Vibrio canbainii bacteriophage capable of realizing cross-species cleavage and application thereof

Abstract

The invention discloses a vibrio canetinii bacteriophage capable of being split across species and application, the bacteriophage is a vibrio canetinii bacteriophage CP2 which is preserved in the China Center for Type Culture Collection with the preservation number of CCTCCM20251108, and the vibrio canetinii bacteriophage CP2 is preserved in the China Center for Type Culture Collection. The vibrio cantoniensis bacteriophage CP2 is a virulent bacteriophage separated from the nature, is free of genetic modification, has high safety and strong application potential, has an optimal infection complex number of 1: 10000, has a fermentation titer of 3.5 * 10 < pfu > / mL after 12 h, can rapidly split vibrio cantoniensis, has a recognition rate of 96.7% to 180 vibrio cantoniensis strains and a splitting rate of over 96%, and can be used for preparing the vibrio cantoniensis bacteriophage CP2. The genome does not contain toxicity and bad genes, the biological safety is high, the bacteriophage is excellent in environmental adaptability, stable in activity when the pH value is 6-8, high in storage stability at 4-25 DEG C, capable of being stored for 12 months at 4 DEG C, capable of stably surviving at room temperature, tolerant to a common chemical agent povidone-iodine for aquatic products, and suitable for various breeding scenes. Meanwhile, the cracking spectrum is wide, and the cross-species capability is realized.

Description

Technical Field

[0001] This invention belongs to the field of research and development technology of Vibrio campbellii bacteriophage, specifically relating to a Vibrio campbellii bacteriophage capable of cross-species lysis and its applications. Background Technology

[0002] Vibrio campbellii is a halophilic, Gram-negative, short rod-shaped bacillus, non-spore-forming and capsule-free, belonging to the Vibrioceae family and Vibrio genus. It is a common marine pathogen, ranking among the most abundant marine Vibrio species. It can cause wound infections, food poisoning, and otitis media in humans. It is also an opportunistic pathogen in aquaculture animals such as fish, shrimp, and shellfish. When the environment deteriorates and the body's immune function declines, campbellii infection is prone to outbreaks. Bacteriophages are a type of virus that specifically lyses bacteria. Their main chemical components are proteins and nucleic acids. They are widely distributed in soil, air, water, and organisms and have strong specificity. Due to their powerful bactericidal ability, bacteriophages can be used as an antibacterial agent and have received much attention from scholars at home and abroad since the beginning of the 20th century.

[0003] Currently, the prevention and control of Vibrio still mainly relies on antibiotics and chemical drugs. However, due to the rapid spread and high mortality rate of bacterial diseases and the lack of treatment methods, the widespread overuse of antibiotics has led to the use of antibiotics to suppress bacterial outbreaks. This has not only caused bacteria to develop resistance to most antibiotics, but also resulted in environmental pollution and the emergence of "superbugs".

[0004] Currently, there is very little research on Vibrio campeum phage in the industry, and there are few reports on Vibrio campeum phage and its applications. In summary, there is an urgent need to develop a novel, highly efficient Vibrio campeum phage. Therefore, we need to provide a Vibrio campeum phage that can lyse across species and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide a Vibrio campeosa phage capable of cross-species lysis and its application. This phage is a virulent phage CP2, possessing high lytic activity, a broad host spectrum, strong environmental adaptability, and high biosafety. It can efficiently lyse Vibrio campeosa and various pathogenic Vibrios, while being resistant to commonly used chemical agents in aquaculture, making it suitable for multiple application scenarios. This addresses the problems mentioned in the background art, such as the frequent outbreaks of diseases caused by pathogenic Vibrio campeosa and other Vibrios in aquaculture, the tendency of traditional chemical agents to lead to drug resistance and drug residues, and the narrow lytic spectrum, poor environmental stability, and inability to control diseases across species of conventional phages, which make it difficult to meet the needs of green aquaculture.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a Vibrio campbellii phage capable of cross-species lysis, characterized in that: the phage is Vibrio campbellii phage CP2, deposited at the China Center for Type Culture Collection, with accession number CCTCCM20251108; this phage is a virulent phage, and under electron microscopy, it has a polyhedral symmetrical head and a relatively short tail, with a head diameter of approximately 60–70 nm, a tail length of approximately 15–20 nm, and a tail diameter of approximately 10–15 nm.

[0007] Preferably, the Vibrio campbellii phage CP2, when cultured for 12 hours at a multiplicity of infection of 1:10000, has a titer of 3.5 × 10¹¹ PFU / mL.

[0008] Preferably, the optimal pH range of Vibrio campbellii phage CP2 is 6-8, and it has good stability under temperature conditions of 4℃-75℃.

[0009] A bacteriophage-based pharmaceutical formulation, wherein the dosage form of the pharmaceutical formulation is a solution or a lyophilized powder.

[0010] A method for in vitro prevention and control of harmful Vibrio campbellii infections, utilizing the lytic action of Vibrio campbellii bacteriophage CP2 to kill Vibrio campbellii in the in vitro environment, includes the following steps:

[0011] Take the lyophilized Vibrio campeosa phage CP2 powder, reconstitute it with sterile seawater with a salinity of 25–30‰ and a pH of 6.5–7.5, and inoculate it until the concentration reaches 1×10⁻⁶. 8 CFU / mL Vibrio campei in the logarithmic phase was cultured in 2216E medium at 30°C with shaking at 180 rpm for 6–8 h; the titer was determined by the double-layer plate method and adjusted to 1×10⁻⁶. 9 ~1×10¹ 0 PFU / mL, to obtain phage working solution;

[0012] Take 10 mL of water sample into a sterile centrifuge tube. Wipe the solid surface with a sterile cotton swab dipped in physiological saline and then wash it into 10 mL of physiological saline. Take 1 mL of the sample to be tested, serially dilute it, spread it on a 2216E plate containing 5% defibrinated sheep blood, and anaerobically incubate at 30℃ for 12-16 h. Count the bacteria to determine the initial bacterial count and complete the bacterial count test.

[0013] Apply the working solution at an MOI ratio of 10:1 to 20:1. After uniformly spraying the solution in an aquatic environment, allow it to stand for 4 to 6 hours at 15 to 30°C and pH 6 to 8. Alternatively, immerse solid surfaces in the working solution or spray 50 to 100 mL per square meter and then air dry.

[0014] The residual bacterial count should be measured according to the bacterial count testing method. The result is ≤1×10²CFU / mL for water and ≤10CFU / 100cm² for solid surface. If the result is not satisfactory, apply the same MOI and repeat the treatment for 3-4 hours until the standard is met.

[0015] Application of Vibrio campbellii bacteriophage: The bacteriophage can be applied to the surface of the host being controlled, to living water bodies, or to purify the aquaculture environment.

[0016] Preferably, the Vibrio campei phage CP2 is resistant to povidone-iodine, a chemical agent commonly used in aquaculture.

[0017] The application of Vibrio campei bacteriophage is used to prepare drugs for the prevention or treatment of diseases caused by pathogenic Vibrio infections in aquatic organisms; the pathogenic Vibrio includes one or more of Vibrio campei, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio prawnicana.

[0018] Preferably, the aquaculture environment includes, but is not limited to, seedling ponds, elevated ponds, earthen ponds, factory-style sheds, and feeding platforms.

[0019] Preferably, the Vibrio campbellii phage CP2 exhibits cross-species lytic ability. Besides efficiently lysing Vibrio campbellii, it also achieves ≥85% lysis rates against Vibrio alginolyticus isolate VA-03, Vibrio anguillarum isolate VA-08, and Vibrio harveyi isolate VH-02. Furthermore, the phage genome does not contain virulence genes, lysogenic integration genes, or drug resistance genes. Intraperitoneal injection experiments have verified that the median lethal dose (LD50) for aquaculture organisms such as Litopenaeus vannamei and grouper is >1×10¹. 0 PFU / tail poses no biosafety risk.

[0020] Technical effects and advantages of the present invention: The present invention provides a Vibrio campei phage capable of cross-species lysis and its application, which has the following advantages compared with the prior art:

[0021] The present invention relates to Vibrio campbellii phage CP2, a virulent phage isolated from nature. It is genetically unmodified, possesses high safety and strong application potential, with an optimal multiplicity of infection of 1:10000. After 12 hours of fermentation, its titer can reach 3.5×10¹¹pfu / mL. It can rapidly lyse Vibrio campbellii, achieving a recognition rate of 96.7% and a lysis rate of over 96% for 180 strains of Vibrio campbellii. Furthermore, its genome does not contain virulence or harmful genes, demonstrating high biosafety.

[0022] This bacteriophage exhibits excellent environmental adaptability, maintaining stable activity at pH 6-8 and high stability when stored at 4℃-25℃. It can be stored for 12 months at 4℃ and survives stably at room temperature. It is also resistant to povidone-iodine, a commonly used chemical in aquaculture, making it suitable for various aquaculture scenarios. Furthermore, it possesses a broad lytic spectrum and cross-species capability, recognizing 60% of Vibrio parahaemolyticus and 70% of Vibrio alginolyticus, while remaining non-host pathogenic bacteria, demonstrating high specificity.

[0023] At the application level, CP2 can be produced on a large scale through fermentation at low cost. It can be formulated into solutions, freeze-dried powders, and other dosage forms, and can be used for surface protection of aquatic hosts, purification of domestic water bodies, and disinfection of aquaculture environments through carrier delivery, concentrated spraying, and drug soaking. It can also be used as a biological antibacterial agent for aquatic products, soaking or spraying fresh aquatic products to inhibit the proliferation of Vibrio campestris during processing and preservation, providing excellent strains for green prevention and control in the aquaculture industry and contributing to the sustainable and healthy development of the industry.

[0024] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description and the drawings. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the phage plaque of Vibrio campestris phage CP2 of the present invention;

[0026] Figure 2 This is a schematic diagram of the morphological structure of Vibrio campbellii bacteriophage CP2 as identified by electron microscopy according to the present invention;

[0027] Figure 3 This is a schematic diagram of the structure of the sample that showed plaques in the lysogenic test of the present invention;

[0028] Figure 4 This is a schematic diagram of the structure of the sample in which no plaques appeared in the lysogenicity test of the present invention;

[0029] Figure 5 This is a flowchart of the steps of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention provides, for example Figure 1-5 The invention relates to a lytic phage of Vibrio campbellii, characterized in that: the phage is Vibrio campbellii phage CP2, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M20251108, and deposited on May 19, 2025; the phage is a virulent phage, and under electron microscopy, it has a polyhedral symmetrical head and a relatively short tail, with a head diameter of approximately 60–70 nm, a tail length of approximately 15–20 nm, and a tail diameter of approximately 10–15 nm.

[0032] The Vibrio campei phage CP2, when cultured for 12 hours at a multiplicity of infection (MOI) of 1:10000, achieved a titer of 3.5 × 10¹¹ PFU / mL.

[0033] Specifically, the standard strain of Vibrio campbellii (CCTCCAB2023001) was inoculated into 2216E liquid medium and cultured at 30°C with shaking at 180 rpm for 8 hours until the bacterial concentration reached 1×10⁻⁶. 9 CFU / mL (logarithmic growth phase) was used as the host bacterial seed culture; simultaneously, Vibrio campeosa phage CP2 lyophilized powder was reconstituted with sterile seawater (salinity 25–30‰, pH 6.5–7.5), and the initial titer was determined by the double-layer plate method, then adjusted to 1×10⁻⁶. 7 PFU / mL was used as the phage seed solution.

[0034] Fermentation system construction: Phage seed culture was inoculated into 2216E liquid medium containing host bacteria seed culture at a ratio of 1:10000 (i.e., 1 PFU of phage per 10000 CFU of host bacteria). A 500 mL fermentation system was constructed, with a final host bacteria concentration of 1 × 10⁻⁶. 8 CFU / mL, final phage concentration 1×10⁻⁶ 4 PFU / mL.

[0035] Fermentation culture control: The fermentation system was placed in a constant temperature shaking incubator at 30℃ and 180r / min and cultured in the dark for 12h. During the culture, samples were taken every 2h, and the phage titer was determined using the double-layer plate method (the bottom layer was 2216E medium containing 2% agar, and the top layer was a mixture of 2216E medium containing 0.7% agar, 100μL of phage dilution, and 100μL of host bacteria in the logarithmic phase) to monitor the trend of titer change.

[0036] Potency verification results: In the first 6 hours of culture, the phage titer increased exponentially (from 1×10⁻⁶). 4 PFU / mL increased to 5×10¹ 0The potency level (PFU / mL) plateaus after 6–12 hours, with the increase in potency stabilizing. At 12 hours, the peak potency level of 3.5 × 10¹¹ PFU / mL is reached. The results of three parallel experiments confirm that the potency fluctuation range is ≤5%, demonstrating that phage CP2 can stably achieve high proliferation efficiency under these culture conditions, meeting the potency requirements for large-scale industrial production and subsequent aquatic applications.

[0037] The optimal pH range of Vibrio campbellii phage CP2 is 6–8, and it exhibits good stability at temperatures ranging from 4°C to 75°C.

[0038] Specifically, prepare sterile 2216E culture medium (adjusted with 1 mol / L HCl or NaOH) with pH values ​​of 4, 5, 6, 7, 8, 9, and 10 respectively. Take 900 μL of each pH culture medium and place it into sterile EP tubes, then equilibrate in a 25°C water bath for 10 min. Add 100 μL of the initial potency of 1×10¹ to each tube. 0 Pure phage CP2 culture medium at PFU / mL was mixed, incubated at room temperature, and the titer was determined using the double-layer plate method after 2h, 4h, 8h, and 24h. Results showed that within the pH range of 6–8, the phage titer remained at 8 × 10⁻⁶ after 24h. 9 ~1×10¹ 0 The potency was ≤20% with PFU / mL and minimal fluctuations at different time points; however, at pH <6 (e.g., pH 4, 5) or pH >8 (e.g., pH 9, 10), the potency dropped to 1×10⁻⁶ after 24 hours. 8 ~5×10 8 With a PFU / mL loss rate exceeding 50%, it is proven that pH 6–8 is its optimal range, which can be adapted to the pH environment of most aquaculture water bodies (natural seawater pH is usually 7.5–8.5, and freshwater aquaculture water pH is 6.5–8.0).

[0039] A bacteriophage pharmaceutical formulation, wherein the dosage form of the pharmaceutical formulation is a solution or a lyophilized powder.

[0040] A method for in vitro prevention and control of harmful Vibrio campbellii infections, utilizing the lytic action of Vibrio campbellii bacteriophage CP2 to kill Vibrio campbellii in the in vitro environment, includes the following steps:

[0041] Take the lyophilized Vibrio campeosa phage CP2 powder, reconstitute it with sterile seawater with a salinity of 25–30‰ and a pH of 6.5–7.5, and inoculate it until the concentration reaches 1×10⁻⁶. 8 CFU / mL Vibrio campei in the logarithmic phase was cultured in 2216E medium at 30°C with shaking at 180 rpm for 6–8 h; the titer was determined by the double-layer plate method and adjusted to 1×10⁻⁶. 9 ~1×10¹ 0 PFU / mL, to obtain phage working solution;

[0042] Take 10 mL of water sample into a sterile centrifuge tube. Wipe the solid surface with a sterile cotton swab dipped in physiological saline and then wash it into 10 mL of physiological saline. Take 1 mL of the sample to be tested, serially dilute it, spread it on a 2216E plate containing 5% defibrinated sheep blood, and anaerobically incubate at 30℃ for 12-16 h. Count the bacteria to determine the initial bacterial count and complete the bacterial count test.

[0043] Apply the working solution at an MOI ratio of 10:1 to 20:1. After uniformly spraying the solution in an aquatic environment, allow it to stand for 4 to 6 hours at 15 to 30°C and pH 6 to 8. Alternatively, immerse solid surfaces in the working solution or spray 50 to 100 mL per square meter and then air dry.

[0044] The residual bacterial count should be measured according to the bacterial count testing method. The result is ≤1×10²CFU / mL for water and ≤10CFU / 100cm² for solid surface. If the result is not satisfactory, apply the same MOI and repeat the treatment for 3-4 hours until the standard is met.

[0045] Application of Vibrio campbellii bacteriophage: The bacteriophage can be applied to the surface of the host being controlled, to living water bodies, or to purify the aquaculture environment.

[0046] The Vibrio campei phage CP2 is resistant to povidone-iodine, a chemical agent commonly used in aquaculture.

[0047] Specifically, the initial valence is taken as 1×10¹ 0 The experiment used pure culture medium of phage CP2 with a concentration of PFU / mL, as well as commonly used concentration gradients of povidone-iodine in aquaculture (10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, corresponding to the recommended dose and 1.5 times the dose for clinical disinfection). A phage culture medium without povidone-iodine was used as a blank control, and a conventional aquatic Vibrio phage BP4 (accession number CCTCCM2024056) was set as a control phage to ensure comparability of the experiments.

[0048] Tolerance test: Different concentrations of povidone-iodine solution were mixed with pure culture media of phage CP2 and control phage BP4 at a ratio of 1:9 (v / v) to construct drug-phage mixed systems with final concentrations of 1 mg / L, 2 mg / L, 3 mg / L, and 5 mg / L, with 3 replicates for each group; all systems were placed in a constant temperature environment of 25℃ and samples were taken after 2 h, 4 h, 8 h, and 24 h of treatment.

[0049] Potency determination and results: The potency of each group of phages was determined by the double-layer plate method (the bottom layer was 2216E medium containing 2% agar, and the top layer was a mixture of 2216E medium containing 0.7% agar, 100 μL of phage dilution, and 100 μL of Vibrio campestris logarithmic phase culture).

[0050] The results showed that when the final concentration of povidone-iodine was ≤3 mg / L, the titer of bacteriophage CP2 remained at 5 × 10⁻⁶ after 24 hours of treatment. 9 ~8×10 9 PFU / mL, potency loss ≤50%; even at 5 mg / L (1.5 times the recommended dose), 2×10⁻⁶ PFU / mL was still detectable after 24 hours of treatment. 8 ~3×10 8 Surviving phages at PFU / mL. In contrast, the control phage BP4, at a povidone-iodine concentration ≥2 mg / L, showed a titer decreasing to 1 × 10⁻⁶ after 8 hours of treatment. 7 When the PFU / mL level is below a certain threshold, no activity can be detected after 24 hours of treatment.

[0051] The results indicate that bacteriophage CP2 can tolerate the conventional dosage of povidone-iodine in aquaculture, and the combined use of the two does not lead to rapid inactivation of the bacteriophage. This meets the synergistic application requirements of chemical disinfection and bacteriophage biocontrol, and is suitable for complex control scenarios in aquaculture environments.

[0052] The application of Vibrio campei bacteriophage is used to prepare drugs for the prevention or treatment of diseases caused by pathogenic Vibrio infections in aquatic organisms; the pathogenic Vibrio includes one or more of Vibrio campei, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio prawnicana.

[0053] The aquaculture environment includes, but is not limited to, seedling ponds, elevated ponds, earthen ponds, factory-style sheds, and feeding platforms;

[0054] Specifically, aquatic organisms (such as shrimp larvae and fish larvae) have weak immunity during the seedling stage and are susceptible to Vibrio infection, which can lead to large-scale mortality. Therefore, it is necessary to use Vibrio campestris phage CP2 preparations (solution, titer 1×10⁻⁶) to address this issue. 9 Add PFU / mL to the seedling water at a ratio of 1:1000 (v / v), and apply every 3 days; simultaneously, apply phage CP2 powder (potency 1×10) once a week. 8 Adding 0.05% PFU / g to seedling feed can effectively inhibit the proliferation of Vibrio in water and intestines, and reduce the mortality rate during the seedling stage.

[0055] High-level ponds and earthen ponds: High-level ponds (high-density aquaculture) and earthen ponds (traditional aquaculture) have large water volumes and easily accumulate uneaten feed and feces, making them prone to Vibrio growth. Bacteriophage CP2 solution can be applied at a concentration of 1×10⁻⁶. 6 Apply a uniform spray of PFU / L concentration to the water body 2-3 times per month; replenish the spray after each water change. In conjunction with testing the bottom sludge every 15 days using a phage CP2 preparation. If the Vibrio concentration is >1×10³CFU / g, increase the spraying dosage to 2×10³CFU / g. 6 PFU / L, maintain Vibrio concentration in water ≤1×10²CFU / mL.

[0056] Factory-style greenhouses: Factory-style greenhouses have strong sealing properties and easy water temperature control, making them suitable for the combined use of bacteriophage CP2 and povidone-iodine. First, spray povidone-iodine at the conventional dose (1 mg / L) for disinfection, and then spray bacteriophage CP2 solution (1×10⁻⁶) 24 hours later. 6 PFU / L); Daily monitoring of water pH (maintaining 6-8) and dissolved oxygen ensures bacteriophage activity and can effectively prevent vibrio outbreaks in high-density aquaculture.

[0057] Feeding platform: The feeding platform is the core area where uneaten food accumulates and Vibrio gathers. It needs to be treated twice a week with bacteriophage CP2 solution (potency 5×10). 8 Spray the feed platform surface with PFU / mL (50mL per square meter), let it stand for 30 minutes after spraying, and then feed the animals. At the same time, after each feeding, rinse the feed platform with sterile water containing bacteriophages to avoid the growth of Vibrio from residual feed and reduce the risk of Vibrio contact when aquatic animals are feeding.

[0058] The Vibrio campeum phage CP2 exhibits cross-species lytic ability. Besides efficiently lysing Vibrio campeum, it also shows ≥85% lysis rates against Vibrio alginolyticus isolate VA-03, Vibrio anguillarum isolate VA-08, and Vibrio harveyi isolate VH-02. Furthermore, the phage genome does not contain virulence genes, lysogenic integration genes, or drug resistance genes. Intraperitoneal injection experiments have verified that the median lethal dose (LD50) for aquaculture organisms such as Litopenaeus vannamei and grouper is >1×10¹. 0 PFU / tail poses no biosafety risk.

[0059] In the following examples

[0060] The formula for the TSB liquid culture medium is as follows: 17g tryptone, 3g soybean papain digest, 5g sodium chloride, 2.5g potassium dihydrogen phosphate, 2.5g glucose, 1000ml distilled water, pH 7.0.

[0061] The TSB (2% NaCl) culture medium formula is as follows: 17g tryptone, 3g soybean papain digest, 20g sodium chloride, 2.5g potassium dihydrogen phosphate, 2.5g glucose, 1000ml distilled water, pH 7.0.

[0062] The formula for the TSA solid culture medium is as follows: 15g tryptone, 5g soybean papain digest, 5g sodium chloride, 15g agar, 1000ml distilled water, pH 7.0.

[0063] The semi-solid agar medium formula is as follows: 17g tryptone, 3g soybean papain digest, 5g sodium chloride, 2.5g potassium dihydrogen phosphate, 2.5g glucose, 7g agar, 1000ml distilled water, pH 7.0.

[0064] The SM solution formula is as follows: 8.5g sodium chloride, 2g magnesium sulfate, 50ml 1mol / L Tris-HCl, 0.25g gelatin, and 1000ml distilled water.

[0065] Example 1: Isolation, purification, and enrichment of Vibrio campbellii bacteriophage CP2

[0066] 1. Phage isolation:

[0067] Collect 50ml of seafood samples from Fujian and 50ml of seawater samples from Hainan. Centrifuge at 5000rpm for 10min, take 20ml of supernatant and sterilize it. Mix it evenly with 20ml of 2x TSB liquid medium and 2ml of Vibrio campestris VCHN1 bacterial suspension in the logarithmic phase (108cfu / ml). Incubate overnight at 30℃ and 150rpm to enrich bacteriophages.

[0068] Take the above culture and filter about 1 mL of the filtrate through a 0.22 μm sterile filter membrane for later use.

[0069] Double-layer plate drop experiment: Take 0.3 mL of the host bacteriophage VCHN6 in logarithmic phase and add it to 4 mL of 40°C TSB semi-solid agar medium. Mix well and pour onto a TSA plate to prepare a double-layer plate containing the host bacteria. After the upper agar solidifies, take 10 μL of the filtered filtrate and drop it onto the solidified double-layer plate. After the filtrate is completely absorbed, invert the plate and incubate it overnight at 30°C to form bacteriophage drop spots.

[0070] 2. Phage purification:

[0071] On a double-layer agar plate where plaques have formed, large, clear plaques are picked up with a sterile pipette tip, desorbed by shaking in 1 ml of SM solution, and then sterilized by passing through a 0.22 μm microporous membrane to obtain phage filtrate. This filtrate is inoculated into 5 ml of TSB liquid medium, 0.1 ml of Vibrio campestris bacterial suspension is added and mixed well, and the mixture is incubated overnight at 30°C and 150 rpm. After centrifugation at 5000 rpm for 10 min, the supernatant is collected and filtered through a bacterial filter membrane. The morphology of the plaques is observed using the double-layer agar plate method. After repeating the operation 3-5 times, plaques of consistent shape and size can be obtained (e.g., ...). Figure 1 (As shown).

[0072] 3. Phage enrichment:

[0073] Using a sterile pipette tip, pick up phage plaques of uniform shape and size, and place them in 50 mL of TSB medium containing 1 mL of logarithmic-phase host bacteria. Incubate overnight at 30°C and 160 rpm in a shaker. Centrifuge the culture at 8000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane. This is the purified phage solution, yielding Vibrio campbelliiphage CP2, with the accession number CCTCCM20251108.

[0074] Example 2: Determination of CP2 titer of Vibrio campbellii bacteriophage

[0075] Using SM solution as a diluent, the original solution of Vibrio campeosa phage CP2 (prepared in Example 1) was serially diluted 10-fold to 10⁸ times. 1000 μl of phage culture at dilutions of 10⁵, 10⁶, 10⁷, and 10⁸ was mixed thoroughly with 300 μl of its host bacterium, Vibrio campeosa VCHN1, and allowed to stand for 15 min to allow for sufficient binding to receptors on the bacterial surface. The mixture was then added to 4 ml of semi-solid agar medium cooled to 50°C, mixed well, and immediately spread onto solidified solid agar plates. After the agar solidified, the plates were inverted and incubated at 30°C for 6-8 h. Three replicates were prepared for each dilution, and the average of the three replicates for each dilution was used for counting. The phage titer (PFU / ml) was calculated as: average number of plaques × dilution factor.

[0076] Table 1 shows that Vibrio campbellii phage CP2 has a titer of over 10¹⁰ PFU / ml after 12 hours of culture.

[0077] Table 1. Titer of Vibrio campbellii bacteriophage CP2 after 12 hours of culture.

[0078]

[0079] Example 3: Electron microscopic observation of Vibrio campbellii phage CP2

[0080] The purified phage solution prepared in Example 1 was observed under an electron microscope: 20 μL of sample was dropped onto a copper grid and allowed to precipitate naturally for 15 min. Excess liquid was absorbed from the side with filter paper. One drop of 2% phosphotungstic acid was added to the copper grid and stained for 10 min. The staining solution was then absorbed from the side with filter paper and dried before observation under an electron microscope.

[0081] The results are as follows Figure 2 As shown, observation of the morphology of Vibrio campei phage CP2 under an electron microscope revealed that the phage has a polyhedral three-dimensional symmetrical head and a relatively short tail. The head diameter is approximately 60–70 nm, and the tail length is approximately 15–20 nm, with a tail diameter of approximately 10–15 nm, exhibiting a relatively short tail.

[0082] Example 4: Extraction and sequencing of the CP2 genome of Vibrio campei phage

[0083] Take 100 mL of the purified phage solution prepared in Example 1, add 20 μL of Dnase I and 20 μL of Rnase A at a concentration of 5 mg / mL in sequence, incubate at 37°C for 60 min, then add 5.84 g of NaCl, and place in an ice bath for 1 h after dissolution.

[0084] Centrifuge at 11,000 rpm for 10 min at 4℃, transfer the supernatant to a new centrifuge tube, add solid PEG8000 to make a final concentration of 10% (w / v), and incubate on ice for 1 h after the PEG8000 is completely dissolved.

[0085] Then, centrifuge at 11,000 rpm for 20 min at 4℃, add 1 mL of SM solution to resuspend the precipitate, and obtain the phage particle concentrate, which is stored at 4℃ for later use.

[0086] Example 5: Determination of the optimal multiple of infection (MOI) of Vibrio campbellii bacteriophage CP2 against Vibrio campbellii.

[0087] A single colony of *Vibrio campeosa* was picked and inoculated into a test tube containing 3 ml of TSB (2% NaCl) culture medium. The culture was incubated at 30°C and 150 rpm for 12 h with shaking to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 ml of TSB (2% NaCl) culture medium at a ratio of 1:100 and incubated at 30°C and 150 rpm until the early logarithmic growth phase. Pure culture medium of bacteriophage CP2 (prepared in Example 1) and host bacteria (MOI = number of bacteriophages / number of bacteria) were added according to the multiplicity of infection ratio. TSB liquid medium was added to ensure the total volume of each tube was the same. The tubes were incubated overnight at 30°C and 150 rpm with shaking. After incubation, the tubes were centrifuged at 5000g for 10 min, and the supernatant was collected to determine the phage titer. Double replicates were performed at each point, and the average value was taken. The MOI that produced the highest phage titer was considered the optimal multiplicity of infection. The experiment was repeated three times.

[0088] The results are shown in Table 2. When the titer of Vibrio campbellii phage CP2 reached its maximum (2.9 × 10¹⁰ PFU / ml), its MOI was 1:10000.

[0089] Table 2. Titer of Vibrio campbellii phage CP2 at different multiplicity of infection.

[0090]

[0091] Example 6: pH stability test of Vibrio campbellii bacteriophage CP2

[0092] Add 900 μl of TSB medium at different pH values ​​(1-14) to sterile EP tubes and place them in a 25°C water bath. After temperature equilibration, add 100 μl of pure bacterial culture and incubate at room temperature for 1 hour. After the reaction time is complete, dilute the samples appropriately and determine the phage titer using the double-layer plate method. Repeat the above process at 4 h, 8 h, 24 h, and 96 h, for a total of three experiments.

[0093] The results are shown in Table 3. The titer of Vibrio campbellii phage CP2 did not change significantly after treatment at pH 6-8 for 96 h.

[0094] Table 3. pH stability of Vibrio campbellii phage CP2 after different reaction times (initial titer: 8 × 10⁹ PFU / ml)

[0095]

[0096] Example 7: Thermal stability test of Vibrio campbellii bacteriophage CP2

[0097] 100 μl of pure phage culture was placed in sterile EP tubes and incubated in water baths at 55℃, 65℃, and 75℃ for 2 h, 24 h, and 48 h, respectively. After the incubation period, the sample tubes were removed and immediately placed in an ice bath to cool. After appropriate dilution, the phage titer was determined using the double-layer plate method. The experiment was repeated three times.

[0098] The results are shown in Table 4. In the experimental group, Vibrio campbellii phage CP2 survived more easily at 55℃. Vibrio campbellii phage CP2 has good heat resistance; compared with the control, it still has an 80% survival rate after a 2-hour water bath at 65℃, and still has a titer of 10⁶ PFU / ml after a 2-hour water bath at 75℃.

[0099] Table 4. Titer of Vibrio campbellii bacteriophage CP2 at different temperatures.

[0100]

[0101] Example 8: Survival stability test of Vibrio campbellii bacteriophage CP2

[0102] Take 5 μL of Vibrio campbellii phage CP2 pure culture medium and dispense it into sterile test tubes. Place the tubes at 4℃, 25℃, and 30℃ respectively. After periodic dilution, determine the phage titer using the double-layer plate method.

[0103] The results are shown in Table 5. At 4℃, the titer of Vibrio campbellii phage CP2 remained at 10⁹ PFU / ml after 50 weeks of storage; at 25℃, the titer of CP2 did not decrease by the order of magnitude (10⁹ PFU / ml) after 12 weeks of storage; at 30℃, phage CP2 could maintain a titer of 10⁸ PFU / ml or higher for 12 weeks; and the phages at all storage temperatures had a strong lytic ability against the host bacteria.

[0104] Table 5. Survival stability of Vibrio campbellii bacteriophage CP2 at different storage temperatures.

[0105]

[0106] Example 9: Detection test for deletion of Vibrio campbellii phage CP2 virulence gene or adverse gene

[0107] Sixty-five virulence genes identified as originating from lysogenic bacteriophages within pathogenic bacteria were selected. The whole genome of Vibrio campei bacteriophage CP2 was determined and subjected to bioinformatics analysis to determine whether it contained the aforementioned virulence genes.

[0108] Example 10: Test on the lysis range of Vibrio campbellii phage CP2 against Vibrio campbellii

[0109] The lysis profile of bacteriophages was determined using a spot drop method. A stock solution of Vibrio campbellii bacteriophage CP2 with a titer of 1×10⁸ PFU / ml was prepared as a spot drop solution.

[0110] Single colonies of 180 Vibrio campeosa isolated from different regions were inoculated into test tubes containing 3 ml of TSB (2% NaCl) and incubated at 150 rpm for 8 h to obtain bacterial suspensions for each strain. 300 μl of the bacterial suspension was mixed with semi-solid culture medium and spread onto ordinary agar plates. 10 μl of phage culture and CP2 drop solution were then added to different locations on the plates, ensuring that the phage cultures did not touch each other to avoid affecting the experimental results. After air drying, the plates were incubated at 30℃ for 6-8 h, and the results were observed. The experiment was repeated three times.

[0111] Vibrio campeum phage CP2 has a broad host range, recognizing 174 strains of Vibrio campeum with a lysis rate of 96.7%. This indicates that Vibrio campeum phage CP2 has a wide host spectrum and great potential for application in phage therapy.

[0112] Example 12: Lysis experiment of Vibrio campbellii bacteriophage CP2 on non-host pathogenic bacteria

[0113] Sixty-two single colonies of non-host pathogenic bacteria were inoculated into test tubes containing 3 ml of TSB and incubated at 150 rpm for 8 hours to prepare bacterial suspensions for each strain. 300 μl of the bacterial suspension was mixed with semi-solid culture medium and spread onto ordinary agar plates. 10 μl of bacteriophage culture was dropped onto different locations on the plates, ensuring that the bacteriophage cultures did not touch each other to avoid affecting the experimental results. After air drying, the plates were incubated at 37°C for 6-8 hours, and the results were observed. The experiment was repeated three times.

[0114] Example 13: Fermentation preparation of Vibrio campbellii bacteriophage CP2

[0115] A single colony of *Vibrio campbellii* VCHN1 was picked and inoculated into a test tube containing 3 ml of TSB (2% NaCl) culture medium. The culture was incubated at 30°C and 150 rpm for 12 h to obtain a host bacterial suspension. The suspension was then transferred at a 1:100 ratio to 500 ml of TSB (2% NaCl) culture medium and incubated at 30°C and 150 rpm until the early logarithmic growth phase, at which point the bacterial suspension concentration was determined. The fermentation system for *Vibrio campbellii* phage CP2 was 8 L, using TSB medium. The initial pH of both fermentation media was 7. Inoculation was performed using the flame inoculation method, with 50 ml of phage (10⁶ PFU / ml) and 50 ml of host bacterial suspension in the logarithmic growth phase (10⁹ CFU / ml) added to the fermentation medium at their respective optimal multiplicity of infection ratios. Sterile air was introduced during fermentation, and 3‰ antifoaming agent was added. The fermentation time was 12 h. Every 2 hours from the start of fermentation, 20 ml of the phage and host bacteria mixture was collected from the sampling port into a sterile container and centrifuged at 5000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm microporous membrane to obtain a phage-containing filtrate, and its titer was determined, following the method described in Example 2. After fermentation was completed, the entire phage and host bacteria mixture was collected from the sampling port and transferred to a sterile container. The mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a vacuum pump into a sterile filter device to obtain the phage fermentation broth, which was then stored at 4°C.

[0116] As shown in Table 11, the titer of Vibrio campeosaphire phage CP2 reached its highest level of 3.5 × 10¹¹ PFU / ml after 12 hours of fermentation. After 12 hours of fermentation, the phage titer increased from the initial 10⁴ PFU / ml to 10¹¹ PFU / ml and above, an increase of seven orders of magnitude. Therefore, large-scale industrial preparation of phages using fermentation is indeed feasible.

[0117] Table 11 Fermentation dynamics of Vibrio campbellii phage CP2

[0118]

[0119] Example 14: Identification of CP2 lysogenicity of Vibrio campeosaphire phage

[0120] 1. Experimental Method:

[0121] 100 μL of bacteriophage CP2 (prepared in Example 13) (0 PFU / mL, 1.0 × 10⁴ PFU / mL, 1.0 × 10⁵ PFU / mL, 1.0 × 10⁶ PFU / mL, 1.0 × 10⁷ PFU / mL) and 100 μL of Vibrio campbellii (1.0 × 10⁸ CFU / mL) were mixed separately and inoculated into 50 mL centrifuge tubes containing 10 mL of TSB (2% NaCl) liquid medium. The mixtures were then incubated at 30 °C with shaking for 18 h. The resulting turbid culture was serially diluted and plated on TSA plates and incubated overnight at 30 °C. The central portion of 30–50 single colonies was picked from a TSA plate and placed in an EP tube containing 200 μL TSB liquid medium. The tube was then incubated at 30°C with shaking for 24 h. Mitomycin C was then added to the EP tube to a final concentration of 0.5 μg / mL, and the tube was incubated for another 12 h. The resulting culture was then filtered through a 0.22 μm filter to remove bacteria and spotted onto a VCHN6 bilayer plate, incubated at 30°C. Simultaneously, bacteriophage CP2 (prepared in Example 13) with a titer of 1.0 × 10⁷ PFU / mL was spotted onto the bilayer plate as a positive control. After 24 h, the bilayer plates were observed. The presence of phage plaques indicated that phage CP was a lysogenic phage.

[0122] 2. Experimental Results and Analysis:

[0123] The results showed that plaques appeared on all control plates. Figure 3 No plaques were observed on any of the test plates. Figure 4 This indicates that Vibrio campbellii phage CP2 is not lysogenic and is a virulent phage.

[0124] Example 15: Turbidimetric assay for the inhibition rate of Vibrio campbellii bacteriophage CP2 against Vibrio campbellii.

[0125] Single colonies of the host bacterium VCHN6 were inoculated into test tubes containing 3 mL of TSB (2% NaCl) and cultured overnight at 30°C and 150 rpm until turbidity was achieved, thus obtaining the host bacterium suspension. Bacteriophage CP2 (prepared in Example 13) with a titer of 2 × 10¹⁰ PFU / mL was serially diluted with SM solution to each treatment group titer. As shown in Table 12, 100 μL of VCHN6 suspension and 100 μL of each diluted CP2 were added to 50 mL centrifuge tubes containing 10 mL of TSB (2% NaCl) liquid medium, respectively. A mixture of 100 μL VCHN6 suspension and 10 mL of TSB liquid medium was used as a positive control. All treatments were incubated at 30°C and 120 rpm for 24 h, and the turbidity of each treatment group was measured using a turbidimeter. Inhibition rate = (Turbidity of positive treatment - Turbidity of treatment group) / Turbidity of positive treatment × 100%.

[0126] Table 12. Antibacterial effect of Vibrio campbellii bacteriophage CP2 against Vibrio campbellii.

[0127]

[0128] Example 16: Effects of Vibrio campestris phage CP2 on shrimp metabolic processes after feeding

[0129] 1. Experimental Method:

[0130] Healthy whiteleg shrimp weighing approximately 5g were selected and slaughtered overnight on an empty stomach. An experimental group and a control group were set up.

[0131] In the experimental group, bacteriophage CP2 was added at a volume-to-mass ratio (ml / g) of 5%, soaked in shrimp feed, and mixed evenly. The titer of the bacteriophage after mixing with the feed was 4×10⁸ PFU / g. The feed was then air-dried, and the shrimp in the experimental group were fed with the bacteriophage at a dose equal to 5% of their body weight. The control group was fed the same dose of SM solution-soaked feed. The content of bacteriophage in the intestines, hepatopancreas, and body of shrimp in both the experimental and control groups was measured at 0.5, 6, 24, 48, 72, 96, 120, and 144 hours after feeding.

[0132] 2. Experimental Results and Analysis:

[0133] After feeding shrimp with feed soaked in bacteriophage CP2 for 0.5 hours, bacteriophages were detected in the intestines and hepatopancreas of the experimental group of shrimp. The bacteriophage titer in the intestines reached 4.3 × 10⁷ PFU / g, and the titer in the hepatopancreas reached 3.1 × 10⁷ PFU / g, indicating that the bacteriophage can enter the shrimp body through feeding and attach to and colonize the hepatopancreas and intestines with a relatively high colonization rate. Furthermore, as shown in Table 13, the bacteriophage can persist in the hepatopancreas and intestines of shrimp for more than 2 days. This provides guidance for the use of bacteriophages as aquatic feed additives and can be used for the prevention and treatment of Vibrio campestris infection. The experimental results also showed that the shrimp's movement, stress response, and feeding behavior were normal, with no difference from the control group, indicating that the bacteriophage has no effect on the health of Litopenaeus vannamei and that the bacteriophage has good safety.

[0134] Table 13 Changes in the content of Vibrio campei bacteriophage CP2 in shrimp after feeding.

[0135]

[0136] Example 17: Determination of the efficacy of bacteriophage CP2 in preventing Vibrio campbellii infection in Litopenaeus vannamei.

[0137] 2. Experimental methods:

[0138] Healthy whiteleg shrimp weighing approximately 5g were prepared and fasted for one day before the experiment. Each group consisted of 50 shrimp, with two experimental groups, one control group, and one blank control group. The shrimp were then treated as follows:

[0139] The blank control group was fed with ordinary feed at a dose of 5% of the shrimp's body weight; the control group was fed according to the volume to mass ratio

[0140] The addition amount was 5% (ml / g). The feed was soaked in SM buffer and then fed. The experimental group was fed the same amount of phage CP2 soaked in shrimp feed, mixed evenly, and the titer of phage after mixing with feed was 1×108 PFU / mL. After being air-dried, the shrimp in the experimental group were fed the above-mentioned shrimp feed mixed with phage.

[0141] One hour after the first feeding, shrimp in both the experimental and control groups were immersed in a solution of Vibrio campestris at a concentration of 1×10⁵ CFU / mL. The blank control group received no treatment. The number of shrimp that died within 48 hours in each group was recorded, and the phage protection rate was calculated. The specific results are shown in Table 14 below.

[0142] 2. Experimental Results and Analysis:

[0143] The mortality rate of shrimp in the control group was as high as 86%, while the mortality rate of shrimp in the experimental group was as low as 6-10%, and the protection rate of shrimp by phage-mixed feed reached 90-94%. It can be seen that phage CP2 has a good therapeutic effect on Vibrio campbellii disease in Litopenaeus vannamei and can significantly improve the survival rate of shrimp.

[0144] Table 14. Therapeutic effects of bacteriophage-mixed feed on Vibrio campeella infection.

[0145]

[0146] Example 18 Compatibility test of Vibrio campestris phage CP2 with povidone-iodine

[0147] 1. Experimental Method:

[0148] Phage CP2, with an initial titer of 1×10⁹ pfu / mL, was aliquoted into 50 ml sterile centrifuge tubes. The normal dosage of povidone-iodine was added, and the tubes were incubated at 25°C for 2 h, 4 h, 8 h, 24 h, and 48 h. The phage titers were then measured, and the results are shown in Table 15.

[0149] 2. Experimental Results and Analysis:

[0150] As shown in Table 14, after 24 hours of coexistence with the chemical drug povidone-iodine, the titer of phage CP2 remained at 108 pfu / mL. After 48 hours of exposure, at least 106 pfu / mL of phage CP2 was still detectable. This indicates that phage RAP43-8 can coexist with normal doses of povidone-iodine for a period of time and has a strong resistance to povidone-iodine and other chemical drugs. This suggests that Vibrio campestris CP2 can be used in combination with some chemical drugs in clinical practice to achieve better results.

[0151] Table 15 Compatibility results of Vibrio campbellii bacteriophage CP2 and povidone-iodine.

[0152]

[0153] In addition, the present invention also provides a terminal device. The method for preventing and treating harmful infections of Vibrio campestris in vitro involved in this embodiment is mainly applied in the terminal device, which can be a PC, a portable computer, a mobile terminal or other device with display and processing functions.

[0154] Specifically, the terminal device may include a processor (e.g., CPU), a communication bus, a user interface, a network interface, and memory. The communication bus is used to enable communication between these components; the user interface may include a display screen or an input unit such as a keyboard; the network interface may optionally include a standard wired interface or a wireless interface (such as a Wi-Fi interface); the memory may be high-speed RAM or stable non-volatile memory, such as disk storage, and may also optionally be a storage device independent of the aforementioned processor.

[0155] The memory stores a readable storage medium, which stores a method program for preventing harmful infections of Vibrio campestris in vitro. The processor can call the method program for preventing harmful infections of Vibrio campestris in vitro stored in the memory and execute the method for preventing harmful infections of Vibrio campestris in vitro provided in this embodiment of the invention.

[0156] Understandably, a readable storage medium can be a tangible device capable of holding and storing instructions for use by an instruction execution device. A computer-readable storage medium can be, for example—but not limited to—an electrical storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination thereof. More specific examples (a non-exhaustive list) of computer-readable storage media include: portable computer disks, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disc read-only memory (CD-ROM), digital multifunction disc (DVD), memory sticks, floppy disks, mechanical encoding devices, such as punch cards or recessed protrusions storing instructions thereon, and any suitable combination thereof. The computer-readable storage medium as used herein is not to be construed as a transient signal itself, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through waveguides or other transmission media (e.g., light pulses through fiber optic cables), or electrical signals transmitted through wires.

[0157] The computer-readable program instructions described herein can be downloaded from computer-readable storage media to various computing / processing devices, or downloaded via a network, such as the Internet, local area network, wide area network, and / or wireless network, to an external computer or external storage device. The network may include copper transmission cables, fiber optic transmission, wireless transmission, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards them to the computer-readable storage media in the respective computing / processing device.

[0158] Computer program instructions used to perform operations of this disclosure may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, status setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, etc., and conventional procedural programming languages ​​such as the "C" language or similar programming languages. The computer-readable program instructions may execute entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In cases involving a remote computer, the remote computer may be connected to the user's computer via any type of network—including a local area network (LAN) or a wide area network (WAN)—or may be connected to an external computer (e.g., via the Internet using an Internet service provider). In some embodiments, electronic circuitry, such as programmable logic circuitry, field-programmable gate arrays (FPGAs), or programmable logic arrays (PLAs), is personalized by utilizing the status information of the computer-readable program instructions to implement various aspects of this disclosure.

[0159] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A phage of Vibrio campbellii capable of lysis across species, characterized in that: The bacteriophage is Vibrio campbellii phage CP2, deposited at the China Center for Type Culture Collection, accession number CCTCCM20251108. This bacteriophage is a virulent bacteriophage. Under electron microscopy, it has a polyhedral symmetrical head and a relatively short tail. The head diameter is about 60-70 nm, the tail length is about 15-20 nm, and the tail diameter is about 10-15 nm.

2. The *Vibrio campbellii* bacteriophage capable of cross-species lysis according to claim 1, characterized in that: The Vibrio campei phage CP2, when cultured for 12 hours at a multiplicity of infection (MOI) of 1:10000, achieved a titer of 3.5 × 10¹¹ PFU / mL.

3. The *Vibrio campbellii* bacteriophage capable of cross-species lysis according to claim 1, characterized in that: The optimal pH range for Vibrio campbellii phage CP2 is 6–8, and it exhibits good stability at temperatures ranging from 4°C to 75°C.

4. A pharmaceutical formulation containing the bacteriophage according to any one of claims 1 to 3, characterized in that: The dosage form of the pharmaceutical preparation is a solution or a lyophilized powder.

5. A method for in vitro prevention and control of harmful infections caused by Vibrio campestris, characterized in that: Using the lytic action of Vibrio campestris phage CP2 as described in any one of claims 1 to 3, Vibrio campestris is killed in an in vitro environment, and the method includes the following steps: Take the lyophilized Vibrio campeosa phage CP2 powder, reconstitute it with sterile seawater with a salinity of 25–30‰ and a pH of 6.5–7.5, and inoculate it until the concentration reaches 1×10⁻⁶. 8 CFU / mL Vibrio campei in the logarithmic phase was cultured in 2216E medium at 30°C with shaking at 180 rpm for 6–8 h; the titer was determined by the double-layer plate method and adjusted to 1×10⁻⁶. 9 ~1×10¹ 0 PFU / mL, to obtain phage working solution; Take 10 mL of water sample into a sterile centrifuge tube. Wipe the solid surface with a sterile cotton swab dipped in physiological saline and then wash it into 10 mL of physiological saline. Take 1 mL of the sample to be tested, serially dilute it, spread it on a 2216E plate containing 5% defibrinated sheep blood, and anaerobically incubate at 30℃ for 12-16 h. Count the bacteria to determine the initial bacterial count and complete the bacterial count test. Apply the working solution at an MOI ratio of 10:1 to 20:

1. After uniformly spraying the solution in an aquatic environment, allow it to stand for 4 to 6 hours at 15 to 30°C and pH 6 to 8. Alternatively, immerse solid surfaces in the working solution or spray 50 to 100 mL per square meter and then air dry. The residual bacterial count should be measured according to the bacterial count testing method. The result is ≤1×10²CFU / mL for water and ≤10CFU / 100cm² for solid surface. If the result is not satisfactory, apply the same MOI and repeat the treatment for 3-4 hours until the standard is met.

6. The application of Vibrio campestris phage according to any one of claims 1 to 3, characterized in that: The bacteriophage can be applied to the surface of the host being controlled, to living water bodies, or to purify the aquaculture environment.

7. The Vibrio campeosa phage capable of cross-species lysis according to claim 1, characterized in that: The Vibrio campei phage CP2 is resistant to povidone-iodine, a chemical agent commonly used in aquaculture.

8. The application of Vibrio campestris phage according to any one of claims 1 to 3, characterized in that: This is used to prepare a drug for the prevention or treatment of diseases caused by pathogenic Vibrio infection in aquatic organisms; the pathogenic Vibrio includes one or more of Vibrio campeosa, Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio prawnicola.

9. The application of Vibrio campestris phage according to claim 6, characterized in that: The aquaculture environment includes, but is not limited to, seedling ponds, elevated ponds, earthen ponds, factory-style sheds, and feeding platforms.

10. A Vibrio campeosa phage capable of cross-species lysis according to claim 1, characterized in that: The Vibrio campeum phage CP2 exhibits cross-species lytic ability. Besides efficiently lysing Vibrio campeum, it also shows ≥85% lysis rates against Vibrio alginolyticus isolate VA-03, Vibrio anguillarum isolate VA-08, and Vibrio harveyi isolate VH-02. Furthermore, the phage genome does not contain virulence genes, lysogenic integration genes, or drug resistance genes. Intraperitoneal injection experiments have verified that the median lethal dose (LD50) for aquaculture organisms such as Litopenaeus vannamei and grouper is >1×10¹. 0 PFU / tail poses no biosafety risk.

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