Method for treating vibrio disease of litopenaeus vannamei larvae by using biofloc

Abstract

The invention belongs to the technical field of prawn culture, and particularly relates to a method for treating vibrio diseases of litopenaeus vannamei seedlings through biological floccules. The method comprises the following steps: (1) preparing runway type biofloc; (2) preparing functional biofloc; and (3) treating vibrio diseases in opossum shrimp and larval shrimp stages. The vibrio disease at the big seedling stage is treated. The invention provides an effective method for treating vibrio diseases of prawn seedlings without using antibiotics, solves the problem of antibiotic abuse in the seedling raising process, and has important significance in promoting green and healthy development of the prawn seedling raising industry.

Description

Technical Field

[0001] The invention belongs to the technical field of shrimp breeding, and in particular relates to a method for treating Vibrio disease of Penaeus vannamei fry with biological flocs. Background Art

[0002] Currently, biofloc is mainly used in the shrimp farming industry. This model is centered on beneficial bacteria and can effectively decompose organic matter such as leftover bait and feces, thereby significantly reducing the accumulation of harmful substances such as ammonia nitrogen and nitrite in the water body, improving the stability and buffering capacity of the water body, alleviating the stress response of shrimp, and reducing the risk of disease caused by water quality deterioration. The beneficial bacteria in biofloc can activate the immune cells in the shrimp body, enhance its nonspecific immunity, and then inhibit the reproduction of pathogens, further reducing the incidence of diseases, so it has broad application prospects in shrimp farming.

[0003] However, biofloc is rarely used in shrimp seedling cultivation, and reports on its use to treat Vibrio diseases in young shrimp are even rarer. During the shrimp seedling cultivation stage, in addition to viral and enterosporidial diseases, Vibrio infection is the most common and extremely harmful problem. Among them, Vibrio harveyi and Vibrio parahaemolyticus are the main pathogenic Vibrio species. Infection with Vibrio harveyi and Vibrio parahaemolyticus can cause lesions in the shrimp seedlings' liver and intestines, seriously impair their digestion and absorption functions, significantly slow down their growth rate, affect their metamorphosis, and lead to different sizes of shrimp seedlings and weak constitutions. In severe cases, it can even cause acute or chronic death. Infection with pathogenic Vibrio has brought huge losses to the shrimp seedling industry.

[0004] Chinese patent 201910857941.X discloses a novel biofloc and a method for applying and utilizing the biofloc to standardize Penaeus vannamei. The biofloc significantly improves the water quality of the Penaeus vannamei standardization pond and improves the survival rate, growth index and immunity of the shrimp.

[0005] Chinese patent 201710048682.7 discloses a method for cultivating bioflocs and its application in the cultivation of Penaeus vannamei. The application of a mixed preparation of Bacillus powder, yeast powder and lactic acid bacteria liquid sprayed in aquaculture promotes the formation of bioflocs in the water body, which is beneficial to the cultivation of economic crops - Penaeus vannamei, improves immunity and disease resistance, inhibits diseases caused by Vibrio, increases food intake and improves the survival rate of Penaeus vannamei.

[0006] However, the above two invention patents only use the function of biofloc to improve water quality and inhibit Vibrio to prevent diseases in the breeding and roughing stages, and do not mention that it can play an effective therapeutic role in the case of Vibrio disease. Moreover, it is not realistic to cultivate biofloc with disease prevention function in time during the seedling process. First of all, because the shrimp seedlings are very small and sensitive, their immune system has not yet fully developed and is extremely sensitive to changes in the external environment. Any slight change in water quality or invasion of pathogens may lead to high mortality. Because the immune system of shrimp seedlings is not yet mature, they are more susceptible to infection by bacteria, viruses and other pathogens. Once a disease occurs, it spreads rapidly and is difficult to treat, often resulting in a large number of deaths. Secondly, the breeding cycle of Penaeus vannamei is short, and it only takes 12 to 15 days to reach the specifications for sale on the market. In such a short period of time, it is difficult to cultivate stable biofloc with the function of improving water quality and inhibiting harmful Vibrio, let alone the treatment of Vibrio disease. It shows that the application of existing biofloc in Penaeus vannamei breeding is not suitable for Penaeus vannamei breeding, and it does not have the ability to treat Vibrio disease in seedlings.

[0007] Therefore, there is an urgent need for a method for treating Vibrio disease in shrimp fry using biofloc. Summary of the invention

[0008] The purpose of the present invention is to provide a method for treating Vibrio diseases of shrimp fry with biofloc, which can successfully treat Vibrio harveyi and Vibrio parahaemolyticus diseases of Penaeus vannamei fry, with significant effect, and can achieve the purpose of antibiotic-free shrimp seedling. At the same time, it can effectively treat and avoid the problems of slow growth and development, uneven size, and even mass death of shrimp fry caused by Vibrio parahaemolyticus and Vibrio harveyi diseases, and achieve the purpose of antibiotic-free shrimp seedling.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A method for treating Vibrio disease of Penaeus vannamei fry with biofloc, comprising the following steps:

[0011] (1) Preparation of raceway biofloc, the operation steps are as follows:

[0012] a. Three days before shrimp fry are released, brown sugar and compound bacteria A are added to the water of the runway pool;

[0013] b. On the 4th day, shrimp fry are released into the water of the runway pool;

[0014] c. In the first month of breeding, add brown sugar at 4 to 6 times the weight of the feed, and add 1 to 2 ppm of compound bacteria A and 0.5 to 1 ppm of nitrifying bacteria every day;

[0015] d. Within the second month of breeding, the water in the raceway pool is allowed to settle in an Inhofe conical tube, and the flocculent precipitate content is within 10 mL;

[0016] e. After 2 months of cultivation, the nitrite content suddenly dropped sharply and finally remained close to 0, indicating that the biofloc had formed a stable form; after precipitation in an Imhof conical tube and centrifugal dehydration, the precipitate A was obtained. The plate count method was used to detect that the Bacillus content was greater than 1.0×106 cfu / g precipitate A;

[0017] f. After the water from the raceway breeding pool is filtered, it is pumped into the empty nursery pool and aerated for standby use to obtain biological flocs in the nursery pool;

[0018] (2) Preparation of functional bioflocs, the operation steps are as follows:

[0019] Add the mixture containing the composite bacteria B to the biological flocs in the nursery pond prepared in step (1), and continue aerating. After the water in the runway pool is allowed to settle in an Inhofe conical tube, the content of the floc sediment is required to reach 10 to 15 mL. Take the floc sediment for testing. After centrifugal dehydration, obtain the sediment B. Use the plate count method to detect that the Bacillus content is greater than 1.0×107 cfu / g sediment B, and obtain functional biological flocs.

[0020] (3) Using the functional biofloc prepared in step (2) to treat Vibrio disease in Litopenaeus vannamei fry at the mysid, larval and fry stages, respectively.

[0021] Furthermore, the method for treating Vibrio disease in mysid shrimp and shrimp larvae specifically comprises the following steps:

[0022] a. In the mysid and shrimp larvae stage, the original pond treatment method is adopted. A drainage net frame is placed in the seedling pond, and the water level of the original seedling pond is lowered to 15-20cm by siphoning;

[0023] b. Pump the functional biofloc into the original seedling pond to increase the water level of the seedling pond by 15-20 cm, and maintain it for 24 hours without adding feed;

[0024] c. After 24 hours, pump the functional biofloc into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time until the water level in the seedling pond reaches 80 to 85 cm, and continue without adding feed;

[0025] d. After 48 hours, take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, normal feeding can be resumed; if the average content of Vibrio parahaemolyticus and Vibrio harveyi is 1.0×101cfu / g~1.0×103cfu / g, use a sieve to concentrate the functional biological flocs with a water level of 10~12cm every 6~8 hours (floc amount = floc content × bottom area × water level), add water and stir well, then pour into the seedling pool;

[0026] e. After 72 hours, take seedling samples for further testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. When the detected content is 1.0×100 cfu / g~1.0×101 cfu / g, the treatment is considered successful. If the detected content is 1.0×102 cfu / g~1.0×103 cfu / g, repeat step d until the treatment is successful.

[0027] Furthermore, the method for treating Vibrio disease at the seedling stage comprises the following steps:

[0028] a. In the large seedling stage, that is, when the shrimp seedlings reach 0.8-1 cm in length, the seedling collection and pond moving treatment method is adopted. A seedling collection frame is placed in the seedling collection pit beside the seedling pond. Water with a height of 30-35 cm is added to the seedling collection frame. The seedling collection net frame is connected to the drainage outlet of the seedling pond. The water in the seedling pond is drained into the seedling collection frame using a siphon until the water level in the seedling pond drops to 15-20 cm. During this process, the large seedlings are scooped into the seedling collection bucket every 5 minutes with a scoop net for temporary care;

[0029] b. After the seedlings are collected, disinfect the seedlings in the seedling collection barrel;

[0030] c. Pump the functional biofloc into the empty seedling pool until the water level is 40-45 cm, move the large seedlings from the seedling collection bucket into the seedling pool, and maintain without feeding for 24 hours;

[0031] d. After 24 hours, pump the functional biofloc into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time until the water level in the seedling pond reaches 80 to 85 cm, and continue without adding feed;

[0032] e. After 48 hours, take out the large seedlings to test the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, the feeding can be resumed without changing the water. If the detected content is 1.0×101cfu / g~1.0×103cfu / g, use a sieve to concentrate the functional biological flocs at a water level of 10~12cm every 6-8 hours (floc amount = floc content × bottom area × water level), add water and stir well, then pour into the seedling pool;

[0033] f. After 72 hours, take seedling samples to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, based on the test results, when the detected content is 1.0×100 cfu / g~1.0×101 cfu / g, it is considered a successful treatment. If the detected content is 1.0×102 cfu / g~1.0×103 cfu / g, repeat step e until the treatment is successful.

[0034] Furthermore, the composite bacteria A comprises Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:(1.2-1.4):(0.5-0.7).

[0035] Furthermore, the components of the mixture containing composite bacteria B and its usage in the biological flocs of the nursery pond are: 1-2 ppm composite bacteria B, 1-2 ppm vitamin C, 10-20 ppm baking soda, 5-10 ppm brown sugar, 2-3 ppm Vibrio harveyi phage freeze-dried powder, 2-3 ppm Vibrio parahaemolyticus phage freeze-dried powder, 1-2 ppm shrimp chips for seedlings, and 1-2 ppm Artemia slurry.

[0036] Furthermore, the composite bacteria B comprises Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of (1.5-1.7):1:(0.2-0.5).

[0037] Furthermore, after 2 months of cultivation in step (1), quicklime, dolomite powder or sodium bicarbonate is added to the aquaculture water according to the pH and total alkalinity test results. For every 10 ppm decrease in total alkalinity, 20 to 30 ppm is added. When it is detected that the nitrite content suddenly drops sharply and finally maintains close to 0, it indicates that the biofloc has formed a stable form.

[0038] Furthermore, after measuring with the Imhof conical tube in step (2), if the biofloc content is lower than 10 mL, continue to add 2 to 5 ppm of brown sugar and 10 to 20 ppm of baking soda.

[0039] Furthermore, in the step (1), the shrimp fry are selected to be thickened and have a body length of 1.5 to 2 cm, and the stocking density is 300 to 500 shrimp fry per cubic meter of water.

[0040] Furthermore, 3 days before the shrimp fry are released in step (1), 5-10 ppm of brown sugar and 2-3 ppm of composite bacteria A are added to the water of the runway pool.

[0041] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0042] (1) The long-term use of antibiotics to treat Vibrio parahaemolyticus and Vibrio harveyi in traditional shrimp seedling cultivation has caused bacterial resistance, increased antibiotic treatment doses, and the emergence of antibiotic abuse. The method of the present invention can perfectly solve this serious problem of treating Vibrio disease. The method of the present invention uses a mixture of composite bacteria A and composite bacteria B, which has a good therapeutic effect on Vibrio parahaemolyticus and Vibrio harveyi.

[0043] (2) In the present invention, as a component of the functional biofloc, it works synergistically with other components, has a low dosage and can proliferate in the nutritional environment provided by the biofloc, ultimately effectively treating Vibrio disease while greatly reducing costs.

[0044] (3) In conventional treatment of Vibrio parahaemolyticus and Vibrio harveyi in shrimp seedlings, bacteriophages are directly sprayed into the seedling pond, which will be affected by the water quality environment of the seedling pond. In the present invention, after bacteriophages are used as components of functional bioflocs, most of the original seedling pond water is discharged during treatment, and the synergistic inhibitory effect of bioflocs on Vibrio provides stable water quality conditions and environment, and the treatment effect is greatly enhanced.

[0045] (4) Conventional biofloc can only improve water quality and prevent Vibrio disease. Although it greatly reduces the probability of disease occurrence, it cannot effectively treat Vibrio disease after it occurs. Therefore, in most cases, it is used for breeding rather than seedling cultivation. However, the functional biofloc particles formed by the functional biofloc of the present invention are ingested by the seedlings and enter the body, thereby achieving the purpose of enhancing nutrition and treatment.

[0046] (5) The present invention provides an effective method for treating Vibrio disease in shrimp seedlings without the use of antibiotics, filling this technical gap and solving the problem of abuse of antibiotics in the seedling cultivation process. The method of the present invention has been used in practice and is a mature biofloc culture technology, which is of great significance in promoting the green and healthy development of the shrimp seedling cultivation industry. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The raw materials used in the present invention are all commercially available products:

[0049] Bacillus pumilus, product number: SHMCC D11043, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0050] Bacillus licheniformis, product number: SHMCC D10742, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0051] Bacillus subtilis, product number: SHMCC D10486, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0052] Bacillus mucilaginosus, product number: SHMCC D12019, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0053] Pichia guilliermong, product number: SHMCC D20457, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0054] Lactobacillus acidophilus, product number: SHMCC D24720, was purchased from Shanghai Baocang Microbiology Co., Ltd.

[0055] Imhof conical tube, brand: German VITLAB Imhof tube. After placing the sample in the Imhof conical tube for 15 minutes, directly read the vertical height of the easily sinking solid.

[0056] Example 1

[0057] This embodiment provides a method for treating Vibrio disease in Litopenaeus vannamei fry with biofloc, comprising the following steps:

[0058] (1) Preparation of raceway biofloc, the cultivation operation steps are as follows:

[0059] a. Three days before shrimp seedlings are released, the water in the runway pool is 60 cm, and an ejector is used to provide high dissolved oxygen and drive the aquaculture water to flow in a circular shape in the runway, with the flow rate controlled at 0.3 m / s; 8 ppm of brown sugar and 3 ppm of compound bacteria A are directly added to the runway pool water and mixed and then sprinkled. Add to the runway once a day for 3 consecutive days; the compound bacteria A includes Bacillus brevis, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:1.3:0.6.

[0060] b. Stocking shrimp fry: select shrimp fry that have been marked and have a body length of 1.5 to 2 cm, and the stocking density is 400 per cubic meter of water;

[0061] c. Within one month of breeding, add brown sugar at 5 times the weight of the feed, and add 2ppm of compound bacteria A and 0.5ppm of nitrifying bacteria every day, and test the ammonia nitrogen and nitrite content in the water every day;

[0062] d. In the second month of breeding, the pH, total alkalinity, ammonia nitrogen and nitrite content of the water body are tested daily, and the total weight of brown sugar added is determined by multiplying 12 times the ammonia nitrogen test value by the total water volume. The floc content is tested daily using an Inhofe conical tube to control the content of biological flocs within 10 mL;

[0063] e. After 2 months of breeding, quicklime was added to the breeding water according to the results of pH and total alkalinity test. For every 10ppm decrease in total alkalinity, 25ppm was added. When the nitrite content suddenly dropped sharply and finally remained close to 0, it indicated that biological flocs were formed. The water from the runway pool was taken and precipitated through an Inhofe conical tube and centrifuged to obtain floc precipitate. The plate count method was used to detect the Bacillus content. The standard GB20287-2006 was implemented. The content was greater than 1.0×10 6 cfu / g flocculent sediment;

[0064] f. The water from the runway breeding pond is filtered through a 120-mesh filter and then pumped into the empty nursery pond with a water level of 1 meter. Nanotube aeration is used for standby use to obtain biological flocs in the nursery pond.

[0065] (2) Preparation of functional bioflocs. The specific steps are as follows:

[0066] Then, a mixture containing composite bacteria B is added to the biological floc water of the nursery pond prepared in step (1), with the ratio of 1.5 ppm vitamin C, 14 ppm baking soda, 7 ppm brown sugar, 1.6 ppm composite bacteria B, 2.3 ppm Vibrio harveyi phage freeze-dried powder (Qingdao Noan Biotech Co., Ltd.), 2.5 ppm Vibrio parahaemolyticus phage freeze-dried powder (Qingdao Noan Biotech Co., Ltd.), 1.3 ppm shrimp chips for seedlings (Boshan Biotech Industrial Co., Ltd.), and 1.6 ppm Artemia slurry (Artemia slurry is hatched for 24 hours, collected, drained and slurried); the composite bacteria B comprises Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of 1.6:1:0.4. Except for Artemia slurry, all other materials need to be rubbed with a 100-mesh silk screen until the mesh size is less than 100, and then poured into the pool with water. Increase the aeration volume of nanotubes and continue aeration for 13 hours. The water body biological floc content is required to be 12mL. If the floc volume is less than 10mL after the Inhofe conical tube test, continue to add 3ppm brown sugar and 15ppm baking soda. Use the plate count method to test the Bacillus content, implement the standard GB20287-2006, and the content requirement is 1.0×10 7 cfu / g or above, and functional bioflocs are obtained.

[0067] (3) The treatment method for Vibrio disease in mysid shrimp and shrimp larvae includes the following steps:

[0068] a. Mysid shrimp and shrimp larvae stage, the seedlings are weak, the original pond treatment method is adopted, a drainage net frame with a mesh size of 60 is placed in the seedling pond, and several 63 diameter # Put the siphon tube into the net frame, use the siphon method to lower the water level of the original seedling pool to 18cm, increase or decrease the number of siphon tubes to control the drainage speed and prevent the seedlings from being sucked onto the drainage net;

[0069] b. Pump the functional biofloc into the original seedling pond to increase the water level of the seedling pond by 17 cm and maintain it for 24 hours without adding feed;

[0070] c. After 24 hours, pump the functional biofloc into the seedling pond every 6 hours, increasing the water level by 10 cm each time until the water level in the seedling pond reaches 80 cm, and continue without adding feed;

[0071] d. After 48 hours, observe the physical condition and vitality of the seedlings with naked eyes, take seedling samples for detection and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi, and arrange the next operation according to the results of vitality and Vibrio detection. If Vibrio is not detected, gradually resume the normal feeding; if the detected content is 1.0×10 1 cfu / g~1.0×10 3 cfu / g, and then collect the functional bioflocs at a water level of 10 cm with a 300-mesh silk screen every 6 hours, add water and stir well, and then pour into the seedling pond;

[0072] e. After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful. If the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure.

[0073] (4) The treatment method of Vibrio disease at the seedling stage includes the following steps:

[0074] a. In the large seedling stage, that is, when the shrimp seedlings are more than 0.8 cm long, the seedling collection and pond transfer treatment method is adopted. A seedling collection frame is placed in the seedling collection pit beside the seedling pond. 30 cm of water is added to the seedling collection frame. The seedling collection frame is connected to the drainage outlet of the seedling pond. At the beginning, three 63-diameter wires are used. # The water is drained into the seedling collection frame through the siphon, and when the water level in the seedling pool drops to 18 cm, the drainage pipe is unplugged. During this process, the large seedlings are scooped into a 500-liter seedling collection bucket every 5 minutes with a 40-mesh scoop net for temporary storage;

[0075] b. After the seedlings are collected, add 1.5ppm povidone-iodine into the 500L seedling collection barrel and disinfect for 2 minutes;

[0076] c. Pump 40cm functional biofloc into the empty seedling pool, then move the large seedlings from the seedling collection bucket into it, and maintain without feeding for 24 hours;

[0077] d. After 24 hours, pump the functional biofloc into the seedling pond every 6 hours, increasing the water level by 10 cm each time until the water level in the seedling pond reaches 80 cm, and continue without adding feed;

[0078] e. After 48 hours, take the large seedlings to observe their physical condition and vitality with the naked eye, take seedling samples to test and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi, and arrange the next operation according to the results of vitality and Vibrio test. If Vibrio is not detected, you can gradually resume feeding without changing water; if the detected content is 1.0×10 1 cfu / g~1.0×10 3cfu / g, and then collect the functional bioflocs at a water level of 10 cm with a 300-mesh silk screen every 6 hours, add water and stir well, and then pour into the seedling pond;

[0079] f. After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure.

[0080] Example 2

[0081] The difference between this embodiment and embodiment 1 is that the composite bacteria A comprises Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:1.2:0.5, and the composite bacteria B comprises Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of 1.7:1:0.2.

[0082] Comparative Example 1

[0083] The difference between this comparative example and Example 1 is that the composite bacteria A comprises Pichia guilleris, Bacillus subtilis and Lactobacillus acidophilus in a weight ratio of 1:1.3:0.6, and the composite bacteria B comprises Bacillus subtilis, Pichia guilleris and Lactobacillus acidophilus in a weight ratio of 1.6:1:0.4.

[0084] Comparative Example 2

[0085] The difference between this comparative example and Example 1 is that the composite bacteria A comprises Pichia guilleriemoniae, Bacillus subtilis and Lactobacillus acidophilus in a weight ratio of 1:1.3:0.6.

[0086] Comparative Example 3

[0087] The difference between this comparative example and Example 1 is that the ratio of the composite bacteria A and the composite bacteria B is different.

[0088] The composite bacteria A comprises Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1.3:0.6:1. The composite bacteria B comprises Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of 0.4:1.6:1.

[0089] Comparative Example 4

[0090] This comparative example provides a method for treating Vibrio disease of Penaeus vannamei fry with biofloc, comprising the following steps:

[0091] (1) Preparation of biofloc in the nursery pond, the cultivation operation steps are as follows:

[0092] a. Three days before shrimp seedlings are released, the water in the runway pool is 60 cm, and an ejector is used to provide high dissolved oxygen and drive the aquaculture water to flow in a circular shape in the runway, with the flow rate controlled at 0.3 m / s; 8 ppm of brown sugar and 3 ppm of powdered composite bacteria A are directly added to the runway pool water and mixed and then sprinkled. Add to the runway once a day for 3 consecutive days; the composite bacteria A includes Bacillus brevis, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:1.3:0.6.

[0093] b. Stocking shrimp fry: select shrimp fry that have been marked and have a body length of 1.5 to 2 cm, and the stocking density is 400 per cubic meter of water;

[0094] c. Within one month of breeding, add brown sugar at 5 times the weight of the feed, and add 2ppm of composite bacteria and 0.5ppm of nitrifying bacteria every day, and test the ammonia nitrogen and nitrite content in the water every day;

[0095] d. In the second month of breeding, the pH, total alkalinity, ammonia nitrogen and nitrite content of the water body are tested daily, and the total weight of brown sugar added is determined by multiplying 12 times the ammonia nitrogen test value by the total water volume. The floc content is tested daily using an Inhofe conical tube to control the content of biological flocs within 10 mL;

[0096] e. After 2 months of breeding, add quicklime, dolomite powder or sodium bicarbonate to the breeding water according to the pH and total alkalinity test results. For every 10ppm decrease in total alkalinity, add 25ppm. When the nitrite content suddenly drops sharply and finally remains close to 0, it indicates that bioflocs have formed. Take the biofloc sediment in the runway pool and use the plate count method to detect the Bacillus content. The standard GB20287-2006 is implemented, and the content requirement is 1.0×10 6 cfu / g or more;

[0097] f. The water from the runway breeding pond is filtered through a 120-mesh filter and then pumped into the empty nursery pond with a water level of 1 meter. Nanotube aeration is used for standby use to obtain biological flocs in the nursery pond.

[0098] (2) The treatment method for Vibrio disease in mysid shrimp and shrimp larvae includes the following steps:

[0099] a. Mysid shrimp and shrimp larvae stage, the seedlings are weak, the original pond treatment method is adopted, a drainage net frame with a mesh size of 60 is placed in the seedling pond, and several 63 diameter # Put the siphon tube into the net frame, use the siphon method to lower the water level of the original seedling pool to 18cm, increase or decrease the number of siphon tubes to control the drainage speed and prevent the seedlings from being sucked onto the drainage net;

[0100] b. Pump the prepared functional bioflocs together with the pond water into the original seedling pond, increase the water level by 17 cm, and maintain it for 24 hours without adding any feed;

[0101] c. After 24 hours, pump the functional bioflocs together with the pond water into the seedling pond every 6 hours, increasing the water level by 10 cm each time until the water level in the seedling pond reaches 80 cm, and continue without adding feed;

[0102] d. After 48 hours, observe the physical condition and vitality of the seedlings with naked eyes, take seedling samples for detection and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi, and arrange the next operation according to the results of vitality and Vibrio detection. If Vibrio is not detected, gradually resume the normal feeding; if the detected content is 1.0×10 1 cfu / g~1.0×10 3 cfu / g, and then collect the functional bioflocs at a water level of 10 cm with a 300-mesh silk screen every 6 hours, add water and stir well, and then pour into the seedling pond;

[0103] e. After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g, if the content is detected to be 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure.

[0104] (3) The treatment method of Vibrio disease at the seedling stage includes the following steps:

[0105] a. In the large seedling stage, that is, when the shrimp seedlings reach a length of 0.8 to 1 cm or more, the seedling collection and pond transfer treatment method is adopted. A seedling collection frame is placed in the seedling collection pit beside the seedling pond. 30 cm of water is added to the seedling collection frame. The seedling collection frame is connected to the drainage outlet of the seedling pond. At the beginning, three 63-diameter wires are used. # The water is drained into the seedling collection frame through the siphon, and when the water level in the seedling pool drops to 18 cm, the drainage pipe is unplugged. During this process, the large seedlings are scooped into a 500-liter seedling collection bucket every 5 minutes with a 40-mesh scoop net for temporary storage;

[0106] b. After the seedlings are collected, add 1.5ppm povidone-iodine into the 500L seedling collection barrel and disinfect for 2 minutes;

[0107] c. Pump 40cm functional biofloc into the empty seedling pool, then move the large seedlings from the seedling collection bucket into it, and maintain without feeding for 24 hours;

[0108] d. After 24 hours, pump the functional biofloc into the seedling pond every 6 hours, increasing the water level by 10 cm each time until the water level in the seedling pond reaches 80 cm, and continue without adding feed;

[0109] e. After 48 hours, take the large seedlings to observe their physical condition and vitality with the naked eye, take seedling samples to test and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi, and arrange the next operation according to the results of vitality and Vibrio test. If Vibrio is not detected, you can gradually resume feeding without changing water; if the detected content is 1.0×10 1 cfu / g~1.0×10 3 cfu / g, and then use a 300-mesh silk screen to concentrate the functional bioflocs at a water level of 10 cm every 6 hours, add water and stir well before pouring into the seedling pond;

[0110] f. After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure.

[0111] Comparative Example 5

[0112] This comparative example uses bacteriophage (Vibrio parahaemolyticus phage and Vibrio harveyi phage in a weight ratio of 1:1, purchased from Qingdao Noan Biotech Co., Ltd.) for treatment. The dosage of phage in the seedling pond is 2.5ppm, twice a day, without draining the seedling pond water, and splashing it once in the original pond in the morning and afternoon for a total of 3 days.

[0113] Performance Testing

[0114] 1. At the Jijia base of Guangdong Haiwei Agricultural Group, the methods of Examples 1-2 and Comparative Examples 1-3 were used to treat Vibrio disease in the seedlings in the nursery pools that had been confirmed to be infected with Vibrio. Each group had 3 parallels (3 nursery pools). D1, D2, and D3 were the average Vibrio content from the first day to the third day after treatment. After the treatment, according to the test results, when the average Vibrio content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment was considered successful if the average Vibrio content was 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure. The results are shown in Table 1.

[0115] Table 1. Treatment results

[0116]

[0117]

[0118] The experimental results show that: in Example 1-2, the method of the present invention has a high success rate for treating seedlings infected with Vibrio for 72 hours through the specific ratio of composite bacteria A and composite bacteria B. In particular, the success rate of Example 1 is higher than 90%. However, in Comparative Examples 1-3, the types or ratios of composite bacteria A and composite bacteria B are changed, and the success rates of treatment are reduced to varying degrees. In Comparative Example 1, the species of composite bacteria A and composite bacteria B are changed, and the treatment fails. In Comparative Example 2, the species of composite bacteria A is changed, and the treatment fails partially, indicating that the composite bacteria A and composite bacteria B of the present invention play a synergistic therapeutic role. In Comparative Example 3, the ratio of composite bacteria A and composite bacteria B is changed, and the therapeutic effect decreases, indicating that the method of the present invention can only achieve a higher treatment success rate by using the synergistic effect of bacteria of specific types and ratios.

[0119] 2. The shrimp seedling treatment application was carried out for two years at the Jijia base of Guangdong Haiwei Agricultural Group. From March to May 2023, Vibrio treatment experiments were carried out using Example 1, Comparative Example 4 and Comparative Example 5.

[0120] After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g was considered as treatment failure.

[0121] The treatment results are shown in Tables 2 and 3.

[0122] Table 2. Treatment results of comparative example 4 from March to May 2023

[0123]

[0124] Table 3. Treatment results of comparative example 5 from March to May 2023

[0125]

[0126] Table 4. Treatment results of Example 1 from March to October 2024

[0127]

[0128]

[0129] Among them, the Vibrio content in the table is the average content of Vibrio parahaemolyticus and Vibrio harveyi in the total infected seed pools, D3 and D4 refer to the average content of Vibrio parahaemolyticus and Vibrio harveyi in the infected seed pools on the third and fourth days of treatment, respectively, the number of pools represents the number of seed pools that failed treatment, the cure rate is the number of seed pools that were successfully treated, and the survival rate is the average survival rate of the successfully cured seed pools.

[0130]

[0131] The results show that from March to May 2023, a total of 30 shrimp seedling ponds were treated using the method of comparative example 4. Before treatment, the Vibrio content was 5.6×10 4 cfu / g, after treatment, the D3 Vibrio content was 5.5×10 2 cfu / g, D4 Vibrio content was 2.0×10 3 cfu / g, cure rate 68.1%, seedling survival rate 62.9%;

[0132] From March to May 2023, a total of 29 shrimp seedling ponds were treated using the method of comparative example 5. Before treatment, the Vibrio content was 6.8×10 4 cfu / g, after treatment, the D3 Vibrio content was 6.8×10 2 cfu / g, D4 Vibrio content was 1.5×10 1 cfu / g, cure rate 81.9%, seedling survival rate 73.5%;

[0133] From March to October 2024, a total of 116 shrimp seedling pools were treated using the method of Example 1. Before treatment, the Vibrio content was 3.8×10 5 cfu / g, after treatment, the content of D3 Vibrio decreased to 9.0×10 2 cfu / g, and the content of D4 Vibrio decreased to 1.5×10 1 cfu / g, the cure rate was 94.7%, and the seedling survival rate was 88.7%.

[0134] The results show that the content of Vibrio decreased to varying degrees after the three treatments were used. The cure rate and survival rate of Comparative Example 4 were the worst. The effect of phage treatment using Comparative Example 5 was better, but there were differences in the treatment effects of batches in different months, and the treatment effect was unstable. The effect of using Example 1 to treat Vibrio was the best, the content of Vibrio decreased exponentially, the cure rate and survival rate were high, and there was no significant difference between batches in different months, indicating that the use of functional bioflocs to treat Vibrio disease in juvenile Penaeus vannamei was effective.

[0135] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for treating Vibrio disease of Penaeus vannamei fry with biofloc, characterized in that: The following steps are involved: (1) Preparation of raceway biofloc, the operation steps are as follows: a. Three days before shrimp fry are released, brown sugar and compound bacteria A are added to the water of the runway pool; b. On the 4th day, shrimp fry are released into the water of the runway pool; c. In the first month of breeding, add brown sugar to the water of the raceway pool, and add 1-2ppm of compound bacteria A and 0.5-1ppm of nitrifying bacteria every day; d. Within the second month of breeding, the water in the raceway pool is allowed to settle in an Inhofe conical tube, and the flocculent precipitate content is within 10 mL; e. After 2 months of cultivation, when the nitrite content suddenly dropped sharply, the water from the runway pool was taken, precipitated in an Inhofe conical tube, and centrifuged to obtain precipitate A. The plate count method was used to detect the Bacillus content, which was greater than 1.0×10 6 cfu / g sediment A; f. After the water from the raceway breeding pool is filtered, it is pumped into the empty nursery pool and aerated for standby use to obtain biological flocs in the nursery pool; (2) Preparation of functional bioflocs, the operation steps are as follows: The mixture containing the composite bacteria B is added to the biological flocs prepared in step (1) and aerated continuously. After the water in the runway pool is allowed to settle in an Inhofe conical tube, the content of the floc sediment is required to reach 10-15 mL. The floc sediment is taken for testing. After centrifugal dehydration, the sediment B is obtained. The Bacillus content is greater than 1.0×10 7 cfu / g sediment B, and functional bioflocs were obtained; (3) Using the functional biofloc prepared in step (2) to treat Vibrio disease in Litopenaeus vannamei fry at the mysid, larval and fry stages, respectively.

2. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: The treatment of Vibrio disease in mysid shrimp and shrimp larvae includes the following steps: a. In the mysid and shrimp larvae stage, the original pond treatment method is adopted. A drainage net frame is placed in the seedling pond to lower the water level of the original seedling pond to 15-20cm; b. Pump the functional biofloc into the original seedling pond to increase the water level of the seedling pond by 15-20 cm, and maintain it for 24 hours without adding feed; c. After 24 hours, pump the functional biofloc into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time until the water level in the seedling pond reaches 80 to 85 cm, and continue without adding feed; d. After 48h, take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, the routine feeding can be resumed. If the average content of Vibrio parahaemolyticus and Vibrio harveyi is 1.0×10 1 cfu / g~1.0×10 3 cfu / g, adding functional biofloc to the seedling pond; e. After 72 hours, take seedling samples for further testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. When the detected content is 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detected content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g, repeat step d until the treatment is successful.

3. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: Treatment of Vibrio disease at the seedling stage includes the following steps: a. In the stage of large seedlings, the seedling collection and pool transfer treatment method is adopted. A seedling collection frame is placed in the seedling collection pit beside the seedling pool, water is added to the seedling collection frame, and the seedling collection net frame is connected to the drainage outlet of the seedling pool. The water in the seedling pool is drained into the seedling collection frame until the water level in the seedling pool drops to 15-20cm. During this process, the large seedlings are scooped into the seedling collection bucket with a scoop net every 5 minutes for temporary care; b. After the seedlings are collected, disinfect the seedlings in the seedling collection barrel; c. Pump the functional biofloc into the empty seedling pool until the water level is 40-45 cm, move the large seedlings from the seedling collection bucket into the seedling pool, and maintain without feeding for 24 hours; d. After 24 hours, pump the functional biofloc into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time until the water level in the seedling pond reaches 80 to 85 cm, and continue without adding feed; e. After 48 hours, take the seedlings to test the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, feed can be resumed without changing water. If the detected content is 1.0×10 1 cfu / g~1.0×10 3 cfu / g, adding functional biofloc to the seedling pond every 6-8h; f. After 72 hours, the seedling samples were taken to continue testing the average content of Vibrio parahaemolyticus and Vibrio harveyi. After the treatment, according to the test results, when the detected content was 1.0×10 0 cfu / g~1.0×10 1 cfu / g, the treatment is considered successful, if the detection content is 1.0×10 2 cfu / g~1.0×10 3 cfu / g, repeat step e until the treatment is successful.

4. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: The composite bacteria A comprises Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:(1.2-1.4):(0.5-0.7).

5. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: The components of the mixture containing the composite bacteria B and the usage amount thereof in the biological flocs of the nursery pond are: 1-2 ppm of composite bacteria B, 1-2 ppm of vitamin C, 10-20 ppm of baking soda, 5-10 ppm of brown sugar, 2-3 ppm of Vibrio harveyi phage freeze-dried powder, 2-3 ppm of Vibrio parahaemolyticus phage freeze-dried powder, 1-2 ppm of shrimp chips for seedlings, and 1-2 ppm of Artemia slurry.

6. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 5, characterized in that: The composite bacteria B comprises Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of (1.5-1.7):1:(0.2-0.5).

7. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: After 2 months of cultivation in the step (1), quicklime, dolomite powder or sodium bicarbonate is added to the aquaculture water according to the pH and total alkalinity test results. For every 10 ppm decrease in total alkalinity, 20 to 30 ppm is added. When it is detected that the nitrite content suddenly drops sharply and finally maintains close to 0, it indicates that the biofloc has formed a stable form.

8. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: After measuring with the Imhof conical tube in step (2), if the biofloc content is less than 10 mL, continue to add 2 to 5 ppm of brown sugar and 10 to 20 ppm of baking soda.

9. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: In the step (1), the shrimp fry are selected after being thickened, with a body length of 1.5 to 2 cm, and the stocking density is 300 to 500 per cubic meter of water.

10. The method for treating Vibrio disease of Penaeus vannamei fry with biofloc according to claim 1, characterized in that: Three days before the shrimp fry are released in step (1), 5-10 ppm of brown sugar and 2-3 ppm of composite bacteria A are added to the water in the runway pool.

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