A strain of Vibrio ostreatus phage, its phage composition and its application

Abstract

The present invention discloses a Vibrio ostrenosum phage, a phage composition thereof and an application thereof. The phage is named vB_VorP_PRC03 and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 30, 2021, with a deposit number of CGMCC No. 22499. The phage has a strong lysis effect on Vibrio ostrenosum, a wide lysis spectrum, strong acid-base stability and high temperature stability. It can be used to prepare drugs, environmental disinfectants, feed additives, aquatic product bio-inhibitors and detection kits for treating or preventing diseases infected by Vibrio ostrenosum, and is used to effectively prevent and treat various aquatic diseases caused by Vibrio ostrenosum infection, greatly reducing the incidence of various diseases caused by Vibrio ostrenosum. The use of the phage is safe and has no side effects, effectively avoiding the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, in particular to a Vibrio ostreatus phage, a phage composition thereof and application thereof. Background Art

[0002] Vibrio ordalii is a Gram-negative bacterium, also known as Vibrio ordalii. Infection with Vibrio ordalii is usually a process that causes sepsis, but it is more specific than other Vibrio species in targeting muscles and skin, with fewer bleeding injuries, but a larger range of surface bleeding lesions. Leukopenia is also obvious, suggesting the presence of leukocyte lytic factors, which is very different from other diseases. Among them, bacteria and pigment-rich macrophages play an important role in the pathogenesis. Vibrio ordalii can cause vibriosis in salmonids such as salmon, red salmon, rainbow trout, etc.

[0003] At present, the existing prevention and control methods mainly use traditional antibiotics to prevent and treat diseases caused by Vibrio ostreatus and other Vibrio infections in seafood. However, excessive use of antibiotics not only easily leads to the production of drug-resistant pathogens, but also causes water pollution. Therefore, it is necessary to find safer and more effective alternatives to antibiotics.

[0004] Bacteriophage (Bacteriophage or Phage) is a general term for microbial viruses that infect bacteria, fungi, actinomycetes or spirochetes. It is small in size, simple in structure, and does not have a cell structure. It is widely distributed, and there are corresponding bacteriophages almost wherever bacteria exist. Due to the strong specificity of bacteriophages, they only target their specific bacterial pathogens as hosts and will not destroy normal flora; and because bacteriophages are host-dependent, they will die as the host clears them and will not remain in the animal's body; and the final degradation products of bacteriophages are amino acids and nucleotides, which have no effect on humans and animals. Therefore, bacteriophages have the advantages of strong specificity, no residue, no toxicity, and safe use. They can be used as an antibiotic substitute to prevent and treat aquatic diseases caused by Vibrio ostreatus infection.

[0005] However, no phage resources that can be used to prevent and treat Vibrio orthosiphon have been found so far. Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a Vibrio ostrenosum phage vB_VorP_PRC03, a phage composition and applications thereof. Based on the phage's high efficiency and specific lysis ability against Vibrio ostrenosum, the phage can be used to prepare drugs, environmental disinfectants, feed additives, food preservatives and detection kits for treating or preventing diseases caused by Vibrio ostrenosum infection. Based on the safety of the phage and its lack of side effects, the phage can solve the Vibrio ostrenosum infection while avoiding the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0007] To solve the above problems, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a Vibrio ostrenosum phage vB_VorP_PRC03, which was isolated from a bay area in Huangdao District, Qingdao City, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on July 30, 2021, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22499.

[0009] Observations under an electron microscope revealed that the purified phage particles of the phage had an icosahedral capsid and a short tail. According to the classification criteria of the 9th report of the International Committee on Taxonomy of Viruses (ICTV), the phage was identified as belonging to the Brachyura family and was named vB_VorP_PRC03.

[0010] This bacteriophage has excellent lysis performance against Vibrio ostrenosum. Experiments have shown that its lysis rate against 90 strains of Vibrio ostrenosum from different sources reached 85.56%. It has a wide lysis spectrum and a wide range of applications, which means that it has good application prospects in killing Vibrio ostrenosum in the environment and preventing and controlling Vibrio ostrenosum disease.

[0011] The phage can maintain its potency unchanged after being treated at a temperature range of 30°C to 60°C for 1 hour, and has good temperature stability. The phage can still maintain its stability after being treated at pH=3 to 11 for 3 hours, has good acid and alkali resistance, and can adapt to harsh acid and alkali environments. Based on its excellent biological characteristics, it has a wide range of adaptability.

[0012] In the present application, the above-mentioned phage vB_VorP_PRC03 includes mutants with a homology of more than 98% or 99% and substantially the same bactericidal activity after point mutation, deletion mutation or addition mutation. Since phages are very prone to mutation during replication, the mutants of the above-mentioned phages are also within the scope of protection requested in the present application. The sequence of phage vB_VorP_PRC03 can be obtained by sequencing the biological materials deposited according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants with similar properties according to the phage provided by the present invention.

[0013] In a second aspect, the present invention also provides a phage composition, comprising the Vibrio ostreatus phage vB_VorP_PRC03 as described above.

[0014] Preferably, in practical applications, in order to further broaden the lysis spectrum of the phage preparation, give full play to the differences in the lysis spectrum of different phages, and complement each other's advantages, the above-mentioned Vibrio ostreatus phage vB_VorP_PRC03 can be used in combination with other phages, such as being combined with other Vibrio ostreatus phages to expand the lysis spectrum of Vibrio ostreatus; in addition, the phage vB_VorP_PRC03 can also be combined with other different types of phages (inhibiting different pathogens that cause the same type of diseases) to improve the prevention and treatment effect of the same type of diseases.

[0015] In a third aspect, the present invention further provides a phage pharmaceutical preparation, the active ingredient of which includes the above-mentioned Vibrio ostreatus phage vB_VorP_PRC03 or the above-mentioned phage composition. Preferably, the phage pharmaceutical preparation also includes other complementary antibacterial or bactericidal active ingredients.

[0016] Optionally, the above-mentioned phage pharmaceutical preparation also includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" used herein refers to a carrier or diluent that does not cause significant stimulation to an organism and does not eliminate the biological activity and characteristics of the active component administered. In order to prepare the pharmaceutical composition as a liquid preparation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol and ethanol. They can be used alone or in any combination thereof. If necessary, other conventional additives can be added, for example, antioxidants, buffers and antibacterial agents, etc. When combined with diluents, dispersants, surfactants, adhesives and / or lubricants, the composition of the present invention can also be prepared into injections (for example, aqueous solutions, suspensions and emulsions), pills, capsules, granules or tablets.

[0017] The dosage form of the bacteriophage drug preparation is a solution, emulsion, suspension, powder, gel, granule or lyophilized agent. Preferably, the bacteriophage drug preparation is a solution. The solution is a method of administering the bacteriophage drug preparation directly into the aquaculture water body by bath administration, which has the advantages of simple operation and good effect.

[0018] In a fourth aspect, the present application also provides the use of the above-mentioned Vibrio ostreatus phage, the above-mentioned phage composition and the above-mentioned phage pharmaceutical preparation in the preparation of drugs, aquatic feed additives or environmental disinfectants for preventing and treating diseases caused by Vibrio ostreatus infection.

[0019] The term "prevention" herein means "prevention and treatment". "Prevention" means all actions including suppressing or delaying the disease by administering the bacteriophage. The term "treatment" herein means all actions including making the disease better or improved by administering the bacteriophage.

[0020] The diseases include various diseases caused by Vibrio ostrenosum infection, etc. Preferably, the disease caused by Vibrio ostrenosum infection is a disease of aquatic products caused by Vibrio ostrenosum infection; more preferably, the septicemia disease of aquatic products caused by Vibrio ostrenosum infection. More preferably, the aquatic products are selected from marine aquatic products. More preferably, the marine aquatic products include farmed species such as salmon, rainbow trout, sweetfish, grouper, and salmon. In the subsequent specific embodiments, only salmon is used as an example for explanation, and the scope of application is not limited to the prevention and treatment of Vibrio ostrenosum disease in salmon.

[0021] Since the above diseases are caused by infection with Vibrio ostrenosum, it is expected that Vibrio ostrenosum phages with a highly efficient and specific lysis effect on Vibrio ostrenosum will have preventive and therapeutic effects on these diseases, and the experimental results also prove this conclusion.

[0022] In a fifth aspect, the present invention also provides an aquatic feed additive, which includes the Vibrio ostrenosum phage or the phage composition as described above. The aquatic feed additive is mixed with aquatic feed and then fed to aquatic animals, thereby achieving the effect of preventing or treating aquatic Vibrio ostrenosum disease.

[0023] Preferably, the titer of each phage in the feed is 1×10 8 PFU / g or above. Under this condition, the effect of feed additives is better.

[0024] In a sixth aspect, the present invention further provides an environmental disinfectant, the active ingredient of which is mainly the aforementioned Vibrio ostreatus phage or the aforementioned phage composition; preferably, the titer of the phage is 1×10 4The water disinfectant may also contain other active ingredients or other adjuvants for inhibiting or eliminating bacteria in the environment; more preferably, the adjuvant includes an adjuvant that can prolong the effective period of bacteriophages.

[0025] The environmental disinfectant can be used for environmental disinfection of aquaculture sites and disinfection and preservation of feed, and can be used to replace antibiotics or traditional disinfection products. The metabolites of the bacteriophage and its environmental disinfectant will not cause harm to humans or other animals.

[0026] In a seventh aspect, the present invention also provides use of the above-mentioned environmental disinfectant in killing Vibrio ostreniformis in the environment.

[0027] The environmental disinfectant can be used to disinfect the breeding environment, feed, feeding equipment, etc. by spraying or soaking. The breeding environment includes the pool wall, water body, etc. The liquid soaking and spraying forms include but are not limited to detergents, disinfectants, and detergents.

[0028] In an eighth aspect, the present invention further provides a detection kit, comprising the Vibrio orthogenes phage or the aforementioned phage composition as described above. Experiments have shown that the phage vB_VorP_PRC03 has a high lysis specificity for the host bacteria. The phage vB_VorP_PRC03 of the present invention can be applied to the rapid detection of Vibrio orthogenes in samples, including but not limited to the detection of pathogens in clinical samples in different forms such as test strips or test strip boxes. The detection method is simple and highly sensitive.

[0029] In a ninth aspect, the present invention also provides use of the above detection kit in detecting Vibrio ostrenosum.

[0030] In a tenth aspect, the present invention also provides a biological antibacterial agent for aquatic products, wherein the active ingredient is mainly the above-mentioned Vibrio orthogenes bacteriophage or bacteriophage composition. The use of the biological antibacterial agent for aquatic products has a disinfection and preservation effect on aquatic products. When used, the biological antibacterial agent can be used to treat fresh aquatic products by soaking or spraying, thereby inhibiting the proliferation of Vibrio orthogenes during product processing, transportation or preservation, and achieving disinfection and preservation effects.

[0031] The present invention has the following beneficial effects:

[0032] 1. The present invention isolates a Vibrio ostreatus phage vB_VorP_PRC03 for the first time. The phage has a strong lysis effect on Vibrio ostreatus, and its lysis rate on Vibrio ostreatus can be as high as 85.56%. It can be used to effectively prevent and control human diseases and various animal (especially aquatic) diseases caused by Vibrio ostreatus in aquaculture farms, greatly reducing the incidence rate of various diseases caused by Vibrio ostreatus; it can also be used for comprehensive disinfection of Vibrio ostreatus in aquaculture farm environments, feeds, water bodies, etc., greatly reducing the morbidity and mortality of aquatic products caused by Vibrio ostreatus; it can also be used for the preservation of fresh aquatic products, with diversified application methods.

[0033] 2. The bacteriophage vB_VorP_PRC03 has the advantages of high acid-base stability and high thermal stability, and has good temperature stability in the temperature range of 30°C to 60°C; the activity of the bacteriophage is stable and high under the condition of pH = 3 to 11, and can adapt to relatively harsh acid-base environments; at the same time, the bacteriophage has strong reproductive ability and is easy to carry out industrial production; the bacteriophage can be widely used in various links in the aquaculture process that are prone to losses due to infection with Vibrio orchester, daily disinfection of the aquaculture environment, and antibacterial of fresh aquatic foods, which is beneficial to the healthy development of the aquaculture industry.

[0034] 3. The bacteriophages of the present invention are obtained from nature. The bacteriophages and bacteriophage compositions are safe to use and have no side effects. They can be effectively used to solve the infection caused by Vibrio orrenyi and the water problems caused by the massive proliferation of the above pathogens, avoiding the problems of antibiotic residues and the induction of drug-resistant Vibrio orrenyi caused by the use of antibiotics. The drugs, disinfectants or preservatives prepared by the bacteriophages also have the advantages of being safe to use and green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is an electron microscope photo of bacteriophage vB_VorP_PRC03;

[0036] Figure 2 The temperature stability results of bacteriophage vB_VorP_PRC03;

[0037] Figure 3 is the pH stability result of bacteriophage vB_VorP_PRC03;

[0038] Figure 4 is the one-step growth curve of bacteriophage vB_VorP_PRC03;

[0039] Figure 5 The results of in vitro lysis experiments of bacteriophage vB_VorP_PRC03;

[0040] Figure 6These are the results of the environmental disinfection experiment of bacteriophage vB_VorP_PRC03. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0042] Example 1 Isolation and culture of bacteriophage

[0043] (I) Preparation of host bacterial suspension

[0044] Vibrio ostreatus is usually grown and preserved in liquid tryptic soy broth (TSB) and tryptic soy agar (TSA). The host bacterium Vibrio ostreatus RC003 is streaked on a TSA plate, and a single colony is picked and inoculated into 5 mL of TSB liquid culture medium. It is proliferated at 170 rpm and 37°C to the logarithmic growth phase to obtain a fresh host bacterium liquid.

[0045] (II) Isolation and purification of bacteriophage

[0046] (1) Isolation of bacteriophage

[0047] The sewage collected from a bay area in Huangdao District, Qingdao City, Shandong Province was centrifuged at 11000r / min for 5 minutes. The supernatant obtained by centrifugation was filtered using a 0.22μm filter membrane, and TSB culture medium and 100μL of host bacterial suspension were added. After culturing at 170rpm and 37°C for 16 hours, it was centrifuged at 11000r / min for 5 minutes, and finally filtered using a 0.22μm filter membrane to obtain the phage stock solution.

[0048] The phage stock solution was diluted 10-fold with 1× PBS solution to obtain 10 -2 , 10 -4 , 10 -6 , 10 -8 After the dilution, take 100 μL of each of the above dilutions of different concentrations and mix them with the prepared host bacterial suspension, incubate at 37°C for 5 minutes, add 5 mL of TSB upper culture medium, mix well and quickly pour into TSA culture medium plate, wait for it to solidify, invert it in a 37°C incubator and culture for 16 hours before observation to obtain plaques.

[0049] (2) Phage purification

[0050] Pick up a single plaque with sterile forceps, place it in 1 mL of PBS solution, and then place it in a shaker at 170 rpm and 37°C for 30 min. Dilute the extract to 10 -2 , mixed with the host bacterial suspension at a ratio of 1:1, incubated at 37°C for 5 minutes, added the mixture to 5 mL of TSB upper medium, mixed well and quickly poured into a TSA medium plate, and after solidification, inverted and cultured in a 37°C incubator for 16 hours to obtain E1 generation phage. Purify 3-5 generations according to the above method to obtain plaques with consistent morphology.

[0051] (III) Determination of phage titer

[0052] The above phage suspension was diluted 10-fold with PBS buffer and diluted to 10 using the double-layer plate method. -6 with 10 -7 The titer of phage suspension was 9.54×10 9 PFU / mL.

[0053] Example 2 Identification of bacteriophage

[0054] (I) Electron microscopy detection of bacteriophage

[0055] 1. Experimental methods:

[0056] Take 20 μl of the titer of 10 9 PFU / mL of phage was dropped onto a copper grid and allowed to settle for 15 min. Excess liquid was removed with filter paper and the grid was stained with 2% phosphotungstic acid for 30 min. After drying, the grid was observed under an electron microscope.

[0057] 2. Experimental results and analysis

[0058] The results are as follows Figure 1 As shown, the phage has an icosahedral capsid with a head length of 60 nm, a head width of 60 nm, and a tail length of about 19 nm. According to the classification standards of the 9th report of the International Committee on Taxonomy of Viruses (ICTV), it can be determined that the phage belongs to the Brachyura family and is named vB_VorP_PRC03.

[0059] (II) Analysis of phage-specific genes

[0060] The phage vB_VorP_PRC03 was sequenced and analyzed, and the analysis results of its specific genes are as follows:

[0061] The highly conserved head-to-tail connector protein gene sequence is shown in sequence 1 in the sequence listing; the highly conserved terminase large subunit protein gene sequence is shown in sequence 2 in the sequence listing; the DNA polymerase gene sequence is shown in sequence 3 in the sequence listing; and the phage exonuclease gene sequence is shown in sequence 4 in the sequence listing.

[0062] Example 3 Determination of biological characteristics of bacteriophage

[0063] (I) Phage lysis spectrum determination

[0064] 1. Experimental subjects: The lysis objects were 90 strains of Vibrio orchester, of which these 90 bacteria were mainly derived from dead fish and water samples in salmon farming areas such as Tumen River in Jilin Province, Fuyuan City in Heilongjiang Province, Huairou District in Beijing, and Yantai in Shandong Province.

[0065] 2. Experimental methods:

[0066] (1) In this experiment, the double-layer plate method was used to determine the lysis spectrum of phages. 100 μL of the phage suspension and each host bacterial suspension were added to a 0.5 mL centrifuge tube and mixed. After incubation at 37°C for 5 min, the mixture was added to 5 mL of the upper culture medium and mixed. The mixture was quickly poured into a TSA culture medium plate. After condensation, the plate was inverted and cultured in a 37°C incubator for 16 h. After the culture was completed, the plate was taken out to observe whether plaques were formed to determine whether the bacteria could be lysed.

[0067] (2) PCR virulence gene detection was performed on these 90 strains to detect whether they carried three virulence genes: heat-labile hemolysin gene (tlh), heat-resistant hemolysin gene (trh), and heat-resistant direct hemolysin (tdh).

[0068] 3. Experimental results and analysis

[0069] (1) The results are shown in Table 1. The bacteriophage vB_VorP_PRC03 can lyse 77 of the 90 strains of Vibrio ostreatus. Its lysis rate against Vibrio ostreatus reaches 85.56%, which has a wide lysis spectrum.

[0070] (2) Among these 90 strains of Vibrio ostrenosum, 22.22% of them carried the gene for heat-labile hemolysin (tlh), 13.33% of them carried the gene for heat-resistant hemolysin (trh), and 10% of them carried the gene for heat-resistant direct hemolysin (tdh).

[0071] Table 1 Lysis spectrum of bacteriophage vB_VorP_PRC03 against Vibrio ostreatus

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] (II) Determination of the optimal infection multiplicity of phage

[0078] 1. Experimental methods:

[0079] The host bacteria concentration was determined by the plate spreading method, and two parallel plates were placed upside down in a 37°C incubator for 16 h. After the culture was completed, the colonies were counted and the host bacteria concentration was calculated.

[0080] Take 100 μL of the host bacterial liquid in 5 mL of TSB liquid culture medium, add the phage suspension at a ratio of 1, 0.1, 0.01, 0.001, and 0.0001 respectively, mix well, shake and culture at 170 rpm and 37 °C until the liquid becomes clear, centrifuge at 11000 rpm for 5 min, and determine the phage titer.

[0081] 2. Experimental results and analysis

[0082] The results are shown in Table 2. When the infection multiplicity was 0.001, the phage titer was the highest, with a maximum of 3.87×10 10 PFU / mL. This shows that the phage can achieve the highest reproduction yield when the initial input is small. This biological performance of the phage shows that it has a competitive advantage in large-scale industrial production and is conducive to reducing the cost of industrial production.

[0083] Table 2 Optimal infection multiplicity of phage

[0084]

[0085] (III) Determination of thermal stability of bacteriophage

[0086] 1. Experimental methods

[0087] The titer was taken as 1×10 9 500 μL of phage suspension with a PFU / mL was placed in a 1.5 mL centrifuge tube and placed in a constant temperature water bath at 30°C, 40°C, 50°C, 60°C, and 70°C, respectively, and the titer was measured at 20 min, 40 min, and 60 min, respectively.

[0088] 2. Experimental results and analysis

[0089] The results are as follows Figure 2 As shown, the titer of bacteriophage vB_VorP_PRC03 did not change significantly after 1 hour at 30℃, 40℃, 50℃ and 60℃; it was basically inactivated after 40 minutes at 70℃, indicating that vB_VorP_PRC03 phage can tolerate a certain degree of high temperature and is relatively stable at 60℃ and below.

[0090] (IV) Determination of pH stability of bacteriophage

[0091] 1. Experimental methods

[0092] Take three sterile test tubes and add 4.5 mL of PBS with different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Place them in a 37°C water bath. After the temperature stabilizes, add 500 μL of 10 9 PFU / mL of phage proliferation solution was mixed and treated in a 37°C water bath for 1h, 2h, and 3h. After the treatment, an appropriate amount of HCl or NaOH was added to the mixture to make the pH value of the mixture about 7, and the phage titer was determined by the double-layer plate method.

[0093] 2. Experimental results and analysis

[0094] The results are as follows Figure 3 As shown, the phage vB_VorP_PRC03 can maintain its stability between pH 3 and 11 and still has lysis ability; however, the phage is less sensitive to acid and alkali and has a wider tolerance range to acid and alkali. Therefore, vB_VorP_PRC03 is relatively stable in acid and alkali environments.

[0095] (V) Determination of one-step growth curve of phage

[0096] 1. Experimental methods

[0097] According to MOI = 0.001, add 1 mL of phage vB_VorP_PRC03 proliferation liquid and its host bacteria Vibrio orthodonticus RC003 logarithmic phase bacterial liquid, mix well and start timing, incubate at 37°C for 5 minutes, centrifuge at 11000rpm for 30 seconds, use a micropipette to remove the supernatant as much as possible, and then wash once with 1 mL of TSB liquid medium (11000rpm, centrifuge for 30 seconds), and discard the supernatant. Use preheated TSB liquid medium to suspend the precipitate (total volume is 5 mL) and mix well, quickly place it in a 37°C shaker with 170rpm shaking culture, take out 150 μL at 0min and every 10min, centrifuge at 11000rpm for 1min, take 100 μl of supernatant and dilute it 10 times with physiological saline, and use the double-layer plate method to determine the phage titer, do 3 replicates, and take the average value of the results. With the infection time as the horizontal axis and the logarithm of the phage titer in the infection system as the vertical axis, a one-step growth curve is drawn to obtain the incubation period and outbreak period of the phage.

[0098] Phage lysis amount = phage titer at the end of lysis / host bacteria concentration at the beginning of infection

[0099] 2. Experimental results and analysis

[0100] like Figure 4 The one-step growth curve of the phage vB_VorP_PRC03 shows that the lysis cycle of the phage is 100 minutes: after the phage infects the host bacteria, the titer is basically stable within 30 minutes, indicating that the phage incubation period is about 30 minutes; within 30-70 minutes after the phage infects the host bacteria, the number of phages increases sharply, and it can be seen that the phage outbreak period is about 40 minutes; in the following 30 minutes, the number of phages remains basically unchanged and enters a stable growth period. The lysis amount of the phage is about 635 (PFU / CFU). This result shows that the incubation period and lysis period of phage vB_VorP_PRC03 are short, and the lysis amount is high, which is suitable for phage therapy.

[0101] (VI) Phage lysis experiment in vitro

[0102] 1. Experimental methods:

[0103] The phage suspension and host bacteria were added to 100 mL TSB liquid culture medium at a ratio of MOI = 0.001. The final concentration of the clinically isolated pathogenic Vibrio ostrenosum strain RC073 was 5.00 × 10 6 CFU / mL, the final concentration of phage is 5.00×10 3 PFU / mL, the control group was added with the same amount of sterile TSA medium as the phage solution, the bacterial solution and phage were mixed and cultured in a shaker at 37°C and 170 rpm, and the OD was measured at regular intervals. 600until the mixture becomes clear.

[0104] 2. Experimental results and analysis

[0105] The results are as follows Figure 5 As shown in the figure, the phage can effectively inhibit the host bacteria. At 2.5h, the OD 600 The OD of the experimental group decreased significantly at 3.5 h, indicating that the bacterial concentration decreased sharply. 600 The OD of the host bacteria in the control group has dropped to 0.17. 600 The value rose to above 1.0 at 3.5 hours; at 5 hours, the test group with the addition of phage suspension had become significantly clarified, while the control group was very turbid.

[0106] Example 4 Metabolic process in salmon after feeding with bacteriophage mixed feed

[0107] 1. Experimental methods:

[0108] Select healthy salmon weighing about 2 kg, a total of 20, fasted overnight. Set up a test group and a control group, 10 salmon in each group, add 5% volume to mass ratio, and mix the bacteriophage vB_VorP_PRC03 with salmon feed. The titer of the bacteriophage after mixing with feed is 5×10 8 PFU / g, the feed was dried in the shade, and then fed to the salmon in the test group at a dose of 5% of the fish body weight, and the control group was fed with the feed soaked in TSB medium at the same dose. The content of bacteriophages in the intestines and gills of the salmon in the test group and the control group was detected at 0.5h, 6h, 24h, 48h, 72h, 96h, 120h, and 144h after feeding.

[0109] 2. Experimental results and analysis:

[0110] The salmon used in the experiment were in good health and had normal activities. No bacteriophage was detected in the salmon of the control group. After feeding the fish feed soaked with bacteriophage vB_VorP_PRC03 for 0.5 h, the presence of bacteriophage was detected in the intestines and gills of the salmon in the experimental group, with a titer of 4.51×10 6 PFU / g. The experimental results show that the bacteriophage can enter the fish body through feeding and can persist in the fish body for more than 2 days. This has a guiding role in the use of bacteriophage as an aquatic feed additive and can be used to prevent and treat Vibrio disease. The experimental results also show that the bacteriophage has no effect on the health of salmon and does not affect the normal activities of fish, indicating that the use of the bacteriophage is highly safe.

[0111] Example 5 Determination of the effectiveness of bacteriophage vB_VorP_PRC03 in preventing Vibrio ostreatus infection in salmon

[0112] 1. Experimental methods:

[0113] Healthy salmon weighing about 2 kg were prepared and fasted for one day before the experiment. A total of 4 groups were set up, with 20 salmon in each group, including two experimental groups, one control group, and one blank control group. In the experimental group, the phage was evenly mixed with the fish feed at a volume-to-mass ratio of 5%. After drying in the shade, the fish were fed with the feed mixed with phage at a dose of 5% of their body weight. The content of phage in the feed was 1×10 6 PFU / g. The control group was fed with feed soaked in TSB medium at the same dose. One hour after the first feeding, salmon in the experimental group and the control group were injected with Vibrio osmodium RC003 at a dose of 2.80×10 6 CFU / fish, and the blank control group did not receive any treatment. The number of dead fish in each group within 48 hours was recorded, and the protection rate of phage was calculated.

[0114] 2. Experimental results and analysis

[0115] The results are shown in Table 3. The protection rate of the test group is higher than 85%, and the protection rate of test group 1 is as high as 90%. This shows that the bacteriophage added to fish feed as an aquatic feed additive can effectively prevent salmon from being infected with Vibrio orthodontii and cause diseases, reduce fish mortality, increase survival rate, and reduce breeding risks.

[0116] Table 3 The preventive effect of bacteriophage mixed feed on Vibrio ostreatus

[0117]

[0118] Example 6 Determination of the effect of bacteriophage vB_VorP_PRC03 in treating salmon infected with Vibrio ostreatus

[0119] 1. Experimental methods:

[0120] Healthy salmon weighing about 2 kg were fasted for one day before the experiment. Each group had 20 salmon, and two experimental groups were set up, one control group and one blank control group. All salmon in each group were injected with Vibrio ostreatus RC003 at a dose of 2.80×10 6 CFU / piece. One hour after the infection, the salmon in the experimental group were fed with feed mixed with bacteriophages. The feed was added at a volume-to-mass ratio of 5% and the content of bacteriophages was 1×10 8 PFU / g, the phage was mixed with fish feed evenly, dried in the shade and fed to fish at a dose of 5% of the fish body weight. In the control group, the feed was soaked in TSB medium and fed at the same dose. The blank control group was not treated. The number of dead fish in each group within 48 hours was recorded, and the protection rate of the phage was calculated.

[0121] 2. Experimental results and analysis

[0122] The experimental results are shown in Table 4. After the salmon were injected with pathogenic Vibrio orbenzene and the challenge treatment was completed, the mortality rate of the salmon was effectively controlled by feeding the feed infiltrated with the bacteriophage of the present invention; compared with the control group (the challenge group not fed with the feed carrying the bacteriophage), the survival rate of the fish in the test group was greatly improved, and the protection rate reached 80-85%. It can be seen that the bacteriophage vB_VorP_PRC03 has a good therapeutic effect on Vibrio orbenzene disease in salmon and can greatly improve its survival rate. The present invention provides a new source for the preparation of bacteriophage preparations and drugs against Vibrio orbenzene, thereby providing a new theoretical basis and practical experience for the treatment of diseases caused by Vibrio orbenzene infection.

[0123] Table 4 The therapeutic effect of bacteriophage mixed feed on Vibrio orthodonticus disease

[0124]

[0125] Example 7 Lysis test of bacteriophage vB_VorP_PRC03 on non-host bacteria

[0126] 1. Experimental methods

[0127] A total of 40 strains of non-host bacteria of different types, including 10 strains of Vibrio alginolyticus, 20 strains of Vibrio parahaemolyticus, and 10 strains of Vibrio harveyi, were selected, and the lysis spectrum of bacteriophage vB_VorP_PRC03 was determined according to the above-mentioned lysis spectrum determination method.

[0128] 2. Experimental results and analysis

[0129] The results showed that no clear plaques were found in the double-layer plates of the phage and 40 strains of non-host bacteria, indicating that the phage vB_VorP_PRC03 could not recognize the above-mentioned non-host bacteria. This shows that the test phage has extremely strong host specificity and has no damaging effect on the microbial community, and can be used to prepare detection kits.

[0130] Example 8 Environmental disinfection test of bacteriophage vB_VorP_PRC03

[0131] 1. Experimental methods

[0132] Take 20L salmon culture water to set up a control group and three experimental groups, each with 5L. The experimental groups were added with a final concentration of 1×10 8 PFU / mL of bacteriophage vB_VorP_PRC03, and the control group was added with an equal volume of PBS buffer. Samples were taken before the experiment and 4h, 8h, 12h, 16h, 20h, and 24h after the addition of bacteriophage, and the bacterial concentration in the water was determined using TCBS plates.

[0133] 2. Experimental results and analysis

[0134] Depend on Figure 6 It can be seen that the bacterial concentration in the control group remained basically consistent before and after the test, with no significant changes; while after the addition of the bacteriophage to the three experimental groups, the bacterial concentration in the water continued to decline, and at 8 hours after the addition, the bacterial concentration in the water decreased rapidly and did not increase within 24 hours. The addition of the bacteriophage showed the effect of purifying the water, which indicates that the bacteriophage vB_VorP_PRC03 has a significant bactericidal and disinfecting effect on the salmon farming environment, and can be used as a water environment improver and disinfectant.

[0135] From the results of the above-mentioned multiple embodiments, it can be seen that the Vibrio oxytoca phage of the present invention has a good preventive and therapeutic effect on Vibrio oxytoca disease in salmon, and the phage can be used to prepare drugs against Vibrio oxytoca, providing new theoretical basis and practical experience for treating diseases caused by Vibrio oxytoca infection. In addition, the phage can also be used to prepare environmental disinfectants and feed additives, etc., and applied to various links of aquaculture, which is beneficial to the healthy development of aquaculture.

[0136] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Vibrio ostrenosum phage, characterized in that: It was named vB_VorP_PRC03 and its deposit number is CGMCC No. 22499.

2. A bacteriophage composition, characterized in that: The invention comprises the Vibrio ostrenii phage as claimed in claim 1.

3. A phage pharmaceutical preparation, the active ingredient of which comprises the Vibrio orwinii phage according to claim 1 or the phage composition according to claim 2.

4. Use of the Vibrio ostreatus phage according to claim 1 or the phage composition according to claim 2 in the preparation of a drug for preventing or treating aquatic diseases caused by Vibrio ostreatus infection, an aquatic feed additive or an environmental disinfectant.

5. An aquatic feed additive, characterized in that: It comprises the Vibrio ostrenosum phage according to claim 1, the phage composition according to claim 2 or the phage pharmaceutical preparation according to claim 3.

6. An environmental disinfectant, characterized in that: The active ingredient comprises the Vibrio ostreatus phage according to claim 1, the phage composition according to claim 2 or the phage pharmaceutical preparation according to claim 3.

7. Use of the environmental disinfectant as claimed in claim 6 in killing Vibrio ostreatus in the environment, characterized in that: The environment includes pool walls, water bodies, breeding environments, feeding tables, breeding equipment, and a circulating water breeding system.

8. A detection kit, characterized in that: The method comprises the Vibrio ostrenosum phage according to claim 1 or the phage composition according to claim 2.

9. A biological antibacterial agent for aquatic products, characterized in that: The method comprises the Vibrio ostrenosum phage according to claim 1 or the phage composition according to claim 2.

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