The Immune Response and Passive Protective Abilities of the Outer Membrane Lipoprotein Slp of Aeromonas hydrophila against the Major Pathogenic Bacteria of Freshwater Aquaculture in Fish

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INTRODUCTION
A eromonas hydrophila is a gram-negative bacillus with a single flagellum that belongs to a species of O n l i n e F i r s t A r t i c l e freshwater aquaculture. They are also zoonotic pathogens and can infect animals and humans (Nayak, 2020;Wang et al., 2020). Antibiotics are the primary drugs used to prevent and treat A. hydrophila and P. fluorescens infections in freshwater aquaculture fish (Bilen and Elbeshti, 2019;González-Renteria et al., 2020), but the abuse of antibiotics will inevitably lead to bacterial resistance, drug residues, and environmental pollution . Many preventative treatments have been researched for fish bacterial diseases, including whole-cell inactivated vaccines, live attenuated vaccines, nucleic acid vaccines, and genetic engineering, but these are still at the experimental stages, and the need to develop new vaccines to prevent and treat aquatic pathogenic bacterial infections in fish.
The outer membrane proteins (OMPs) of Gramnegative bacteria are located in the outermost layer of the bacteria and are important virulence factors , helping to maintain cellular integrity, signal transduction, transport nutrients, metabolize energy, promote the formation of biofilms (Egan, 2018;Silva et al., 2020), and contribute to the adhesion and colonization of bacteria in the host (Bonsor and Sundberg, 2019;Vaca et al., 2020). OMPs are also relatively easily recognized by the host's immune system, and stimulate an immune response for locating on the surface of the bacteria (Cole et al., 2021). Thus, many OMPs have high immunogenicity for potential candidate vaccine proteins, including A, C, F, and H bacterial OMPs (Pore and Chakrabarti, 2013;Liko et al., 2018;Diao et al., 2020;Nie et al., 2020), which can improve the resistance of animals to bacterial infection.
The outer membrane lipoprotein Slp of A. hydrophila is located in the outermost layer of the bacteria and has an eight-stranded beta-barrel domain (Hooda et al., 2017), which contributes to adhesion in the host (Fedorchuk et al., 2019), bacterial antibiotic resistance, and bacterial virulence (Hooda et al., 2016;Price and St-John, 2000). Slp may also be a prime candidate for vaccine development (Hooda et al., 2017), but its immune responses and passive protective abilities are unclear.
In the current study, A. hydrophila Slp protein was obtained by molecular cloning, expression, and purification, and Slp mouse serum was prepared and its immune function analyzed. The passive protective activity was evaluated by immunizing Carassius auratus with Slp mouse serum, challenging with A. hydrophila and P. fluorescens, and analyzing the protection rate, immunerelated factors, inflammation-related genes, antioxidantrelated factors, and pathological sections. This research is expected to provide the theoretical basis for Slp vaccine development in fish.

Animals and bacterial strains
C. auratus were purchased from Shanghai Original Ecological Aquarium Co. Ltd. (Shanghai, China), and Kunming mice were purchased from Chongqing Tengxin Biotechnology Co. Ltd. (Chongqing, China). All animal procedures were performed in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Ethics Committee, Shaanxi University of Technology, Hanzhong, China (No. 2020-08).
A. hydrophila ATCC7966, P. fluorescens ATCC13525, Escherichia coli BL21, E. coli DH5α and pET32a plasmid were all preserved in the biochemistry and molecular laboratory of Shaanxi University of Technology.

Construction, expression and purification of Slp
The slp gene recombinant strain was constructed by molecular cloning. Briefly, according to the slp gene sequence (GenBank: ABK38004.1) from the NCBI database, primers were designed to amplify the slp gene using the PCR method: F-slp: AGGGAATTCATGTTGAAGCGTGCAATG; R-slp: T A A C T C G A G C T A G C G G C G C AGCGGCCGT (the underlined position is the restriction site of EcoR I and Xho I, respectively). The genome of A. hydrophila was extracted according to the instructions of the bacteria genomic DNA extraction kit (Takara, Dalian, China), which was used as a template to amplify the slp gene by PCR following the instructions of the rTaq polymerase kit (Takara, Dalian, China). The PCR process consisted of 30 cycles of pre-denaturing for 3 min at 94 °C, denaturing for 30 s at 94 °C, annealing for 45 s at 55 °C and extension for 90 s at 72 °C, followed by abundant extension for 10 min at 72 °C. The PCR samples were digested with EcoR I and Xho I and connected to pET-32a plasmid by T4-DNA ligase (Takara, Dalian, China). Then, they were transformed to E. coli DH5a to amplify slp recombinant plasmid, which was further identified by restriction enzyme digestion and gene sequencing identify O n l i n e F i r s t A r t i c l e (Vosun Biotechnology Co. Ltd., Xian, China). Finally, the recombinant plasmid was transformed into E. coli BL21 to construct a slp gene prokaryotic expression strain (Liu et al., 2018). The Slp protein was expressed and purified as previously described (Chen et al., 2019). Briefly, the recombinant Slp strain was cultured overnight at 37℃ and transferred to 600 mL fresh LB medium until bacterial concentration reached 0.6 (OD 600 nm). Then, isopropylβ-D-thiogalactoside (IPTG) (Sigma, St. Louis, USA) was added and induced at 20 ℃ for 24 h to express Slp protein. After centrifugation, bacteria were disrupted with ultrasonic crushing, and Slp was purified with Ni-NTA flow resin (Sigma, St. Louis, USA).

The optimal expression conditions of Slp
The expression conditions of Slp was optimized by an orthogonal experimental design L 9 (3 4 ) as previously described (Chen et al., 2019). Briefly, the four-factor parameters are the strain-inducing concentration of OD 600 value (A), IPTG inducing concentration (B), inducing time (C) and inducing temperature (D); the three-level parameters are shown in Table II. According to the L 9 (3 4 ) model, once bacteria were cultured to the designed OD 600 value, different concentrations of IPTG were added to induce Slp expression at settings temperature and time. Next, 2×SDS protein loading buffer (300 μL) was added after the bacterial solution (1 mL) was harvested by centrifugation; it was then heated for 5 min in boiling water. Next, 10 μL samples were used for SDS-PAGE gel electrophoresis, and the Slp expression was visualised by G-250 staining. The density of Slp bands was analysed using Phoretix 1D software to assess the density dataset. Range analysis and SPSS software were employed to analyse optimal expression conditions.

Preparation, specificity, titre and immune-related factors analysis of Slp polyclonal antibody
Mice were immunized with Slp (50 µg per mouse) to prepare the polyclonal antibody. Freund's complete adjuvant (Sigma, St. Louis, USA) was used in the first immunization, and the second and third immunizations consisted of Freund's incomplete adjuvant (Sigma, St. Louis, USA). The second immunization occurred at 14-day intervals, and the third immunization occurred at 7-days intervals. Seven days after the third immunization, mice plasma was collected from the eyeballs under anaesthesia. The serum was harvested with 3000 r/min centrifugation and stored at 4 ℃ overnight.
The specificity and titre of the polyclonal antibody were analysed using Western blotting as previously described . Briefly, A. hydrophila was harvested overnight culture for SDS-PAGE gel electrophoresis, and the proteins were transferred from SDS-PAGE gel to the NC membrane at 80 V for 1 h. Skim milk (5%) was used to block the NC membrane for 2 h at room temperature. The serum of different dilutions (1: 800, 1: 1600, 1: 3200) was added to the NC membrane and incubated at 37 °C for 1 h, and received blank serum (NC) as a control. Then, the NC membrane was incubated with secondary goat anti-mouse antibodies (Sigma, St. Louis, USA), and a dimethylaminoazobenzene (DAB) substrate system (Sigma, St. Louis, USA) was employed to visualise bands.
The immune-related factors of lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) were assessed in serum according to the manufacturer's instructions (Jiancheng Institute of Biotechnology Co. Ltd., Nanjing, China).

Analysis of immune recognition between antiserum and bacteria
The recognition between the antiserum and bacteria was identified using ELISA (Liu et al., 2021). Briefly, bacteria were harvested after being cultured overnight and were inactivated with oxymethylene (1%, W/V), and the bacterial concentration was adjusted to 6×10 8 CFU/ mL with normal saline. The bacteria and antiserum at the various dilutions were mixed and added to 1.5 mL tubes to incubate at 37 °C for 1 h. After the second antibody was added, the samples were suspended with PBS solution (20 μL) and transfer to a 96-well enzyme-linked plate. After the addition of colouration liquid (50 µL TMB and 50 µL H 2 O 2 ) and 50 μL stop solution (2 M H 2 SO 4 ) to the wells, the absorbance values were analysed using a microplate reader at OD 450 nm (Bio-Rad, Hercules, USA).

Passive protective ability of Slp mouse serum in C. auratus
The passive protective ability of Slp mouse serum was evaluated using C. auratus immunized with serum and challenged with A. hydrophila and P. fluorescens (Liu et al., 2018). Briefly, C. auratus were divided into four groups with 15 fish per group. Groups 1 and 2 were intraperitoneally immunized with 30 μL Slp serum, and groups 3 and 4 received blank serum (NC) as a control. After 2 h, fish of groups 1 and 3 were challenged with A. hydrophila (3 × 10 8 CFU), and groups 2 and 4 with P. fluorescens (1.5 × 10 9 CFU). Then, fish mortality was counted for 14 days, and the immune protection rate (RPS) of C. auratus was calculated using the formula RPS (%) = [1-(% vaccinated mortality/% control mortality)] × 100. SPSS software was used to analyse the statistical significance.  Leukocyte phagocytosis of plasma Leukocyte phagocytosis was performed as follows: 2 mL of S. aureus (6×10 8 CFU/mL) and C. auratus plasma were incubated at 25℃ for 60 min; 10 µL mixture solution was drawn to glass slide; and methanol was added. After Giemsa dyeing solution performed for 30 min, the slide was washed and air dried for observation with an oil microscope. The phagocytic percentage (PP %) was obtained using the following formula: WBCs involved phagocytosis of 100 WBCs / 100 ×100%, and phagocytic index (PI %): Bacteria phagocytised/ WBCs phagocytising bacteria. The results were analysed the ANOVA method, and the significance was determined using SPSS19.0 software.

Analysis of inflammation-related gene expression using qRT-PCR
After 2 days of challenging with bacteria, mRNA was obtained from the kidney, spleen and gill tissues of C. auratus using an RNA isolation kit (Takara, Tokyo, Japan), and qRT-PCR was performed as previously described (Liu et al., 2021). A commercial kit (Takara, Tokyo, Japan) was utilised to prepare cDNA with mRNA reverse transcription, and qRT-PCR was implemented using a qRT-PCR system (ABI Applied Biosystems, Waltham, USA) with an SYBR ® Green Permix Pro Taq HSqPCR kit (Takara, Tokyo, Japan) and synthetic primers (Vosun Biotechnology Co. Ltd.) ( Table I). The 2 -(ΔΔCt) method was employed to analyse the mRNA expression with the internal control gene GAPDH.

Analysis of antioxidant index of C. auratus
After 2 days of challenging with bacteria, C. auratus serum was harvested at 3000 r/min and was used to analyse the antioxidant index of visceral organs using malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) according to the kit instructions (Jiancheng Institute of Biotechnology Co. Ltd., Nanjing, China).

Histopathological observation of visceral organs
Pathological sections of the kidneys, spleens and intestines of the C. auratus were prepared with H&E staining . Briefly, the kidneys, spleens and intestines were obtained after 2 days of challenging with bacteria and were dehydrated for 1 h with an alcohol gradient and added to alcohol: xylene mixture (1:1, V/V) for 25 min, xylene for 10 min; xylene: paraffin solution (1:1, V/V) for 30 min and paraffin at 60℃ for 1 h. Next, slices of roughly 5 μm thickness were cut, stained with H & E, observed and photographed with a microscope (Leica, Wetzlar, Germany).

Homologous and phylogenetic analysis of Slp
Based on the Slp amino acid sequences of the different bacteria, homologous analysis showed that the species of bacteria have homology (Fig. 1A); and the phylogenetic trees showed that the relationship among species of Enterobacteria (E. coli, E. hormaechei and S. boydii) was closer than other kinds of bacteria, and the relationship O n l i n e

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Immune Response and Passive Protective Abilities of Slp 5 between A. hydrophila and P. fluorescens was closer than others (Fig. 1B). Therefore, anti-Slp serum may provide cross-protection against A. hydrophila and P. fluorescens infection in animals.

Construction, expression and purification of recombinant Slp
The slp gene was amplified from the A. hydrophila genome with PCR. DNA electrophoresis showed there was a single band at about 500 bp ( Fig. 2A), which was consistent with the theoretical value. The recombinant slp plasmid was digested by EcoR I and Xho I, and a 500 bp band was obtained (Fig. 2B). The results of gene sequencing showed the target gene is consistent with the slp sequence of the NCBI database.  After the recombinant strain of Slp was induced with IPTG, the results of SDS-PAGE electrophoresis showed the target protein was about 39 kDa, including 19 kDa of the target protein and 20.4 kDa of the fusion protein, which was consistent with the theoretical value. The Slp was purified with Ni-NTA flow resin (Fig. 2C).

The optimal expressing conditions of Slp
According to the L 9 (3 4 ) orthogonal design SDS-PAGE electrophoretic maps were obtained (Fig. 3), and the optical density of the bands was analysed using Phoretix 1D software. The mean value analysis showed the optimal Slp expressing conditions were A1, B1, C2 and D2, which means a strain OD 600 value of 0.5, IPTG concentration of 0.1 mmol/L, inducing time of 8 h and inducing temperature of 32 ℃ (Table II).

Immune activity of Slp in mice
In order to assess the effects of nonspecific immunity, the immune-related factors of LZM, AKP and ACP in Slp mouse serum were determined and the results showed that LZM and AKP increased (p < 0.05) and ACP unchanged (Figure 4), which means Slp could activate nonspecific immunity in mice. Fig. 4. The immune-related factors in Slp mouse serum. **p < 0.01 (compared with control). LZM and AKP increased (p < 0.01) and ACP was unchanged, meaning that Slp may activate nonspecific immunity in mice.
The recognition of Slp mouse serum to A. hydrophila and P. fluorescens in vitro was simulated by the ELISA method. With the increases in serum dilution, the ability of SLP serum to recognize A. hydrophila and P. fluorescens decreased gradually, and the recognition between Slp serum and these two kinds of bacteria was evaluated with the dilution of 1: 12 800 (Fig. 5A). Western blotting results showed that Slp serum was specifically combined with Slp and that the negative control (blank antiserum) had no band. Slp could stimulate the mice to produce a specific antiserum with a titre of 1: 3200 (Fig. 5B). These results suggest that Slp may have antigenicity.

Passive protective and passive cross-protective rate of Slp mouse serum in C. auratus
In order to assess the effects of passive protective and passive cross-protective abilities, C. auratus was immunized with Slp mouse serum and challenged with P. fluorescens and A. hydrophila. C. auratus showed obvious toxic symptoms after being challenged with the bacteria, including less food intake and sluggish activity. The majority of the C. auratus in the control group died within 5 days; the remainder survived and gradually recovered after 6 days (Fig. 6). The passive protective rate of Slp mouse serum against A. hydrophila was 42.5 % (p < 0.05), and the passive cross-protective rate of Slp mouse serum against P. fluorescens infection was 18.6%. Therefore, the Slp mouse serum has passive protective and passive crossprotective abilities.

Immune-related factors and leukocyte phagocytosis of C. auratus plasma to evaluate passive protective abilities
The immune-related factors of LZM, AKP and ACP and the leukocyte phagocytosis of C. auratus plasma were determined on 2 days after the C. auratus were passive immunized with Slp mouse serum. The results showed that LZM, AKP and ACP increased (p < 0.01) (Fig. 7), and the leukocyte phagocytosis indexes of PP and PI were higher than those of the NC group (p < 0.01) (Table III). These results suggest that passive immunization with Slp mouse serum can activate a nonspecific immunity in C. auratus.

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Immune

Inflammation-related gene expression to evaluate passive protective abilities
In order to assess the passive protective and passive cross-protective abilities of Slp mouse serum, the mRNA expression of inflammation-related genes (IL-6, IL-8, IL-1β and TNF-α) were analysed in the kidneys and spleens on 2 days after the C. auratus were immunized with Slp mouse serum and challenged with bacteria. Compared to the NC groups, most of the mRNA expression of IL-6, IL-8, IL-1β and TNF-α decreased (p < 0.05) in kidneys and spleens after being challenged with A. hydrophila (Fig.  8A). In addition, after being challenged with P. fluorescens, most of the mRNA expression of IL-6, IL-8, IL-1β and TNF-α decreased (p < 0.05) in kidneys and spleens (Fig.  8B). The results suggest that passive immunization with Slp mouse serum could reduce the inflammatory reaction induced by A. hydrophila and P. fluorescens infection in C. auratus. Fig. 7. The immune-related factors of C. auratus plasma after passive immunization with Slp mouse serum. **p < 0.01 (compared with control). LZM, AKP and ACP increased (p < 0.01), meaning that passive immunization with Slp mouse serum may activate a nonspecific immunity in C. auratus.

Levels of antioxidant-related factors to evaluate passive protective abilities
In order to assess the antioxidant effects of passive immunization with Slp mouse serum, the antioxidantrelated factors of MDA, SOD, GSH-PX and CAT in C. auratus serum were determined on 2 days after immunizing with Slp serum and challenging with bacteria. The results showed that MDA, SOD, CAT and GSH-PX were no significant change in C. auratus serum after challenging with A. hydrophila and P. fluorescens (Fig. 9). These results suggest that passive immunization with Slp mouse serum has no antioxidative effect against A. hydrophila and fluorescens infection in C. auratus.

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Histopathology of C. auratus to evaluate passive protective abilities
In order to assess the protective abilities of Slp mouse serum for viscera against bacterial infection, sections of the kidneys, spleens and intestines were prepared on 2 days after C. auratus were immunized with Slp serum and challenged with A. hydrophila and P. fluorescens to observe any injury. Compared to the kidneys of C. auratus immunized with Slp serum, the kidneys of C. auratus that received NC appeared to have glomerular atrophy, nuclear apoptosis and incomplete organisational structure ( Fig.10Aa and 10Ac); the spleens that received NC appeared to less nuclear density and loose organisational structure ( Fig. 10Ba and 10Bc); the intestines that received NC appeared to have abscission of intestinal villous epithelial cells and myometrial cell apoptosis and degeneration ( Fig. 10Ca and 10Cc). Therefore, Slp serum can reduce the injury to the kidneys, spleens and intestines in C. auratus caused by P. fluorescens and A. hydrophila.

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Immune Response and Passive Protective Abilities of Slp 9 Fig. 10. Histopathology of kidneys, spleens and intestines of C. auratus after immunization and bacteria challenge (H & E×400 magnification). A, B and C represent the sections of the kidneys, spleens and intestines, respectively. C. auratus were passive immunization NC (blank serum) and challenged with A. hydrophila (a) and P. fluorescens (c). C. auratus were passive immunization Slp mouse serum and challenged with A. hydrophila (b) and P. fluorescens (d). After bacteria challenge, glomerular atrophy, nuclear apoptosis and incomplete organisational structure were observed (Aa and Ac); less nuclear density and loose organisational structure (Ba and Bc); and abscission of intestinal villous epithelial cells, myometrial cells apoptosis, and degeneration were observed (Ca and Cc). Thus, passive immunization with Slp mouse serum can reduce the injury to the kidney, spleen and intestine.
A. hydrophila and P. fluorescens are major freshwater aquaculture etiologic agent in fish, and protein vaccines are particularly important (Circella et al., 2020;Dong et al., 2020). A. hydrophila Slp is located in the outermost layer of the cell membrane and belongs to an OMP protein; it interacts with the host and has immune activity (Hooda et al., 2017). In current research, the recombinant protein of A. hydrophila Slp was constructed by molecular cloning and purified with Ni-NTA flow resin. The optimal expressing conditions of Slp were strain OD 600 of 0.5,

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IPTG concentration of 0.1 mmol/L, induction temperature of 32 °C and time of 8 h. We found a low concentration of IPTG is beneficial to the expression of Slp, which may be related to the cytotoxicity of IPTG, and is consistent with our previous study showing that protein expression needs a low concentration of IPTG (Chen et al., 2019). These results lay the foundation for Slp immune function. Mice polyclonal antibodies have been widely used in the analysis of immune protein function due to their convenience and shorter preparation time, relatively simple preparation compared to monoclonal antibodies (Beigel, 2018). The research of protein immune function usually needs to be prepared its antibody. In previous studies, we have obtained OMPs antisera of OmpA, OmpC, OmpK, and found they have an immune protective function (Liu et al., 2018;Chen et al., 2019). In current research, a specificity Slp mouse serum was prepared with a titre of more than 3200 times. Slp serum could recognize A. hydrophila and P. fluorescens to form the compound of Slp antibodies-bacteria in vitro, and the complex may provide an opportunity for antigen presentation and phagocytic removal of bacteria in fish (Nelson et al., 2020). In addition, we found that anti-Slp serum may provide cross-protection against A. hydrophila and P. fluorescens infection in animals by bioinformatics. The immune-related factors of LZM, AKP and ACP can improve the immune ability of animals and promote the elimination of bacteria and viruses (Zhong et al., 2019;Niu et al., 2020). In this research, we found that the immune-related factors increased (p < 0.05) by immunizing mice with Slp. These results suggest that Slp can activate the immune activity of mice.
Mice have a more sophisticated immune system than fish and can activate a higher antiserum titre, and passive immunization of fish with mouse antiserum can be used to screen for protective immunogens to assess immune protein ability (Li et al., 2010;Peng et al., 2016Peng et al., , 2018. Peng et al. (2016) have been used passive immunization antiserum method to identify 17 OMPs of Vibrio parahaemolyticus, and VP2309, VP0887, VPA0548 and VP1019 have been found to have efficiently protective immunogens. In this research, the passive protection rate of Slp mouse serum against A. hydrophila infection was 42.5 % (p < 0.05), and the passive cross-protective rate of Slp mouse serum against P. fluorescens infection was 18.6 % in C. auratus. In addition, passive immunization with Slp mouse serum enhanced the immune-related factors expressing and the leukocyte phagocytosis of C. auratus, indicating that Slp serum might activate a nonspecific immunity in C. auratus. The results suggest that Slp mouse serum has passive protection abilities (against A. hydrophila) and passive cross-protection abilities (against P. fluorescens) in C. auratus.
Passive protective abilities can also be assessed by inflammation-related genes, antioxidant factors and visceral organ injury after passive immunization with animal antiserum and challenging with pathogenic bacteria (Samblas et al., 2019;Hoseinifar et al., 2020;Solana et al., 2020;Liu et al., 2021). OmpA protein is located in the outermost layer of E. coli, and we found that OmpA nanoparticles can decrease the expressing of inflammation-related genes and decrease visceral organ injury in mice (Liu et al., 2021). In current research, passive immunization with Slp serum to C. auratus was shown to decrease the expression of the inflammationrelated genes IL-6, IL-8, IL-1β and TNF-α after being challenged with A. hydrophila and P. fluorescens; and the histopathological sections showed that Slp serum had protective abilities to visceral structure integrity (kidneys, spleens and intestines) against A. hydrophila and P. fluorescens infection. Therefore, Slp serum can reduce the inflammatory reaction induced by A. hydrophila and P. fluorescens infection in C. auratus. These results suggest that Slp mouse serum has passive protective and passive cross-protective abilities.

CONCLUSIONS AND RECOMMENDATIONS
Slp was analysed by bioinformatics and obtained by molecular cloning, expression and purification, and the expressing conditions of Slp were optimized. Slp can activate nonspecific immunity in mice, and a high titre of Slp mouse serum was prepared that recognized to A. hydrophila and P. fluorescens. Slp mouse serum has the passive protective and passive cross-protective abilities against A. hydrophila and P. fluorescens infection, and can activate a nonspecific immunity in C. auratus. Passive immunization with Slp serum can down-regulate the expression of inflammation-related genes, and reduce visceral organ injury caused by A. hydrophila and P. fluorescens in C. auratus. This study contributes to the development of a Slp polyvalent protective immunogen that can be used to boost resistance to infection with major freshwater aquaculture etiologic agents in fish.

Funding
This research was supported by the Natural Science Basic Research Program of Shaanxi Province (2022JM-120) and Project of Shaanxi University of Technology (SLGQD1803), China.

IRB approval and ethical statement
All animal procedures were performed in accordance with the guidelines in the Guide for the Care and Use of O n l i n e

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Laboratory Animals and were approved by the Institutional Animal Ethics Committee, Shaanxi University of Technology, Hanzhong, China (No. 2020-08).

Data availability statement
The data that support the findings of this study are available on request from the corresponding author.

Statement of conflict of interest
The authors have declared no conflict of interest.