CN116987674A

CN116987674A - Vibrio parahaemolyticus phage and antibacterial agent prepared from same and application of antibacterial agent

Info

Publication number: CN116987674A
Application number: CN202310816989.2A
Authority: CN
Inventors: 何增国; 付静芸; 汤伟; 吴春光; 唐涛; 孙晓雯; 乔晓妮
Original assignee: Qingdao Bioantai Biotechnology Co ltd; Qingdao Marine Biomedical Research Institute Co Ltd
Current assignee: Qingdao Bioantai Biotechnology Co ltd; Qingdao Marine Biomedical Research Institute Co Ltd
Priority date: 2023-07-05
Filing date: 2023-07-05
Publication date: 2023-11-03

Abstract

The invention discloses a phage capable of cracking vibrio parahaemolyticus and application thereof in prawn culture, and the phage has the characteristics of wide cracking spectrum, strong proliferation capacity and short incubation period, can crack various vibrio parahaemolyticus and vibrio alginolyticus, and can quickly kill and prevent diseases caused by pathogenic vibrio. The phage has strong vibrio control capability and good safety, can improve the activity of alkaline phosphatase in the prawn serum, and can improve the immunity of the prawn; has good temperature and acid-base tolerance, and has important application potential in the prevention and control aspect of vibrio parahaemolyticus of aquiculture animals.

Description

Vibrio parahaemolyticus phage and antibacterial agent prepared from same and application of antibacterial agent

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacterial phage for knowing parahaemolytic arcs and application of the bacterial phage in prawn culture.

Technical Field

Shrimp farming is the primary seafood product in international trade, and half of the total shrimp yield comes from aquaculture. Over the past 30 years, shrimp farming has progressed rapidly worldwide, in part because of the adoption of high-feed density farming practices. However, high density breeding patterns are often accompanied by the occurrence of diseases caused by certain pathogenic bacteria. One of the major threats is vibriosis, which is caused by bacteria of the genus vibrio. Vibrio is very abundant in aquatic environments around the world. Although some are not pathogenic, many can have serious health effects on humans and aquatic organisms. Among them, vibrio parahaemolyticus is one of the most important causes of diseases, death and economic losses in the shrimp farming industry. It can cause acute hepatopancreatic necrosis (AHPND) or early death syndrome (EMS), which is one of the most serious diseases in prawn culture, and the death rate is nearly 100% within one week after the first symptom of cultured prawns. The disease shrimp farming causes devastating economic losses, especially in asia. The causative agent of the disease has been identified as two PirA-like toxins, pirA and PirB, encoded by plasmids. Plasmids have also been found to transfer between other vibrios, increasing the chances of disease transmission in regions and worldwide, and pose a serious threat to shrimp farming worldwide. This bacterium is also pathogenic to humans, which is considered to be a major problem for human health, and when people eat raw or uncooked seafood contaminated with this bacterium, acute gastroenteritis can result.

Traditionally, various antibiotics have become the primary means of managing the problem of Vibrio parahaemolyticus. However, the frequent use of antibiotics has led to the striking emergence of multi-drug resistant strains in the global environment and has become a significant therapeutic challenge. In order to reduce the risk of development and transmission of drug-resistant pathogens, there is an increasing need to develop alternative non-antibiotic approaches to combat bacterial infections.

Phages are viruses found in large numbers in the marine environment and are known as natural killers of bacteria. Phage therapy is to control bacterial infection by utilizing phage, and has the characteristics of high efficiency, specificity, environmental friendliness and the like. Phage therapy has attracted renewed interest as a non-antibiotic approach in human infections, food safety, agricultural, veterinary applications and environmental fields due to the increasing incidence of antibiotic resistance in bacterial pathogens. Also, phage therapy has great potential for pathogenic bacteria in aquaculture. Many reports in the past have demonstrated that phage therapy is an effective method for preventing and treating vibriosis caused by vibrio infection in the aquaculture industry, including a treatment that reduces morbidity and mortality using phage.

The invention separates a strain of lytic vibrio parahaemolyticus phage from aquaculture wastewater in the aquaculture market by taking the toxic vibrio parahaemolyticus preserved in the laboratory as host bacteria, researches the biological property and practical application of preventing and controlling vibrio parahaemolyticus diseases, provides methodology reference and phage materials for developing phage preparations for preventing and controlling vibrio parahaemolyticus, and lays a certain foundation for developing antibiotic substitutes for preventing and controlling and treating vibrio parahaemolyticus.

Disclosure of Invention

Aiming at the problems, the invention provides a vibrio parahaemolyticus bacteriophage with cracking property and application thereof in prawn culture, the bacteriophage has wide cracking spectrum, strong proliferation capability and short incubation period, has strong cracking and quick killing effects on various vibrio parahaemolyticus and vibrio alginolyticus ATCC17749, has good safety, has no acute and subacute toxic effects on prawns, can properly improve the activity of alkaline phosphatase and acid phosphatase in prawn serum, improves the immunity of prawns, and has good temperature and acid-base tolerance capability, and can adapt to more severe application environments. The phage is applied to preventing and treating infection of vibrio parahaemolyticus in the process of culturing prawns, can effectively prevent and treat infection caused by vibrio parahaemolyticus, reduce morbidity and improve survival rate, can quickly and effectively inhibit growth of vibrio parahaemolyticus in a prawn culture pond, effectively reduce content of vibrio parahaemolyticus, prevent vibrio parahaemolyticus diseases, has important application value in preventing and controlling vibrio parahaemolyticus diseases of aquaculture prawns, and provides phage sources for developing antibacterial preparations for preventing and controlling vibrio parahaemolyticus.

The technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a phage for lysing vibrio parahaemolyticus, the host of which is vibrio parahaemolyticus VP31, the vibrio parahaemolyticus phage being designated as vb_vpas_pp39, isolated from an aquatic market in Qingdao, shandong province, which has been deposited at the China general microbiological culture Collection center, location: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is CGMCC No.45570.

Further, the phage head was observed under electron microscopy to be oblong, approximately 79.54nm long and approximately 31.25nm wide, with a long tail, approximately 125.13nm long, and was a tailed phage virus.

Furthermore, the phage has good temperature tolerance, can be stored for a long time under the condition of-80 ℃ and has stable potency; stable activity at 60deg.C, and potency no less than 10 ⁹ PFU/mL。

Furthermore, the phage has good pH tolerance, and can act for 2 hours within the pH range of 5-11, the activity is stable, and the potency is not less than 10 ⁹ PFU/mL; at pH 4 and 12, phage also survived, and the titer was not less than 10 ⁴ PFU/mL。

Further, the phage has a broad lysis spectrum, and among 32 strains of Vibrio parahaemolyticus which have been isolated and identified, 18 strains thereof can be lysed, the lysis rate is about 55% -60%, and Vibrio alginolyticus ATCC17749 can be lysed.

Further: the phage has strong proliferation capacity, and the MOI of infection complex is at least 0.00001.

Further: the phage had a short incubation period of 10min.

In a second aspect of the present invention, there is provided the use of vibrio parahaemolyticus phage vB_ValP_PP39 in the preparation of a vibrio parahaemolyticus antibacterial agent.

Further, the vibrio parahaemolyticus phage also has the purpose of preparing a prophylactic or therapeutic agent for vibrio alginolyticus ATCC17749.

Further, the antibacterial agent has an effect of increasing alkaline phosphatase activity in serum.

Further, the use of the vibrio parahaemolyticus antimicrobial in shrimp culture.

The phage has no acute toxicity to prawn, and does not influence the survival of prawn. The phage has no subacute toxic effect on prawns, does not influence survival and feeding of the prawns, does not influence the activity of related enzymes in serum, and does not influence the normal structure of hepatopancreas.

In a third aspect of the present invention, there is provided an antibacterial agent for preventing or treating Vibrio parahaemolyticus, which comprises an effective amount of Vibrio parahaemolyticus phage vB_ValP_PP39.

Further, the antibacterial agent is a vibrio parahaemolyticus phage purified solution.

Further, the titer of phage vB_ValP_PP39 in the phage purification liquid is not less than 10 ¹⁰ PFU/mL。

The invention has the beneficial effects that:

1. in the invention, phage vB_VpaS_PP39 has wide lysis spectrum, strong proliferation capacity and short incubation period, can lyse various vibrio parahaemolyticus and vibrio alginolyticus ATCC17749, has the lowest complex MOI of infection of 0.00001 and has incubation period of 5-15min, and can strongly lyse and quickly kill host pathogenic bacteria.

2. In the present invention, phage vB_VpaS _P39 has stable activity at 60deg.C, and has potency of not less than 10 ⁹ PFU/mL; the pH is 5-12 for 2h, the activity is stable, and the potency is not lower than 10 ⁹ PFU/mL, has good temperature and pH tolerance, and can adapt to more severe environments.

3. In the invention, the phage vB_VpaS_PP39 has good safety and has no acute and subacute toxic effects on prawns.

4. In the invention, phage vB_VpaS_PP39 can properly improve the activity of alkaline phosphatase in the prawn serum and improve the immunity of the prawn.

5. In the invention, the phage vB_VpaS_PP39 can effectively prevent and treat infection caused by vibrio parahaemolyticus, reduce the incidence rate and improve the survival rate.

6. In the invention, the phage vB_VpaS_PP39 has strong cracking effect on vibrio parahaemolyticus in the shrimp culture environment, can quickly and effectively reduce the content of the vibrio parahaemolyticus, has important application value in the prevention and control aspect of the vibrio parahaemolyticus diseases in the shrimp culture, is a potential safe and effective antibiotic substitute, and provides phage sources for industrial production for preventing and controlling the vibrio parahaemolyticus phage in the culture environment.

Description of the drawings:

FIG. 1 is a transmission electron microscope image of phage vB_VpaS_PP39.

FIG. 2 shows the results of the optimal multiplicity of infection assay for phage vB_VpaS_PP39.

FIG. 3 shows the one-step growth curve measurement of phage vB_VpaS_PP39.

FIG. 4 shows the results of the temperature stability measurement of phage vB_VpaS_PP39.

FIG. 5 shows the pH stability measurement results of phage vB_VpaS_PP39.

FIG. 6 shows the survival rate of prawns in acute toxicity experiments.

FIG. 7 shows the survival rate and total weight gain of the prawns in the subacute toxicity test, and FIG. 7-A shows the survival rate of the prawns; FIG. 7-B total weight gain of prawns.

FIG. 8 shows HE staining results of hepatopancreatic tissue sections of prawns in subacute toxicity experiments, magnification 400×, and FIG. A shows hepatopancreatic tissue sections of prawns in a control group; panel B is a view of a section of liver and pancreas tissue of a shrimp of the experimental group.

FIG. 9 shows the results of phage vB_VpaS_PP39 used for preventing and treating diseases caused by infection of Vibrio parahaemolyticus in prawn culture, and FIG. A shows the survival rate of prawns in different prevention groups; panel B shows the survival rate of the shrimps in the different treatment groups.

FIG. 10 shows the result of phage vB_VpaS_PP39 for controlling Vibrio parahaemolyticus in the water body of a prawn culture plant, and FIG. A shows the result of controlling Vibrio parahaemolyticus in a prawn culture plant; FIG. B shows the result of controlling Vibrio parahaemolyticus in a cultivation plant for penaeus vannamei.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments.

The experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer; materials, reagents, etc. not noted are commercially available products. Percentages and parts are by weight unless otherwise indicated.

Example 1, strain and growth conditions

Here, 32 strains of Vibrio parahaemolyticus including Vibrio parahaemolyticus ATCC17802 (purchased from Guangdong microorganism culture collection center) and other 31 strains of Vibrio parahaemolyticus isolated and identified from pond water and hepatopancreas of sick shrimps in different regions of China were used (Table 1). These Vibrio were stored in 2216E liquid medium (Haibo biotechnology Co., ltd.) containing 30% glycerol in an ultra-low temperature refrigerator at-80 ℃. They were passaged from-80℃strain onto 2216E agar plates (2216E liquid medium containing 2% agar) and passaged at 37℃for at least 24h. Standard conditions for culturing the vibrio parahaemolyticus strain are: liquid 2216E medium, 220rpm,37 ℃.

TABLE 1 detection of Vibrio parahaemolyticus virulence factor and Vibrio parahaemolyticus lytic ability of bacteriophage vB_VpaS_PP39

Note that: a++, clear and transparent spot; ++, slightly blurry spots; ++, blurry spots; -, no spots

Example 2 detection of Vibrio parahaemolyticus virulence Gene

To examine whether or not the Vibrio parahaemolyticus used in this study carries the virulence gene PirA, a 336bp PriA gene DNA fragment (F: 5'-ATGAGTAACAATATAAAACA TGAAAC-3' and R:5'-GTGGTAATAGATTGTACA GAA-3') was amplified by the PCR method. The PCR amplification conditions are 94 ℃ initial denaturation for 5min,94 ℃ 30s,58 ℃ 60s,72 ℃ 45s and 35 cycles; amplified PCR products were stained with ethidium bromide on a 1% agarose gel and run at 90V for 25min. The gel imaging system by gel electrophoresis showed a corresponding target band to confirm whether the PirA gene was present or not, and PCR was repeated 3 times. The results are shown in Table 1.

EXAMPLE 3 isolation, purification and propagation of phages

1. Preparation of fresh bacterial liquid

According to the results of Table 1, 5 strains of Vibrio parahaemolyticus harboring virulence genes PirA, VP5, VP7, VP8, VP15 and VP43, respectively, were selected, and single colonies of these 5 strains were inoculated into 5mL 2216E liquid medium, respectively, and were subjected to shaking culture at 37℃and 170rpm for about 4 hours, to obtain fresh bacterial solutions in the logarithmic growth phase.

2. Isolation and purification of phages

A sample of the cultivation wastewater from an aquatic market in Qingdao, shandong was centrifuged at 11000rpm for 5min, and the supernatant was filtered and sterilized with a 0.22 μm filter. 5mL of the filtrate and 5mL of fresh host bacteria were added together to 50mL of sterilized 2216E liquid medium, and the mixture was subjected to shaking culture at 37℃and 170rpm overnight to proliferate phage which may be present. Taking 5mL of culture solution, centrifuging at 11000rpm for 10min, filtering the supernatant with a 0.22 μm filter membrane to obtain phage proliferation stock solution, and storing at 4deg.C.

Separating phage by double-layer agar plate method, diluting phage proliferation stock solution by 10 times, mixing 100 μl of diluted solution with 100 μl of host bacteria solution, incubating at 37deg.C for 5min, placing the mixed solution on upper agar (agar concentration of 0.7%) with temperature of about 50deg.C, mixing, rapidly pouring onto lower agar (agar concentration of 1.5%) plate, and shaking gently. After cooling, the cells were incubated at 37℃overnight with inversion. The following day, the plaque formation was observed, and if phage were present, transparent, regular circular plaques, i.e., plaques, were present on the bilayer plates.

Individual plaques were picked from double-layered plates with obvious individual plaques in 1mL PBS buffer and bathed at 42 ℃ for 30min to obtain phage leachate. The leachate was centrifuged at 12000rpm for 5min, the supernatant was sterilized with 0.22 μm filter, and 10-fold gradient diluted with PBS buffer, and the gradient to obtain individual plaques was selected to prepare a double-layered plate. The steps are repeated for 4 to 5 times until plaque with consistent size, morphology and definition is obtained.

3. Determination of proliferation and titers of phages

Individual plaques of the purified phage were picked up in 1mL of PBS buffer and bathed at 42 ℃ for 30min to give phage extract. 100. Mu.L of the extract and 100. Mu.L of the host bacteria liquid were cultured in 5mL of liquid 2216E medium at 37℃with shaking at 170rpm until the liquid became clear, to obtain a phage multiplication liquid.

Taking phage multiplication liquid 5mL, centrifuging at 12000rpm for 5min, sterilizing supernatant with 0.22 μm filter membrane, and performing 10-fold gradient dilution to 10 with PBS buffer ⁹ Multiple of 10 times ⁷ 、10 ⁸ And 10 ⁹ Double-layer plates were prepared from 100. Mu.L of the dilution of the double dilution and 100. Mu.L of the host bacteria solution, respectively, and the titer of phage was determined. Phage titer (PFU/mL) =number of plaques x dilution x 10.

The results showed that the vibrio parahaemolyticus phage vB_VpaS_PP39 forms a uniform, regular circular, transparent plaque with a diameter of about 1mm on a double-layered plate of its host VP43 after 4 generations of isolation and purification. After the phage had proliferated, the titers were measured at about 8.84X10 ⁹ PFU/mL。

Example 4 electron microscopic observations of phages

20 mu L of phages were taken by phosphotungstic acid negative stainingBody proliferation liquid (potency 10) ⁹ PFU/mL) is dripped on a copper net, after standing and precipitating for 15min, redundant liquid is sucked by filter paper, 15 mu L of 2% phosphotungstic acid staining solution is dripped for 5min, redundant staining solution is sucked, and after natural drying, phage morphology is observed by a transmission electron microscope.

As shown in FIG. 1, the head of phage vB_VpaS_PP39 under electron microscope is prolate, has a length of about 79.54nm and a width of about 31.25nm, has a long tail, and has a length of about 125.13nm, and is a tailed phage virus.

The vibrio parahaemolyticus phage was designated vb_vpas_pp39 and deposited at the China general microbiological culture Collection center, location: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is CGMCC No.45570.

Example 5 detection of the cleavage spectrum and host Range of phages

1. Determination of the cleavage Rate

The sample application method is adopted. 100 mu L of each vibrio bacterial suspension is added into 5mL of upper agar culture medium, fully and uniformly mixed, and then quickly spread on a lower agar plate, and horizontally placed for cooling. Taking 2 mu L of the phage multiplication liquid to a double-layer plate containing each vibrio parahaemolyticus, culturing for 8-12h at 37 ℃, and if a transparent circle appears on the plate, indicating that the phage can lyse the vibrio parahaemolyticus.

As a result, as shown in Table 1, for 32 strains of Vibrio parahaemolyticus, phage vB_VpaS_PP39 was able to lyse 18 strains with a lysis rate of about 56.25% (Table 1).

2. Detection of host range

1 strain of Vibrio alginolyticus ATCC17749 (purchased from Guangdong microorganism culture collection center) and 4 strains of Vibrio alginolyticus, 4 strains of Vibrio cholerae, 2 strains of Vibrio vulnificus and 3 strains of non-Vibrio bacteria are selected to detect the host range of the phage, and the determination method is the same as above.

As a result, as shown in Table 2, phage vB_VpaS_PP39 also lyses Vibrio alginolyticus ATCC17749.

TABLE 2 lytic ability of phage vB_VpaS_PP39 against other bacteria

Example 6 optimal multiplicity of infection MOI determination of phage

The multiplicity of infection MOI is the ratio of the number of phages to the number of bacteria at the time of infection, and the optimal MOI is the ratio of phages reaching optimal growth conditions. To determine the optimal MOI, stock solutions of phages (8.84X 10 ⁹ PFU/mL) was diluted in a 10-fold ratio, and phage dilutions of appropriate concentrations and logarithmic growth phase bacterial liquids (7.2X10) of host bacteria VP43 were selected ⁷ CFU/mL) was added to 5mL fresh 2216E liquid medium to make the infection ratio 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 37 ℃,220rpm shaking culture until the liquid was clear and bacterial fragments were visible. Phage titers in each tube were then determined using the double-layer plate method. The MOI corresponding to the highest titer is taken as the optimal MOI.

As a result, as shown in FIG. 2, phage vB_VpaS_PP39 was the highest in titer, which was 2.26X10 when MOI=0.01 ¹⁰ PFU/mL, the optimum MOI for phage is 0.01; its minimum infection complex can reach 0.00001.

Example 7 one-step growth Curve determination of phage

Stock solutions of phages (8.84×10 ⁹ PFU/mL) and logarithmic phase bacterial liquid (7.2X10) of host bacterium VP43 ⁷ CFU/mL) was added to 5mL of 2216E liquid medium at a multiplicity of infection MOI of 1, incubated at 37℃for 10min, centrifuged at 12000r/min for 30s, the supernatant removed, re-suspended and washed 2 times with 2216E liquid medium to remove unadsorbed phage, then re-suspended and precipitated with micro 2216E liquid medium, the re-suspension was transferred to 2216E which was preheated to 50mL of 37℃and immediately placed at 37℃for 220rpm shaking culture, defined as t=0 min, and samples were collected every 10min in 180min using double-layer agar platesThe titers of each sample were determined by the method. The burst size of the phage is the ratio of the final number of free phage particles to the initial number of infected bacterial cells.

As shown in FIG. 3, the phage vB_VpaS_PP39 had a latency of 10min and an outbreak of about 156 PFUs/infected cells, indicating that the phage has the characteristics of short latency and large outbreak.

Example 8 temperature stability experiment of phage

Will be filled with 1mL phage stock solution (8.84X 10) ⁹ PFU/mL), water baths at 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for 20min, 40min, 60min, respectively, followed by double-layer plating with host bacteria VP43 to determine phage titers.

As can be seen from FIG. 4, phage vB_VpaS_PP39 is stable in activity at 60℃and has a potency of not less than 10 ⁹ PFU/mL,70 ℃ complete inactivation; in addition, the phage can be stored for a long time at-80 ℃ and the potency is kept stable.

Example 9 pH stability experiment of phage

Phage stock solution (8.84×10) ⁹ PFU/mL) was mixed with 2216E liquid medium having pH values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, respectively, in a water bath at 37℃for 2 hours, and after completion of the reaction, a proper amount of 1mol/L HCl or 1mol/L NaOH was added to the mixture to a pH value of about 7, and then the mixture was subjected to phage titer measurement by laying a bilayer plate with host bacteria VP 43.

As can be seen from FIG. 5, phage vB_VpaS_PP39 was allowed to act for 2h at pH 5-11, the activity was kept stable, and the titer was not lower than 10 ⁹ PFU/mL, also has phage survival at pH 4 and 12, titers of no less than 10 ⁴ PFU/mL shows that the phage has acid-base tolerance and can adapt to more severe environment.

Example 10 preparation of phage purification liquid

The liquid preparation of phage vB_VpaS_PP39 was prepared as follows:

(1) Inoculating host bacteria VP43 into 2216E liquid culture medium, culturing at 37deg.C and 220rpm for 3-4 hr to obtain logarithmic phase host bacteria liquid;

(2) Simultaneously inoculating the phage stock solution and the host bacterial solution into 2216E liquid culture medium according to the optimal infection complex number of 0.01, culturing at 37 ℃ and 220rpm for 4-6h to obtain phage seed solution;

(4) Simultaneously inoculating phage seed liquid and host bacteria liquid into a fermentation tank filled with a sterilized liquid culture medium according to the optimal infection complex number of 0.01, culturing for 10-12h under the aeration condition at 37 ℃ with stirring at 70rpm to obtain a fermentation mixed liquid, and paving a double-layer flat plate for measuring titer;

(5) And (3) centrifuging and membrane filtering the fermentation mixed solution to remove host bacteria VP43, thus obtaining the purified solution of the phage, namely the liquid preparation of the phage, and paving a double-layer flat plate for measuring the titer.

The fermentation broth titer of phage vB_VpaS_PP39 was determined to be 2.8X10 ¹⁰ PFU/mL, the titer of the purified solution was 1.17X10 ¹⁰ PFU/mL。

Example 11 phage safety experiments

Before practical application, the application safety of the phage is evaluated through acute and subacute toxicity tests of the prawns.

The acute toxicity of phage to prawn is discussed by observing the influence of phage to short-term survival rate of prawn and related serum index in serum. 180 healthy penaeus vannamei boone (weight of 6+/-0.5 g) are cultivated in a 20L glass jar under proper conditions, the water temperature in the jar is kept at 25+/-1 ℃, and the penaeus vannamei boone is equally divided into a control group and an experimental group. Each group of 90 prawns is divided into 3 parallel subgroups with 30 tails each. Then, the control group and the experimental group were injected with sterile physiological saline and phage purified solution, respectively (10 ¹⁰ PFU/mL), 100 μl per tail, the injection site is the subcutaneous muscle between the second and third sections of the shrimp abdomen. Each group of prawns was fed at a timing of 4% of the body weight in the morning and evening (about 8:00 and 20:00) with half of the sea water changed each day. The death of each group of shrimps was recorded daily, and the status of each group of shrimps was observed and monitored. The survival rate of each group of prawns is counted at 24h, 48h, 72h, 96h and 120h respectively, 3 tail prawns are randomly selected from each repetition of each group at the time point of 120h, all selected prawns are sterilized by 70% ethanol for about 3 minutes, and haemolymph is collected. 1.0mL sterile syringe was removed from the shrimp headThe back of the chest plate is inserted into the pericardial cavity to extract pericardial haemolymph. The supernatant was centrifuged at 5000r/min for 10min at 4℃to determine the activity of biochemical markers of serum, including aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), total serum protein (TP), total serum cholesterol (T-CHOL), triglyceride (TG) and alkaline phosphatase (AKP). The measurement of the index activity adopts a kit for Nanjing built biological research institute, and the measurement steps are carried out according to the specification of the kit.

The subacute toxic effect of phage on the prawns is detected by observing the influence of phage on the long-term survival rate of the prawns, serum biochemical index detection and hepatopancreatic tissue section observation. The experiment was grouped, fed and operated as above, but the experimental period was 30d. The total weight of each group of prawns was weighed on day 0 and day 30 of the experiment, respectively, and the total weight gain of each group was calculated. The death of each group of shrimps was recorded daily and the status of each group of shrimps was monitored. After the experiment was completed, the survival rate of each group of prawns was counted, then each group randomly selected 3 prawns from each repetition, all selected prawns were sterilized with 70% ethanol for about 3 minutes, and then their haemolymph was collected and their hepatopancreas was extracted. The blood sampling method is the same as above, dissecting after blood sampling, separating liver pancreas, and fixing with 10% formalin. And (3) dehydrating, transparentizing and waxing the fixed specimen, embedding in paraffin, continuously slicing, HE dyeing and sealing by glue. Tissue sections were observed using a nikon 80i multifunctional microscope.

The results of the acute toxicity experiments show that the control group and the experimental group have no prawn death in 24h, 48h, 72h, 96h and 120h, and the survival rate is 100 percent (figure 6); the results of the serum biochemical markers are shown in Table 3, and the activities of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), serum Total Protein (TP), serum total cholesterol (T-CHOL) and Triglyceride (TG) in the control group and the experimental group were not significantly different at 120h of phage injection, but the activities of alkaline phosphatase (AKP) in the two groups were significantly different. Alkaline phosphatase is an enzyme related to immunity in serum, when phage liquid is injected into prawn body for 120h, the AKP enzyme activity of the experimental group reaches 10.41+/-0.91U/L, which is more than 2 times of that of the control group, and the AKP enzyme activity of the experimental group is obviously different from that of the control group (P < 0.05). This shows that the vibrio parahaemolyticus phage vB_VpaS_PP39 can effectively increase the AKP content in the prawn serum, and plays a positive role in enhancing the immunity of prawns. The results of the acute toxicity experiments indicate that the phage vB_VpaS_PP39 has no acute toxic effect on prawns.

TABLE 3 serum Biochemical index detection result of acute toxicity experiment (120 h)

The results of the subacute toxicity experiments show that the prawns in the control group and the experimental group normally survive, the prawns do not die at the end of the experiments, the survival rate is 100% (figure 7-A), and the total weight gain of the prawns in each group is not significantly different (figure 7-B); the results of the two groups of serum biochemical indicators showed (Table 4) that there was no significant difference in activity of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), total serum protein (TP), total serum cholesterol (T-CHOL), triglyceride (TG) and alkaline phosphatase (AKP); HE staining of the hepatopancreas of both groups of prawns showed that the hepatopancreas of the control group (FIG. 8-A) and the experimental group (FIG. 8-B) had intact structure, the hepatic tubules were aligned in a star shape, and no pathological symptoms were found, indicating that the number of pathological symptoms was about 10 ⁸ At the dosage of PFU/g, the phage purified solution has no acute toxicity and subacute toxicity to prawn, and the phage vB_VpaS_PP39 is preliminarily verified to have application safety.

TABLE 4 serum Biochemical index detection results of subacute toxicity experiments

Example 12 phage control of Vibrio parahaemolyticus infection in prawn culture

The experiment aims to verify the prevention and treatment effects of phage on pathogenic bacterial infection in practical application. Prior to the experiment, a high concentration of phage was obtained. The specific operation is as follows: phage particles were precipitated with 10% polyethylene glycol 8000 (PEG 8000) overnight at 4deg.C, centrifuged at 10000r/min for 15min, and then suspended in PBS buffer. The suspension was diluted to the desired concentration with PBS buffer prior to use. Adding the diluted suspension into pellet feed, stirring thoroughly, and soaking for at least 30min.

The experiment adopts vibrio parahaemolyticus VP8 to attack the penaeus vannamei. To prepare vibrio parahaemolyticus VP8 cells, the log phase culture was removed, washed 3 times with sterile physiological saline, and the optical density (OD ₆₀₀ ) And coating TCBS plate counting to obtain a concentration of 4×10 heavy suspension ⁶ CFU/mL。

In the phage prophylaxis test, 540 healthy prawns were equally divided into 6 groups of 90 tails each, and into 3 parallel subgroups of 30 tails each. Each group of prawns was fed at a timing of 4% of the body weight in the morning and evening (about 8:00 and 20:00) with half of the sea water changed each day. Groups 1 and 3 were fed untreated pellet feed and group 2 was fed phage-impregnated pellet feed with MOI of 10. Group 4 was fed with doxycycline-impregnated pellet feed with doxycycline concentration of 5mg/L. Groups 5 and 6 were fed phage-impregnated pellet feed with MOI 1 and 10, respectively. After feeding each group for 7d, the other 4 groups were injected with a syringe into the Vibrio parahaemolyticus VP8 suspension (4X 10) except for 60. Mu.L of sterile physiological saline for 1 group and 2 groups, respectively ⁶ CFU/mL). Each group was then fed untreated pellet feed, each group status was monitored and mortality recorded. After 7 days of culture, the survival rate of each group of prawns was counted.

In phage therapy application experiments, the grouping and feeding conditions were as above. The difference is bacterial challenge time and dosing time. Groups 1 and 2 were fed untreated pellet feed and phage-impregnated pellet feed with MOI of 10, respectively, after injection of sterile physiological saline for 4h. After injection of VP 84 h, group 3 was fed untreated pellet feed. After VP 84 h of injection of group 4, doxycycline-impregnated pellet feed was fed with doxycycline at a concentration of 5mg/L. Groups 5 and 6 were fed phage-impregnated pellet feed with MOI 1 and 10, respectively, after VP 84 h injection. The death of each group of shrimps was recorded daily and the status of each group of shrimps was monitored. After 7d feeding, the survival rate of each group of prawns is counted.

As shown in FIG. 9, the survival rate of the prawns in the positive control group to which only VP8 bacteria was added was 48.34% (G-3 in FIGS. 9-A and 9-B); in the prophylactic experiments with phages, the survival rates of the phage groups were 85% and 88.34% (G-5 and G-6 in FIG. 9-A), respectively, and were better than 81.67% of the doxycycline group (G-4 in FIG. 9-A). In phage therapy experiments, the survival rates of the phage groups were 78.34% and 81.67% (G-5 and G-6 in FIG. 9-B), respectively, and were better than 75% (G-4 in FIG. 9-B) of the doxycycline group.

To exclude adverse effects of vb_vpas_pp39 on prawns, individual phages were used in both experiments, group 2. The survival rate of group 2 was 100% (G-2 in FIGS. 9-A and 9-B), which was the same as that of group 1 (G-1 in FIGS. 9-A and 9-B), indicating that the phage itself did not adversely affect the prawns.

Example 13 phage control of Vibrio parahaemolyticus content in prawn culture Water

The experiment aims to verify the evaluation of the prevention and control effect of phage on vibrio parahaemolyticus in the water body of a culture factory in practical application. Experiments were performed in two penaeus vannamei farms in the sun-shine and smoke-counter markets, respectively, in Shandong province.

In a solar prawn culture factory, the average weight of prawn is 8+ -0.6 g, the water temperature is 26 ℃, the salinity is 25%, and the culture pond size is 30m ³ Feeding for a fixed time every day, and changing water for 20% every day. In order to control the reproduction of pathogenic vibrio, reagents such as povidone iodine, compound iodine, chlorine dioxide, probiotics and the like are added into the pool regularly and stopped at least 72 hours before phage experiments. The experiment was divided into 3 groups, and each group of culture ponds was tested for vibrio content by coating TCBS plates before starting. Group 1 was a blank group, and no treatment was performed. 2 groups are sprayed with iodine disinfectant, and the concentration is 5mg/L.3 groups were spiked with phage purified solution with MOI 1. The experiment was repeated 3 times for each group, pool water samples were taken every 12 hours, and the vibrio content was checked.

The cultivation conditions of prawns (average weight=6±0.5 g) in a certain farm were the same as those in a sunshine farm, except that the water in the farm was changed by 30% per day. The experimental setup was the same as for the solar farming plant except that the pond water was sampled every 8 hours and the vibrio content was detected.

As a result, as shown in FIG. 10, it can be seen that the Vibrio content in each of the control groups remained unchanged or increased over time (G-1 in FIGS. 10-A, 10-B), which reflects the challenge presented by Vibrio without intervention. Group 2 treated with iodine disinfectant was effective for the first few hours, but then vibrio was continuously rebound and increased to the same level as the control group at about 24 hours (G-2 in fig. 10-a, 10-B). In contrast, the number of Vibrio in phage-bearing farms remained on a decreasing trend throughout the experiment (G-3 in FIGS. 10-A, 10-B). The result of the action on vibrio (in particular vibrio parahaemolyticus) in the water body of the culture plant proves that phage vB_VpaS_PP39 can be used as an effective biological agent for controlling vibrio in actual culture.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. The vibrio parahaemolyticus phage vB_VpaS_PP39 is characterized in that the vibrio parahaemolyticus phage vB_VpaS_PP39 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 45570 at 5-month-16 of 2023.

2. The vibrio parahaemolyticus phage vb_vpas_pp39 of claim 1, wherein the temperature tolerance of the vibrio parahaemolyticus phage is in the range of-80 ℃ to 60 ℃.

3. The vibrio parahaemolyticus phage vb_vpas_pp39 of claim 1, wherein the vibrio parahaemolyticus phage has a pH tolerance range of 4 to 12.

4. Use of the vibrio parahaemolyticus phage vb_vpas_pp39 as claimed in claim 1 for the preparation of a vibrio parahaemolyticus antibacterial agent.

5. The use according to claim 4, wherein the use is in the prevention or treatment of vibrio parahaemolyticus and/or vibrio alginolyticus ATCC17749.

6. The use according to claim 4, wherein the use is for increasing alkaline phosphatase activity in serum.

7. The use according to claim 4, wherein the use is in the cultivation of prawns.

8. An antibacterial agent for preventing or treating vibrio parahaemolyticus, characterized in that the antibacterial agent comprises an effective dose of vibrio parahaemolyticus phage vb_vpas_pp39.

9. The antibacterial agent of claim 8, wherein the antibacterial agent is a vibrio parahaemolyticus phage purified solution.

10. The antibacterial agent according to claim 8, wherein the phage vb_vpas_pp39 has a potency of not less than 10 ¹⁰ PFU/mL。