Vibrio phage XHX-V-54P with wide temperature and pH tolerance and application thereof

Abstract

The application discloses a Vibrio phage XHX-V-54P with wide temperature and pH tolerance and an application thereof, and relates to the technical field of microorganisms.The application discloses a Vibrio phage XHX-V-54P with wide temperature and pH tolerance, and the preservation number is CCTCC No: M 2025489.The phage can keep stable lytic activity in a wide temperature and pH range, has excellent environmental adaptability, and has the characteristics of wide lytic spectrum, short latent period, high lytic efficiency and the like.The phage can significantly reduce the mortality of Vibrio alginolyticus disease in shrimp culture and has high biological safety.The phage can be widely applied to the preparation of pharmaceutical preparations for preventing and treating Vibrio alginolyticus disease of aquatic animals, aquatic feed additives and breeding environment disinfectants and the like, and provides a safe, efficient and environment-friendly Vibrio disease prevention and control scheme for the aquaculture industry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Vibrio phage XHX-V-54P with wide temperature and pH tolerance and its applications. Background Technology

[0002] Vibrio alginolyticus is one of the major pathogens in aquaculture, infecting fish, crustaceans, and shellfish, leading to decreased farming efficiency. It can also infect humans, causing gastroenteritis and even septicemia. In recent years, this disease has become increasingly serious, posing a significant challenge to the sustainable development of aquaculture.

[0003] Currently, aquaculture production relies heavily on antibiotics to control Vibrio alginolyticus infection. However, long-term use has led to the emergence of drug-resistant strains, increasing the difficulty of treatment. Furthermore, drug-resistant genes may spread through the food chain, threatening human health. Simultaneously, conventional disinfection measures are insufficient to completely eliminate Vibrio alginolyticus from the environment, as this bacterium can form biofilms on the surfaces of aquaculture facilities, causing recurrent outbreaks. Therefore, there is an urgent need to develop safe and highly effective novel antibacterial agents.

[0004] Bacteriophages are a class of viruses that specifically infect and lyse bacteria. They possess advantages such as high species specificity, environmental friendliness, and the ability to self-replicate, showing promising application prospects in the control of pathogenic bacteria in aquatic organisms. However, existing Vibrio alginolyticus bacteriophages generally suffer from narrow host range and poor environmental stability, especially being easily inactivated under acidic, alkaline, or high-temperature conditions. This makes them difficult to use stably in the complex and variable aquaculture environment, and also presents challenges for large-scale production and formulation development.

[0005] In practical aquaculture applications, bacteriophages may face a variety of extreme environments. For example, during the hot summer months, the water temperature in aquaculture ponds can rise above 30°C, and in southern regions, the water temperature can even reach 35-40°C. This requires bacteriophages to maintain stable activity over a wide temperature range. In addition, the pH of aquaculture water often fluctuates drastically due to factors such as bottom sediment acidification, algal growth, acidic rainwater runoff from heavy rains, or disinfectant residues. The pH can be as low as 3.0-4.0 or as high as 10.0 or above. If bacteriophages cannot tolerate a wide pH range, they will quickly become inactive after actual application or addition to feed, making it difficult for them to exert a sustained bactericidal effect.

[0006] Therefore, discovering a Vibrio alginolyticus phage with a broad lysis spectrum and excellent temperature and pH tolerance, and developing corresponding drug formulations, feed additives and environmental disinfection products, is of great significance for green prevention and control in aquaculture.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention 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 aforementioned technical problems, embodiments of the present invention provide a Vibrio phage XHX-V-54P with wide temperature and pH tolerance and its applications, thereby resolving the issues raised in the background art.

[0009] A Vibrio alginolytic phage with wide temperature and pH tolerance, wherein the phage is a Vibrio phage ( Vibrio alginolyticus phage The specimen, named *Vibrio phage* XHX-V-54P, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China. Accession number: CCTCC No.: M 2025489. Classification: Vibrio phage. Vibrio alginolyticus phage The deposit date is March 17, 2025.

[0010] Preferably, the gene sequence of the phage Vibrio phage XHX-V-54P is shown in SEQ ID NO.1.

[0011] The use of the Vibrio alginolyticus phage described above in the preparation of a drug for the prevention or treatment of Vibrio alginolyticus infection in aquatic animals.

[0012] An application of the Vibrio alginolyticus phage described above in the preparation of aquatic feed or aquatic feed additive.

[0013] An application of the Vibrio alginolyticus bacteriophage described above in the preparation of a disinfectant for aquaculture environments.

[0014] An aquatic feed additive comprising the aforementioned Vibrio phage XHX-V-54P, which exhibits wide temperature and pH tolerance.

[0015] A pharmaceutical preparation for aquaculture includes the Vibrio phage XHX-V-54P, which has wide temperature and pH tolerance, as described above, and pharmaceutically acceptable excipients.

[0016] Preferably, the dosage form of the aquaculture drug preparation is a spray solution, a bath solution, or an injection solution.

[0017] A disinfectant for aquaculture environments, comprising the aforementioned Vibrio phage XHX-V-54P, which exhibits wide temperature and pH tolerance.

[0018] A feed composition comprising a base feed and the aforementioned aquatic feed additive.

[0019] The present invention provides a Vibrio phage XHX-V-54P with wide temperature and pH tolerance and its application, which has the following beneficial effects: (1) The bacteriophage of the present invention can maintain stable activity after treatment in a wide temperature range and can adapt to the temperature changes of aquaculture pond water in the high temperature season of spring and summer. At the same time, after incubation in a wide pH range from slightly acidic to slightly alkaline, the potency of the bacteriophage of the present invention is not significantly different from the initial potency. It can adapt to the pH fluctuations of aquaculture water caused by factors such as bottom sediment acidification and algae reproduction. It can maintain stable bactericidal activity when applied on site, which greatly improves the environmental adaptability in practical applications.

[0020] (2) The bacteriophage of the present invention has a broad lysis spectrum and can lyse a variety of Vibrio alginolytic strains with a lysis rate of over 90%. It can effectively lyse Vibrio alginolytics from different sources and with different genotypes, and has the potential for broad-spectrum bactericidal application.

[0021] (3) The bacteriophage of the present invention has a short incubation period and high lysis efficiency. It can rapidly proliferate and enter the lysis period in a short time, and its titer is significantly improved, which is conducive to quickly controlling the spread and transmission of pathogens during an outbreak.

[0022] (4) The results of the treatment experiment on Vibrio alginolyticus disease in shrimp larvae of Litopenaeus vannamei showed that after spraying the bacteriophage of the present invention, the mortality rate of the treatment group was significantly lower than that of the challenge group, the relative protection rate was higher, and the shrimp larvae fed normally and moved actively, indicating that the bacteriophage of the present invention has obvious therapeutic and preventive effects on Vibrio alginolyticus disease.

[0023] (5) The results of the mouse safety experiment showed that after continuous oral administration of high doses of the phage of the present invention, the mice were healthy and their daily behavior was normal. No tissue or organ damage was found during the autopsy. Moreover, the phage was completely cleared from the digestive tract within a short time after the oral administration was stopped, with no residue. This indicates that the phage of the present invention has high biosafety and no toxic side effects. Attached Figure Description

[0024] Figure 1 Photograph of a plaque of phage Vibrio phage XHX-V-54P.

[0025] Figure 2 Transmission electron micrograph of bacteriophage Vibrio phage XHX-V-54P.

[0026] Figure 3 This represents the optimal multiplicity of infection assay result for Vibrio phage XHX-V-54P.

[0027] Figure 4The first-step growth curve of bacteriophage Vibrio phage XHX-V-54P is shown.

[0028] Figure 5 The effect of temperature on the activity of phage Vibrio phage XHX-V-54P.

[0029] Figure 6 The effect of pH on the activity of bacteriophage Vibrio phage XHX-V-54P.

[0030] Figure 7 The effect of bacteriophage Vibrio phage XHX-V-54P on the growth of Litopenaeus vannamei larvae infected with Vibrio alginolyticus.

[0031] Figure 8 The effect of bacteriophage Vibrio phage XHX-V-54P on the survival of Litopenaeus vannamei larvae infected with Vibrio alginolyticus. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0033] To address the aforementioned technical problems, embodiments of the present invention provide a Vibrio phage XHX-V-54P with wide temperature and pH tolerance and its applications, thereby resolving the issues raised in the background art.

[0034] I. Experimental Materials and Reagents The host bacterium TW9 involved in the examples is Vibrio alginolyticus, which was isolated from Jiangsu Province and preserved by the Jiangsu Academy of Agricultural Sciences.

[0035] 1 mol / L sterile CaCl2 solution (1L): Weigh 111g of CaCl2 solid using a balance, pour it into a beaker, add water to dissolve it, pour the solution into a 1L volumetric flask, and rinse the beaker 2-3 times with distilled water. Pour the rinsing solution into the volumetric flask as well, add distilled water to the volumetric flask until the mark is reached, mix well, and autoclave for later use.

[0036] TSA solid culture medium (400mL): Weigh 16g of TSA solid powder into 400mL of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and cool for later use.

[0037] 0.6% LB agar medium / plate (200mL): Weigh 2g tryptone, 2g sodium chloride, 1g yeast powder and 1.2g agar into 200mL distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and cool for later use.

[0038] 1% TSB liquid culture medium (400mL): Weigh 12g of TSB solid powder and 4g of sodium chloride into 400mL of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and cool for later use.

[0039] SM solution (1L): Weigh 6.055g Tris and dissolve it in 20mL of distilled water. Adjust the pH to 7.5 with concentrated hydrochloric acid and bring the volume to 50mL. Then add 5.8g NaCl and 2g MgSO4, dissolve them, and bring the volume to 1L. Autoclave at 121℃ for 15min and cool for later use.

[0040] 1×PBS buffer (1L): Weigh 99g of PBS solid powder into 1L of distilled water, heat to boiling until completely dissolved to prepare 10×PBS buffer. Take 100mL of 10×PBS solution and add it to 900mL of distilled water. Autoclave at 121℃ for 15min and cool for later use.

[0041] TCBS solid culture medium (500mL): Weigh 44.5g of TCBS solid powder into 500mL of distilled water, heat to boiling until completely dissolved, cool to 50℃, and pour into sterile Petri dishes for later use. II. Specific Implementation Methods Example 1: Isolation and preparation of bacteriophages The host bacteria were streaked onto TCBS solid medium and cultured overnight. Single clones were then picked and inoculated into 1 ml of 1% TSB liquid medium and cultured at 37°C with shaking for 5-6 hours to obtain the host bacteria culture for later use.

[0043] In April 2025, wastewater samples were collected in Nantong City, Jiangsu Province, China. The supernatant was filtered through double-layer filter paper, centrifuged at 10,000 rpm for 20 min, and then filtered again through a 0.22 μm filter membrane. 10 mL of the filtered supernatant was taken, and 0.5 mL of overnight host bacterial culture was added. Then, sterile CaCl2 stock solution was added to a final concentration of 1.25 mM, and the mixture was stirred. 20 mL of 1% TSB liquid medium was added, and the mixture was incubated at room temperature for 30 min, followed by incubation at 37℃ for 6-8 h. The culture was then centrifuged at 12,000 rpm at 4℃ for 30 min, and the supernatant was collected. 10 mL of this supernatant was taken, and 0.5 mL of overnight host bacterial culture was added again. Sterile CaCl2 stock solution was added to a final concentration of 1.25 mM, and the mixture was stirred. 20 mL of TSB liquid medium was then added. LB liquid medium was cultured and centrifuged according to the above method to obtain the enriched supernatant; the supernatant was enriched again according to the above experimental method, and the supernatant enriched three times was filtered through a 0.22 μm filter membrane to form the phage stock solution.

[0044] Divide the TSA agar plate into two areas: take 0.1 mL of the above host bacterial culture and drop it into the center of the plate, then spread the bacterial solution evenly with a spreader; after it dries, take 10 mL of the above phage stock solution and drop it into one of the areas; after it dries naturally, place it in a 37℃ incubator for 10 h, and then observe whether there are any empty plaques in the area where the phage was added.

[0045] If plaques form, it indicates the presence of bacteriophages. Take 100 mL of the phage stock solution and perform a series of 10-fold dilutions. Take 10... -2 10 -4 and 10 -6 Mix 0.1 mL of each diluent with 0.1 mL of the host bacterial culture. After incubating at room temperature for 15 min, add about 4 mL of melted 0.6% LB agar medium, mix well, and quickly pour onto the top layer of TSA medium plates. Shake well and let stand flat for 10 min until solidified. Incubate at 37℃ for 12 h and observe to obtain double-layer plates with single phage plaques.

[0046] Example 2: Phage amplification and purification On the bilayer plate from which plaques were formed in Example 1, a single plaque with a larger diameter was picked up with the tip of a pipette and inoculated into 3-5 ml of 1% TSB liquid medium. 0.1 mL of phage host culture was added, mixed, and incubated at room temperature for 15 min, then at 37°C for 10-14 h. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was collected and 0.3% chloroform was added. The bilayer experiment was repeated, and single plaques were picked up 4-5 times in this manner to purify the phage into plaques of the same size.

[0047] Take 1 mL of freshly cultured host bacteria and add 0.3 mL of phage lysis buffer (at ratios of 1:1, 1:10, and 1:100 for single phage culture to host bacteria, respectively). Incubate at 37°C for 20 min to allow phage particles to adsorb onto the host bacteria; add 100 mL of LB liquid medium, then add CaCl2 stock solution to a final concentration of 1.25 mM, and incubate at 37°C with shaking for 12–16 h. Centrifuge at 12,000 rpm at 4°C for 10 min, and collect the supernatant, which is the phage lysis buffer.

[0048] PEG purification: Add RNase A and DNase I to 50 mL of lysis buffer to a final concentration of 1 μg / mL and incubate at 37 °C for 30 min; add 9.3 g PEG 8000 and 5.8 g NaCl, shake well until dissolved, and incubate on ice for 1 h or at 4 °C overnight; centrifuge at 10000 rpm for 10 min at 4 °C and discard the supernatant; add 2 mL of SM solution, wash the precipitate thoroughly, and incubate at room temperature for 1 h; add an equal volume of chloroform for extraction and gently shake for 30 s; centrifuge at 5000 rpm for 10 min at 4 °C to separate the organic and hydrophilic phases, recover the hydrophilic phase containing phage particles, and obtain purified phage.

[0049] CsCl isodense gradient centrifugation purification: Slowly add 10 mL of 1.6 gm / cc CsCl to the bottom of the centrifuge tube, followed by 10 mL of 1.4 gm / cc CsCl, 5 mL of 25% sucrose, and 10 mL of phage lysis buffer, and equilibrate. Place the mixture into a centrifuge tube sleeve and slowly suspend it in the rotor. Turn on the ultracentrifuge (Optima L-80 XP Ultracentrifuge, Beckman), set the speed to 30,000 rpm, time to 120 min, and temperature to 18℃. After centrifugation, wait for the vacuum to drop to 0, then open the chamber door, remove the sample, and turn off the centrifuge. A white band will appear at the bottom of the sample, between the 1.4 gm / cc and 1.6 gm / cc concentrations. Carefully aspirate this band using a fine needle inserted from the side. Approximately 5-8 mL of 20 mL sample will be obtained. Place the sample in a dialysis bag and dilute with 10 mM... Dialysis was performed using Tris-HCl, pH 7.4, and 100 mg / mL MgCl2 buffer, 2 mL at a time (10⁻¹⁴ kDa). The final sample volume was aspirated to approximately 10 mL, and the phage titer was determined.

[0050] The phage titer was determined using the double-layer agar plate method: The purified phage solution was serially diluted 10-fold. 0.1 mL of each dilution was mixed thoroughly with 0.1 mL of the host bacterial solution, and the mixture was plated onto double-layer agar plates and incubated at 37°C for approximately 10 hours. Plaques were counted on each agar plate. Plates showing approximately 100-200 plaques were selected, and the initial phage concentration calculated based on the dilution factor was used to determine the phage titer. The purified phage was as follows: Figure 1 As shown, bacteriophages can form clear plaques in TSA agar medium with no surrounding halo and clear, regular edges.

[0051] The purified phage was named *Vibrio phage* XHX-V-54P and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China. Accession number: CCTCC No.: M2025489. Classification: Vibrio phage. Vibrio alginolyticus The phage is deposited on March 17, 2025.

[0052] Example 3: Transmission electron microscopy observation of bacteriophage Vibrio phage XHX-V-54P Figure 2 This image shows the morphology of a Vibrio phage sample obtained using a Hitachi transmission electron microscope (TEM). The observation acceleration voltage was 80.0 kV, the magnification was 60,000x, the image calibration was 1.083 nm / pixel, and the scale bar was 100 nm. The nanoscale structure of the phage was clearly distinguishable. A complete Vibrio phage particle was visible in the center of the field of view, with a regularly spherical head and clear surface texture. The outer periphery showed a dark contrast boundary due to negative staining, and the structure was intact and undamaged. A short tail structure was visible on one side of the phage, with morphological characteristics consistent with typical short-tailed phages. A small amount of lipid vesicle-like impurities were distributed in the background, and there was no large-area phage aggregation. The sample was well dispersed, directly confirming the presence and structural integrity of the target Vibrio phage in this formulation.

[0053] Example 4: Determination of the optimal multiple of infection for phage Vibrio phage XHX-V-54P Host bacteria were inoculated into fresh 1% TSB medium and incubated at 37°C with shaking until the logarithmic growth phase. Phage lysates were mixed with host bacterial suspensions at different MOIs: 100, 10, 1, 0.1, 0.01, and 0.001. 200 μL of each mixture was added to 5 ml of fresh 1% TSB liquid medium. The mixtures were incubated at 37°C and 200 rpm for 4 h with shaking. After centrifugation at 8000 rpm for 10 min, the supernatant was collected, filtered, and the phage titer was determined using the bilayer plate method. The MOI at which the phage titer was highest was considered the optimal multiplicity of infection for that phage. Results are as follows: Figure 3 As shown, the valence is highest when MOI = 0.1, reaching 4.9 × 10⁻⁶. 9 PFU / mL, i.e., the optimal MOI for phage Vibrio phage XHX-V-54P is 0.1.

[0054] Example 5: Determination of the one-step growth curve of bacteriophage Vibrio phage XHX-V-54P Adjust the host bacteria and bacteriophage to 500 μL each according to the optimal multiplicity of infection ratio, incubate at 37°C for 10 min, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend in 1 ml of fresh 1% TSB liquid medium. Repeat the above steps of incubation, centrifugation, discarding supernatant, and resuspension three times. Finally, resuspend in 20 ml of fresh 1% TSB liquid medium. Take samples every 10 min starting from 0 min, for a total of 160 min. The results are as follows: Figure 4 As shown, the titer of bacteriophage Vibrio phage XHX-V-54P remained essentially unchanged during the first 10 minutes of growth, then rapidly increased by five orders of magnitude between 10 and 120 minutes, entering a stationary phase after 120 minutes. The titer of Vibrio phage XHX-V-54P reached 3.9 × 10⁻⁶ when it reached the end of lysis at 120 minutes. 9 PFU / mL.

[0055] Example 6: Temperature and acid-base tolerance test of bacteriophage Vibrio phage XHX-V-54P Take the purified bacteriophage (8.3 × 10⁻⁶) obtained in Example 2. 9 500 μL of PFU / mL was added to an EP tube and incubated in a water bath at 30℃-90℃ for 30 min. After cooling the sample, its titer was measured to examine the thermal stability of the phage. The pH of the SM buffer was adjusted to 2.0-13.0 using NaOH or HCl. 900 μL of each buffer at different pH values ​​was added to a sterile EP tube, and 100 μL of the purified phage obtained in Example 2 was added to the EP tube. After incubation at 37℃ for 2 h, its titer was measured to examine the stability of the phage at different pH values.

[0056] Temperature detection results as follows Figure 5 As shown, the activity of bacteriophage Vibrio phage XHX-V-54P did not change significantly after being treated at 30-60℃ for 30 min; the activity decreased significantly at 70℃; and no bacteriophage survived after being treated at 90℃ for 30 min.

[0057] pH test results are as follows Figure 6 As shown, no bacteriophages were detected at pH 2.0; at pH 11.0, the titer differed significantly from the initial titer, reaching 6.2 × 10⁻⁶. 5 When the pH is 3.0-10.0, there is no significant difference in potency compared to the initial potency; however, when the pH is greater than 12.0, no bacteriophages can be detected.

[0058] Example 7 Host profile analysis of bacteriophage Vibrio phage XHX-V-54P The titer of the phage Vibrio phage XHX-V-54P obtained in Example 2 was adjusted to 1×10⁻⁶. 9 PFU / mL available.

[0059] Forty strains of Vibrio alginolyticus isolated from this study were selected to analyze the host spectrum of bacteriophage Vibrio phage XHX-V-54P. The specific procedures were as follows: 100 μL of overnight culture from each of the 40 Vibrio strains was added to the center of a TSA agar plate, and spread evenly using a spreader. Each plate was then divided into two equal areas. In one area, 10 μL of Vibrio phage XHX-V-54P was added to the surface of the bacterial growth, while in the other area, 10 μL of physiological saline was added as a control. After the droplets dried, the plates were inverted and incubated at 37°C for 12-16 hours. The results were observed; the presence of plaques was marked as "+", otherwise as "-". The results are shown in Table 1. In Table 1, strains 1-40 are pathogenic strains named by the applicant.

[0060] Table 1. Host spectrum analysis of phage Vibrio phage XHX-V-54P 1 GY-13 + 2 ZYD-15 + 3 HW5 + 4 GY-18 + 5 JS-7 + 6 ZYD-12 + 7 ZYD-13 + 8 ZYD-14 + 9 ZYD-25 + 10 LN1 + 11 LN2 + 12 LN3 + 13 LN4 + 14 LN5 + 15 LN6 + 16 LN12 + 17 LN13 - 18 LN14 + 19 HW1 + 20 HW2 - 21 HW3 + 22 HW10 + 23 ZYD-3 + 24 TW6 + 25 Q1 + 26 Q2 + 27 Q3 + 28 Q4 + 29 Q5 + 30 Q6 + 31 Q7 + 32 HP7 + 33 FQ9 + 34 FQ12 + 35 FQ15 + 36 JM1 + 37 JM11 + 38 JM12 + 39 JM17 + 40 JM18 - As shown in Table 1, the bacteriophage Vibrio phage XHX-V-54P lysed 37 out of 40 Vibrio strains, with a lysis rate of 92.5%, indicating that the bacteriophage has a broad lysis spectrum.

[0061] Example 8: Whole genome sequencing, assembly, and annotation of bacteriophage Vibrio phage XHX-V-54P Genomic DNA was extracted from Vibrio phage using a phage genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). Genome sequencing was performed by Qingdao Qingke Co., Ltd. (Qingdao, China) using an Illumina HiSeq 2500 sequencer. Sequencing was then performed, and the genome was assembled using SPAdes genome assembly software (v3.11.1) and A5-miseq (v20160825). The Vibrio phage XHX-V-54P read length was 43121 bp; the average sequencing depth was 100.3-fold.

[0062] The putative protein-coding sequences (CDS) were predicted using the bakta_v1.11.4 software. The prediction of coding sequences (CDS) was performed using Prodigal (v2.6.3); tRNA and rRNA genes were identified using tRNAscan-SE (v2.0.11) and Infernal (v1.1.5) based on the Rfam (v14.9) database, respectively. Functional annotation was performed by comparing UniProtKB (release 2024_01), NCBI RefSeq, and Pfam (v36.0) databases. Bakta annotation revealed that the Vibrio phage XHX-V-54P genome contains a large number of Tail protein and Spike protein, whose protein characteristics specifically recognize membrane proteins. This genome has a wide range of binding sites for recognizing Vibrio membrane proteins. In summary, after genome annotation, Vibrio phage XHX-V-54P was found to have the characteristic of specifically recognizing and lysing Vibrio.

[0063] Example 9: Plotting the phylogenetic tree of Vibrio phage XHX-V-54P To identify homologous sequences for phylogenetic analysis, target nucleotide sequences were used as query sequences. BLASTn (v2.13.0) was used to search the NCBI non-redundant nucleotide (nr / nt) database, with an E-value threshold of 1e-5 and a minimum query coverage of 70%. Sequences with nucleotide identity ≥ 80% were selected for subsequent analysis. All statistical analyses and phylogenetic reconstructions were performed in R software (v4.3.1), using the following R packages: ape (v5.7), phangorn (v2.11), seqinr (v4.2), ggtree (v3.10), ggplot2 (v3.4), reshape2 (v1.4), and rentrez (v1.2). Homologous nucleotide sequences obtained from the NCBI nr / nt database using BLASTn were imported and processed using the seqinr package. Multiple sequence alignment was performed using the msa or DECIPHER packages. The phylogenetic tree was constructed using the maximum likelihood (ML) method via the phangorn package. The optimal nucleotide substitution model was determined by calculating the Akaike Information Criterion (AIC) for each candidate model using the modelTest() function, and the model was selected based on the minimum AIC principle. Node support was evaluated using 1,000 bootstrap replicates. The final phylogenetic tree was visualized, annotated, and enhanced using the ggtree package, and further graphically refined using ggplot2. The reshape2 package was used for data reconstruction before plotting related heatmaps or multiple combination plots.

[0064] Based on second-generation whole-genome sequencing analysis, we extracted the whole-genome information of the Vibrio phage XHX-V-54P phage. Through contig sequence assembly, we determined that Vibrio phage XHX-V-54P is 43121 bp in length and identified it as a lytic phage using phabox. Protein annotation tools revealed that this phage possesses multiple tail proteins with strong binding capabilities to bacterial membrane proteins or lipopolysaccharides. To identify its specificity, after comparison with databases such as NCBI, we constructed its phylogenetic tree using meta-analysis software and the online function of itol, determining its uniqueness within the Vibrio phage population and understanding its evolutionary relationships. While its characteristics are present in the Vibrio phage population and widely distributed in tail fibrous proteins, specifically recognizing Vibrio membrane proteins, no Vibrio phage sequences with largely similar or identical sequence characteristics were found in currently known databases. The significant functional characteristic of this phage in recognizing Vibrio, coupled with its sequence similarity not found in currently published databases, is the main innovation of this work.

[0065] Example 10: The therapeutic effect of bacteriophage Vibrio phage XHX-V-54P on Vibrio alginolyticus infection. Healthy and vigorous shrimp were selected and three groups were set up: a blank control group, a Vibrio alginolyticus challenge group, and a treatment group. Each group was housed in a separate container, with 30 shrimp (20L of seawater) in each container. During the experiment, normal management was maintained, and water temperature, salinity, and pH were controlled within suitable ranges. The control group received no treatment other than normal feeding. The challenge group had a Vibrio concentration of 10... 6 CFU / mL, after challenge with the phage group, in addition to normal feeding, 2ppm Vibrio phage XHX-V-54P was applied four times a day for 72 hours. During the experiment, water samples were taken from the aquaculture pond daily at 6h, 24h, 48h, and 72h, and appropriate amounts of seawater were evenly spread on TCBS medium for Vibrio classification and counting experiments. The condition of the shrimp was monitored daily, checking whether the hepatopancreas was dark and shiny, whether the intestines were full, and whether the hepatic tubules were clear. The number of dead shrimp in the experimental group and the control group was counted to compare the mortality rate.

[0066] The test results show that the number of Vibrio bacteria (CFU / mL) per day after the start of the experiment is shown in Table 2: Table 2. Vibrio content in seawater at different time points (CFU / mL)

[0067] The condition of each group of shrimp at each time point after the start of the experiment is as follows: Figure 7 As shown, the shrimp in the blank control group grew well; the challenged group ate little or nothing, had empty stomachs, and moved slowly; compared with the challenged group, the phage group had milder symptoms and ate normally.

[0068] The dynamic data results of dead shrimp during the experiment are as follows: Figure 8 As shown, compared with the control group, the mortality rate of the challenge group was 53.33%, the mortality rate of the bacterial phage group was 20%, and there were no deaths in the control group; indicating that the bacteriophage Vibrio phage XHX-V-54P has significant therapeutic and preventive effects against Vibrio alginolyticus infection and can be used as a biological antibacterial agent for the prevention and treatment of Vibrio alginolyticus in aquatic animals.

[0069] Example 11 Safety test of bacteriophage Vibrio phage XHX-V-54P Forty female SPF-grade BALB / c mice, aged 6-8 weeks and with an average weight of 27±2g, were purchased from the Comparative Medicine Center of Yangzhou University. The mice were randomly divided into two groups of 20 each; one group received oral administration of Vibrio phage XHX-V-54P10. 8PFU / 0.25mL / mouse (provided in Example 2); the control group was orally administered an equal volume of PBS. After 14 days of continuous oral administration, 5 mice in each group were euthanized by cervical dislocation, and changes in internal organs, digestive tract and mucosa were observed; the remaining 15 mice in each group continued to be fed, and their feces were collected daily to detect changes in phage count.

[0070] The results showed that this dose of phage had no effect on the health and daily behavior of mice, no abnormalities were found during autopsy, and no phage was detected in the feces of mice 7 days after the end of oral administration of phage.

[0071] Therefore, it is believed that this Vibrio phage XHX-V-54P can be used to prepare drugs for the prevention and treatment of Vibrio alginolyticus infection.

[0072] In practical applications, Vibrio phage XHX-V-54P can also be used to kill Vibrio in the environment, including farm environments and spatial environments.

[0073] It should be noted that, in order to facilitate an accurate understanding of the common meanings of the English abbreviations in this invention document, the following explanations are provided in this invention document, specifically referring to the English abbreviations and their Chinese meanings comparison table shown in Table 3.

[0074] Table 3: Comparison of English Abbreviations and Chinese Meanings pH pH g Grams (unit of mass) mol / L Moles per liter (unit of concentration) L Liter (a unit of volume) <![CDATA[CaCl2]]> Calcium chloride TSA Tryptone soybean agar mL milliliters (unit of volume) min minutes (unit of time) ℃ Celsius (a unit of temperature) SM bacteriophage suspension buffer Tris Tris(hydroxymethyl)aminomethane NaCl Sodium chloride <![CDATA[MgSO4]]> Magnesium sulfate 1× 1x working concentration 10× 10 times the working concentration PBS Phosphate-buffered saline TCBS Thiosulfate-citrate-cholesterol-sucrose agar h Hour (unit of time) rpm revolutions per minute (rpm) mM millimoles per liter LB Luria-Bertany medium TSB Tryptone Soy Broth PEG polyethylene glycol RNase A Ribonuclease A DNase I Deoxyribonuclease I μg / mL micrograms per milliliter (unit of concentration) CsCl cesium chloride gm / cc grams per cubic centimeter (a unit of density) Optima L-80 XP Ultracentrifuge, Beckman Beckman Optima L-80 XP Ultracentrifuge kD 1,000 Daltons μm micrometer s Seconds (unit of time) Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride kV kilovolt (volt, a unit of voltage) nm nanometer pixel Pixels nm / pixel Image calibration coefficients r / min Revolutions per minute (rpm) is the unit of rotational speed of a shaking machine. MOI Multiple infection PFU / mL Plaque formation units / mL, phage titer standard units Illumina HiSeq 2500 Whole-genome high-throughput next-generation sequencer (Inmeina) SPAdes Bacterial genome assembly tool (v3.11.1) A5-miseq Bacterial genome assembly tools read Bacterial genome sequence size unit (150 bp) bp The smallest unit of a base bakta Gene and protein annotation tools (v1.11.4) Prodigal Gene and protein annotation tools tRNA transfer RNA rRNA Ribosomal RNA tRNAscan-SE Software tool for finding and annotating tRNA (transfer RNA) genes (v2.0.11) Rfam Databases (transfer RNA and ribosomal RNA) Infernal Software suite for searching and comparing structural RNA (such as tRNA and rRNA) sequences. UniProtKB Protein Database NCBI RefSeq American Bioinformatics Reference Database ape Phylogenetic tree algorithm R package (v5.7) phangorn Phylogenetic tree algorithm R package (v2.11) seqinr Sequence analysis and alignment R package (v4.2) ggtree Phylogenetic tree plotting R package (v3.10) ggplot2 Plotting R package (v3.10) reshape2 Data processing R package (v1.4) rentrez Data processing R package (v1.2) Tail Protein Tail fibroin CFU / mL Colony formation units / mL, standard units of bacterial viable concentration ppm Parts per million concentration SPF No specific pathogen BALB / c Mouse strain name, retain the English name directly: Balbu mouse (commonly used for immunization modeling). The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A Vibrio alginolyticus phage with wide temperature and pH tolerance, characterized in that, The bacteriophage is a vibrio bacteriophage ( Vibrio alginolyticus phage The specimen, named *Vibrio phage* XHX-V-54P, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No: M 2025489.

2. The Vibrio alginolyticus phage with wide temperature and pH tolerance according to claim 1, characterized in that, The gene sequence of the phage Vibrio phage XHX-V-54P is shown in SEQ ID NO.

1.

3. The use of the Vibrio alginolyticus phage according to claim 1 or 2 in the preparation of a medicament for the prevention or treatment of Vibrio alginolyticus infection in aquatic animals.

4. The application of Vibrio alginolyticus phage according to claim 1 or 2 in the preparation of aquatic feed or aquatic feed additive.

5. The application of Vibrio alginolyticus phage according to claim 1 or 2 in the preparation of disinfectants for aquaculture environments.

6. An aquatic feed additive, characterized in that, Includes the Vibrio phage XHX-V-54P, which has wide temperature and wide pH tolerance as described in claim 1 or 2.

7. A pharmaceutical preparation for aquaculture, characterized in that, Includes the Vibrio phage XHX-V-54P with wide temperature and pH tolerance as described in claim 1 or 2, and pharmaceutically acceptable excipients.

8. The aquaculture pharmaceutical preparation according to claim 7, characterized in that, The dosage form of the aquaculture drug preparation is a spray solution, a bath solution, or an injection solution.

9. A disinfectant for aquaculture environments, characterized in that, Includes the Vibrio phage XHX-V-54P, which has wide temperature and wide pH tolerance as described in claim 1 or 2.

10. A feed composition, characterized in that, Includes basic feed and the aquatic feed additives as described in claim 6.

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