A lytic vibrio alginolyticus bacteriophage with high efficiency and a composition and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a highly efficient lysing Vibrio alginolyticus phage derived from abalone, its composition, and its applications. The phage, named RKP-VA24471, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46426. The phage has a titer of not less than 1 × 10⁻⁶. ^10 With a PFU / mL concentration and an optimal MOI of 0.01, this phage exhibits advantages such as a short latency period, high outbreak rate, good genetic stability, and a wide adaptability to temperature and pH. Lysis spectrum experiments show that this phage has broad-spectrum lytic activity against various Vibrio species, including *Vibrio alginolyticus* and *Vibrio parahaemolyticus*, with an overall lysis rate of 80.6%. Animal experiments have demonstrated that this phage significantly improves the survival rate of abalone infected with *Vibrio alginolyticus*, outperforming antibiotics and fish-derived phages. The phage and its composition provided by this invention can be used for the prevention and treatment of vibriosis in aquatic animals such as abalone, offering a highly efficient and environmentally friendly biological control solution as an alternative to antibiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a highly efficient lysing Vibrio alginolyticus phage derived from abalone, its composition, and its applications. Background Technology

[0002] Abalone farming is an important part of my country's aquaculture industry. However, bacterial diseases caused by pathogens during the farming process, such as vibriosis, have always been a key factor restricting the healthy development of the industry. Among them, *Vibrio alginolyticus*... (Vibrio) alginolyticus) As a common opportunistic pathogen, it is highly susceptible to outbreaks in high-temperature, high-density aquaculture environments, causing symptoms such as ulcers and foot erosions in abalone. It is highly contagious, has a high mortality rate, and causes significant economic losses to abalone farmers.

[0003] Currently, the aquaculture industry primarily relies on antibiotics for the prevention and control of bacterial diseases. However, the long-term and widespread use of antibiotics has led to the emergence of multidrug-resistant strains such as Vibrio alginolyticus, resulting in a declining effectiveness of antibiotic treatment. Furthermore, the overuse of antibiotics can leave drug residues in water and organisms, disrupting the microecological balance of the aquaculture environment and posing food safety risks. Therefore, finding a green prevention and control technology that can replace antibiotics has become an urgent need in this field.

[0004] Bacteriophages, as viruses capable of specifically infecting and lysing bacteria, are considered potential antibiotic alternatives due to their strong host specificity and low likelihood of developing drug resistance. Currently, research is attempting to isolate bacteriophages targeting Vibrio alginolyticus from natural environments (such as sewage, fish, or other aquatic animals) and explore their applications in aquaculture.

[0005] However, the application of these existing bacteriophage technologies in abalone farming still faces challenges. The physiological environment of abalone (such as temperature, salinity, pH, and mucus environment within the intestines and tissues) differs significantly from that of seawater or fish hosts. Bacteriophages isolated from non-abalone environments may struggle to adapt effectively to the unique microenvironment within abalone, resulting in lower-than-expected survival rates, colonization capabilities, and lysis efficiency, thus affecting the stability and effectiveness of prevention and control.

[0006] Therefore, there is an urgent need in this field for a bacteriophage resource that can better adapt to the abalone farming environment, has high efficiency in lysing Vibrio alginolyticus from abalone, and has stable performance, in order to overcome the shortcomings of existing prevention and control methods.

[0007] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a highly efficient lysing Vibrio alginolyticus phage derived from abalone, its composition, and its applications.

[0009] The technical solution of this invention is as follows: On the one hand, the present invention provides a highly efficient lysing Vibrio alginolyticus phage derived from abalone, wherein the phage is a Vibrio alginolyticus phage. (Vibrio alginolyticus bacteriophage) RKP-VA24471 was deposited on March 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46426.

[0010] On the other hand, the titer of the bacteriophage is not less than 1×10⁻⁶. ^10 PFU / mL.

[0011] On the other hand, the optimal multiplicity of infection (MOI) of the phage was 0.01, and after 6 hours of fermentation, the phage titer reached 8.41 × 10⁻⁶. 11 PFU / mL.

[0012] On the other hand, the phage has a latency period of 20 min, an outbreak period of 60 min, and a burst rate of 207 PFU / cell.

[0013] On the other hand, the bacteriophage can also lyse Vibrio parahaemolyticus. (Vibrio parahaemolyticus) Vibrio harveyi (Vibrio harveyi) and Vibrio cholerae ( Vibrio cholerae) One or more plants in the group.

[0014] On the other hand, the present invention provides a composition comprising the above-mentioned Vibrio alginolyticus phage RKP-VA24471, and a pharmaceutically or aquaculture-acceptable carrier.

[0015] On the other hand, the dosage form of the composition is a liquid formulation, a lyophilized powder formulation, or a microcapsule formulation.

[0016] The present invention provides the use of the above-mentioned Vibrio alginolyticus phage RKP-VA24471 or composition in the preparation of articles for the prevention and / or treatment of Vibrio infections.

[0017] On the other hand, the Vibrio infection is caused by Vibrio bacteria that can be lysed by the bacteriophage RKP-VA24471.

[0018] On the other hand, the Vibrio alginolyticus infection is caused by Vibrio alginolyticus. (Vibrio alginolyticus) Vibrio parahaemolyticus (Vibrio parahaemolyticus) Vibrio harveyi (Vibrio harveyi) Or Vibrio cholerae (Vibrio) cholerae) cause.

[0019] On the other hand, the objects of the prevention and / or treatment are aquatic animals.

[0020] On the other hand, the aquatic animal mentioned is abalone.

[0021] On the other hand, the methods of administration include oral administration, soaking, or injection.

[0022] The beneficial effects achieved by this invention are as follows: 1. The bacteriophage RKP-VA24471 of this invention has an extremely short latency period (20 minutes) and a huge burst rate (approximately 207 PFU / cell), indicating that once it infects the host bacteria, it can rapidly replicate and produce a large number of progeny bacteriophages, achieving rapid clearance of pathogens. The bacteriophage of this invention can achieve extremely high titers (not less than 1×10⁻⁶) during culture. ^10 PFU / mL, up to 10 ^11 With a PFU / mL level and an optimal multiple of infection (MOI) as low as 0.01, this means that extremely high yields can be obtained with less phage seed culture in production, resulting in high production efficiency and significant cost advantages. 2. This invention's bacteriophage breaks with the conventional understanding that bacteriophages are usually highly specific, exhibiting strong lytic ability against a variety of Vibrio species, including Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi. The overall lysis rate against 207 clinically isolated Vibrio strains in the strain library reached 80.6%, with lysis rates exceeding 87% for abalone and shrimp strains. This broad-spectrum characteristic allows a single bacteriophage strain to handle cross-infection with multiple Vibrio species, broadening its application range and increasing its practical value. 3. The bacteriophage of this invention maintains good activity within a temperature range of 20℃ to 50℃ and a pH range of 5 to 10, demonstrating strong adaptability to fluctuations in abalone farming environments. After 30 consecutive generations of subculturing, its titer remains stable, proving its reliable genetic performance with no obvious gene degeneration or functional decline, making it highly suitable for large-scale industrial fermentation production and long-term storage. 4. The abalone-derived bacteriophages described in this invention are significantly more effective than non-abalone-derived bacteriophages isolated from fish hosts in treating vibriosis in abalone. Specifically, they significantly improve the survival rate of infected abalone, clear pathogens from abalone hemolymph and hepatopancreas more quickly, and synergistically enhance the host's immune response (e.g., better recovery of lysozyme and SOD activity). 5. Animal treatment experiments have shown that the use of the bacteriophage of this invention for prevention and treatment is more effective than traditional enrofloxacin antibiotic treatment. Through a combined strategy of "preventive feeding + post-infection injection," the survival rate of infected abalone can be increased from 13.3% to 86.7%, and the bacterial load in tissues can be significantly reduced. The specific lytic action of the bacteriophage can avoid disrupting the aquatic microecological balance, fundamentally reducing antibiotic use and alleviating the development of bacterial resistance, thus achieving both economic and ecological benefits. Attached Figure Description

[0023] Figure 1 This describes the colony morphology of Vibrio alginolyticus derived from abalone on TCBS plates.

[0024] Figure 2 This is the colony morphology of Vibrio alginolyticus derived from abalone in this invention on a Vibrio chromogenic plate.

[0025] Figure 3 This invention relates to phage plaques of host bacteria created by Vibrio alginolyticus phage derived from abalone.

[0026] Figure 4 This is an electron micrograph of the abalone-derived Vibrio alginolyticus phage of the present invention.

[0027] Figure 5 The titer of Vibrio alginolyticus phage from abalone under different infection multiplicity conditions.

[0028] Figure 6 This is a one-step growth curve of the abalone-derived Vibrio alginolyticus phage of the present invention.

[0029] Figure 7 This is the optimal growth temperature curve for the abalone-derived Vibrio alginolyticus bacteriophage of the present invention.

[0030] Figure 8 This is the pH stability curve of the abalone-derived Vibrio alginolyticus phage of the present invention.

[0031] Figure 9 This is the genetic stability curve of the abalone-derived Vibrio alginolyticus phage of the present invention.

[0032] Figure 10 This is a survival rate graph comparing the bactericidal effects of Vibrio alginolyticus bacteriophages from different sources. Note: *** indicates a significant difference (P<0.001), ** indicates a significant difference (P<0.01), and * indicates a significant difference (P<0.05). Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0034] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, *Vibrio alginolyticus* VA24471 from abalone is referred to as "host VA24471" or simply "VA24471," *Vibrio alginolyticus* phage RKP-VA24471 from abalone is referred to as "phage RKP-VA24471" or simply "RKP-VA24471," and *Vibrio alginolyticus* phage RKP-VA24471 from abalone is referred to as "phage RKP-VA24471" or simply "RKP-VA24471."

[0035] Example 1: Phage Screening and Identification 1.1 Isolation and Identification of Vibrio alginolyticus from Abalone Abalone samples exhibiting typical symptoms were collected from aquaculture farms. Visceral tissue was extracted and placed in LB liquid medium (3% NaCl) and incubated at 37°C with shaking for 12 hours. The culture was then streaked onto TCBS plates and incubated overnight at 37°C with the plates inverted. Colony morphology was observed, and yellow colonies were selected for further streaking on TCBS plates for purification three times to obtain uniformly morphologically uniform colonies. Figure 1 As shown. A dominant single colony was selected and inoculated into 3 mL of LB liquid medium, and cultured with shaking at 160 rpm and 37°C for 4 h. Subsequently, it was inoculated onto a Vibrio chromogenic plate and incubated upside down at 37°C for 12 h, where creamy colonies were observed. Figure 2 As shown.

[0036] The prepared bacterial suspension was identified by 16S rRNA gene sequencing technology. The strain was identified as Vibrio alginolyticus and named VA24471. The bacterial suspension was then preserved by streaking it onto LB solid medium plates and incubating at 37℃ for 12 hours. The bacterial growth was then scraped off and placed in 2216E liquid medium. After mixing, it was mixed with 60% glycerol broth at a 1:1 ratio and stored at -80℃.

[0037] 1.2 Isolation and purification of Vibrio alginolyticus bacteriophage derived from abalone (1) Wastewater treatment: Abalone culture water and sludge were mixed and filtered, centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a membrane (0.22 μmol). The filtrate was mixed with the host bacterial culture in the logarithmic growth phase at a ratio of 1:1 and cultured in a shaker at 37℃ for 16 h. The supernatant was then collected and filtered through a membrane (0.22 μmol). The filtrate ① was stored at 4℃ for later use. (2) Phage enrichment: Take 0.1 mL of Vibrio alginolyticus suspension from abalone and 1 mL of filtrate ① and add it to 5 mL of LB liquid medium (3% NaCl). Incubate at 160 rpm and 37 °C for 12 h on a shaker. Then centrifuge at 12,000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter. This is filtrate ②. Repeat the above operation to enrich the phages a second time. Store filtrate ③ at 4 °C for later use. (3) Phage isolation: Phages were isolated using the double plate method. 0.1 mL of host bacterial suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 50 μL of filtrate ③ was spotted onto the plate and allowed to stand until the mixture was absorbed. After incubation at 37°C for 6 h, a transparent phage plaque was picked up with an inoculation loop and placed in 1 mL of SM buffer. The plaque was shaken for 15 s to allow the phage to be completely released, thus obtaining the abalone-derived Vibrio alginolyticus phage. (4) Phage purification: Phages were isolated using the double-plate method. 0.1 mL of phage extract and 0.1 mL of host bacterial suspension were mixed thoroughly. Soft agar cooled to approximately 50°C was added and mixed again. The mixture was then plated on double plates and incubated at 37°C for 6 hours. A single clear plaque with smooth edges was picked and placed in 1 mL of SM buffer. After thorough shaking, 0.1 mL of the extract was mixed with 0.1 mL of the bacterial suspension. Soft agar cooled to approximately 50°C was added and mixed thoroughly. The mixture was then plated on double plates and incubated at 37°C for 6 hours. This step was repeated 3-5 times until uniformly sized, smooth-edged phage plaques were purified. Figure 3 As shown, the purification of the bacteriophage is now complete; (5) Preparation of bacteriophage: Take 0.1 mL of Vibrio alginolyticus suspension from abalone and 1 mL of bacteriophage liquid and add it to 35 mL of LB broth. Shake and incubate at 160 rpm and 37 °C for 6 h. Then centrifuge at 12,000 rpm for 10 min. Filter the supernatant through a membrane (0.22 μmol). The bacteriophage filtrate is used for later use. (6) Preservation of bacteriophage: One abalone-derived Vibrio alginolyticus bacteriophage was isolated. The bacteriophage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80℃.

[0038] 1.3 Electron microscopic observation of bacteriophages Take 20 μL of liquid containing coarse phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 min, then blot away excess liquid with filter paper from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min. Then blot away the staining solution with filter paper from the side. After the sample dries, observe the phage morphology using an electron microscope. Figure 4 As shown: Bacteriophage RKP-VA24471 has a polyhedral head with three-dimensional symmetry, which encapsulates nucleic acid and has a diameter of 58 nm and a tail length of about 84 nm.

[0039] 1.4 Phage whole genome sequencing 1.4.1 Experimental Methods: The library was constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit method; the specific steps are as follows: 1) Construct a library starting with 1 μg of phage genomic DNA; 2) Covaris M220 ultrasonically breaks down DNA to 300-500 bp; 3) Fill in the 3' end with an A and connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit); 4) Library enrichment, PCR amplification for 8 cycles; 5) 2% agarose gel recovery target band (Certified Low Range Ultra Agarose); 6) TBS380 (Picogreen) quantitative analysis: mix according to the data ratio and run on the machine; 7) Bridged PCR amplification was performed on the cBot solid-phase support to generate clusters; 8) Illumina Hiseq sequencing platform, performing 2×150bp sequencing.

[0040] 1.4.2 Experimental Results: The complete genome of bacteriophage RKP-VA24471, as shown in SEQ ID NO.1, is 41551 bp in size. Predictive analysis indicates that the genome of bacteriophage RKP-VA24471 contains 57 coding sequences. It is a strictly lytic bacteriophage and does not contain integrase genes, repressor protein genes, or any known bacterial virulence genes or antibiotic resistance genes.

[0041] Example 2 Fermentation Performance Analysis 2.1 Determination of phage titer Take 0.1 mL of the host bacterial suspension and the concentrated phage RKP-VA24471 solution, respectively, and add them to 10 mL of LB semi-solid medium (dissolved at high temperature and cooled to room temperature). Mix well and pour the mixture onto LB solid agar, ensuring complete coverage of the 10 mL LB solid agar plate (9 cm in diameter). Incubate at 37°C for 6 h. Then, collect a single plaque and place it in 1 mL of SM buffer, shake thoroughly, and filter through a 0.22 μm filter membrane. Take 0.1 mL of the filtrate and serially dilute it 10-fold with sterile water. Then, take 0.1 mL of the bacterial suspension and the diluted phage solution, respectively, and add them to 10 mL of LB semi-solid medium (dissolved at high temperature and cooled to room temperature). Mix well and pour the mixture onto LB solid agar. Incubate at 37°C for 6 h. Select countable plates, count the phage plaques, and calculate the phage titer (PFU / mL) = plaque count × dilution factor / sample volume (mL).

[0042] The titer of bacteriophage RKP-VA24471 was determined to be 2.74 × 10⁻⁶. 11 PFU / mL.

[0043] 2.2 Determination of Optimal Multiple of Infection (MOI) for Bacteriophages The concentration of the host bacteria cultured to the stable phase was adjusted to 10. 8 CFU / mL. MOIs were set at 0.001, 0.01, 0.1, 1, 10, and 100. Phage solution with determined titers was added to the host bacterial culture in the specified proportions, 0.1 mL each. The mixture was incubated at 200 rpm and 37°C with shaking for 6 h. Then, it was centrifuged at 12,000 rpm for 5 min, and the phage titer was measured. The highest multiplicity of infection (MOI) was considered the optimal MOI.

[0044] like Figure 5 As shown, when the multiplicity of infection (MCI) was 0.01, phage RKP-VA24471 exhibited the highest titer, 2.1 × 10⁻⁶. 11 PFU / mL.

[0045] 2.3 One-step growth curve of bacteriophage Take phage fluid (4.86×10⁻⁶) separately. 10 PFU / mL) and host bacterial culture (2.35×10) 8Mix 0.5 mL of phage (CFU / mL) at the ratio required for the optimal multiple of infection (MOI = 0.01), incubate at room temperature for 10 min, then centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash with LB liquid medium, and centrifuge again at 12,000 rpm for 1 min. Repeat the above operation, washing the precipitate 3 times, then add 10 mL of LB liquid medium preheated to 37°C, mix thoroughly, and quickly incubate at 37°C with shaking. Take a sample every 10 min initially and thereafter, aspirating 0.2 mL, then centrifuge at 12,000 rpm for 2 min, and aspirate 0.1 mL of the supernatant for phage titer determination. Finally, plot a one-step growth curve with infection time on the x-axis and the logarithm of the phage titer in the infection system on the y-axis.

[0046] like Figure 6 As shown, within 20 minutes of phage infection of the host bacteria, the number of phages did not increase significantly, indicating a phage latency period of 20 minutes. From 20 to 80 minutes after infection, the number of phages increased rapidly; this period is the phage burst phase, approximately 60 minutes, with a burst dose of about 207 PFU / cell. The phage number remained constant for the following 40 minutes, reaching the stationary growth phase. Phage RKP-VA24471 has a short latency and lysis period, a large burst dose, and good lysis efficiency, making it suitable for phage therapy.

[0047] 2.4 Determination of the optimal growth temperature for bacteriophages Take a sterile 2mL centrifuge tube and add 1mL of phage stock solution (initial titer 4.86×10⁻⁶). 10 The phage (PFU / mL) was incubated at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively. After the incubation period, the sample tubes were removed, diluted to an appropriate concentration, and 0.1 mL of the treated phage solution and the host bacterial solution were taken respectively. The phage titer was determined using the double-plate method.

[0048] like Figure 7 As shown, bacteriophage RKP-VA24471 survives relatively well at 50℃, but its activity decreases significantly at 60℃, and it is completely inactivated at 70℃ and above. This bacteriophage strain exhibits good stability at 20–50℃ and can be stably stored.

[0049] 2.5 Determination of pH stability of bacteriophages Add 3 mL of LB liquid medium to each 5 mL sterile test tube, and adjust the pH to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 respectively. Then place them in a 37°C constant temperature water bath. After temperature equilibration, add 0.1 mL of phage stock solution (4.86 × 10⁻⁶) to each tube. 10 Phage titers (PFU / mL) and host bacterial culture were incubated at 37°C, and determined using the double-layer plate method at incubation times of 1 h, 2 h, and 3 h. The pH was adjusted to approximately 7.0 using hydrochloric acid or sodium hydroxide solution before measurement.

[0050] like Figure 8 As shown, bacteriophage RKP-VA24471 retained its lytic ability after treatment at pH 5-10 for 3 hours, and maintained a pH of 10. 8 ~ 10 10 High PFU / mL titer. The phage is completely inactivated under strongly acidic or alkaline conditions with a pH below 2 or above 13.

[0051] 2.6 Genetic experiments on phage stability Take phage fluid (4.86×10⁻⁶) separately. 10 PFU / mL) and host bacterial culture (1.2×10) 8 Mix 0.5 mL of phage (CFU / mL) at the ratio required for the optimal multiple of infection (MOI = 0.01) and incubate at 37°C for 30 min. Then add 10 mL of LB liquid medium preheated to 37°C, mix thoroughly, and immediately place in a shaker at 37°C and 160 rpm for 6 h. Afterward, centrifuge at 12,000 rpm for 10 min to remove bacterial fragments, and collect the phage supernatant by 0.22 μm filtration (denoted as P1). Repeat the above steps to infect fresh host bacteria with the previous generation of phage, and continue passage for 30 generations (P1-P30). The phage titer is determined using the double plate method.

[0052] Depend on Figure 9 It can be seen that the titer of bacteriophages remains stable throughout the passage process, indicating that bacteriophages have good biological genetic stability and are an excellent choice for the production process.

[0053] Example 3: Fragmentation Spectrum Experiment The lysis effect of abalone-derived Vibrio alginolyticus phage RKP-VA24471 on 111 Vibrio alginolyticus strains, 69 Vibrio parahaemolyticus strains, and 27 other Vibrio strains preserved in the strain library was determined using the double-layer plate method. The bacterial suspensions of the Vibrio strains to be tested were revived, and then 0.1 mL of the bacterial suspension and phage suspension were taken separately, mixed, and added to LB semi-solid medium. The mixture was then quickly poured into LB solid medium plates, allowed to cool and solidify, and incubated at 37°C for 6 hours. The appearance of phage plaques was observed to determine the success of lysis.

[0054] The 207 Vibrio strains in the strain library were isolated from shrimp, fish, sea cucumber, abalone, water, and mud samples collected from Qingdao, Shandong; Zhangzhou, Fujian; and Lianyungang, Jiangsu.

[0055] Table 1. Lysis effect of Vibrio alginolyticus phage RKP-VA24471 derived from abalone

[0056] Where: "+" represents cleavage, and "-" represents non-cleavage. As shown in Table 1, the lysis effect of the abalone-derived Vibrio alginolyticus phage RKP-VA24471 on these 207 Vibrio strains was as follows: 167 strains were functionally lysed, with a lysis rate of 80.6%. Among them, 111 strains of Vibrio alginolyticus were lysed, with a lysis rate of 86.49%; 67 strains of Vibrio parahaemolyticus were lysed, with a lysis rate of 89.86%; and 27 other Vibrio strains were lysed, with 9 strains lysed, including 5 Vibrio species, 3 Vibrio harveyi species, and 1 Vibrio cholerae species. Statistically, the lysis rate of this phage was 87.10% for abalone-derived strains, 87.27% for shrimp-derived strains, and 90% for strains isolated from water sources.

[0057] Example 4: Comparative Experiment on the Bactericidal Effects of Vibrio alginolyticus Bacteriophages from Different Sources Healthy abalone with a body length of 5±0.5 cm were selected. They were temporarily housed in a circulating water system with a salinity of 32‰, 24±1℃, and pH 7.8 for 14 days to acclimatize. On day 15, the abalone were challenged with Vibrio alginolyticus VA24471, exposed to a Vibrio alginolyticus suspension (1×10⁻⁶). 8 CFU / mL was administered for 24 hours until typical symptoms (foot ulcers, decreased vitality) appeared. Subsequently, treatment was administered with abalone-derived Vibrio alginolyticus phage RKP-VA24471 and fish-derived Vibrio alginolyticus phage RKP-VA24363, respectively, via intramuscular injection of phage suspension (1×10⁻⁶ CFU / mL) into the foot. 8The positive control group was injected with an equal volume of sterile PBS buffer. Hemolymph was collected on days 1, 3, 5, and 7 post-infection, spread on TCBS agar plates, and CFU of Vibrio alginolyticus were counted. On day 7 post-infection, lysozyme and superoxide dismutase (SOD) activities in the hemolymph were measured to assess the abalone's immune status.

[0058] Table 2. Bacterial load in hemolymph (CFU / mL)

[0059] Note: In the table, " / " indicates that the abalone is dead, and "-" indicates that no bacteria were detected.

[0060] Table 3 Changes in blood lymphocyte immune markers

[0061] Depend on Figure 10 It was found that the survival rate of abalone after bacterial infection was only 25%. After phage treatment, the survival rate of abalone was significantly improved, and the survival rate of the abalone-derived phage group was significantly higher than that of the fish-derived phage group (P<0.05). The bacterial load in the abalone-derived phage group decreased significantly on the 3rd day (see Table 2), which was faster than that in the fish-derived phage group. Furthermore, Table 3 shows that the lysozyme and SOD activities in the abalone-derived phage group recovered to levels closer to healthy levels. These findings indicate that abalone-derived phage can significantly improve the survival rate of infected abalone, accelerate bacterial clearance, and synergistically enhance the host's immune response.

[0062] Example 5: Treatment Experiment of Bacteriophage on Abalone Healthy abalone with a body length of 5±0.5 cm were selected. They were temporarily housed in a circulating water system with a salinity of 32‰, 24±1℃, and pH 7.8 for 14 days to acclimatize. On the 15th day, the abalone were challenged with Vibrio alginolyticus VA24471 (0.1 mL of bacterial solution was injected intramuscularly into the foot). 6 Intramuscular challenge with phage suspension (CFU / mL) was performed to establish an infection model. 0.1 mL of phage suspension (10 CFU / mL) was injected intraperitoneally 24 hours post-infection. 8 Phage therapy was administered using PFU / mL and a water temperature maintained at 25°C. Simultaneously, a phage prevention group was established: 48 hours prior to bacterial infection, the patient was fed a diet containing phage microcapsules (10 PFU / mL). 8 The group receiving the first group of infections received phage suspension via peritoneal injection after infection (PFU / g); the antibiotic treatment group received enrofloxacin (10 mg / kg) via peritoneal injection 24 hours after infection; a blank control group was also set up: uninfected individuals received 0.1 mL sterile PBS via intramuscular injection in the foot.

[0063] Table 4 Group Design of Phage Therapy Experiments

[0064] Mortality was recorded every 12 hours after challenge, and morbidity was monitored for 96 hours. Abalone were considered dead when they detached due to weak adhesion. During the experiment, the abalone's morphology was observed, including typical symptoms such as foot ulceration, mantle shrinkage, shell detachment, and cessation of feeding. Hepatopancreatic tissue was collected from dying abalone for bacterial load determination (qPCR detection of the vscC gene copy number).

[0065] Bacterial load detection method: Aseptically remove hepatopancreatic tissue from dying abalone, weigh 0.1 g, and wash three times in pre-cooled PBS (to remove surface contaminants). Add 1 mL of sterile PBS, flash freeze in liquid nitrogen, and homogenize (180 rpm, 3 min). Centrifuge (4℃, 12,000×g, 10 min) and collect the supernatant. Add 0.2 mL of lysozyme (final concentration 10 mg / mL) to the homogenate and incubate at 37℃ for 30 min. Extract DNA using a bacterial genomic DNA extraction kit (e.g., TIANamp Bacteria DNA Kit) and perform qPCR detection.

[0066] The target gene is the Vibrio alginolyticus virulence gene vscC (type III secretion system): Forward primer: 5'-GCTGAAGTGATGGGTTACGA-3' Reverse primer: 5'-CGGTCAAGGTTCTGGTAGGT-3' (amplified fragment 152 bp) Table 5. Bacteriophage treatment experiments on abalone

[0067] Table 5 shows that the mortality rate of infected abalone was as high as 86.7%, and the abalone exhibited foot ulceration and weakened adhesion. Different prevention and treatment methods alleviated the disease to varying degrees. The phage control group, which involved feeding abalone with feed containing phage microcapsules before infection and then injecting phage after infection, effectively improved the survival rate of infected abalone and reduced the bacterial load in the hepatopancreas. Furthermore, phage treatment was more effective than antibiotic treatment. This indicates that using abalone-derived Vibrio alginolyticus phage RKP-VA24471 can reduce the incidence of bacterial diseases caused by Vibrio alginolyticus in abalone. Moreover, the combined intervention strategy of "preventive feeding + post-infection injection" significantly improved the survival rate of abalone and inhibited pathogen proliferation, demonstrating superior efficacy compared to traditional antibiotic treatment.

[0068] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A highly efficient lysing Vibrio alginolyticus phage derived from abalone, characterized by: The bacteriophage is Vibrio alginolyticus bacteriophage (Vibrio alginolyticus bacteriophage) RKP-VA24471, which was deposited with the China General Microbiological Culture Collection Center on March 7, 2025, and has the accession number CGMCC No. 46426.

2. The lysozyme-competent Vibrio alginolyticus bacteriophage of claim 1, wherein: The phage has a titer of not less than 1 x 10 ^10 PFU / mL.

3. The lysozyme-competent Vibrio alginolyticus bacteriophage of claim 1, wherein: The optimal multiplicity of infection MOI of the phage is 0.01, and the phage titer can reach 8.41x10 11 PFU / mL after 6 hours of fermentation culture.

4. The lysozyme-competent Vibrio alginolyticus bacteriophage of claim 1, wherein: The phage latency period was 20 min, the outbreak period was 60 min, and the phage outbreak rate was 207 PFU / cell.

5. The lysozyme-competent Vibrio alginolyticus bacteriophage of claim 1, wherein: The bacteriophage can also lyse one or more of Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio cholerae.

6. A composition, characterized in that: It comprises Vibrio alginolyticus phage RKP-VA24471 as described in any one of claims 1 to 5, and a pharmaceutically or aquaculture-acceptable vector.

7. The use of Vibrio alginolyticus phage RKP-VA24471 according to any one of claims 1 to 5 or the composition according to claim 6 in the preparation of articles for the prevention and / or treatment of Vibrio infections.

8. The application according to claim 7, characterized in that: The Vibrio infection is caused by Vibrio bacteria that can be lysed by the bacteriophage RKP-VA24471.

9. The application according to claim 7 or 8, characterized in that: The objects of the prevention and / or treatment are aquatic animals.

10. The application according to claim 9, characterized in that: The aquatic animal in question is abalone.

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