Application of piperine in preparation of drugs and feed additives for preventing and treating vibriosis of aquatic animals

Abstract

The invention provides application of piperine in preparation of drugs and feed additives for preventing and treating vibriosis of aquatic animals, and belongs to the technical field of prevention and control of bacterial diseases of the aquatic animals. According to the invention, piperine is applied to prevention and treatment of vibriosis of aquatic animals for the first time, especially vibriosis harveyi of pearl gentian groupers, the survival rate of pearl gentian groupers infected with vibriosis harveyi can be increased within an effective dose, and the relative protection rate reaches 49.99%.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial disease prevention and control of aquatic animals, and particularly relates to application of piperine in preparing drugs and feed additives for preventing and treating vibriosis in aquatic animals. Background Art

[0002] Vibrio disease, a disease caused by bacteria of the genus Vibrio, is currently widespread in marine aquaculture farms around the world. Vibrio disease is mostly characterized by rapid onset, strong infectivity, high morbidity and mortality, and has caused huge economic losses to the marine aquaculture industry. Among them, Vibrio harveyi is the main pathogen of vibrio disease. Vibrio harveyi can infect farmed animals such as Penaeus vannamei, turbot, and grouper. The main symptoms of infected fish include blackening of body color, protruding eyes, ulcers on the surface of the body, congestion of liver and spleen tissues, and enteritis. At present, the prevention and treatment of vibrio disease in marine aquaculture animals is still mainly based on immunity and antibiotics. However, serological diversity and genetic variation limit the development of vibrio vaccines and reduce their application effects. The long-term use and abuse of antibiotics and chemical drugs have made vibrio resistant to these drugs, the resistance spectrum has been continuously expanded, and the effect of medication has been continuously reduced, resulting in farmers having to increase the dosage of drugs, resulting in negative effects such as drug residues, environmental pollution, imbalance of the microecology of the aquaculture environment, and the spread of resistance factors to human and animal pathogens, which seriously threatens public health safety and public health. Natural compounds are secondary metabolites or endogenous active substances in organisms, and have the advantages of being green and safe, low residue, low toxicity, multifunctional and low drug resistance.

[0003] The pathogenicity of pathogenic microorganisms mainly depends on their virulence factors, including adhesion, colonization, infection, transmission, diffusion, immune escape and drug resistance. Biofilm is one of the main virulence factors of Vibrio harveyi. Vibrio harveyi survives in water bodies mainly by aggregating and sticking together to secrete sticky substances to form biofilms. The main components of its biofilm are polysaccharides, nucleic acids, lipids and proteins. The functions of Vibrio harveyi biofilm mainly include: enhancing the resistance of Vibrio harveyi to antibiotics, external environmental pressure and host immunity; reducing the nutritional consumption of Vibrio harveyi and prolonging its survival time; enhancing the response ability of Vibrio harveyi to external environmental conditions such as pH, osmotic pressure, nutrition and gas exchange; and improving the drug resistance and anti-body immunity of Vibrio harveyi. Studies have shown that the drug resistance rate of bacteria on biofilms is as high as 1,000 times that of planktonic bacteria. Therefore, inhibiting the formation of Vibrio harveyi biofilms or degrading the formed Vibrio harveyi biofilms is an important strategy to reduce its pathogenicity and drug resistance. Summary of the invention

[0004] The technical problem solved by the invention is to provide a natural compound for preventing and treating Vibrio harveyi disease in grouper, namely, piperine.

[0005] Piperine, an alkaloid, is an off-white to yellow-brown crystalline powder with the molecular formula: C 17 H 19 NO3, which exists in the fruits of many Piper plants, is one of the spicy ingredients, and its structural formula is shown in Formula I:

[0006]

[0007] Piperine has been found to have antioxidant, immunomodulatory, anti-tumor and other effects. The present invention found that piperine can significantly inhibit the formation of biofilm of pathogenic factors of Vibrio harveyi, and has great application potential in the prevention and treatment of fish vibriosis.

[0008] The technical solution of the present invention is as follows:

[0009] The invention provides application of piperine in preparing a medicine for preventing and treating vibriosis in aquatic animals.

[0010] The piperine was piperine having a purity of HPLC 98%.

[0011] In a specific embodiment of the present invention, the pathogenic bacteria of Vibrio disease in aquatic animals is Vibrio harveyi; and the aquatic animal is pearl grouper.

[0012] In a specific embodiment of the present invention, piperine prevents and treats Vibrio disease by inhibiting Vibrio biofilm.

[0013] The present invention found that piperine can effectively inhibit the formation of biofilm, a key virulence factor of Vibrio harveyi.

[0014] In a specific embodiment of the present invention, the effective concentration of piperine for inhibiting Vibrio biofilm is 20-200 μg / mL.

[0015] EC of piperine against Vibrio harveyi biofilm 50 It is 29.99±6.46μg / mL.

[0016] In a specific embodiment of the present invention, the dosage form of the drug is a mixed liquid, microemulsion, nanoemulsion, medicinal bath liquid, capsule, oral capsule coating, powder, granule, pellet, micropill or different dosage forms of drugs prepared with pharmaceutically acceptable carriers and excipients.

[0017] The dosage form is preferably a mixture of 2 g piperine dissolved in anhydrous ethanol solution to prepare a mixing liquid (2 g / 50 mL), and then sprayed onto the feed at a ratio of 50:1 (mL:kg) to prepare the medicated feed. It can also be a different dosage form of medicine prepared together with a pharmaceutically acceptable carrier and excipient.

[0018] The invention also provides the use of piperine in preparing a feed additive for preventing and treating vibriosis in aquatic animals.

[0019] In a specific embodiment of the present invention, the pathogenic bacteria of Vibrio disease in aquatic animals is Vibrio harveyi; and the aquatic animal is pearl grouper.

[0020] The invention also provides feed for preventing and treating vibriosis of aquatic animals, comprising the feed and piperine.

[0021] In a specific embodiment of the present invention, the mass ratio of piperine to feed is: 2-4g:1kg.

[0022] In a specific embodiment of the present invention, the pathogenic bacteria of Vibrio disease in aquatic animals is Vibrio harveyi; and the aquatic animal is pearl grouper.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention is the first to apply piperine to the prevention and treatment of Vibrio disease in marine aquaculture animals, especially Vibrio harveyi disease in grouper. Within an effective dose, the survival rate of grouper infected with Vibrio harveyi can be increased, and the relative protection rate reaches 49.99%.

[0025] 2. The piperine in the present invention has the advantages of wide source, low residue, no pollution, green and safe, industrial production, controllable quality, stable efficacy, etc.

[0026] 3. The natural compound piperine disclosed in the present invention for preventing and treating Vibrio disease in marine aquaculture animals, in vitro experiments show that the minimum inhibitory concentration (MIC) of piperine against Vibrio harveyi is above 1024 μg / mL. The median effective concentration (EC) of piperine against Vibrio harveyi biofilm is 1024 μg / mL. 50 ) is 29.99±6.46μg / mL. This means that at the appropriate dose, piperine can effectively inhibit the synthesis of biofilm without killing Vibrio harveyi, thereby greatly reducing its pathogenicity and preventing bacteria from developing drug-resistant mutations due to survival pressure. Piperine is expected to be a leading compound for the development of new pathogen virulence inhibitors, effectively solving the current problem of drug resistance of Vibrio and even bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the growth curve and biofilm formation curve of Vibrio harveyi in Example 1.

[0028] Figure 2 Symptoms of pearl grouper infected with Vibrio harveyi in Example 3. In the figure, A: enlarged head and kidney; B: red and swollen liver; C: enlarged spleen; D: urethral effusion.

[0029] Figure 3 This is a graph showing the cumulative mortality of pearl grouper infected with different concentrations of Vibrio harveyi in Example 3.

[0030] Figure 4 This is a diagram showing the effect of feeding piperine mixed with feed in Example 3 to prevent Vibrio harveyi infection in pearl grouper. DETAILED DESCRIPTION

[0031] The present invention is further described by the following specific examples, but the embodiments of the present invention are not limited thereto. The pearl grouper used in the examples was purchased from a farm in Wenchang City, Hainan Province. Vibrio harveyi is a pathogen isolated and preserved from diseased grouper in Hainan, and was identified as Vibrio harveyi by morphology and 16S rDNA molecular biology methods.

[0032] Example 1 Effect of piperine on biofilm formation of Vibrio harveyi

[0033] 1. Preparation of Vibrio harveyi Suspension

[0034] After the activation of the Vibrio harveyi strain stored at -80℃, inoculate it with a new broth (Luria-Bertani, LB) liquid culture medium at a 1% inoculum volume and culture it overnight at 30℃ and 180r / min. Centrifuge at 5000r / min for 5min to collect the bacteria, wash them with PBS (0.01M, pH 7.4), centrifuge at 5000r / min for 5min, collect the bacteria, wash them, and centrifuge and wash them 3 times. Finally, resuspend the bacterial suspension with PBS to adjust the concentration to OD 600nm = 1, the bacterial concentration is about 1×10 9 CFU / mL, as the test bacterial suspension, stored at 4℃ for later use.

[0035] 2. Preparation of Piperine Solution

[0036] 10 mg of piperine was weighed and dissolved in 1 mL of anhydrous ethanol to prepare a 10 mg / mL piperine ethanol solution, and 5 μL was added to 5 mL of autoclaved LB liquid culture medium to prepare an LB liquid culture medium with a final piperine concentration of 10 μg / mL and an ethanol content of 0.1%.

[0037] 3. Plotting of the growth curve and biofilm formation curve of Vibrio harveyi

[0038] After adding 195 μL of LB liquid culture medium to a 96-well cell culture plate, 5 μL of the bacterial suspension prepared in Example 11 (1×10 9CFU / mL) and mix well. Fill a circle around the 96-well plate with sterile water and culture for 4, 8, 16, 24, 36, 48, 60 and 72h respectively. After the culture is completed, use a pipette to remove the LB culture medium in the well, use sterile PBS (0.01M, pH 7.4) to gently wash each well three times to wash away the floating bacteria, and put the 96-well plate in a 55°C oven for 30min to dry. Add 200μL of 0.1% crystal violet stain to each well, and evenly stain the biofilm at room temperature at 100r / min decolorization shaker for 10min. Use a pipette to suck out the dye in each well, wash off the residual dye with double distilled water, wash three times, and put the 96-well plate in a 55°C oven to dry for 30min. After complete drying, add 200 μL of 33% glacial acetic acid to each well. After decolorization for 15 min on a decolorization shaker at 100 r / min, use an ELISA reader to measure the absorbance value of each well at 570 nm. Six parallels are used for each time point, and LB liquid culture medium is used as a negative control.

[0039] The results are as follows Figure 1 As shown, Vibrio harveyi began to form biofilm after 4 hours of culture, and the amount of biofilm formation reached a peak at 48 hours, after which the amount of biofilm began to decline. 24h to 48h is the logarithmic formation period of Vibrio harveyi biofilm. Therefore, 36h of culture was selected to investigate the effect of drugs on biofilm formation of Vibrio harveyi.

[0040] 4. Effect of piperine concentration on Vibrio harveyi biofilm

[0041] The 10 mg / mL piperine ethanol solution prepared in Example 12 was diluted with sterile LB medium to obtain LB medium with final concentrations of 4, 8, 16, 32, 64 and 128 μg / mL piperine. 5 μL of the bacterial suspension prepared in Example 11 (about 1×10 9CFU / mL), 195 μL of LB liquid medium containing a final concentration of 4, 8, 16, 32, 64, and 128 μg / mL piperine, a circle of sterile water was filled around the well plate, and cultured at 30°C for 36 hours, the culture medium was aspirated, and the wells were gently washed three times with sterile PBS (0.01M, pH 7.4), and the 96-well plate was placed in a 55°C oven for 30 minutes for drying, 200 μL of 0.1% crystal violet dye solution was added, and the biofilm was uniformly stained at room temperature for 10 minutes on a 100r / min decolorization shaker. Use a pipette to aspirate the excess dye in each well, rinse three times with double distilled water, and place the 96-well plate in a 55°C oven for about 30 minutes. After drying, 200 μL of 33% glacial acetic acid was added to each well, and decolorized for 15 minutes under a decolorization shaker (100r / min), and the absorbance value of each well at 570nm was measured using an enzyme marker. Six parallels were set up in each group, and the blank control group consisted of 5 μL bacterial suspension and 195 μL LB solution in each well. The inhibition rate of each concentration group of piperine on Vibrio harveyi biofilm was calculated using the formula in Case 1.4, and the half effective concentration (EC 50 ).

[0042] Biofilm inhibition rate calculation formula: IRBF i =(1-OD 570nmi / OD 570nm0 )×100%

[0043] Where, IRBF i is the biofilm formation inhibition rate, OD 570nm0 OD of blank control 570nm , O.D. 570nmi OD of piperine group 570nm .

[0044] The results showed that the inhibition rates of piperine at 4, 8, 16, 32, 64 and 128 μg / mL on Vibrio harveyi biofilm were 12.44±4.83%, 15.62±3.28%, 35.69±5.67%, 45.55±7.49%, 69.36±1.56% and 78.10±1.27%, respectively. 50 is 29.99±6.46μg / mL, y=0.83–0.76 / (1+(x / 26.12) 1.64 ), x is the concentration of piperine, y is the biofilm inhibition rate, R 2 =0.99.

[0045] Example 2 Minimum inhibitory concentration (MIC) of piperine against Vibrio harveyi

[0046] The MIC of piperine against Vibrio harveyi was determined according to the broth microdilution method in the Microbiological Clinical Laboratory Standards and Operation Specifications (M07-A9) established by the National Committee for Clinical Laboratory Standards (NCCLS, 2009).

[0047] 1. Inoculate and streak Vibrio harveyi onto hydrolyzed casein (Mueller-Hinton, MH) solid medium and culture at 30°C overnight.

[0048] 2. Weigh 20 mg of piperine and dissolve it in 1 mL of anhydrous ethanol to prepare a 20 mg / mL piperine mother solution. Take 256 μL of piperine mother solution and add it to 5 mL of MH liquid medium to obtain a MH liquid medium with a final concentration of 1024 μg / mL piperine. Use the MH liquid medium two-fold gradient dilution method to obtain a final concentration of 512, 256, 128, 64, 32, 16, 8, 4 and 2 μg / mL piperine MH liquid medium for the test.

[0049] 3. Pick a single colony from Example 21 and inoculate it into LB liquid culture medium, shake and culture it at 30°C, 180r / min overnight. Collect the bacterial solution, centrifuge it at 5000r / min for 5min, remove the supernatant, resuspend the collected bacteria in PBS, centrifuge it again, collect the bacteria by centrifugation, wash and resuspend them, repeat the operation 3 times, and adjust the OD 600nm =1(about 1×10 9 CFU / mL). The bacterial suspension at this time was centrifuged at 5000r / min for 5min, the supernatant was discarded, and the suspension was resuspended with an equal volume of MH liquid culture medium. 200μL of MH liquid culture medium containing 1024, 512, 256, 128, 64, 32, 16, 8, 4, and 2μg / mL piperine was added to each well, and the bacterial solution prepared above was inoculated at 1%. At the same time, a negative control group of 200μL of culture medium containing the above concentrations of piperine and a positive control group inoculated with 1% bacterial suspension in 200μL MH liquid culture medium were set up, and six replicates were set up for each group. After the sample preparation was completed, the absorbance value of each group at 600nm was measured by an enzyme marker and recorded as A0. After static culture at 30℃ for 16h, the 96-well culture plate was taken out and the absorbance value of each group at 600nm was measured by an enzyme marker again and recorded as A i . A i The lowest piperine concentration that was not significantly different from A0 was recorded as MIC.

[0050] Piperine is an alkaloid. If the concentration is too high, it may combine with the protein in the MH medium to form a complex, which will interfere with the interpretation of the MIC determination results. Therefore, a negative control without adding piperine to the bacterial solution was performed in this experiment to eliminate the interference of the piperine complex. The results showed that the MIC of piperine against Vibrio harveyi exceeded 1024μg / mL.

[0051] Example 3 Effect of piperine on preventing Vibrio harveyi infection in pearl grouper

[0052] 1. Preparation of injectable bacterial suspension: according to Example 11. Preparation of Vibrio harveyi bacterial suspension.

[0053] 2. Preparation of intragastric administration solution: Weigh an appropriate amount of piperine into a 15 mL centrifuge tube, add 10% ethanol PBS (0.01 M, pH 7.4) solution to aid dissolution, and prepare 10% ethanol PBS solution of piperine with final concentrations of 205, 102.5, 10.25 and 1.025 mg / mL respectively.

[0054] 3. Acute toxicity test of piperine on pearl grouper

[0055] A total of 180 healthy fish with a body length of 15.82±2.21cm and a body weight of 61.56±4.35g were selected and randomly divided into 12 aquariums (125L, 0.5m×0.5m×0.5m), 15 fish in each tank, and randomly divided into 6 groups, including 4 experimental groups, 1 blank control group and 1 solvent control group, with 2 parallel aquariums in each group. The fish were fed with 5% of the basic feed of fish body weight every day and raised for 7 days to adapt to the density environment (raising conditions: water temperature 28±2℃, dissolved oxygen>6.5mg / L, water was changed every 2 days, and the amount of water changed each time was 1 / 4 of the total water body to keep the water environment continuously stable. Adaptability After the feeding, the fish in each group were fasted for 24 hours, and the fish were gavaged with piperine solution at 0.15mL / (61.56g sample fish). After 4 hours, the fish in the experimental group received oral gavage of 5, 50, 500 and 1000mg / kg (piperine / fish weight), respectively. The solvent control group was gavaged with the same dose of 10% ethanol PBS solution, and the blank control group was gavaged with the same dose of sterile PBS (0.01M, pH7.4). After gavage, the groupers were still raised according to the original feeding conditions, and the symptoms were continuously observed for 14 days. The dying groupers were promptly removed and dissected to observe the symptoms.

[0056] The results showed that no fish died in each group, and their swimming and reactions were normal, and there was no abnormality in eating. After the observation period, all the fish were fished out and their body surfaces were observed. There were no blood spots, ulceration, dephosphorization, etc., and the eyes were not cloudy or congested. All the fish were dissected and there was no fluid accumulation in the abdominal cavity. The organs were bright in color, without ulceration or swelling, and there was no water accumulation in the stomach and abdominal cavity.

[0057] 4. Establishment of the model of artificial infection of Vibrio harveyi in pearl grouper

[0058] 120 healthy pearl groupers with a body length of 14.10±1.15cm and a body weight of 37.13±2.33g were selected and randomly divided into 10 aquariums (125L, 0.5m×0.5m×0.5m), with 12 fish in each tank. The 10 tanks were randomly divided into 4 experimental groups and 1 control group (see Table 1), with two parallel aquariums in each group, and were temporarily reared for 7 days. Basic feed was fed during the temporary rearing period and the entire experimental period. The water temperature of the aquarium was maintained at 28±2℃ and the dissolved oxygen was greater than 6.5mg / L. The water was changed once every 2 days, and 1 / 2 of the water was changed each time. The groupers were fasted for 24h. Different doses of Vibrio harveyi suspension were intraperitoneally injected into the pearl groupers according to the scheme in Table 1. The culture conditions during the temporary rearing were continued for 14d, and the activity of the experimental fish was observed. The number of fish deaths in each group was counted every day, and the dead fish were fished out in time, and the pathogens of the newly dead fish were isolated.

[0059] Table 1 Establishment of the disease model of Vibrio harveyi

[0060] Group (n=2) Challenge dose feed Control group PBS (0.01 M, pH 7.4) Basic feed Experimental Group 1 <![CDATA[2.7×10 5 CFU / kg]]> Basic feed Experimental Group 2 <![CDATA[2.7×10 6 CFU / kg]]> Basic feed Experimental Group 3 <![CDATA[2.7×10 7 CFU / kg]]> Basic feed Experimental Group 4 <![CDATA[2.7×10 8 CFU / kg]]> Basic feed

[0061] Note: Injection volume: 0.1 mL / (37.13 g fish body weight).

[0062] Symptoms of pearl grouper after artificial infection Figure 2 As shown, the liver, spleen, and head kidney of fish infected with Vibrio harveyi were swollen, and there was urethral effusion. After injection, the fish in the experimental group breathed rapidly at the bottom of the aquarium, swam slowly, had a slow stress response, and lost their appetite. The control group did not eat on the day of injection, but started eating on the second day. The experimental group did not eat for 1 to 4 days after injection, but started eating on the fifth day. After the onset of the disease, white feces appeared in the tank, the fish's abdominal cavity was swollen, and some fish had ulcers on their bodies.

[0063] The cumulative mortality curves of groupers in each group after infection are as follows Figure 3 The cumulative mortality bacteria concentration curve was drawn and the curve was fitted to obtain the fitting equation y = 0.317 × LG (x) - 1.612 (R 2 =0.976), and the median lethal dose (LD50) of Vibrio harveyi to pearl grouper was calculated according to the fitting equation. 50 ) and 80% lethal dose (LD 80 ) are respectively

[0064] 4.6×10 6 CFU / kg and 4.1×10 7 CFU / kg. The pathogens isolated from the dead fish were all identified as Vibrio harveyi by 16S-rDNA.

[0065] 5. Feeding with piperine to prevent pearl grouper from being infected with Vibrio harveyi

[0066] 120 healthy groupers with a body length of 13.33±1.56cm and a body weight of 35.30±2.07g were selected and randomly assigned to 8 aquariums (125L, 0.5m×0.5m×0.5m) for grouping, with 15 fish in each group. These aquariums were randomly divided into 4 groups, namely 1 blank group, 1 control group and 2 drug-treated groups, with two parallel aquariums in each group. After 7 days of feeding with basic feed to adapt to the environment, after the adaptive feeding, they were fed according to the scheme in Table 2 for 14 days. 4 hours after the last feeding, the groupers in each group were injected according to the scheme in Table 2. After injection, they were cultured for 14 days according to the previous culture conditions, and the activities of the experimental fish were observed. The number of fish deaths in each group was counted every day, and the dead fish were fished out in time, and the pathogens of the newly dead fish were isolated.

[0067] RPS=[1-(P T / P CON )]×100%

[0068] Where: RPS is the relative protection rate; P T is the mortality rate of the piperine mixed feed group; CON is the mortality rate of the control group.

[0069] Table 2 Experimental scheme for preventing pearl grouper from being infected with Vibrio harveyi by feeding with piperine mixed feed

[0070]

[0071] Note: The volume of bacterial suspension injected intraperitoneally for artificial infection is 100 μL / (35.30 g fish body weight).

[0072] Survival curves are shown in Figure 4 , 2g / kg (piperine: feed) piperine mixed with feed and fed to pearl gentian grouper can reduce the probability of death of grouper infected with Vibrio harveyi disease.

[0073] The RPS of each group was calculated according to the formula. The results are shown in Table 3:

[0074] Table 3 The preventive effect of piperine mixed feed on Vibrio harveyi infection in pearl grouper

[0075] Group Death amount (number) Cumulative mortality rate (%) Relative protection rate (%) Blank Group 0 0 - 0g / kg 16 53.33 - 2g / kg 8 26.67 49.99 4g / kg 12 40 25.00

[0076] 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. Application of piperine in the preparation of drugs for preventing and treating Vibrio disease in aquatic animals.

2. The use of piperine according to claim 1 in preparing a drug for preventing and treating vibriosis in aquatic animals, characterized in that: the pathogenic bacteria of vibriosis in aquatic animals is Vibrio harveyi; and the aquatic animal is pearl grouper.

3. The use of piperine according to claim 1 in the preparation of a drug for preventing and treating vibriosis in aquatic animals, characterized in that piperine prevents and treats vibriosis by inhibiting vibrio biofilm.

4. The use of piperine according to claim 1 in the preparation of a drug for preventing and treating Vibrio disease in aquatic animals, characterized in that the effective concentration of piperine in inhibiting Vibrio biofilm is 20-200 μg / mL.

5. The use of piperine according to claim 1 in preparing a drug for preventing and treating vibriosis in aquatic animals, characterized in that: The dosage form of the medicine is mixed liquid, microemulsion, nanoemulsion, medicinal bath liquid, capsule, oral capsule coating, powder, granule, pellet, micropill or different dosage forms of medicine prepared with pharmaceutically acceptable carriers and excipients.

6. Application of piperine in the preparation of feed additives for preventing and treating Vibrio disease in aquatic animals.

7. The use of piperine according to claim 6 in preparing a feed additive for preventing and treating vibriosis in aquatic animals, characterized in that: The pathogenic bacteria of vibriosis in aquatic animals is Vibrio harveyi; the aquatic animal is pearl grouper.

8. A feed for preventing and treating Vibrio disease in aquatic animals, characterized in that: Includes feed and piperine.

9. The feed for preventing and treating Vibrio disease in aquatic animals according to claim 8, characterized in that: The mass ratio of piperine to feed is: 2-4g:1kg.

10. The feed for preventing and treating Vibrio disease in aquatic animals according to claim 8, characterized in that: The pathogenic bacteria of vibriosis in aquatic animals is Vibrio harveyi; the aquatic animal is pearl grouper.

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