Method for detecting, preventing and treating pomfret diseases in industrial aquaculture mode

Abstract

The invention aims to provide the method for detecting, preventing and treating common diseases of the pomfret in the industrial aquaculture mode, so that the problem that the pomfret is easily infected by parasites, bacteria and viruses in a high-density aquaculture environment to cause large-scale death is solved, and the survival rate of artificially cultured pomfret is increased. According to the method, the disease condition of the bred pomfret can be simply and efficiently detected in a time-saving and labor-saving mode, a large number of experimental equipment is not needed, and operation can be conducted on the first line of pomfret breeding. Disease conditions can be found in the first time, control measures are taken in time, and the breeding survival rate is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to a method for detecting and preventing diseases of silver pomfret in a factory-based breeding model. Background Art

[0002] Silver pomfret is one of my country's major commercial marine fish species, known for its delicious meat, high nutritional value, and market demand. Domestic researchers have begun to develop artificial breeding of silver pomfret. Ningbo University has achieved breakthroughs in silver pomfret aquaculture, completing large-scale aquaculture. However, high-density aquaculture makes it susceptible to infection by various parasites, bacteria, and viruses, leading to serious diseases and significant economic losses. Common parasitic diseases in silver pomfret aquaculture include the dinoflagellate Amylolyticus and Cryptocaryon irritans. The former primarily parasitizes the gills, damaging the gill filaments and thus affecting the fish's respiratory function; the latter primarily parasitizes the gills, skin, fin rays, and tongue. Bacterial diseases such as Nocardia and Photobacterium mermaidii are also common in silver pomfret aquaculture, causing symptoms such as internal bleeding and skin ulcers, respectively. Currently, due to the relatively backward disease detection facilities and equipment in silver pomfret aquaculture and seedling production facilities, the primary reliance is on transporting farmed silver pomfret from farms to laboratories for testing. Disease prevention and control plans are then developed based on the test results. This is time-consuming, labor-intensive, and inefficient. Often, by the time the laboratory detects a pathogen, an outbreak has already occurred in the production line. Therefore, a simple method for disease detection and prevention in silver pomfret production is needed. This method can quickly and accurately diagnose silver pomfret diseases and enable effective prevention and control measures. While maintaining high-density farming, it can also reduce the likelihood of disease, improve survival rates, and promote the industrialization of silver pomfret aquaculture. Therefore, establishing an efficient disease detection and prevention technology system for silver pomfret production is crucial to ensuring successful aquaculture. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for detecting and preventing common diseases of silver pomfret in a factory farming model, thereby solving the problem that silver pomfret is susceptible to parasitic, bacterial and viral infections in a high-density farming environment, leading to large-scale mortality, and improving the survival rate of artificially cultured silver pomfret.

[0004] The present invention provides a method for detecting and preventing diseases of silver pomfret in a factory-based breeding mode, comprising the following steps: 1) Randomly sample silver pomfret from the aquaculture pond. Remove the gills and spread them flat on a glass slide for pest and disease testing. The random sampling is carried out once a day during the high temperature period and once every 2-5 days when the water temperature is low and there is no mortality; The pests and diseases include amylodinium, Cryptocaryon irritans, trichodinia or trypanosoma; Going a step further, use formaldehyde baths to treat Dinoflagellates amylolyticus or Cryptocaryon irritans; 2) Use culture media for hemolytic assays, identification and isolation of Vibrio bacteria, bacterial load testing, and culture and testing of dominant bacteria; The culture medium includes TSA solid medium, 20ng / mL ampicillin-resistant TSA solid medium, TSB liquid medium, sheep blood agar plate and TCBS medium; The hemolytic test is to detect whether the liver and kidney of the fish have hemolytic activity; 3) Conduct drug susceptibility testing and combine the bacterial load test results to determine the best antibiotic to use; The drug sensitivity test is used to detect the drug sensitivity of florfenicol, neomycin sulfate, enrofloxacin, doxycycline and co-trimoxazole; 4) Drug treatment: Different drug treatment methods are adopted according to the identification results of different diseases in the silver pomfret farming process. For parasites, formaldehyde bath is used; for bacteria, antibiotics are mixed into the feed; and for wheelworms, Sophora flavescens powder is used for bath treatment.

[0005] The present invention also provides an antimicrobial peptide having good inhibitory activity against drug-resistant Vibrio harveyi and Aeromonas hydrophila, and the sequence thereof is GKTCPLSCYGGSCRRWRFCSKKGAPRCPVNHVCL (SEQ ID NO: 1).

[0006] The present invention provides a method for detecting and preventing diseases of silver pomfret in a factory-based aquaculture model. This method can save time and effort, simply and efficiently detect diseases in aquacultured silver pomfret, and can be performed on the front lines of silver pomfret aquaculture without requiring extensive laboratory equipment. This method can detect diseases immediately, allowing for timely prevention and control measures and improving the survival rate of aquacultured silver pomfret. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The results of Cryptocaryon irritans parasitism on the gills of silver pomfret Figure 2 The microscopic examination results of the silver pomfret gills in Example 1 are shown in FIG. Figure 3 The results of bacterial hemolytic test on the kidney and liver tissues of silver pomfret in Example 1 are shown in FIG. Figure 4 The results of the bacterial load test of the kidney and liver tissues of silver pomfret in Example 1 are shown in FIG. Figure 5 1 is a graph showing the drug sensitivity test results of the dominant bacteria in Example 1. In the graph, L represents the liver, K represents the kidney, 1 is enrofloxacin, 2 is neomycin sulfate, 3 is cotrimoxazole, 4 is florfenicol, and 5 is doxycycline.

[0008] Figure 6Figure A shows the inhibitory performance of the antimicrobial peptides against strains, and Figure B shows the inhibitory performance against Aeromonas hydrophila. DETAILED DESCRIPTION

[0009] The present invention provides a method for detecting and preventing diseases of silver pomfret in a factory-based aquaculture model, comprising microscopic examination, culture medium streak detection, drug sensitivity test, and drug treatment, wherein: (1) Microscopic examination: Randomly sample the silver pomfret that is normally active in the breeding pond, once a day during the high temperature period, and once every 2-5 days if there is no death during the low water temperature period. Take 2-3 fish from each breeding pond, including the fish and water, to prevent the death of fish before microscopic examination from affecting the observation results. Take out the fish gills and spread them flat on a slide, spread them out with tweezers to avoid overlapping, and observe under a ten-fold objective lens. The starch egg dinoflagellate is also known as the starch egg whipworm, which is the pathogen of "white spot disease". The gill filaments and body mucus of the fish parasitized by the starch egg dinoflagellate can be observed to be grayish-dark. The parasitic worm body is oval and about 20-150μm in size. The starch egg dinoflagellate has an attachment device with a pseudo-root-like protrusion at one end of its sphere. The irritant cryptorchid is oval or spherical in shape, slightly larger in size than the starch egg dinoflagellate, and its body surface is covered with a large number of short cilia, such as Figure 1 . It can be seen rotating slowly under an optical microscope. Formaldehyde bath is more effective for these two parasites. In addition, during the breeding process, wheelworms and trypanosomes may also parasitize. The wheelworm body looks like a felt hat from the side and a round disc from the back. It moves like a wheel. Trypanosomes are usually slender and spiral, with slightly pointed ends, and the body length usually ranges from 15-50μm. The former can be observed through the above-mentioned microscopic examination of the silver pomfret gills, while the latter requires blood smear staining, that is, a small amount of blood is collected from the tail vein or heart of the fish, a blood smear is prepared, and the Giemsa staining method is used for observation under a microscope. The nucleus and kinetochores of the stained blood trypanosomes are usually dark purple or blue-purple, while the cytoplasm is light blue, and the flagella and undulating membrane can also be stained and observed. If wheelworms are found to be parasitic, use Sophora flavescens powder for continuous medicinal bathing for 1-3 days. Since trypanosomes are parasitic in the blood, the pond needs to be thoroughly cleaned and disinfected before stocking to kill leeches in the environment.

[0010] (2) Culture medium streak check: The culture media that need to be prepared in advance are: TSA solid culture medium, 20ng / mL ampicillin-resistant TSA solid culture medium, TSB liquid culture medium, sheep blood agar plate and TCBS culture medium.

[0011] A. Hemolysis Test: First, dissect the sampled fish, remove the liver and kidneys, pierce the kidneys and liver with a disposable inoculating loop, and inoculate the tissue fluid onto a sheep blood agar plate. Incubate at 24°C for 1-3 days. Finally, observe under appropriate lighting for the presence of hemolysis (clear areas appearing at the edges of cells).

[0012] B. Identification and Isolation of Vibrio Species: Similarly, use a disposable inoculating loop to puncture the kidney and liver, and inoculate the tissue fluid onto TCBS medium. Incubate in a constant temperature incubator at 35-37°C for 18-24 hours to identify and isolate any Vibrio flavus and Vibrio viridis.

[0013] C. Bacterial load test: Remove the liver and kidneys of the fish, cut them open with surgical scissors, pierce the cross section of the organs with a disposable inoculation loop, inoculate the tissue fluid into TSA solid culture medium, and culture at 24°C for 24-96 hours. Inoculate as soon as possible after removing the internal organs to prevent the tissue fluid from drying out and affecting the results of the plate. Inoculating with a disposable inoculation loop is equivalent to inoculating 10μm of tissue fluid. After counting the number of colonies, multiply it by 100 to get the bacterial load in CFU / ml. Nocardia is suitable for detection in ampicillin-resistant TSA solid culture medium. If the final bacterial load is on the order of 10 4 This indicates that the fish's internal organs have a certain degree of bacterial infection, and a small amount of antibiotics must be used for prevention and treatment, and disease supervision and testing must be strengthened; if the bacterial load reaches 10 5 If the infection is severe, administer appropriate antibiotics and increase water changes. Test the bacterial load in the pond 5-7 days after using antibiotics.

[0014] D. Cultivation and Submission of Dominant Bacteria: Based on the bacterial load test, continue culturing on the same TSA solid medium and observe the dominant bacteria in the medium. Dominant bacteria typically form larger colonies due to their large numbers and rapid adaptation to the environment. Isolate the dominant bacteria by diluting them with 100-300μL of TSB liquid medium and grinding them with a pestle. Take 100μL of the bacterial suspension and inoculate it onto TSA solid medium. Incubate at 28°C for 24-96 hours before sending them to the laboratory for molecular testing and identification.

[0015] (3) Drug sensitivity test: Place drug sensitivity paper strips for florfenicol, neomycin sulfate, enrofloxacin, doxycycline, and cotrimoxazole on TSA culture medium and streak them. Based on the hemolytic test, separate the dominant bacteria in the sheep blood culture medium, dilute them with 100-300 μL of TSB liquid culture medium, grind them with a pestle, take 100 μL of the bacterial solution, spread it evenly with a disposable spreader, and incubate it at 28°C for 24-96 h. Observe the inhibitory plaques on different paper strips and determine the best antibiotic based on the bacterial load test results.

[0016] (4) Drug treatment: Different drug treatment methods are adopted according to the identification results of different diseases in the silver pomfret breeding process. For parasitic diseases (such as amylolytic dinoflagellates and Cryptocaryon irritans), first lower the water level and use 50-100ppm formaldehyde bath according to the different sizes of silver pomfret. The formaldehyde is evenly sprinkled into the breeding pond and sprayed on the pond wall. During the drug treatment, oxygen is added to the maximum to prevent the fish from dying due to lack of oxygen. After the drug treatment, a large water change of 100% is required to remove the remaining formaldehyde. For bacterial diseases (such as Nocardia and Photobacterium mermaidii), select appropriate antibiotics based on drug sensitivity tests and mix them into the feed. The general dosage is 10-30mg / kg fish body weight and is used continuously for 5-7 days. After use, the culture medium is streaked again to observe the bacterial load in the silver pomfret's viscera. If a large number of Vibrio is found, lactic acid bacteria or Clostridium butyricum can be mixed into the feed to improve the intestinal flora structure and reduce the harm of Vibrio infection. If wheelworms are found, use Sophora flavescens powder, 2-3ppm, and continue to bathe for 1-3 days.

[0017] The present invention further provides an antimicrobial peptide having good inhibitory activity against Vibrio harveyi and Aeromonas hydrophila; the sequence of the antimicrobial peptide is: GKTCPLSCYGGSCRRWRFCSKKGAPRCPVNHVCL (SEQ ID NO: 1).

[0018] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0019] Example 1 In July 2024, a study was conducted at a farm in Ninghai County, Zhejiang Province. To effectively prevent and control various diseases that are prone to occur in silver pomfret during the summer, the following prevention and control methods were adopted: (1) Detection and prevention of parasites: The water temperature in the culture pond on that day was 25.8℃, and the inlet water temperature was 24.8℃. At this water temperature, the reproduction rate of starch dinoflagellates was relatively fast.

[0020] A. Microscopic examination: In the morning, three silver pomfret juveniles were randomly sampled from the breeding ponds. Microscopic examination revealed that about 30 to 40 starch-egg dinoflagellates were parasitizing the gills of the silver pomfret in two breeding ponds. Figure 1 .

[0021] B. Medical Treatment: Stop feeding for the day, lower the water level in the aquaculture pond by approximately 50cm, and evenly spray formaldehyde diluted with seawater throughout the pond. Simultaneously, spray the pond walls to disinfect the area. Use a 65ppm formaldehyde concentration, soak for 2 hours, and continue for two days. During the formaldehyde bath, enable aeration to allow the formaldehyde to fully penetrate. Also, increase oxygenation to maximum to prevent larvae from hypoxia and death. During the bath, check the larvae every 10 minutes to ensure they are moving normally. If large areas of floating or capsizing occur, add water immediately to remedy the situation. Due to formaldehyde's strong oxidizing properties and its denaturing effects on proteins, parasite attachment apparatus and certain key proteins can be damaged, causing them to fall off the gills. Formaldehyde also has certain side effects on fish, so a 100% water change is required after the bath.

[0022] C. Pond Management: First, isolate and disinfect fishing gear in the infested pond to avoid mixing and cross-infection. When the water temperature is low and no parasites are found, perform a water change daily, changing approximately 60% of the water volume. When the water temperature is above 22°C, perform water changes twice daily, changing 80% to 100% of the water volume. Frequent and large-scale water changes maintain water quality. Deteriorating water quality can weaken fish immunity and increase the risk of infection. When changing water, first lower the water level to 80 cm, then open the inlet and outlet, allowing water to flow to 40 cm, and finally add water to 1.4 m. If a large number of juvenile fish are found congregating at the outlet during water flow, disperse them by splashing water. Maintain daily pond cleaning. Record water temperature, dissolved oxygen, ammonia nitrogen concentration, and other data daily to monitor water quality.

[0023] D. Control effect: The farm has put into use two 100m2 2 The silver pomfret breeding ponds in the two ponds are numbered 639 and 626. Due to the increase in water temperature, microscopic inspections were carried out once a day during the breeding process. Both breeding ponds were found to be infected with the disease of starch egg dinoflagellate when the water temperature was above 25℃. During the infection period, each pond lost 30 to 50 fish per day. Before drug treatment, microscopic examination revealed that 20 to 30 starch egg dinoflagellates were parasitic on a single gill. After two days of formaldehyde bathing, only a small amount of starch egg dinoflagellates were found on some gills, including some that had been structurally damaged but had not yet fallen off. The daily loss in each pond was also greatly reduced, with a loss of about 10 fish per day. After stopping the formaldehyde bath for one day to alleviate the damage, the drug treatment was carried out for another two days to completely eliminate the starch egg dinoflagellate and Cryptocaryon irritans in the water.

[0024] (2) Detection and prevention of bacteria: In late July, a total of 6 100m 2The silver pomfret breeding ponds in the county were numbered 639, 626, 63A, 6311, 6313, and 6314. On that day, high juvenile losses were observed in ponds 63A and 6311, ranging from 20 to 30 fish. The normal ammonia nitrogen levels for that day were 0.2 mg / L and 0.1 mg / L, respectively. Microscopic examination revealed no parasites on the fish gills, suggesting a degree of bacterial infection in ponds 63A and 6311.

[0025] A. Culture medium streak inspection: In the morning, 3 fish were randomly sampled from pools 63A and 6311, and the fish bodies were dissected. Since the spleen of the fish was too small at this stage, only the liver and kidney were taken for plating. First, use surgical scissors to cut the internal organs in the middle, and use a disposable inoculation loop to penetrate the cross section of the internal organs and stir gently. After taking it out, the tissue fluid was inoculated on the pre-prepared sheep blood plate, TSA solid culture medium and TCBS culture medium. When opening the disposable inoculation loop, be sure to operate in a sterile environment to avoid contaminating the end of the loop. It is best to perform streaking in a clean workbench to ensure that the inoculation loop is operated in a sterile environment. The same internal organs of the juvenile fish in the same breeding pond are smeared on a piece of culture medium and marked with a marker. After dissection, inoculation and streaking should be performed as soon as possible to prevent the tissue fluid from drying up and affecting the growth of the colony. After inoculation, the culture medium is incubated at 24°C for 24-96 hours, and the colony growth is observed at irregular intervals every day. The results of the bacterial hemolytic test of kidney and liver tissues are as follows: Figure 2 , some bacterial load test results are as follows Figure 3 After the culture was completed, the colonies in the TSA culture medium were counted. The colony counts on the liver plates of the juvenile fish in pool 63A were 143, 159, and 148, respectively; and the colony counts on the kidney plates were 234, 257, and 261, respectively. One inoculation with a disposable inoculation loop is equivalent to 10 μL of tissue fluid. Finally, the bacterial load in the liver was calculated to be approximately 1.5*10 4 CFU / mL, the bacterial load in the kidney is about 2.5*10 4 CFU / mL. The colony counts on the liver of juvenile fish in pool 6311 were 101, 83, and 86; the colony counts on the kidney of juvenile fish were 132, 117, and 126. The final calculated bacterial load in the liver was approximately 9.0*10 3 CFU / mL, the bacterial load in the kidney is about 1.25*10 4 CFU / mL. The bacterial load results indicate bacterial infection in pools 63A and 6311, requiring antibiotic susceptibility testing and prompt treatment with antibiotics. Furthermore, significant numbers of Vibrio flavus and Vibrio chlororaphis were isolated from TCBS medium. Probiotics can be added to the feed in appropriate amounts while concurrently with antibiotic treatment.

[0026] B. Antimicrobial Susceptibility Testing: Isolate the dominant bacteria from the sheep blood culture medium, dilute it with 100-300μL of TSB liquid medium, grind it with a pestle, and evenly spread 100μL of the bacterial solution onto TSA medium with a drug susceptibility paper strip using a disposable applicator. Incubate at 28°C for 24 hours. The next day, on both plates tested for drug susceptibility, there was no obvious inhibitory plaque around the cotrimoxazole susceptibility paper strip. However, inhibitory plaques of varying sizes appeared around the other four antibiotics, with the largest plaque around florfenicol. The results are as follows: Figure 4 This indicates that florfenicol has the best antibacterial effect on this bacterium.

[0027] C. Drug treatment: Combined with the results of the drug sensitivity test, florfenicol antibiotics are preferred for treatment. The dosage of florfenicol is 30mg / kg of fish, mixed with feed and fed at noon and in the evening for three days. At the current stage, the total weight of silver pomfret fry in each pond is about 56kg, so 1.68g of florfenicol needs to be mixed with feed for one meal. At the same time, Clostridium butyricum is added in stages to improve the intestinal flora of silver pomfret, and the addition amount is 1% of the feed. During the feeding process, always pay attention to the feeding status of silver pomfret and maintain a small and frequent feeding rhythm. Three days later, the bacterial load of silver pomfret in pools 63A and 6311 was tested again. The results showed that the bacterial count in the liver of the fish in pool 63A was significantly reduced, about 2.1*10 3 CFU / mL, the amount of bacteria in the kidneys was significantly reduced to about 1.0*10 3 CFU / mL. The bacterial load on the fish in pool 6311 also decreased, with the bacterial load in the liver being approximately 2.0*10 2 CFU / mL, kidney is about 2.5*10 3 CFU / mL. This shows that through this series of disease detection and prevention measures, the bacterial load is effectively controlled. With the use of florfenicol antibiotics, bacteria may show a certain degree of drug resistance. According to the results of the drug sensitivity test, enrofloxacin antibiotics can be replaced for treatment, and the enrofloxacin dosage is 30mg / kg fish body for mixing and feeding. Florfenicol and neomycin sulfate can also be mixed at the same time, with a florfenicol: neomycin sulfate ratio of 5:4, that is, 35mg / kg florfenicol and 28mg / kg neomycin sulfate.

[0028] D. Molecular Testing: After incubation, observation revealed that the bacteria isolated from the blood plates in both pools exhibited a degree of hemolysis and were morphologically suspicious for Photobacterium mermanii, necessitating further molecular testing. Nocardia was isolated using TSA plates supplemented with 20 ng / mL ampicillin. After three days of incubation, a single colony suspected of Nocardia emerged, also requiring molecular confirmation. These samples were sent to the laboratory for testing.

[0029] Example 2 This study was conducted at a farm in Xiangshan County, Zhejiang Province, in August 2024. The farm operated four silver pomfret aquaculture ponds in August, numbered 1, 2, 3, and 4. The average water temperature that month was approximately 28.3°C. Disease incidence and drug use were analyzed at the farm in August.

[0030] Water temperatures were generally high in August. To ensure disease detection and prevention, random microscopic sampling of silver pomfret was performed daily in each aquaculture pond. Table 1 summarizes the incidence of white spot disease in four aquaculture ponds. The frequency and intervals of occurrence of white spot disease varied considerably across the ponds. However, with strict disease prevention and control measures, the average interval between occurrences of white spot disease became regular, occurring approximately every four to five days. During the August aquaculture period, parasitic parasites were observed on the gills of fish in all four ponds for three consecutive days. During this period, aquaculture equipment was isolated, and each pond was treated with a 65 ppm formaldehyde bath for two hours for three consecutive days. The activity of silver pomfret was observed every 10 minutes during the baths, and the water was immediately changed after the baths, with a 100% water change. The number of starch-egg dinoflagellates on individual gills of the fish was counted during these three days.

[0031] Table 1: Occurrence of white spot disease in each breeding pond in August

[0032] Table 2: The number of single gill starch dinoflagellates obtained by microscopic examination during the formaldehyde bath in each culture pond

[0033] Microscopic examination on the first day revealed a severe infestation of 30 to 40 dinoflagellates on the gills of fish in all four ponds. After treatment on the first day, the number of parasites decreased significantly, and by the third day, the parasites had almost disappeared from the gills, with only a few remaining in pond 3. Treatment was then discontinued for a day to alleviate the damage, followed by a formaldehyde bath, which essentially completely eliminated the dinoflagellates from the water. Daily losses of silver pomfret also decreased significantly during this period, from approximately 40 on the first day, when the disease was most severe, to only 8 on the third day. This demonstrates that this treatment method is highly effective in preventing and controlling parasitic diseases.

[0034] In early August, it was discovered that both No. 1 and No. 2 breeding ponds had a certain degree of bacterial infection. The antibiotic medication cycle is generally about 7 days. The bacterial load in the viscera of the two breeding ponds within 7 days and the medication situation are statistically analyzed. The statistical results are shown in Table 3. Considering that no strong bacterial resistance was found during this stage, a lower concentration of florfenicol was used for treatment. The two ponds were first fed with florfenicol 10mg / kg mixed fish feed for three days. After three days, the bacterial load was significantly reduced, and the bacterial load order of magnitude decreased from 10 4 Reduced to 10 3Two days after stopping the drug, it was found that the bacterial load had a slight growth trend. In the next two days, 15mg / kg of florfenicol was used to mix with fish feed and the bacterial load in the fish culture pond was further reduced. In conjunction with the subsequent bacterial load test, it was found that after one cycle of drug treatment, the bacterial load in the two culture ponds could be stabilized and controlled at 10 3 This level of bacteria does not pose a significant threat to the survival of silver pomfret. This medication method is ideal for the prevention and treatment of bacterial diseases. If continued drug treatment has little effect on the bacterial load, it is necessary to reselect the antibiotic mix and feed based on the most recent drug sensitivity test.

[0035] Table 3: Bacterial load and drug treatment status of some aquaculture ponds within one week

[0036] Example 3 During the breeding process, it was found that after a period of application, the survival rate of silver pomfret fry decreased, and the bacterial load in the liver and kidney increased significantly. It was speculated that due to long-term use of drugs, the pathogenic bacteria developed drug resistance. Therefore, beneficial bacteria with inhibitory effects on drug-resistant bacteria were screened in the breeding water of silver pomfret.

[0037] The water and intestinal contents of silver pomfret were collected from three different farms, and five parallel samples were collected from each farm. The water samples were filtered through a 0.45 μm filter membrane and then graded diluted (10 -1 -10 -6 The intestinal contents were homogenized with sterile saline and serially diluted. Culture was performed using 2216E marine medium and TCBS selective medium at 25°C for 48-72 hours. Colony morphology was observed and purified.

[0038] Drug-resistant Vibrio harveyi and Aeromonas hydrophila were isolated and identified from diseased silver pomfret. The candidate strains were spotted on LB agar plates, incubated at 37°C for 24 hours, and then placed in an Oxford cup. 200 μL of indicator bacteria suspension (1×10 6 CFU / mL), the inhibition zone diameter ≥ 10 mm was considered positive, and the average value was obtained by three measurements.

[0039] The peptide AMP-B3 with inhibitory effect on drug-resistant Vibrio harveyi and Aeromonas hydrophila was screened from the silver pomfret antimicrobial peptide database established in the laboratory. Among them, the antimicrobial peptide with the sequence GKTCPLSCYGGSCRRWRFCSKKGAPRCPVNHVCL (SEQ ID NO: 1) had an inhibition zone diameter of 16.5±0.8mm and 14.2±0.6mm against drug-resistant Vibrio harveyi and Aeromonas hydrophila, respectively ( Figure 6 ).

[0040] The MIC of the antimicrobial peptide AMP-B3 was determined by the CLSI-recommended broth microdilution method. Eight concentration gradients were set to determine the inhibitory performance of the antimicrobial peptide against drug-resistant bacteria. The MIC against drug-resistant Vibrio harveyi was 4 μg / mL, and the MIC against drug-resistant Aeromonas hydrophila was 8 μg / mL. The results are shown in Table 4.

[0041] Table 4: MIC of antimicrobial peptide AMP-B3 (μg / mL)

[0042] The antimicrobial peptide was added to the fish feed at a rate of 10 mg / kg, and the control method in Example 2 was used. The survival of silver pomfret fry in the breeding pond was observed and the bacterial load in the viscera was detected. Comparison of the data in Table 5 with Table 3 shows that the bacterial load in the viscera of the breeding pond was significantly reduced after the addition of the antimicrobial peptide, and the duration of the antibacterial effect was significantly prolonged, indicating that the antimicrobial peptide has a good antibacterial effect.

[0043] Table 5: Bacterial load in viscera of some aquaculture ponds after addition of Bacillus Velezii B3

[0044] This method is applicable to all stages of silver pomfret farming, particularly during high-temperature seasons. It enables real-time monitoring of silver pomfret diseases and timely implementation of appropriate drug treatments for disease prevention and control. The present invention provides a disease detection and prevention method for silver pomfret production lines. Using this method, the survival rate of silver pomfret juveniles in high-temperature seasons can reach 76.5%. This method can meet the needs of disease detection and prevention at all stages of silver pomfret farming.

Claims

1. A method for detecting and preventing diseases of silver pomfret in a factory farming model, characterized in that: The method comprises the following steps: 1) Randomly sample silver pomfret from the aquaculture pond. Remove the gills and spread them flat on a glass slide for pest and disease testing. 2) Use culture media for hemolytic assays, identification and isolation of Vibrio bacteria, bacterial load testing, and culture and testing of dominant bacteria; 3) Conduct drug susceptibility testing and combine the bacterial load test results to determine the best antibiotic to use; 4) Drug treatment: Different drug treatment methods are adopted according to the identification results of different diseases in the silver pomfret farming process. For parasites, formaldehyde bath is used; for bacteria, antibiotics are mixed into the feed; and for wheelworms, Sophora flavescens powder is used for bath treatment.

2. The method according to claim 1, wherein The random sampling in 1) is carried out once a day during the high temperature period and once every 2-5 days when the water temperature is low and there is no mortality.

3. The method according to claim 1, wherein The pests and diseases mentioned in 1) include dinoflagellates, Cryptocaryon irritans, Trichodina or Trypanosoma spp.

4. The method according to claim 1, wherein The culture medium in 2) includes TSA solid culture medium, 20 ng / mL ampicillin-resistant TSA solid culture medium, TSB liquid culture medium, sheep blood agar plate or TCBS culture medium.

5. The method according to claim 1, wherein The hemolytic test in 2) is to detect whether the liver and kidney of the fish have hemolytic activity.

6. The method according to claim 1, wherein The drug sensitivity test in 3) is to detect the drug sensitivity of florfenicol, neomycin sulfate, enrofloxacin, doxycycline or co-trimoxazole.

7. The method according to claim 1, wherein Antimicrobial peptides are also added to the feed in 4).

8. The method according to claim 7, wherein The amino acid sequence of the antimicrobial peptide is SEQ ID NO:

1.

9. An antimicrobial peptide, characterized in that The amino acid sequence of the antimicrobial peptide is SEQ ID NO:

1.

10. Use of the antimicrobial peptide according to claim 9 as a feed additive.

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