization, immunization frequency analysis, and validation experiments to assess protection against \u003cem\u003eS. agalactiae\u003c/em\u003e HN016 infection in tilapia.\u003c/p\u003e \u003cp\u003eIn the dose-selection experiment, fish were immunized once by immersion with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e at concentrations of 1.5 \u0026times; 10\u0026sup2;, 1.5 \u0026times; 10\u0026sup3;, and 1.5 \u0026times; 10⁴ CFU/mL. Unimmunized fish served as the positive control, and fish immunized with \u003cem\u003eE. coli\u003c/em\u003e DH5α served as the bacterial control. At 35 days post-immunization, fish were challenged by intraperitoneal injection with 3 \u0026times; 10⁸ CFU/fish of GBS. Results showed that all \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u0026ndash;immunized groups exhibited significant protective effects compared with controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with protection increasing in a dose-dependent manner. The high-dose group achieved an RPS of 73.12%, whereas the DH5α control group showed an RPS of 0, confirming the absence of anti-GBS activity in the control strain.\u003c/p\u003e \u003cp\u003eBased on the optimal dose, the effect of immunization frequency was evaluated. Fish received 1, 2, or 3 immersion immunizations at 14-d intervals. The corresponding RPS values were 60.00%, 53.30%, and 48.00%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although a declining trend in protection was observed with increasing immunization frequency, statistical analysis indicated no significant differences among groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the validation experiment, a single immersion immunization at 1.5 \u0026times; 10⁴ CFU/mL resulted in an RPS of 65.30% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which was consistent with the dose- and frequency-optimization results and showed no significant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, these results demonstrate that \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e confers effective protection against GBS infection in tilapia, with a single immersion immunization at 1.5 \u0026times; 10⁴ CFU/mL representing the optimal and reliable regimen.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtective efficacy of different immersion doses of \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e live vaccine against \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e in tilapia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable vaccine concentration (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImmersion time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunization times (times)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003edose/fish\u003c/p\u003e \u003cp\u003e(CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003etime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. dead/ No.\u003c/p\u003e \u003cp\u003etotala\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eMortality/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRPS/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDH5α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8/31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e56.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6/31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e67.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5/31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e73.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea: Fish were challenged 35-days post-immunization with 3\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/fish of HN016 strain and monitored for 14 days post challenge. RPS: Relative percent survival. PC:Positive Control Group. NC:Negative Control Group,fish were intraperitoneally injected with 0.2 mL of sterile PBS.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtective efficacy of \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e live vaccine against \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e in tilapia with different immersion immunization frequencies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable vaccine concentration (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImmersion time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunization times (times)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003edose/fish\u003c/p\u003e \u003cp\u003e(CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003etime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. dead/ No.\u003c/p\u003e \u003cp\u003etotala\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eMortality/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRPS/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25/36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e69.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDH5α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22/33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10/36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e60.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12/37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e53.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13/36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e36.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e48.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea: Fish were challenged 7 d post-final immunization with 3\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/fish of HN016 strain and monitored for 14 d post challenge. RPS: Relative percent survival. PC:Positive Control Group. NC:Negative Control Group,fish were intraperitoneally injected with 0.2 mL of sterile PBS.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtective efficacy of \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e live vaccine at 1.5\u0026times;10⁴ CFU/mL with single immersion against \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e in tilapia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable vaccine concentration (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImmersion time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eImmunization times (times)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003edose/fish\u003c/p\u003e \u003cp\u003e(CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChallenge\u003c/p\u003e \u003cp\u003etime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. dead/ No.\u003c/p\u003e \u003cp\u003etotala\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eMortality/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRPS/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19/33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e57.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e3\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7/35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e65.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea: Fish were challenged 15 d post-immunization with 3\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/fish of HN016 strain andmonitored for 14 days post challenge. RPS: Relative percent survival. PC:Positive Control Group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHematological and serum biochemical characteristics\u003c/h3\u003e\n\u003cp\u003eHematological and serum biochemical analyses were performed to compare physiological responses between the immunized group (MY) and the control group (CK). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, significant differences were observed in platelet-related parameters (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Platelet counts (PLT) in the MY group increased by 161.40% compared with the CK group, accompanied by increases in monocyte percentage (+\u0026thinsp;56.01%) and basophil percentage (+\u0026thinsp;88.74%), indicating activation of immune responses. In contrast, erythrocyte-related parameters showed only minor fluctuations, with hemoglobin (HGB) and red blood cell counts (RBC) decreasing by 10.14% and 5.92%, respectively.\u003c/p\u003e \u003cp\u003eSerum biochemical parameters displayed coordinated functional changes (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the MY group, globulin (GLO) levels increased by 25.90%, and alanine aminotransferase (ALT) activity increased by 31.62%, suggesting enhanced immune protein synthesis and hepatic metabolic activity. Meanwhile, cholesterol (CHOL) levels decreased by 16.54%, and glucose (GLU) exhibited only minor variation, indicating a metabolic shift toward supporting immune demands. Although some parameters, such as total protein (TP) and albumin (ALB), showed slight decreases, the overall pattern of immune- and metabolism-associated indicators suggests that immunization activated immune defenses while maintaining general physiological stability in tilapia.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematological parameters(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMY (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYM%(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.24\u0026thinsp;\u0026plusmn;\u0026thinsp;16.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.55\u0026thinsp;\u0026plusmn;\u0026thinsp;12.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMON%(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.04\u0026thinsp;\u0026plusmn;\u0026thinsp;6.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.78\u0026thinsp;\u0026plusmn;\u0026thinsp;15.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNEU%(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.77\u0026thinsp;\u0026plusmn;\u0026thinsp;22.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.41\u0026thinsp;\u0026plusmn;\u0026thinsp;21.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEOS%(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBASO%(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLYM#(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMON#(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5662\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.614\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2583\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNEU#(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEOS#(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBASO#(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC(10\u003csup\u003e12\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHGB(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106.83\u0026thinsp;\u0026plusmn;\u0026thinsp;8.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCT(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCV(fL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e122.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e119.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCH(pg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHC(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e375.00\u0026thinsp;\u0026plusmn;\u0026thinsp;16.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e365.83\u0026thinsp;\u0026plusmn;\u0026thinsp;12.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRDW-CV(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePLT(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMPV(fL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: CK group : Blank Control Group; MY group: Single immersion immunization with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e ( 1.5 \u0026times; 10⁴ CFU/mL).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum biochemical parameters(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCK (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMY (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e30.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALB(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLO(g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.45\u0026thinsp;\u0026plusmn;\u0026thinsp;5.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTBIL(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e39.00\u0026thinsp;\u0026plusmn;\u0026thinsp;18.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e51.33\u0026thinsp;\u0026plusmn;\u0026thinsp;27.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALP(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBUN(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRE(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e39.50\u0026thinsp;\u0026plusmn;\u0026thinsp;17.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e38.83\u0026thinsp;\u0026plusmn;\u0026thinsp;12.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2201.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1040.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2432.83\u0026thinsp;\u0026plusmn;\u0026thinsp;884.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMY(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;12.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLU(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHOL(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP(umol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: CK group : Blank Control Group; MY group: Single immersion immunization with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e ( 1.5 \u0026times; 10⁴ CFU/mL).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMeasurement of antioxidant indices\u003c/h3\u003e\n\u003cp\u003eThe superoxide dismutase (SOD) activities in the PC group and MY+Challenge group were the highest, and were significantly higher than those in the CK group and MY group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The malondialdehyde (MDA) content in the positive control group was significantly higher than that in the CK group and MY group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the MDA content in the MY+Challenge group was lower than that in the PC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePC: Positive Control Group; CK: Blank Control Group; MY: Single immersion immunization group treated with \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e (1.5 \u0026times; 10⁴ CFU/mL); MY+Challenge: Single immersion immunization group immunized with DH5α-ORF4-GFP (1.5\u0026times;10⁴ CFU/mL) and challenged with HN016. (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\n\u003ch3\u003eHistopathological analysis\u003c/h3\u003e\n\u003cp\u003eHistological examination revealed no marked differences in tissue integrity between the MY and CK groups. In both groups, hepatic sinusoidal structures were clearly defined, and no obvious pathological alterations were observed in renal glomeruli or tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Consistent with these observations, serum biochemical indices related to liver and kidney function (e.g., ALT, creatinine[ CRE]) showed no significant abnormalities. These results indicate that vaccination did not induce detectable liver or kidney damage, demonstrating favorable biosafety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eScale bar =\u0026thinsp;20 \u0026micro;m. (HE, \u0026times;400)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of vaccination on intestinal microbiota\u003c/h3\u003e\n\u003cp\u003eGiven that vaccination can modulate intestinal microbial communities and influence host health[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], 16S rRNA gene sequencing was performed to examine the effects of \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e vaccination on the gut microbiota of tilapia. These sequence data have been submitted to the GSA databases under accession number CRA036623. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, no significant differences were observed in Chao or Shannon indices between the MY and CK groups, indicating comparable microbial richness and diversity. However, principal coordinate analysis (PCoA) revealed a clear separation between the two groups, suggesting significant differences in community structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eMicrobial composition was further analyzed at the phylum and genus levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). At the phylum level, the CK group was dominated by Fusobacteriota (45.8%) and Bacillota (27.5%). Compared with the CK group, the MY group showed a reduced abundance of Fusobacteriota (27.3%) and an increased abundance of Pseudomonadota (23.9%). At the genus level, the relative abundance of \u003cem\u003eCetobacterium\u003c/em\u003e decreased from 45.8% (CK group) to 27.3% ( MY group).\u003c/p\u003e \u003cp\u003eLinear discriminant analysis effect size (LEfSe) identified differentially enriched taxa between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In the CK group, o_Enterobacterales, f_Enterobacteriaceae, and g_Ligilactobacillus were significantly enriched, whereas in the MY group, c_Alphaproteobacteria, p_Actinomycetota, and c_Actinobacteria were significantly enriched.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, a CRISPR/Cas9-based strategy was first employed to generate a recombinant \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; strain expressing the ScpB-derived ORF4 antigen, and the successful expression and biological stability of the recombinant strain were confirmed at both the molecular and phenotypic levels.\u003cem\u003eScpB\u003c/em\u003e is a highly conserved virulence determinant among all \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e serotypes, and the encoded C5a peptidase facilitates bacterial invasion by degrading complement C5a and thereby suppressing host immune responses, making it an attractive target for vaccine development[18, 24].In this study, ORF4\u0026mdash;encoding the major subunit of the C5a peptidase\u0026mdash;was predicted to be surface-exposed and immunogenic. Importantly, ORF4 is a truncated form rather than the full-length C5a peptidase, which may reduce potential virulence-associated risks[21]. The functional ORF4 fragment was amplified using specific ORF4-F/R primers and expressed in \u003cem\u003eE.coli\u003c/em\u003e DH5\u0026alpha; via CRISPR/Cas9-mediated genome editing. Compared with conventional homologous recombination or plasmid-based transformation, the high targeting precision of CRISPR/Cas9 reduces unintended events, while GFP labeling enables rapid identification of positive clones. Consequently, the construction cycle was shortened to 5 days, outperforming the 7\u0026ndash;10 days typically required by traditional methods [25], and providing a practical basis for scalable vaccine preparation.\u003c/p\u003e\n\u003cp\u003eConsistent with the immunization and challenge results, the \u003cem\u003eDH5\u0026alpha;-ORF4-GFP\u003c/em\u003e vaccine conferred moderate but stable protection via a single immersion immunization, highlighting the feasibility of non-invasive delivery for GBS control in tilapia. Evaluation of protective efficacy is central to vaccine development. Relative to existing strategies, the \u003cem\u003eDH5\u0026alpha;-ORF4-GFP\u003c/em\u003e vaccine exhibited several advantages. Although inactivated vaccines are generally considered safe, their RPS values are often limited to ~\u0026thinsp;50\u0026ndash;65%[26, 27] and typically rely on injection, which is labor-intensive and may induce stress responses in fish. Live attenuated vaccines can achieve RPS values\u0026thinsp;\u0026gt;\u0026thinsp;75% [28], yet concerns regarding reversion to virulence constrain their use, particularly under high-density farming conditions. In contrast, \u003cem\u003eDH5\u0026alpha;-ORF4-GFP\u003c/em\u003e delivered by immersion conferred a maximal RPS of 73.12%, and the validation assay yielded a stable RPS of 65.26% (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), without reversion risk, while remaining operationally simple and cost-manageable for large-scale aquaculture. Notably, the protection level was lower than that reported for the attenuated vaccine developed by Zhang et al. (RPS\u0026thinsp;=\u0026thinsp;90.47%) [8]. Future work may explore adjuvant strategies (e.g., fucoidan or IL-11) to enhance antigen presentation and further improve protective performance [27, 29, 30].\u003c/p\u003e\n\u003cp\u003eAn observation warranting further discussion is that during immunization frequency optimization, a \u0026ldquo;single-dose outperforming multiple-dose\u0026rdquo; trend was noted (RPS: 1 dose, 60.00%; 2 doses, 53.30%; 3 doses, 48.00%), although differences were not statistically significant. This pattern may be linked to mucosal immunity characteristics. Immersion immunization primarily stimulates mucosa-associated lymphoid tissues (MALT). Repeated antigen exposure at short intervals may promote CD103\u003csup\u003e+\u003c/sup\u003e dendritic cells to secrete TGF-\u0026beta; and retinoic acid, thereby driving regulatory T cell (Treg) expansion [31, 32]. Such responses can dampen effector T-cell activation, resulting in a gradual decline in protective immunity. These findings suggest that mucosal vaccines in aquaculture should avoid a potential \u0026ldquo;immune tolerance trap\u0026rdquo; and that increasing booster frequency does not necessarily improve protection. In future studies, antibody titers and memory lymphocyte proportions at 7, 14, and 30 days post-immunization should be monitored to define an optimal immunization window, thereby enabling evidence-based vaccination schedules for farming practice.\u003c/p\u003e\n\u003cp\u003eHematological, biochemical, antioxidant indices and histopathological analysis further confirmed that the DH5\u0026alpha;-ORF4-GFP vaccine was biosafe under the optimized immunization protocol. In teleost immunity, thrombocytes contribute not only to hemostasis but also to innate defense via pathogen adhesion and the release of antimicrobial peptides and chemotactic mediators [33]. In the present study, only platelet counts (PLT) were significantly elevated in the MY group ( 161.40% increase vs. CK group; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while other parameters showed no significantly differences.In the present study, the SOD activity and MDA content in the PC group were significantly increased compared with the CK group, which was consistent with the typical changes of oxidative stress in tilapia infected with Streptococcus agalactiae[34]. In addition, the MDA content in the MY+Challenge group was lower than that in the PC group, while the SOD activity remained at a high level comparable to the PC group, suggesting that vaccination could enhance the stress resistance of tilapia by regulating oxidative balance.Meanwhile, there were no significant differences in MDA content and SOD activity between the MY group and the CK group, indicating that the recombinant vaccine itself had no adverse effects on the oxidative balance of tilapia and did not induce additional oxidative stress. Together with histological observations of no overt pathological lesions in the liver and kidney, these data support the vaccine\u0026rsquo;s biosafety and suggest no apparent adverse impact on tilapia health.\u003c/p\u003e\n\u003cp\u003eAlthough the \u003cem\u003eDH5\u0026alpha;-ORF4-GFP\u003c/em\u003e vaccination did not significantly alter overall microbial richness or diversity, the observed shift in community structure suggests a potential indirect interaction between vaccination and intestinal microbial composition.Accumulating evidence indicates that the gut microbiota is closely associated with fish immune function, and enrichment of beneficial taxa can indirectly enhance host resistance via competitive exclusion, reinforcement of mucosal barriers, and modulation of local immune responses [35\u0026ndash;37]. In this study, vaccination did not significantly affect alpha diversity (richness/diversity) but significantly reshaped community composition (distinct PCoA separation). The MY group showed increased relative abundance of taxa such as c_Alphaproteobacteria and p_Actinomycetota, implying that \u003cem\u003eDH5\u0026alpha;-ORF4-GFP\u003c/em\u003e may not only elicit antigen-specific immunity but also positively modulate the intestinal micro-ecosystem via a \u0026ldquo;microbiota\u0026ndash;immunity\u0026rdquo; axis, thereby providing an additional protective barrier [37, 38].\u003c/p\u003e\n\u003cp\u003eDespite demonstrating the potential of a CRISPR/Cas9-edited vaccine strategy, several limitations should be acknowledged. First, the observation period for protective efficacy was relatively short; the durability of vaccine-induced immune memory should be evaluated in long-term farming trials (\u0026ge;\u0026thinsp;3\u0026ndash;6 months), with challenge tests and antibody monitoring at multiple time points to determine long-lasting protection. Second, mechanistic investigations did not include immune-related gene expression (e.g., \u003cem\u003eIL-1\u0026beta;, TNF-\u0026alpha;, IgM\u003c/em\u003e), limiting molecular-level interpretation of immune regulation. Future studies integrating transcriptomics and proteomics could clarify the signaling pathways by which this vaccine activates innate and adaptive immunity in tilapia. Third, laboratory conditions differ from the complex environments of production ponds (water quality fluctuations, climate, and diverse microbial communities); therefore, field-scale performance should be validated under commercial settings. Fourth, protection was assessed only against \u003cem\u003eS. agalactiae\u003c/em\u003e strain HN016, and cross-protection against other serotypes remains unknown. Given the high conservation of \u003cem\u003eScpB\u003c/em\u003e among serotypes [18], broader protection is plausible; nevertheless, challenges with multiple serotypes are required to define the breadth of cross-protection and to enhance practical value.\u003c/p\u003e"},{"header":"Conclusions","content":" \u003cp\u003eUsing CRISPR/Cas9-mediated genome editing, we successfully constructed recombinant \u003cem\u003eE. coli\u003c/em\u003e DH5α-ORF4-GFP expressing the \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e ScpB-derived ORF4 antigen, with the optimal immunization regimen identified as a single immersion at 1.5 \u0026times; 10⁴ CFU/mL. This regimen elicited effective protection against GBS infection in tilapia. Hematological, biochemical and gut microbiota analyses collectively confirmed the feasibility and application potential of this recombinant strain as a vaccine candidate against \u003cem\u003eS. agalactiae\u003c/em\u003e in tilapia aquaculture, providing an alternative technical strategy for the immunoprophylaxis of streptococcosis.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e strain HN016 (GenBank accession no. CP011325.1), \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α (GenBank accession no. CP026085), recombinant ORF4 protein, and mouse-derived polyclonal antibodies against ORF4 were provided by the Clinical Laboratory of the College of Animal Science and Technology, Guangxi University. Plasmid pBAV1K-T5-sfGFP was purchased from HonorGene (HG-VYH1347). Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-Donor26D were obtained from Shandong Qibang Biotechnology Co., Ltd. Nile tilapia (\u0026ldquo;Hainan No. 1\u0026rdquo;; average body weight\u0026thinsp;\u0026asymp;\u0026thinsp;20 g) were purchased from a commercial hatchery in Nanning, Guangxi, China. Fish were confirmed to be free of bacterial infection by bacteriological examination of brain and kidney tissues and were acclimated in disinfected tanks (7.0 m \u0026times; 1.5 m \u0026times; 1.2 m) with continuous aeration and daily feeding of a commercial diet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eRecombinant plasmids were generated using the pEASY\u0026reg;-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). DNA polymerases and PCR reagents were purchased from Takara Bio (Beijing, China). The sgRNA was designed using the sgRNAcas9 v3.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.biootools.com/\u003c/span\u003e\u003cspan address=\"https://www.biootools.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Primer were designed using Primer Premier 5.0, and primer sequences are listed in Supplementary Material Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Primer synthesis, gene synthesis, and DNA sequencing were conducted by Tsingke Bio (Beijing, China) and BGI Genomics (Guangdong, China).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe pSC101-PBAD-sgRNA-ORF4-GFP vector was constructed according to the method described by Huang et al.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. genomic DNA from \u003cem\u003eS. agalactiae\u003c/em\u003e HN016 was used as a template to amplify a 408 bp promoter fragment ( primers QDZ-F/R) and a 2432 bp ORF4 fragment ( primers ORF4-F/R). Genomic DNA from \u003cem\u003eE. coli\u003c/em\u003e DH5α was used to amplify a 512 bp left homology arm (LHA) and a 539 bp right homology arm (RHA) with primers LHA-F/R and RHA-F/R, respectively. The GFP gene (757 bp) was amplified from plasmid pBAV1K-T5-sfGFP using primers GFP-F/R. Five additional fragments (2127, 367, 301, 1118, and 341 bp) were amplified from pSC101-PBAD-sgRNA-Donor26D using primers yw1-5. All ten fragments were purified and assembled by isothermal assembly to generate the final plasmid pSC101-PBAD-sgRNA-ORF4-GFP. Plasmid sequences are provided in Supplementary Material 1.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of recombinant strains\u003c/h2\u003e \u003cp\u003eThe construction of recombinant strains followed the procedure illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Competent \u003cem\u003eE. coli\u003c/em\u003e DH5α cells were prepared according to the protocol described by Rachel et al.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Plasmids p15A-PBAD-Cas9-PT5-Redγβα and pSC101-PBAD-sgRNA-ORF4-GFP were co-transformed into competent cells via heat shock at 42\u0026deg;C. Following recovery in LB medium at 30\u0026deg;C for 1 h, transformed cells were plated onto LB agar containing ampicillin (100 \u0026micro;g/mL) and kanamycin (50 \u0026micro;g/mL) and incubated at 30\u0026deg;C for approximately 48 h.\u003c/p\u003e \u003cp\u003eInduction and screening of positive clones were performed with slight modifications to the method described by Huang et al.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Single colonies were cultured in LB medium, induced with IPTG and L-arabinose, and subsequently screened on kanamycin-containing plates. Colonies exhibiting green fluorescence were selected for PCR verification. The successfully edited strain was designated \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGrowth curve assay\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe target strains were cultured in LB broth at 37\u0026deg;C with shaking (180 rpm) to the exponential phase, inoculated (1%, v/v) into fresh LB, and incubated under the same conditions. Samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, and 24 h. Optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was measured using uninoculated LB as a blank. Three independent replicates were performed for each time point, and mean values were used for analysis.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and Western blotting\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBacterial cells were harvested by centrifugation (6000 \u0026times;g, 10 min, 4\u0026deg;C), washed, and resuspended in 1\u0026times; PBS. Cells were lysed by ultrasonication and clarified by centrifugation (10,000 \u0026times;g, 10 min, 4\u0026deg;C). Total protein concentration was determined using the Bradford method. Samples were mixed with 6\u0026times; loading buffer and denatured at 95\u0026deg;C for 10 min, separated by SDS\u0026ndash;PAGE (4% stacking gel; 80 V, 2 h), and transferred onto nitrocellulose (NC) membranes using a semi-dry transfer system. Membranes were blocked with 5% (w/v) non-fat milk and incubated with mouse polyclonal anti-ORF4 antibody, followed by HRP-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (ABclonal, China). Signals were visualized using BeyoECL Star (Beyotime, China).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunization and challenge experiments\u003c/h2\u003e \u003cp\u003eBased on the preliminary experimental results, no significant difference was observed in the protective efficacy among intraperitoneal injection, oral gavage, and immersion administration routes (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Meanwhile, bacterial virulence tests revealed that immunization with the vaccine at a concentration of 1.5\u0026times;10⁵ CFU/mL resulted in mortality in a small number of tilapia (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Therefore, the immersion administration route was adopted for all subsequent experiments. All animal experiments were conducted in strict compliance with standardized procedures and obtained approval from the Animal Ethics Review Committee of Guangxi University (Approval No.: GXU-2025-282).\u003c/p\u003e \u003cp\u003e \u003cb\u003e1. Dose optimization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTilapia were acclimated for 5 d in a recirculating system (28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C; dissolved oxygen\u0026thinsp;\u0026ge;\u0026thinsp;5 mg/L; pH 7.0\u0026ndash;7.5; ammonia nitrogen\u0026thinsp;\u0026le;\u0026thinsp;0.05 mg/L) and fasted for 24 h prior to immunization. Fish were assigned to six groups (\u0026ge;\u0026thinsp;30 fish/group): three \u003cem\u003eDH5α-ORF4-GFP\u003c/em\u003e immersion-immunized groups (1 h) at concentrations of 1.5 \u0026times; 10\u0026sup2;, 1.5 \u0026times; 10\u0026sup3;, or 1.5 \u0026times; 10⁴ CFU/mL; one \u003cem\u003eE. coli\u003c/em\u003e DH5α immersion-immunized group (1 h) at 1.5 \u0026times; 10\u0026sup3; CFU/mL (empty vector control);a positive control (unimmunized, challenged); and a blank control (unimmunized, unchallenged). At 35 d post-immunization, all groups except the blank control were challenged by intraperitoneal injection of GBS (1.5 \u0026times; 10⁹ CFU/mL; 0.2 mL/fish). Mortality was recorded for 14 d, and cumulative mortality and relative percent survival were calculated (RPS = [1 \u0026minus; (mortality of immunized group/mortality of positive control)] \u0026times; 100%).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. Immunization frequency\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUnder the same rearing conditions and immersion procedure, fish were allocated to six groups (\u0026ge;\u0026thinsp;30 fish/group):three DH5α-ORF4-GFP immunized groups receiving 1, 2, or 3 immersion vaccinations at 14-d intervals (1.5 \u0026times; 10⁴ CFU/mL; 1 h); one DH5α immunized group receiving a single immersion vaccination (1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU/mL; 1 h); and corresponding controls (positive and blank). At 7 d after the final immunization, fish (except the blank control) were challenged as described above, and mortality was monitored for 14 d to calculate RPS.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Confirmation of the optimal regimen\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFish were divided into an immunized group (single immersion at 1.5 \u0026times; 10⁴ CFU/mL) and a blank control (\u0026ge;\u0026thinsp;30 fish/group). After 14 d of rearing at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, six fish per group were sampled. The remaining fish were then challenged as described above, and mortality was recorded for 14 d to calculate RPS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHematology and serum biochemistry\u003c/h2\u003e \u003cp\u003eFor the groups in Confirmation of the optimal regimen, fish were anesthetized by immersion in MS-222 (Tricaine Methanesulfonate, 100 mg/L) prior to blood collection from the caudal vein as previously described[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. For hematology, whole blood was mixed with EDTA-K2 anticoagulant and analyzed within 2 h using an automated five-part differential hematology analyzer. For serum biochemistry, 200 \u0026micro;L serum was transferred to sterile tubes and analyzed using an automated biochemical analyzer (Celercare\u0026reg; M5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of antioxidant indices\u003c/h2\u003e \u003cp\u003eSerum superoxide dismutase (SOD) and malondialdehyde (MDA) in tilapia were measured using commercial kits, which were purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. Serum samples were collected as described in the previous section (6 samples per group). All detection procedures were performed strictly in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eHistological procedures followed Liu et al.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, liver and kidney tissues (three fish per group from the optimal regimen group) were collected after euthanasia by overdose immersion in MS-222 (200 mg/L) until opercular movement ceased, then fixed in 4% paraformaldehyde, paraffin-embedded, sectioned at 6 \u0026micro;m, and stained with hematoxylin and eosin (H\u0026amp;E). Images were acquired using a digital slide scanner (LG-S80, Servicebio, China). Histological evaluation was performed using a semi-quantitative scoring system adapted from Bernet et al.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and Rey et al.[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Liver and kidney sections were examined for the presence and severity of pathological alterations, including hepatocellular vacuolation, necrosis, inflammatory infiltration, renal tubular degeneration, and glomerular abnormalities. Each lesion was scored on a scale of 0 to 3 (0\u0026thinsp;=\u0026thinsp;absent, 1\u0026thinsp;=\u0026thinsp;mild, 2\u0026thinsp;=\u0026thinsp;moderate, 3\u0026thinsp;=\u0026thinsp;severe) as described previously in tilapia studies[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota analysis\u003c/h2\u003e \u003cp\u003eIntestinal contents were collected from six fish per group (from the optimal regimen group) following euthanasia by overdose immersion in MS-222 (200 mg/L). Total genomic DNA was extracted using the E.Z.N.A.\u0026reg; Soil DNA Kit (OMEGA). The V3\u0026ndash;V4 region of bacterial 16S rRNA genes was amplified using primers 338F (5\u0026prime;-ACTCCTACGGGAGGCAGCA-3\u0026prime;) and 806R (5\u0026prime;-GGACTACHVGGGTWTCTAAT-3\u0026prime;). Amplicons were quantified using a Quant-iT PicoGreen dsDNA assay (Microplate reader; BioTek FLx800) and sequenced on an Illumina NovaSeq platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.) for absolute abundance profiling. Paired-end reads were processed using QIIME 2. Downstream analyses, including alpha diversity, principal coordinate analysis (PCoA), and linear discriminant analysis (LDA), were conducted using the Majorbio Cloud(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.majorbio.com\u003c/span\u003e\u003cspan address=\"https://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed in GraphPad Prism 8.0 (GraphPad Software, USA). Differences among groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple-comparisons test. Values with different lowercase letters in the same row indicate significant differences among treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAbbreviation\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFull name\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS-222\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTricaine Methanesulfonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA/G\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlbumin globulin ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlbumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlkaline phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine amiotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSerum amylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBas#\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAbsolute value of basophils\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBas%\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasophil proportion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCalcium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHOL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatine kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEos#\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAbsolute value of eosinophils\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEos%\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEosinophil ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlobulin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematocrit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHemoglobin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLym#\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAbsolute value of lymphocytes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean corpuscular hemoglobin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean corpuscular hemoglobin concentratiom\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean corpuscular volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMon#\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonocyte absolute value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMon%\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonocyte ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMean platelet volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeu#\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutrophil\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeu%\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutrophil ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlateletocrit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet distribution width\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet count\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRed blood cell count\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRDW-CV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eErythrocyte variation coefficient\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRDW-SD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation of distribution width\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal bilirubin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhite blood cell count\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eAll animal experiments were conducted in strict compliance with standardized procedures and obtained approval from the Animal Ethics Review Committee of Guangxi University (Approval No.: GXU-2025-282). All the methods and procedures of this study comply with the requirements of the ARRIVE Guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e The manuscript has been given publication permission by all authors.Not applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship and publication of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe work was supported by grants from the Fangchenggang Science and Technology Program (AB22013007) and Guangxi Science and Technology Program (AA17204081-1).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**MH** : Conceptualization, Methodology, Investigation, Writing \u0026ndash; original draft. **XL** : Resources, Validation. **TP** : Formal analysis, Data curation. **DW** : Formal analysis, Data curation. **GL** : Investigation, Visualization. **WW** : Supervision, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the staff of the College of Animal Science and Technology, Guangxi University, for their technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics \u0026amp; Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA036623) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA036623.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu DH, Shoemaker CA, Klesius PH. 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VACCINES-BASEL. 2023;11(12):1811.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChai R, Sun W, Xu Z, Yao X, Chen S, Wang H, Guo J, Zhang Q, Yang Y, Li T, et al. Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria. INT J BIOL MACROMOL. 2024;278:135065.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamos-Espinoza FC, Cueva-Quiroz VA, Yunis-Aguinaga J, de Moraes JRE. A comparison of novel inactivation methods for production of a vaccine against Streptococcus agalactiae in Nile tilapia Oreochromis niloticus. \u003cem\u003eAQUACULTURE\u003c/em\u003e 2020, 528:735484.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWangkahart E, Thongsrisuk A, Vialle R, Pholchamat S, Sunthamala P, Phudkliang J, Srisapoome P, Wang T, Secombes CJ. Comparative study of the effects of Montanide\u0026trade; ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus). FISH SHELLFISH IMMUN. 2023;134:108563.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa T, Li F, Wu C, Lin J, Chen M, Zhang Y, Zeng H. Evaluation of MarR-deleted Streptococcus agalactiae as a live-attenuated vaccine candidate for Nile Tilapia (Oreochromis niloticus). \u003cem\u003eAQUACULTURE\u003c/em\u003e 2026, 612:743195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuha R, Lakshmi S, Krebs T, Schroers VJ, Adamek M, Elumalai P. Efficacy of a novel oral bivalent vaccine with fucoidan as adjuvant against Aeromonas hydrophila and Edwardsiella tarda infections in Nile tilapia aquaculture. FISH SHELLFISH IMMUN. 2026;169:111074.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSunthamala P, Wang T, Phadee P, Luang-In V, Srisapoome P, Zou J, Secombes CJ, Wangkahart E. IL-11 acts as a vaccine adjuvant in Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae, enhancing both cellular and humoral immune responses. FISH SHELLFISH IMMUN. 2026;168:110917.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim KS, Surh CD. Induction of Immune Tolerance to Dietary Antigens. ADV EXP MED BIOL. 2015;850:93\u0026ndash;118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoombes JL, Siddiqui KRR, Arancibia-C\u0026aacute;rcamo CV, Hall J, Sun C, Belkaid Y, Powrie F. A functionally specialized population of mucosal CD103\u0026thinsp;+\u0026thinsp;DCs induces Foxp3\u0026thinsp;+\u0026thinsp;regulatory T cells via a TGF-β\u0026ndash; and retinoic acid\u0026ndash;dependent mechanism. J Exp Med. 2007;204(8):1757\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtiz M, Esteban M\u0026Aacute;. Biology and functions of fish thrombocytes: A review. FISH SHELLFISH IMMUN. 2024;148:109509.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Li D, Xiao W, Yu J, Chen B, Zou Z, Yang H. Survival, serum biochemical parameters, hepatic antioxidant status, and gene expression of three Nile tilapia strains under pathogenic Streptococcus agalactiae challenge. FISH SHELLFISH IMMUN. 2024;155:110030.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinmeyer S, Lee K, Jayaraman A, Alaniz RC. Microbiota Metabolite Regulation of Host Immune Homeostasis: A Mechanistic Missing Link. CURR ALLERGY ASTHM R. 2015;15(5):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmes E, Li JV, Athanasiou T, Ashrafian H, Nicholson JK. Understanding the role of gut microbiome\u0026ndash;host metabolic signal disruption in health and disease. TRENDS MICROBIOL. 2011;19(7):349\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Q, Feng Y, Wang H, Zhu K, Teng L, Yue M, Li Y. Gut microbiota as a regulator of vaccine efficacy: implications for personalized vaccination. Gut Microbes. 2025;17(1):2563709.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutra J, Hashim AM, Yusof MT, Al-Saari N, Nasruddin NS, Saad MZ, Zahaludin A, Yasin ISM, Azmai MNA. Dynamics of the gut microbiome of Asian seabass (Lates calcarifer) following oral vaccination and challenge with virulent Vibrio harveyi. \u003cem\u003eAQUACULTURE\u003c/em\u003e 2025, 603:742420.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang C, Guo L, Wang J, Wang N, Huo Y. Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering. APPL MICROBIOL BIOT. 2020;104(18):7943\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang S, Li H, Zhang L, Mu W, Zhang Y, Chen T, Wu J, Tang H, Zheng S, Liu Y, et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. NUCLEIC ACIDS RES. 2025;53(D1):D1670\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen R, Rogers EJ. Transformation of Chemically Competent \u003cem\u003eE.coli\u003c/em\u003e. Methods in Enzymology. Volume 529. Elsevier; 2013. pp. 329\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamos-Espinoza FC, Cueva-Quiroz VA, Yunis-Aguinaga J, Alvarez-Rubio NC, Paganoti De Mello N, Engr\u0026aacute;cia, De Moraes JR. Efficacy of two adjuvants administrated with a novel hydrogen peroxide-inactivated vaccine against Streptococcus agalactiae in Nile tilapia fingerlings. \u003cem\u003eFISH SHELLFISH IMMUN\u003c/em\u003e 2020, 105:350\u0026ndash;358.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Zhang Y, Ma J, Jiang N, Fan Y, Zhou Y, Cain K, Yi M, Jia K, Wen H, et al. Determination of a novel parvovirus pathogen associated with massive mortality in adult tilapia. PLOS PATHOG. 2020;16(9):e1008765.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernet D, Schmidt H, Meier W, Burkhardt-Holm P, Wahli T. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J FISH DIS. 1999;22(1):25\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRey AL, As\u0026iacute;n J, Ruiz Zarzuela I, Luj\u0026aacute;n L, Iregui CA, de Blas I. A proposal of standardization for histopathological lesions to characterize fish diseases. REV AQUACULT. 2020;12(4):2304\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahboub HH, Rahman ANA, Elazab ST, Abdelwarith AA, Younis EM, Shaalan M, Aziz EK, Sobh MS, Yousefi M, Ismail SH, et al. Nano-chitosan hydrogel alleviates Candida albicans-induced health alterations in Nile tilapia (Oreochromis niloticus): antioxidant response, neuro-behaviors, hepato-renal functions, and histopathological investigation. BMC VET RES. 2025;21(1):159.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CRISPR-Cas9, Streptococcus agalactiae, tilapia, vaccine, immune protection","lastPublishedDoi":"10.21203/rs.3.rs-9209433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9209433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStreptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant E. coli DH5α-ORF4-GFP (targeting GBS ScpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimal regimen was single immersion at 1.5×10⁴ CFU/mL, with a maximum RPS of 73.12% and stable 65.26% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and enhanced globulin synthesis, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not change intestinal microbial richness but optimized its structure, enriching beneficial taxa like Alphaproteobacteria via the microbiota-immunity axis, enhancing host’s defense capacity via the \"microbiota-immunity\" axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study successfully developed a highly effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.\u003c/p\u003e","manuscriptTitle":"CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 19:59:22","doi":"10.21203/rs.3.rs-9209433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-20T12:07:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T08:55:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T07:36:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T03:38:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171195590887114144390088918328270064773","date":"2026-04-10T01:12:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211337583658030725086859644613485445774","date":"2026-04-06T22:56:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297274817930854722083979448646967045057","date":"2026-04-04T04:54:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-03T07:45:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T07:32:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-31T17:03:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T10:15:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-03-30T10:04:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27b3bc20-0527-4d9c-92a0-062e7fe9b078","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T05:54:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 19:59:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9209433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9209433","identity":"rs-9209433","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}