A vibrio harveyi inactivated vaccine for east asian seaperl moribundity disease, and a preparation method and application thereof

Abstract

The application discloses a Vibrio harveyi inactivated vaccine for East Asian seabream body ulcer disease, a preparation method and application thereof, and belongs to the field of aquatic animal immunology. The Vibrio harveyi inactivated vaccine comprises a Vibrio harveyi VH2409105311 antigen and a pharmaceutically acceptable adjuvant, and the preservation number of the Vibrio harveyi VH2409105311 is CCTCC NO: M2026590. The application first separates a new Vibrio harveyi strain VH2409105311 from East Asian seabream and prepares the inactivated vaccine. The relative protection rate of the vaccine to East Asian seabream reaches 91.22%, the antibody induced by the vaccine can have a cross agglutination reaction with 56 Vibrio harveyi strains, has a good cross protection effect, can effectively stimulate specific humoral immune response, and overcomes the problem of insufficient immunogenicity of existing strains, thereby providing a safe and efficient vaccine product for preventing and controlling East Asian seabream body ulcer disease.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal immunology, and in particular to an inactivated Vibrio harveyi vaccine against aquatic spotted rot disease, its preparation method, and its application. Background Technology

[0002] Leopard-gill spiny perch ( Plectropomus leopardus The grouper, also known as the Eastern Star Grouper, is a prized marine aquaculture fish. Its delicate flesh and vibrant coloration give it high economic and ornamental value. In recent years, due to high-density aquaculture and environmental changes, the Eastern Star Grouper has become susceptible to various bacterial diseases, particularly Vibrio harveyi (Vibrio harveyi). Vibrio harveyi The most severe form of this disease is rotting, caused by [unspecified pathogen]. In its early stages, the disease manifests as pinpoint hemorrhages or ulcers on the body surface, which then enlarge, leading to skin ulceration and, in severe cases, exposure of muscle and bone. If not controlled promptly, the pathogen enters the body through wounds, causing septicemia and internal organ infection, resulting in mass mortality rates exceeding 90% and significant economic losses. Currently, traditional prevention and control methods mainly rely on antibiotics, but the resulting drug resistance problem is becoming increasingly prominent. Developing safe and effective vaccines has become an important direction for replacing antibiotics in the control of this disease.

[0003] Inactivated vaccines, due to their high safety and relatively controllable cost, have been proven suitable for the control of bacterial diseases in various fish species. Research on inactivated vaccines against Vibrio harveyi has been reported, but current technologies mainly focus on other farmed fish (such as grouper and flounder). No inactivated Vibrio harveyi vaccines specifically for the disease of the red snapper have been reported. This is because Vibrio harveyi from different fish sources or geographical locations exhibit significant differences in antigenicity, pathogenicity, and immunogenicity, and not all isolates are suitable for preparing highly effective vaccines. In practice, it has been found that Vibrio harveyi isolated from diseased red snapper lesions often exhibits unstable virulence, altered antigenic phenotypes after in vitro culture, and poor immunoprotective effects, making it difficult for conventionally inactivated bacteria to elicit an ideal immune response. Therefore, screening for pathogenic strains with stable immunogenicity suitable for vaccine preparation has long been a technical challenge in this field.

[0004] The current technology lacks an inactivated Vibrio harveyi vaccine against spotted eczema, primarily due to the difficulty in obtaining a suitable specific pathogen for vaccine preparation. This difficulty stems from two main factors: firstly, the host adaptability and strain diversity of Vibrio harveyi make it difficult for universal vaccines to provide effective protection against spotted eczema; secondly, strains isolated from infected spotted eczema are prone to antigenic variation during in vitro passage, resulting in insufficient immunogenicity to induce specific immune memory after inactivation. These issues have led to a long-standing lack of safe, stable, and effective vaccine products in this field, and antibiotics remain the primary means of controlling the disease. Summary of the Invention

[0005] The purpose of this invention is to provide an inactivated Vibrio harveyi vaccine against spotted rotten skin disease (S. septemlobus), its preparation method, and its application, thereby addressing the problems existing in the prior art. This invention is the first to isolate a novel Vibrio harveyi strain, VH2409105311, from spotted rotten skin disease and prepare it into an inactivated vaccine. This vaccine achieves a relative protection rate of 91.22% against spotted rotten skin disease, effectively eliciting a specific humoral immune response, overcoming the problem of insufficient immunogenicity of existing strains, and providing a safe and efficient vaccine product for the prevention and control of spotted rotten skin disease.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Vibrio harveyi ( Vibrio harveyi VH2409105311, this strain was deposited at the China Center for Type Culture Collection on April 2, 2026, with accession number CCTCC NO: M2026590.

[0007] The present invention also provides the application of Vibrio harveyi VH2409105311 in the preparation of an inactivated vaccine for the prevention of stellate rottenness disease, wherein the inactivated vaccine is an inactivated vaccine capable of inducing cross-protection against multiple strains of Vibrio harveyi.

[0008] The present invention also provides an inactivated vaccine capable of inducing cross-protection against multiple strains of Vibrio harveyi, the inactivated Vibrio harveyi vaccine comprising Vibrio harveyi VH2409105311 antigen and a pharmaceutically acceptable adjuvant; the Vibrio harveyi VH2409105311 has the accession number CCTCC NO: M2026590.

[0009] Optionally, the Vibrio harveyi VH2409105311 antigen is a formaldehyde-inactivated whole-bacterial antigen.

[0010] Optionally, the volume ratio of the Vibrio harveyi VH2409105311 antigen to the adjuvant is 3:7.

[0011] Optionally, the bacterial concentration of the whole-cell antigen in the Vibrio harveyi inactivated vaccine before inactivation is 4.3 × 10⁻⁶. 10 CFU / mL.

[0012] The present invention also provides a method for preparing the Vibrio harveyi inactivated vaccine, comprising the following steps: (1) The Vibrio harveyi VH2409105311 described in claim 1 is revived and cultured on a large scale; (2) Collect the bacterial cells, add formaldehyde for inactivation treatment, and obtain inactivated antigen; (3) The inactivated antigen is mixed with the adjuvant and emulsified to obtain the Vibrio harveyi inactivated vaccine.

[0013] Optionally, the final volume fraction of formaldehyde in step (2) is 0.3%, the inactivation temperature is 30°C, and the inactivation time is 24 h.

[0014] The present invention also provides the use of the Vibrio harveyi VH2409105311 or the Vibrio harveyi inactivated vaccine in the preparation of products for the prevention of Vibrio harveyi infection.

[0015] The present invention also provides the use of Vibrio harveyi VH2409105311 or the Vibrio harveyi inactivated vaccine in the preparation of products for the prevention of scolic dermatitis.

[0016] The present invention discloses the following technical effects: This invention marks the first time that a novel Vibrio harveyi strain, VH2409105311 (CCTCC NO: M2026590), with good immunogenicity has been isolated and screened from diseased spotted lesions. After inactivation, this strain was combined with an adjuvant to successfully prepare an inactivated Vibrio harveyi vaccine against spotted lesions. Immunoprotection assays showed that the vaccine achieved a relative protection rate of 91.22% against spotted lesions, significantly reducing mortality after challenge and effectively preventing septicemia caused by Vibrio harveyi infection.

[0017] The inactivated vaccine prepared in this invention can effectively stimulate a specific humoral immune response in Vibrio harveyi. Serum antibody titer assays showed that the antibody titer reached its peak 2 weeks post-immunization and remained at a high level until 4 weeks post-immunization. Cross-agglutination experiments indicated that the antibodies induced by the prepared inactivated vaccine not only efficiently recognized and bound Vibrio harveyi strain VH2409105311, but also exhibited cross-agglutination reactions with 56 other Vibrio harveyi strains, demonstrating good cross-protective ability. This invention overcomes the difficulties of unstable virulence of strains isolated from diseased Vibrio harveyi, altered antigenic phenotypes after in vitro culture, and insufficient immunogenicity after inactivation, providing a safe, efficient, and scalable vaccine product that can be used to replace antibiotics in the prevention and control of Vibrio harveyi infection, showing promising industrial application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Phylogenetic tree of strain VH2409105311; Figure 2The survival rate of live bacteria challenged with *Gnaphalium affine* within 14 days after vaccine immunization; Figure 3 The serum antibody titers of spotted scorpion were measured at 7, 14, 21, and 28 days post-vaccination. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Isolation, Screening and Identification of Strains (1) Screening and isolation of pathogens Fish exhibiting typical symptoms of body rot disease were collected from an aquaculture area in Hainan Province. The average body length of the diseased fish was 10 cm, and the average weight was 12 g. Under aseptic conditions, the fish were dissected, and liver, spleen, and kidney tissues were collected. Samples were taken using a sterile inoculation loop and streaked onto TSB, 2216E, and TCBS agar media, and incubated at 28°C for 24 h. After incubation, colony growth on each medium was observed. Dominant single colonies were selected and continuously streaked on the same medium for purification. This process was repeated three times until morphologically consistent pure cultures were obtained. The purified strain was inoculated into 2216E liquid medium and cultured at 28°C with shaking until the logarithmic growth phase. The bacterial cells were collected, and sterile 20% glycerol was added as a protectant. The strain was then aliquoted and stored at -80°C for later use, designated as strain VH2409105311.

[0026] (2) Identification of pathogens The above-mentioned frozen bacterial strains were inoculated into 2216E liquid medium and incubated at 28°C for 24 h. Genomic DNA was extracted from the strains using a bacterial genomic DNA extraction kit. Using the extracted DNA as a template, PCR amplification was performed on the toxR gene of *Vibrio harveyi*. The PCR reaction system and amplification conditions were set according to standard methods. After detection by 1% agarose gel electrophoresis, the amplified products were sent to a sequencing company for bidirectional sequencing. After splicing and quality control, the toxR gene sequence was obtained, and its length is shown in SEQ ID NO.1.

[0027] SEQ ID NO.1: .

[0028] (3) Phylogenetic analysis of pathogens BLAST homology comparisons were performed in the NCBI database to construct a phylogenetic tree for strain VH2409105311. The phylogenetic tree was constructed using MEGA software with neighbor-joining (bootstrap repeated 1000 times). The results are as follows: Figure 1As shown. Strain VH2409105311 and multiple strains of Vibrio harveyi (… Vibrio harveyi Reference strains (WXL538, 345, fish10, SB1, XH2145, A2) clustered into the same monophyletic clade, with a Bootstrap support of 80%, indicating that the clustering results were stable and reliable. Combined with the TOXR gene sequence homology comparison results, strain VH2409105311 was identified as *Vibrio harveyi*. Vibrio harveyi This strain was deposited on April 2, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026590.

[0029] (4) Pathogenicity detection Healthy grouper (average body length 10 cm, average weight 12 g) were provided by Wanning Linlan Aquaculture Co., Ltd., and raised in a 0.2 m... 3 Twenty fish were stocked in a round plastic container under the following conditions: temperature 28-30℃, salinity 28-32‰, with water changed twice daily. Ten healthy grouper were randomly selected, and liver, spleen, and kidney tissues were aseptically collected and streaked onto 2216E and TCBS agar media, respectively. No other bacteria were detected after incubation, indicating that the experimental fish were free of potential pathogens and met the requirements for a healthy experiment.

[0030] The isolated, purified, and identified Vibrio harveyi strain VH2409105311 was used for injection infection. The experimental group received an intraperitoneal injection of 0.1 mL, resulting in a bacterial concentration of 7.1 × 10⁻⁶. 3 CFU / mL, while the control group was injected with an equal volume of sterile saline.

[0031] The experimental results showed that the experimental grouper gradually developed body rot symptoms after injection, with a cumulative mortality rate of 55%; the control group showed no mortality and exhibited normal activity and feeding. The dead fish in the experimental group were dissected, and liver and spleen tissues were aseptically collected and streaked onto 2216E and TCBS agar media. After incubation at 28°C for 24 h, dominant bacterial colonies were isolated. DNA was extracted from these colonies, and toxR gene PCR amplification and sequencing identification were performed. The results were consistent with the initially isolated strain VH2409105311, confirming that the pathogen causing the death of the grouper was indeed Vibrio harveyi VH2409105311.

[0032] Example 2: Preparation of an inactivated vaccine against Vibrio harveyi VH2409105311 1. Strains' recovery and propagation The Vibrio harveyi strain VH2409105311 isolated, identified, and frozen at -80℃ in Example 1 was thawed under aseptic conditions and inoculated into TSB liquid medium containing 2% NaCl. The culture was then incubated at 30℃ for 16 h. After incubation, the bacterial culture was transferred to TSB liquid medium containing 2% NaCl at a 1% (v / v) inoculation rate, expanding the culture to a total volume of 500 mL. The culture was then incubated at 30℃ and 200 rpm with shaking for 16 h to allow the bacteria to reach the late logarithmic phase.

[0033] 2. Determination of bacterial concentration The cultured bacterial suspension was centrifuged at 5000 rpm for 10 min at room temperature to collect the bacterial cells. An appropriate amount of the bacterial cells was resuspended in sterile physiological saline, and the optical density (OD) of the bacterial suspension was measured spectrophotometrically at a detection wavelength of 600 nm. 600 The bacterial concentration was calculated using a pre-established standard curve. Based on the measurement results, the bacterial concentration was adjusted to the desired value using sterile physiological saline.

[0034] 3. Bacterial inactivation Add formaldehyde solution to the adjusted bacterial suspension to a final volume fraction of 0.3%, mix thoroughly, and incubate at 30°C for 24 hours to inactivate the bacteria. After inactivation, take an appropriate amount of the inactivated bacterial suspension and spread it on a TSA solid plate containing 2% NaCl. Incubate at 30°C for 48 hours and observe for colony growth. If no colonies appear after incubation, it indicates that the bacteria have been completely inactivated and there are no surviving individuals.

[0035] 4. Antigen collection and washing The inactivated bacterial suspension was centrifuged at 5000 rpm for 10 min at room temperature. The supernatant culture medium was discarded, and the inactivated bacterial precipitate was collected. Sterile physiological saline was added to the precipitate to resuspend the bacteria, and the suspension was centrifuged again under the same conditions. The supernatant was discarded, and the washing was repeated twice to remove residual formaldehyde and culture medium components. After washing, the inactivated bacterial cells were collected, which is the Vibrio harveyi inactivated antigen.

[0036] 5. Vaccine preparation The inactivated Vibrio harveyi antigen prepared above was mixed with Montanide™ ISA 763 A VG adjuvant (manufactured by Seppic, France) at a volume ratio of antigen:adjuvant = 3:7. Following the manufacturer's instructions, the mixture was emulsified under aseptic conditions until a homogeneous and stable water-in-oil emulsion was formed. After emulsification, samples were taken and observed under a microscope for droplet morphology and uniformity to confirm the absence of stratification and demulsification. After dilution with physiological saline, the antigen content in the vaccine was determined using plate count or spectrophotometry, ultimately preparing a vaccine with a pre-inactivation bacterial concentration of 4.3 × 10⁻⁶. 10A monovalent inactivated vaccine with a concentration of CFU / mL. The prepared vaccine is dispensed into sterile glass vials, sealed, and stored at 4°C in the dark for later use.

[0037] 6. Vaccine administration method This vaccine is suitable for the immunization and prevention of healthy red grouper. The specific method of administration is as follows: Select healthy red grouper with a body length of 10–12 cm, and withhold food for 24 hours before immunization. At the time of immunization, remove the vaccine from 4℃, allow it to warm to room temperature, and gently shake well. Administer via intraperitoneal injection, with a dosage of 0.1 mL per fish. After immunization, continue to raise the red grouper under normal aquaculture conditions, observe the health status of the fish, and record any abnormal reactions.

[0038] Example 3: Immunoprotection test of the vaccine 1. Experimental grouping and immunity Healthy grouper, measuring 10–12 cm in length, were selected and randomly divided into a vaccine-immunized group and a control group. The vaccine-immunized group consisted of 300 fish, divided into three replicates of 100 fish each. The vaccine was administered via intraperitoneal injection. Each fish in the vaccine-immunized group received 0.1 mL of the inactivated Vibrio harveyi vaccine prepared in Example 2; the control group received an equal volume of sterile saline solution per fish. After immunization, all experimental fish were kept under identical conditions at a water temperature of 28–30°C.

[0039] 2. Immunization cycle sampling The immunization cycle was set at 28 days. On days 0, 7, 14, 21, and 28 post-immunization, experimental fish were randomly selected from each parallel group, and liver, spleen, kidney tissue, and blood samples were collected under aseptic conditions for subsequent detection of immune-related indicators.

[0040] 3. Preparation of bacterial suspension for viral challenge Live *Vibrio harveyi* strain VH2409105311 was inoculated into TSB liquid medium containing 2% NaCl and cultured at 30°C for 16 h. The culture was then expanded to the logarithmic growth phase at a 1% (v / v) inoculum. The bacterial cells were collected, washed three times with sterile physiological saline, and the bacterial concentration was adjusted to 2 × 10⁻⁶. 4 The concentration of CFU / mL was temporarily stored at 4°C for later use, and the challenge injection was completed within 2 hours.

[0041] 4. Virus challenge experiment The challenge experiment was conducted on day 29 post-immunization. Ninety fish from the vaccine-immunized group were challenged, divided into three replicates of 30 fish each. Simultaneously, 90 fish from the blank control group were challenged, also divided into three replicates of 30 fish each. The challenge method was intraperitoneal injection, with each fish receiving 0.1 mL of the Vibrio harveyi bacterial suspension prepared in step 3 (i.e., a challenge dose of 2 × 10⁻⁶ fish per fish).3 (CFU). After challenge, all experimental fish continued to be raised under the same conditions and observed for 14 consecutive days. Mortality was recorded daily for each group, and dead fish were promptly removed and their numbers recorded.

[0042] 5. Calculation of relative protection rate Based on the cumulative mortality rate of each group within 14 days, the relative protection rate (RPS) is calculated according to the following formula: RPS = (1 - mortality rate of the vaccine immunization group / mortality rate of the blank control group) × 100%.

[0043] 6. Experimental Results Survival was recorded for 14 days after challenge. Results showed that in the blank control group, *Stellaria media* began dying on the first day after challenge, with a final survival rate of 36.67%; in the vaccine-immunized group, mortality began on the second day, but the number of deaths was significantly reduced, with a final survival rate of 94.44%. Figure 2 Compared with the blank control group, the mortality rate of the vaccine-immunized group was significantly reduced. According to the formula, the relative protection rate of the Vibrio harveyi monovalent inactivated vaccine against *Scutellaria baicalensis* was 91.22%. These results indicate that the Vibrio harveyi inactivated vaccine prepared in Example 2 can effectively protect *Scutellaria baicalensis* from Vibrio harveyi infection and can be used as an effective vaccine for preventing *Scutellaria baicalensis* infection in *Scutellaria baicalensis*. Example 4: Serum Specific Antibody Titer Determination 1. Sample Collection Following the immunization protocol of Example 3, experimental fish were randomly selected from both the vaccine-immunized group and the blank control group on days 0, 7, 14, 21, and 28 post-immunization. Blood samples were collected from the tail vein or heart. The collected blood was placed in sterile centrifuge tubes and allowed to stand at room temperature for 1–2 hours to allow for natural coagulation. Then, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C to separate the supernatant serum. The separated serum was aliquoted into sterile centrifuge tubes, labeled, and stored at -20°C for later use.

[0044] 2. Antigen coating The titer of specific antibodies in serum was determined using enzyme-linked immunosorbent assay (ELISA). The specific procedure was as follows: Vibrio harveyi inactivated antigen was taken, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶ using coating buffer (carbonate buffer, pH 9.6). 8CFU / mL. Add 200 μL of the above antigen coating solution to each well of a 96-well microplate and incubate at room temperature in the dark for 1 h. After incubation, discard the coating solution, add 200 μL of PBST washing buffer (phosphate buffer containing 0.05% Tween-20, pH 7.4) to each well, wash 3 times, shaking for 3 min each time, and drain any remaining liquid from the wells.

[0045] 3. Antigen fixation Add 200 μL of fixative to each well after washing and fix at 30°C for 15 min. After fixation, discard the fixative and wash three times with PBST as described in step 2.

[0046] 4. Enclosed Add 250 μL of blocking buffer (PBS solution containing 5% skim milk powder, w / v) to each well and incubate at 37°C in the dark for 2.5 h. After incubation, discard the blocking buffer and wash three times with PBST.

[0047] 5. Serum incubation Serum samples from the vaccine-immunized group and blank control group collected in Example 3 were removed from -20°C and thawed at 4°C. Each serum sample was diluted 50-fold with diluent (phosphate-buffered saline containing 2% BSA). 100 μL of the diluted serum sample was added to each well of the blocked ELISA plate, with three replicates per sample. The plates were incubated at 30°C for 1 h. After incubation, the serum diluent was discarded, and each well was washed three times with 200 μL of PBST.

[0048] 6. Primary antibody incubation Dilute rabbit anti-fish IgM antibody with PBST at a ratio of 1:1000 and mix thoroughly. Add 100 μL of the diluted rabbit anti-fish IgM antibody to each well and incubate at 37°C for 1 h. After incubation, discard the antibody solution and wash three times with PBST as described above.

[0049] 7. Secondary antibody incubation Dilute goat anti-rabbit IgG-HRP-labeled antibody with PBST at a ratio of 1:5000 and mix thoroughly. Add 100 μL of the diluted secondary antibody to each well and incubate at 37°C for 1 h. After incubation, discard the secondary antibody solution and wash three times with PBST.

[0050] 8. Color Development and Termination Add 100 μL of TMB chromogenic solution to each well and incubate at 37°C in the dark for 10 min. After chromogenic development, add 100 μL of stop solution (2 mol / L H2SO4) to each well and gently shake to mix and terminate the enzymatic reaction.

[0051] 9. Results Reading and Data Analysis The absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 Using the absorbance values ​​of the blank control wells as a background reference, the corrected OD of each sample well was calculated. 450 value.

[0052] 10. Measurement Results like Figure 3 As shown, at each time point after immunization, the antibody titers in the vaccine-immunized group were significantly higher than those in the blank control group (P<0.01). One week after immunization (day 7), the antibody titer in the vaccine-immunized group rapidly increased; two weeks after immunization (day 14), the antibody titer reached its peak, the highest level during the entire testing period; three weeks after immunization (day 21), the antibody titer slightly decreased from the peak but remained at a high level; four weeks after immunization (day 28), the antibody titer in the vaccine-immunized group did not show a significant decrease and remained at a stable level near the peak. These results indicate that the inactivated Vibrio harveyi vaccine prepared in Example 2 can effectively stimulate a specific humoral immune response in Vibrio harveyi, and the immunization effect has good persistence and stability.

[0053] Example 5: Serum Antibody Cross-Agglutination Reaction 1. Sample Collection The serum isolated in Example 4 was used.

[0054] 2. Antigen recovery and amplification Vibrio harveyi was cultured and collected at room temperature. The specific procedure was as follows: 56 strains isolated from different species of *Symplocos halofae* were taken from the laboratory and, following the identification procedure described in Example 1, were sequentially subjected to strain resuscitation culture, genomic DNA extraction, toxR gene PCR amplification, electrophoresis detection, and gene sequencing analysis. Based on sequence alignment results, all 56 strains were identified as *Vibrio harveyi* (numbered VH01-VH56). After thawing, the 56 *Vibrio harveyi* strains were inoculated into TSB liquid medium containing 2% NaCl and cultured at 30°C for 16 h. After resuscitation, the bacterial culture was transferred to TSB liquid medium containing 2% NaCl at a 1% (v / v) inoculation rate, and cultured to a volume of 5 mL per strain. The strains were labeled and cultured at 30°C and 200 rpm with shaking for 16 h to allow the bacteria to reach the late logarithmic growth phase.

[0055] 3. Antigen collection and concentration determination The 56 bacterial strains cultured above were centrifuged at 5000 rpm for 10 min at room temperature to collect the bacterial cells. An appropriate amount was resuspended in sterile physiological saline, and the optical density (OD) of the 56 bacterial strains was measured spectrophotometrically at a detection wavelength of 600 nm. 600 The bacterial concentration was calculated using a pre-established standard curve. Based on the measurement results, the bacterial solution was adjusted to a specific concentration using sterile physiological saline.

[0056] 4. Antigen washing The 56 bacterial strains with adjusted bacterial concentrations were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was collected. Sterile physiological saline was added to the pellet to resuspend the bacteria, and the mixture was centrifuged again under the same conditions. The supernatant was discarded, and the washing process was repeated twice to remove residual extracellular proteins and culture medium components. After washing, the bacterial cells were collected; this yielded the desired Vibrio harveyi antigen.

[0057] 5. Antigen-antibody cross-agglutination test Serum samples from the vaccine-immunized group and blank control group collected in Example 4 were removed from -20℃, thawed at 4℃, and diluted 2-fold. Cross-agglutination reactions were performed on glass slides using 56 washed strains of Vibrio harveyi, with two replicates for each sample. The specific procedure was as follows: 10 μL of antibody serum and 10 μL of antigen solution were added to a clean glass slide, mixed with a pipette, and allowed to stand for 5 minutes. Agglutination was observed under light. The control experiment was performed by repeating the above procedure. To eliminate the influence of complement present in the serum, the 2-fold diluted serum samples were heat-inactivated by heating in a 56℃ water bath for 30 minutes, and the above procedure was repeated after cooling.

[0058] 6. Measurement Results As shown in Table 1, all 56 strains of Vibrio harveyi and the antibodies in the vaccine immunization group exhibited varying degrees of agglutination reactions, and agglutinated particles could be observed under light. The serum results after heating in a 56°C water bath for 30 minutes were consistent with the results before heating, eliminating the influence of complement in the serum. These results indicate that the inactivated Vibrio harveyi vaccine prepared in Example 2 exhibits high cross-reactivity in inducing immunity against *Vibrio harveyi* strains and can effectively combat different strains of Vibrio harveyi.

[0059] Table 1. Agglutination reaction of antibodies from 56 strains of Vibrio harveyi to the vaccine-immunized group. Note: The more "+" signs in the table, the stronger the aggregation reaction.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Vibrio harveyi ( Vibrio harveyi VH2409105311, characterized in that, This strain was deposited at the China Center for Type Culture Collection on April 2, 2026, with accession number CCTCC NO: M2026590.

2. The use of Vibrio harveyi VH2409105311 as described in claim 1 in the preparation of an inactivated vaccine for the prevention of Orthopaedic scabies, characterized in that, The inactivated vaccine is an inactivated vaccine that can induce cross-protection against multiple strains of Vibrio harveyi.

3. An inactivated vaccine capable of inducing cross-protection against multiple strains of Vibrio harveyi, characterized in that, The inactivated vaccine contains Vibrio harveyi VH2409105311 antigen and a pharmaceutically acceptable adjuvant; the Vibrio harveyi VH2409105311 has the accession number CCTCC NO: M2026590.

4. The inactivated vaccine according to claim 3, characterized in that, The Vibrio harveyi VH2409105311 antigen is a formaldehyde-inactivated whole-bacterial antigen.

5. The inactivated vaccine according to claim 3, characterized in that, The volume ratio of Vibrio harveyi VH2409105311 antigen to adjuvant is 3:

7.

6. The inactivated vaccine according to claim 4, characterized in that, The bacterial concentration of the whole-cell antigen in the Vibrio harveyi inactivated vaccine before inactivation is 4.3 × 10⁻⁶. 10 CFU / mL.

7. A method for preparing an inactivated vaccine according to any one of claims 3-6, characterized in that, Includes the following steps: (1) The Vibrio harveyi VH2409105311 of claim 1 is revived and cultured on a large scale; (2) Collect the bacterial cells, add formaldehyde for inactivation treatment, and obtain inactivated antigen; (3) The inactivated antigen is mixed with the adjuvant and emulsified to obtain the Vibrio harveyi inactivated vaccine.

8. The preparation method according to claim 7, characterized in that, The final volume fraction of formaldehyde in step (2) is 0.3%, the inactivation temperature is 30℃, and the inactivation time is 24 h.

9. The use of the Vibrio harveyi VH2409105311 of claim 1 or the inactivated vaccine of any one of claims 3-6 in the preparation of a product for the prevention of Vibrio harveyi infection.

10. The use of Vibrio harveyi VH2409105311 as described in claim 1 or the inactivated vaccine as described in any one of claims 3-6 in the preparation of a product for the prevention of Orthopaedic scabies.

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