Application of resveratrol in preparation of medicine or feed additive for preventing and treating fish streptococcus iniae disease

Abstract

The invention provides application of resveratrol in preparation of drugs or feed additives for preventing and treating fish streptococcus iniae diseases. The resveratrol has the remarkable activity of inhibiting the main virulence factor phosphoglucomutase (PGM) of streptococcus iniae, and the inhibition rate of the resveratrol to the activity of the PGM at the low concentration of 10 mu g / mL reaches 27.3%. A low-concentration natural compound is used as a virulence factor inhibitor to research and develop a medicine, is more economical, green and efficient, and is expected to be researched and developed into a natural green medicine for replacing antibiotics in aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of research and development of drugs for preventing fish from bacterial diseases, and particularly relates to application of resveratrol in the preparation of drugs or feed additives for preventing and treating fish streptococcal disease. Background Art

[0002] Streptococcus iniae is one of the most important pathogens in the world. It is a Gram-positive bacterium that can infect humans, livestock, and fish, and can cause inflammation and sepsis. The main symptoms of fish infected with Streptococcus iniae are protruding eyes, blackened body surface, abnormal swimming, congestion and swelling of internal organs, and effusion. Streptococcus iniae can also co-infect with other pathogens, spreading rapidly and causing large-scale deaths. Streptococcus iniae disease in fish has the characteristics of rapid onset, high incidence, long duration of the disease, wide host, easy recurrence and high mortality, which seriously endangers the development of fish farming. The pathogenicity of Streptococcus dolphinus is closely related to its virulence factors. The main virulence factors of Streptococcus dolphinus have been found to be hemolysin, phosphoglucomutase (PGM), M protein, capsular polysaccharide, CAMP factor, C5a peptidase and α-enolase. These virulence factors play an important role in the occurrence of diseases such as pathogen colonization, invasion, immune escape, stimulating inflammation and damaging the host. They usually cause disease by destroying host tissues, inhibiting the immune system or interfering with the normal function of host cells. At present, the prevention and control of Streptococcus dolphinus disease mainly relies on drugs such as antibiotics or chemical drugs that inhibit and kill pathogens. However, the repeated use and abuse of such drugs induce pathogen resistance, environmental pollution and drug residues.

[0003] Natural compounds are compounds derived from natural organisms. They have the advantages of wide sources, low prices, good biocompatibility, environmental friendliness and low probability of drug resistance. They are a good choice for the development of new green, safe and efficient drugs or feed additives for the prevention and treatment of fish dolphin streptococcal disease. Resveratrol is a natural compound isolated from plants such as Vitaceae Juss, Liliaceae, and Polygonaceae. It appears as white needle-shaped crystals, is insoluble in water, and is easily soluble in organic solvents. Its relative molecular mass is 228.25 Da, and its molecular formula is C 12 H 14 O3, the structural formula is shown in formula (1):

[0004]

[0005] Resveratrol has the following pharmacological activities and effects: (1) It regulates and inhibits the release of tumor cell death signal molecules, thereby playing an anti-cancer role; (2) It repairs cell membrane lipid peroxidation and DNA damage caused by reactive oxygen species (ROS); (3) It has an anti-inflammatory effect and has a good effect on both acute and chronic inflammation; (4) It has a good inhibitory effect on Pseudomonas aeruginosa, Shigella flexneri, and human skin pathogenic fungi and bacteria; (5) It inhibits virus-induced activation of cell signal molecules; (6) It relaxes the cardiovascular system, reduces myocardial ischemia-reperfusion injury, and resists atherosclerosis; (7) It protects nerve cells, reduces nerve damage, and improves cognitive ability; (8) It regulates immunity and regulates intestinal H + -K + -ATPase. Summary of the invention

[0006] The present invention provides the use of resveratrol in the preparation of a drug or feed additive for preventing and treating dolphin streptococcal disease in fish. In the present invention, the natural compound resveratrol has a significant activity of inhibiting the activity of PGM, the main virulence factor of dolphin streptococcus. PGM is a key enzyme for the synthesis of bacterial capsule polysaccharides and can maintain the integrity of the cell wall. The present invention also found that resveratrol can enhance the sensitivity of dolphin streptococcus to lysozyme and H2O2, making it easier for the host immune system to eliminate dolphin streptococcus. The results of the efficacy experiment show that the protection rate of resveratrol on tilapia and marine medaka suffering from dolphin streptococcal disease can reach up to 53.57% and 65.38% respectively.

[0007] The technical solution of the present invention is as follows:

[0008] The invention provides application of resveratrol in preparing a medicine for preventing and treating dolphin streptococcal disease in fish, wherein the medicine does not produce drug resistance.

[0009] In a specific embodiment of the present invention, the fish is tilapia or medaka.

[0010] In a specific embodiment of the present invention, the iniae streptococcal disease is prevented and treated by inhibiting the phosphoglucomutase activity of the iniae streptococcus.

[0011] In the present invention, resveratrol achieves the purpose of treating Streptococcus iniae disease by inhibiting the activity of phosphoglucomutase of Streptococcus iniae. Therefore, resveratrol can be used for any host infected with Streptococcus iniae, not just limited to fish.

[0012] In a specific embodiment of the present invention, the concentration of resveratrol that effectively inhibits the activity of phosphoglucomutase of Streptococcus iniae is 1-25 μg / mL.

[0013] In the present invention, when the concentration of resveratrol is 1-25 μg / mL, it can effectively inhibit the activity of phosphoglucomutase of Streptococcus iniae, while not affecting the growth of Streptococcus iniae, thus not generating drug resistance.

[0014] In a specific embodiment of the present invention, the concentration of resveratrol that effectively inhibits the activity of phosphoglucomutase of Streptococcus iniae is 10 μg / mL.

[0015] The invention also provides the use of resveratrol in preparing the inhibitor of phosphoglucomutase activity of fish Streptococcus iniae.

[0016] In a specific embodiment of the present invention, the concentration of resveratrol in the inhibitor is 1-25 μg / mL.

[0017] In a specific embodiment of the present invention, the fish is tilapia or medaka.

[0018] The invention discloses an application of resveratrol in the preparation of a feed additive for preventing and treating dolphin streptococcal disease in fish. The feed additive is added into the feed to make the concentration of resveratrol be 300-900 mg / kg.

[0019] Feed was given twice a day, morning and evening, at 2% of the fish body weight each time.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The main ingredient resveratrol used in the present invention for the prevention and treatment of dolphin streptococcal disease in fish has a significant inhibitory effect on the main virulence factor PGM of dolphin streptococcus, significantly reducing the pathogenicity of dolphin streptococcus, thereby achieving the effect of preventing and treating dolphin streptococcal disease in fish. Because it has almost no effect on the growth of pathogens, and enhances the sensitivity of pathogens to H2O2, improves the ability of host immunity to eliminate them, and effectively avoids the resistance of pathogens due to growth selection pressure, it is an effective strategy to solve the problem of pathogen resistance.

[0022] (2) The resveratrol used in the present invention has a wide range of sources, low price, good biocompatibility, is easily degraded in the environment, and has a low probability of residue, and is very suitable for development into a new drug for preventing and treating dolphin streptococcal disease in fish.

[0023] (3) The present invention is the first to use resveratrol as a drug for the prevention and treatment of dolphin streptococcal disease in fish, and can improve the survival rate of diseased tilapia and marine medaka within a safe and effective dose. The RPS of tilapia infected with dolphin streptococcus was 14.29%, 32.14% and 53.57% respectively when mixed with feed containing 300, 600 and 900 mg / kg of resveratrol; the RPS of marine medaka infected with dolphin streptococcus was 26.92%, 50% and 65.38% respectively. The half effective dose (EC 50 ) were 881.35 mg / kg and 586.54 mg / kg, respectively, indicating that resveratrol has a good therapeutic effect on dolphin streptococcal disease in fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the standard curve for the determination of phosphoglucomutase (PGM) activity;

[0025] Figure 2 It is a bar graph showing the inhibition rate of natural compounds on PGM of Streptococcus iniae;

[0026] Figure 3 The effect of different concentrations of resveratrol on the sensitivity of Streptococcus india to lysozyme. Note: EG and CG in the figure represent the resveratrol-treated group and the non-treated group; μor t d Expressed as mean ± SD (n = 5);

[0027] Figure 4 The sensitivity of Streptococcus iniae to different concentrations of hydrogen peroxide before and after resveratrol treatment. Note: EG and CG in the figure represent the resveratrol-treated group and the non-treated group, and * indicates a significant difference (p<0.05); μor t d Expressed as mean ± SD (n = 5);

[0028] Figure 5 is the cumulative mortality curve of tilapia infected with different concentrations of S. iniae;

[0029] Figure 6 The effect of different concentrations of resveratrol in feed on the survival curve of tilapia infected with Streptococcus iniae. Note: "*" indicates that there are significant differences between the survival curves of fish in different experimental groups and the control group, *p < 0.05; "**" indicates that there are extremely significant differences, **p < 0.01;

[0030] Figure 7 The cumulative mortality curve of medaka fish infected with different doses of S. iniae;

[0031] Figure 8The effect of different concentrations of resveratrol in feed on the survival curve of medaka infected with Streptococcus iniae. Note: "*" indicates significant differences between the survival curves of fish in different experimental groups and control group, *p < 0.05; "**" indicates extremely significant differences, **p < 0.01. DETAILED DESCRIPTION

[0032] The present invention is further described below with specific examples, but the embodiments of the invention are not limited thereto. The pathogen used in the examples is Streptococcus iniae, which was isolated and purified by the patent applicant from diseased oval pomfret in Qiaotou Town, Lingao County, Hainan Province, and was identified as Streptococcus iniae by morphology and 18S rDNA molecular biology methods.

[0033] Example 1: Effect of resveratrol on the activity of PGM of Streptococcus iniae

[0034] 1. Determination of PGM activity of Streptococcus iniae

[0035] The PGM activity of Streptococcus dolphinus was determined according to the instruction manual of the microbial PGM enzyme-linked immunosorbent assay kit (Jiangsu Enzyme-Free Biotechnology Co., Ltd.), that is, the microplate was coated with purified PGM antibodies to prepare solid-phase antibodies, and PGM standards were added to the coated microwells in sequence, and then horseradish peroxidase (HRP)-labeled PGM antibodies were added to bind to form antibody-antigen-enzyme-labeled antibody complexes. After thorough washing, the substrate tetramethylbenzidine (TMB) was added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The absorbance value (A) was measured at a wavelength of 450nm using an enzyme reader. 450nm ), A 450nm The size is positively correlated with the activity of the PGM standard. The activity of the PGM standard is taken as the horizontal axis. 450nm The standard curve is drawn with the value as the vertical axis ( Figure 1 ), and fitting the standard curve to obtain the standard equation (a):

[0036] Y=0.0222x-0.0582R 2 =0.9836 (a)

[0037] In formula (a), Y is A 450nm value, x is the activity of PGM standard, R 2 is the correlation coefficient.

[0038] 2. Determination of PGM Activity

[0039] (1) Preparation of Streptococcus dolphinus suspension: Take out Streptococcus dolphinus from -80℃ and thaw at room temperature, streak on brain heart infusion (BHI) solid culture medium, and culture at 30℃ for 24h. Pick a single colony and inoculate it into BHI liquid culture medium, and culture it in a shaker at 30℃ and 180r / min for 24h. Centrifuge at 5000r / min for 5min, collect the cells, wash them three times with phosphate buffered saline (PBS, pH7.4, 0.01mol / L), and adjust the concentration of the bacterial suspension with PBS to an OD of 620nm =1.0(about 2.5×10 9 CFU / mL) and kept at 4℃ for later use.

[0040] (2) Adding resveratrol to bacterial suspension: Dissolve 0.01 g of resveratrol in 10 mL of pure dimethyl sulfoxide (DMSO), sterilize with a 0.22 μm microporous filter to obtain a resveratrol solution with a concentration of 1000 μg / mL, and take 1 mL and add it to 100 mL of BHI liquid medium to make the final concentration of resveratrol in the BHI liquid medium reach 10 μg / mL. Inoculate the test bacterial suspension into the medium containing resveratrol at a 5% inoculum. Add 1 mL of DMSO to the BHI liquid medium and inoculate 5% of the bacterial suspension as a control. Each group has 3 parallels, culture at 30°C for 12 h, centrifuge at 5000 r / min for 5 min, collect the bacteria, wash twice with PBS buffer, and resuspend the bacteria to adjust the OD 620nm The concentration of 1.0 was set at 4°C for later use.

[0041] (3) PGM activity determination: 1 mL of bacterial suspension from the resveratrol-treated experimental group and the untreated control group was taken into 1.5 mL centrifuge tubes, and 5 μL of 20 mg / mL lysozyme was added to each tube. The tubes were incubated at room temperature for 15 min and centrifuged at 3000 r / min for 5 min. The supernatant of the bacterial suspension was taken and the PGM activity of Streptococcus iniae was determined according to the method for determining the PGM activity of Streptococcus iniae in Example 1, and the determination was repeated 3 times.

[0042] The measured data were used to calculate the activity of the PGM of the S. iniae strain using the standard curve formula to obtain the PGM activity of the S. iniae strain cultured with resveratrol, and the inhibition rate (IRP) of resveratrol on the PGM activity of the S. iniae strain was calculated using equation (b).

[0043] IRP=(1-PGM / PGM0)×100% (b)

[0044] PGM is the PGM activity of the experimental group, and PGM0 is the PGM activity of the control group.

[0045] The final calculated IRP of 10 μg / mL resveratrol on the PGM activity of S. iniae was 27.30%.

[0046] Under the same conditions, the IPR of rhein, forsythin, apigenin, allicin, osthole, sorbitol, baicalin, soy isoflavones, trans-anethole, kaempferol, quercetin, salicylic acid, and gallic acid on the PGM activity of S. iniae was also tested. Figure 2 shown.

[0047] Compound English name Compound English name Resveratrol Resveratrol Baicalin Baicalin Rhein Rhein Soy Isoflavones Soyisoflavone Forsythiaside Forsythin trans-anethole trans-Anethole Apigenin Apigenin Kaempferol Kaempferol Allicin Garlic Quercetin Quercetin Osthole Osthole Salicylic acid Salicylic acid Sodium sorbate Sodium Sorbate gallic acid Gallicacid

[0048] 3. Determination of the minimum inhibitory concentration (MIC) of resveratrol using the modified broth microdilution method

[0049] Take a 96-well plate, fill the wells around it with sterile deionized water, dilute the BHI liquid medium containing a final concentration of 100 μg / mL resveratrol into dilutions of different concentrations by two-fold dilution method (so that the final concentration of resveratrol reaches 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μg / mL), replace the control group with DMSO, add 200 μL to the experimental wells and control wells respectively, and then inoculate 20 μL OD 620nm = 0.1 of the Streptococcus india suspension, incubate at 30°C for 12 h, take out the 96-well plate and place it in an ELISA reader to detect the OD of each well 620nm value.

[0050] (1) Preparation of bacterial suspension: The preparation method of Streptococcus iniae suspension is the same as that of Example 12. PGM activity determination method (1). 620nm = 1.0 bacterial suspension was diluted with PBS solution to OD 620nm =0.1;

[0051] (2) Add resveratrol solution to BHI liquid culture medium: Dissolve 0.01g resveratrol powder in 1mL pure DMSO solvent, and filter through a 0.22μm microporous filter to prepare a resveratrol solution with a concentration of 10mg / mL. Take 8 test tubes, add 9.9mL BHI liquid culture medium to the first test tube, add 100μL of 10mg / mL resveratrol solution, so that the final concentration of resveratrol in the bacterial suspension is 100μg / mL, and dilute it with BHI liquid culture medium to dilute it into resveratrol solutions with concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78μg / mL as the experimental group. In addition, take 8 test tubes for the control group, add 100μLDMSO and 9.9mL BHI liquid culture medium to the first test tube for a two-fold gradient dilution, so that the DMSO concentration in the BHI liquid culture medium is consistent with that of the experimental group;

[0052] (3) Transfer the sample solutions of the experimental group and the control group from low to high into a 96-well plate and add them to each well. Each concentration is parallel to three wells, 180 μL per well, and then add 20 μL of the prepared OD 620nm =0.1 of Streptococcus iniae suspension.

[0053] (4) Place them in a constant temperature incubator at 30°C. After culturing for 12 hours, observe them under a black background without indirect reflected light and arrange them from high to low according to drug concentration. The lowest concentration of sterile growth visible to the naked eye is the MIC of resveratrol against S. iniae.

[0054] The final MIC of resveratrol against Streptococcus iniae was 250 μg / mL.

[0055] 4. Effect of resveratrol on the growth curve of Streptococcus iniae

[0056] The method for preparing the suspension of Streptococcus iniae and the method for treating the suspension with resveratrol are the same as (1) and (2) in the method for determining the activity of PGM in Example 12. The method for determining the growth curve of the test bacteria by resveratrol is to add 270 μL of fresh BHI culture medium to the microplate, and divide it into two groups, an experimental group and a control group, respectively. 30 μL of bacterial suspension treated with resveratrol at a final concentration of 10 μg / mL is added to the wells of the experimental group, and 30 μL of Streptococcus iniae suspension treated with 0.1% DMSO (the same DMSO concentration as in the experimental group) is added to the control group, and 5 replicates are set. The microplate with the sample added is placed in an automatic growth curve instrument, and the absorbance value is detected every 1 hour at 30° C. and a wavelength of 620 nm, and the monitoring is continuous for 24 hours.

[0057] The growth curve of Streptococcus iniae was fitted using Origin 2022b software.

[0058] y=B2+(B1-B2) / (1+(x / x0)p) (c)

[0059] y is OD 620nm , x is the culture time, B1, B2, x0 and p are constants.

[0060] Calculate the specific growth rate (μ) and bacterial doubling time (t d )

[0061] μ=(dy / dx) / y (d)

[0062] t d =0.693 / μ (e)

[0063] The test results of the effect of 10 μg / mL resveratrol on the growth of S. iniae strains are shown in Table 1. μ and t of S. iniae cultured in medium containing and not containing 10 μg / mL resveratrol d There were no significant differences.

[0064] Table 1 Effect of 10 μg / mL resveratrol on the growth of Streptococcus iniae

[0065]

[0066] Note: μor t d Mean ± SD (n = 5)

[0067] 5. Effect of resveratrol on the sensitivity of Streptococcus iniae to lysozyme and H2O2

[0068] The same method as in Example 12. PGM activity determination method (1) was used to prepare bacterial suspensions. 240 μL of lysozyme (A: 0.25 mg / mL; B: 0.5 mg / mL; C: 1.0 mg / mL; D: 2.0 mg / mL; E: 0 μg / mL) or H2O2 (A: 0 mol / mL; B: 5×10 -5 mol / mL; C: 1×10 -4 mol / mL; D: 2×10 -4 mol / mL; E: 4×10 -4 mol / mL) BHI liquid medium, add 60 μL of the prepared bacterial suspension at a ratio of 20%, put the microplate into the automatic growth curve instrument, adjust the temperature to 30°C, and then shake culture it for 24 hours. 620nm The absorbance was measured every 1 h at the wavelength, and 5 parallel wells were set for different concentrations. The growth curve was fitted according to the fitting equation (c), and the specific growth rate (μ) and bacterial doubling time (t d ).

[0069] Effects of resveratrol-treated Streptococcus iniae on lysozyme and H2O2 sensitivity Figure 3 and Figure 4 .from Figure 3 It can be seen that there is no significant difference between the growth rate of S. iniae treated with 10 μg / mL resveratrol and the growth rate of untreated S. iniae in the medium containing different concentrations of lysozyme. Figure 4 It can be seen that when the H2O2 concentration in the culture medium is lower than 1×10 -4 mol / mL, μ and t of resveratrol-treated and untreated Streptococcus iniae dThere was no significant difference; when the H2O2 concentration in the culture medium was higher than 1×10 -4 mol / mL, the growth of Streptococcus iniae was inhibited, indicating that the resveratrol treatment group increased the sensitivity of Streptococcus iniae to H2O2.

[0070] Example 2: Acute toxicity test of resveratrol on tilapia

[0071] 1. Preparation of resveratrol solution

[0072] 1.00, 0.50 and 0.25 g of resveratrol were weighed into 25 mL centrifuge tubes, 1 mL of anhydrous ethanol was added for dissolution, and 9 mL of PBS buffer was used to dilute the resveratrol to a final concentration of 100, 50 and 25 mg / mL for later use.

[0073] 2. Tilapia

[0074] The experimental tilapia was purchased from Guangzhou Yufeng Aquatic Seedling Base and was temporarily raised in a glass tank in the aquaculture system. The aquaculture system was equipped with an aeration device and a water pump (the water temperature was controlled at 30±2℃, pH 7±0.5), the DO was higher than 6.5mg / L, and the tilapia basic feed was fed twice a day at a fixed point.

[0075] 3. Tilapia intragastrically administered with resveratrol solution

[0076] 128 healthy tilapia with a body length of 8±2cm were selected and randomly assigned to 8 aquariums (0.8m×0.50m×0.55m), with 16 fish in each aquarium. The 8 aquariums were randomly divided into 4 groups, namely 3 experimental groups and 1 control group, with 2 aquariums in parallel in each group. The water temperature of the aquarium was maintained at 30±2℃, and the dissolved oxygen (DO) in the water was greater than 6.5mg / L. They were temporarily kept for 7 days, and half of the water was changed every 2 days. Fasting for 12h before gavage, each fish was gavaged twice according to the group, with a gavage interval of 4h, and each gavage volume was 0.2mL / 20g, so that the dose of resveratrol in each fish reached 2.00g / kg, 1.00g / kg, and 0.50g / kg. The control group was gavaged with PBS solution containing 10% anhydrous ethanol and continued to be kept under the original feeding conditions for 14d, and the behavior of the experimental fish was observed and their health status was recorded. The results showed that the fish in the experimental group and the control group did not show any symptoms, their behavior remained good, and their feeding was normal. The viscera of tilapia were dissected and observed, and there was no pathological phenomenon in the organs, and no water accumulation, sticky substances, or swelling was found in the abdomen. The brain, liver, spleen, and kidney tissues were homogenized and spread on BHI solid culture medium plates, and placed in a 30℃ constant temperature incubator for 24 hours, and no bacterial growth was found. This shows that oral resveratrol is safe for tilapia.

[0077] Example 3: Efficacy of resveratrol on tilapia streptococcal infection

[0078] 1. Experimental fish

[0079] The fish used in the experiment were from the same batch as the tilapia in Example 2.

[0080] 2. Preparation of Streptococcus iniae suspension

[0081] Referring to (1) in Example 12, the method for determining the activity of PGM, a suspension of Streptococcus iniae was prepared and diluted with PBS to different concentrations of the suspension (see Table 2).

[0082] 3. Establishment of Tilapia Disease Model Infected with Streptococcus iniae

[0083] 240 healthy tilapia with a body length of 8±2cm were selected and randomly assigned to 12 aquariums (0.8m×0.50m×0.55m), with 20 fish in each aquarium. The 12 aquariums were divided into 6 groups, namely A, B, C, D, E and F (see Table 2), with 2 aquariums in each group. The water temperature of the aquarium was maintained at 30±2℃, DO was greater than 6.5mg / L, and half of the water was changed every 2 days. Tilapias were fasted for more than 12h before infection. Groups A to E were intraperitoneally injected with india Streptococcus indica, and group F was intraperitoneally injected with PBS buffer. The injection dose is shown in Table 2. After that, they were raised under the original feeding conditions for 14d. The behavior of the experimental fish was observed, and their health status and the dead fish in the experiment were recorded.

[0084] Table 2 Experimental scheme for establishing a tilapia model infected with Streptococcus iniae

[0085]

[0086] Note: OD of S. iniae 620nm =1, that is, 2.5×10 9 CFU / mL; injection volume 0.1mL / 20g.

[0087] The results showed that after intraperitoneal injection of different concentrations of Streptococcus dolphins, the movement of tilapia showed obvious abnormalities, such as slow swimming, solitary swimming, stagnation, loss of balance, wild swimming on the water surface, cloudy eyes, dark body color, bleeding and ulcers on the body surface. The pathogen was isolated and identified from the dying tilapia, and the dominant bacteria was identified as Streptococcus dolphins.

[0088] Cumulative mortality curve of tilapia in each group after infection Figure 5 The relationship between mortality and bacterial concentration was fitted according to the death situation: y = 0.205 × Lgx - 1.1266 (R 2 =0.9617). The LD of tilapia is obtained from the equation 80 1.25×10 10CFU / kg. Pathogen identification results The strains isolated and purified from tilapia were all Streptococcus indiae after 16S-rDNA detection. From the cumulative death curves of tilapia infected with different concentrations of Streptococcus indiae, it can be concluded that the higher the injection dose, the more tilapia deaths.

[0089] 4. Resveratrol mixed feed fed to tilapia artificially infected with Streptococcus iniae

[0090] 160 healthy tilapia with a body length of 8±2cm and a body weight of 10±2g were selected and randomly distributed into 10 aquariums (0.80m×0.50m×0.55m), with 16 fish in each aquarium. The 10 aquariums were randomly divided into 5 groups, namely A, B, C, D and E (see Table 3), with 2 aquariums in each group. The aquarium temperature was maintained at 30±2℃ and the DO was greater than 6.5mg / L. The fish were fed with a 7-day basic feed twice a day, at 9:00 am and 15:00 pm, respectively, according to 2% of the fish body weight. Half of the water was changed every 2 days. According to the scheme in Table 3, 1.25×10 10 CFU / kg Streptococcus iniae liquid (LD 80 ), fasting for 12 h before injection, feeding 2 h after injection, and continuing to feed for 14 days under the original feeding conditions, observing the behavior of the experimental fish, recording their health status and the number of dead fish, and calculating the RPS% of each group according to formula (c): RPS% = (1-(mortality rate of experimental group / mortality rate of control group) × 100%. (c)

[0091] The results are as follows Figure 6 As shown in the figure, the RPS of tilapia fed with 300, 600 and 900 mg / kg (the concentration of resveratrol in each kilogram of feed) resveratrol was 14.29%, 32.14% and 53.57% respectively. The equation of the curve fitting between the logarithm of the resveratrol dose and the RPS of tilapia is y=0.7985×Lg(x)-1.8517, where x is the logarithm of the resveratrol dose, y is the RPS of the infected tilapia, R 2 =0.9595. The EC is obtained by the fitting equation 50 It is 881.35mg / kg.

[0092] Table 3 Study on the therapeutic effect of resveratrol on tilapia streptococcal disease

[0093]

[0094] Note: OD of S. iniae 620nm =1, that is, 2.5×10 9 CFU / mL; injection volume 0.1mL / 20g.

[0095] Example 4: Efficacy of resveratrol on streptococcal infection in medaka

[0096] 1. Saltwater Killifish

[0097] The experimental fish were marine medaka, purchased from the Yellow Sea Fisheries Research Institute. After purchase, they were temporarily kept in a glass tank (0.80m×0.50m×0.55m). The culture system was equipped with an aeration device for continuous aeration (water temperature was controlled at 25±2℃, pH 7±0.5), DO was higher than 6.5mg / L, and basic feed was fed at fixed points at 9:00 am and 16:00 pm every day.

[0098] 2. Preparation of Bacterial Suspension

[0099] The preparation method is the same as that in Example 12. PGM activity determination method (1) Prepare a suspension of Streptococcus iniae, and then dilute the suspension with PBS buffer to a concentration of 2.5×10 9 , 2.5×10 8 , 2.5×10 7 , 2.5×10 6 and 2.5×10 9 The concentration of CFU / mL was measured and used for later use.

[0100] 3. Establishment of the streptococcal disease model in medaka fish

[0101] 120 healthy marine medaka fish were selected and randomly divided into 12 aquariums (0.80m×0.50m×0.55m), with 10 fish in each aquarium. The 12 aquariums were randomly divided into 6 groups, namely A, B, C, D, E and F (see Table 4), with 2 aquariums in parallel in each group. The aquarium temperature was maintained at 25±2℃ and the DO was greater than 6.5mg / L. The basal feed was fed for 7 days, and half of the water was changed every 2 days. Before the experiment, the medaka fish in the five experimental groups were fasted for 12h, and 10mL of bacterial suspension of different concentrations was added to it. They were immersed in the water for infection for 30min. The control group F was added with 10mL of sterile PBS. The rest of the operations were the same as those of the infection groups A, B, C, D and E. They were raised under the original feeding conditions for 14d, and the behavior of the experimental fish was observed, and their health status and the number of dead fish were recorded.

[0102] Table 4 Grouping and dosage of medaka infected with Streptococcus iniae

[0103]

[0104] Note: OD of S. iniae 620nm =1, that is, 2.5×10 9 CFU / mL

[0105] The infected medaka swam slowly, lost balance, had partial abdominal swelling, and had skin bulges and ulcers. The cumulative mortality curves of the infected medaka in each group are as follows: Figure 7 The curve of challenge dose VS cumulative mortality was drawn, and the fitting equation was y = 0.2000 × Lg (x) - 0.6596 (R 2 =0.9804), and the LD of Streptococcus dolphinus against medaka was calculated. 80 2.0×10 7 CFU / mL.

[0106] 4. The therapeutic effect of resveratrol on dolphin streptococcal disease in medaka

[0107] 160 healthy marine medaka fish were selected and randomly distributed to 10 aquariums (0.80m×0.50m×0.55m), with 16 fish in each aquarium. They were randomly divided into 5 groups, namely A, B, C, D and E. The aquarium temperature was maintained at 25±2℃ and the DO was greater than 6.5mg / L. The fish were fed with a 7-day basic feed twice a day, at 9:00 am and 15:00 pm, respectively, according to 2% of the fish body weight. Half of the water was changed every 2 days. The water was changed every 2 days, and 10mL of iniae Streptococcus LD was added to the aquariums of groups A, B, C and D. 80 The fish were immersed in the water for 30 minutes at the same dose. Group F was the blank control and 10 mL of sterile PBS was used instead. The fish were fasted for 12 hours before immersion and infection. 2 hours after immersion and infection, the fish were fed with resveratrol-containing feed (concentrations shown in Table 5). Other conditions remained unchanged. The symptoms were observed for 14 days, the behavior of the experimental fish was observed, and their health status and the number of dead fish were recorded.

[0108] Table 5 Grouping and resveratrol dosage of medaka fish infected with Streptococcus iniae

[0109]

[0110] Note: OD of S. iniae 620nm =1, that is, 2.5×10 9 CFU / m

[0111] The RPS% of each group was calculated according to formula (c). The results are as follows: Figure 8 As shown in the figure, feeding 300, 600 and 900 mg / kg (reveratrol concentration per kilogram of feed) resveratrol feed to marine medaka infected with Streptococcus dolphinus caused RPS of 26.92%, 50% and 65.38% respectively. The equation of the curve fitting between the logarithm of resveratrol dose and RPS of medaka is y=0.8019×Lg(x)-1.7199, where x is the logarithm of resveratrol dose, y is the RPS of infected medaka, R 2=0.9988. The EC is obtained by this fitting equation 50 It is 586.54mg / kg.

[0112] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. The use of resveratrol in the preparation of a drug for preventing and treating dolphin streptococcal disease in fish, characterized in that: The drug does not develop resistance.

2. The use of resveratrol according to claim 1 in the preparation of a drug for preventing and treating dolphin streptococcal disease in fish, characterized in that: The fish is tilapia or marine medaka.

3. The use of resveratrol according to claim 1 in the preparation of a drug for preventing and treating dolphin streptococcal disease in fish, characterized in that: Prevention and treatment of Streptococcus iniae disease by inhibiting the phosphoglucomutase activity of Streptococcus iniae.

4. The use of resveratrol according to claim 3 in the preparation of a drug for preventing and treating dolphin streptococcal disease in fish, characterized in that: The concentration of resveratrol that effectively inhibits the phosphoglucomutase activity of Streptococcus iniae is 1-25 μg / mL.

5. The use of resveratrol according to claim 4 in the preparation of a drug for preventing and treating dolphin streptococcal disease in fish, characterized in that: The concentration of resveratrol that effectively inhibits the phosphoglucomutase activity of Streptococcus iniae is 10 μg / mL.

6. Application of resveratrol in the preparation of inhibitors of phosphoglucomutase activity of Streptococcus dolphinus in fish.

7. The use of resveratrol according to claim 6 in the preparation of an inhibitor of phosphoglucomutase activity of Streptococcus iniae, characterized in that: In the inhibitor, the concentration of resveratrol is 1-25 μg / mL.

8. The use of resveratrol according to claim 6 in the preparation of an inhibitor of phosphoglucomutase activity of Streptococcus iniae, characterized in that: The fish is tilapia or marine medaka.

9. The use of resveratrol in the preparation of a feed additive for preventing and treating dolphin streptococcal disease in fish, characterized in that: Feed additives are added to the feed so that the concentration of resveratrol is 300-900 mg / kg.

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