Manufacturing method of retention type ichthyophthiriasis immune offspring seeds of Malus salicina

Abstract

The invention relates to a production method of retention type ichthyophthiriasis immune fry of Malus salicina, and belongs to the technical field of aquaculture. The technical problem that a large number of fishes or all fishes die due to the fact that retention type Malus salmonilio is extremely prone to being infected with ichthyophthiriasis under the normal culture condition is solved. The preparation method comprises the following steps: firstly, putting healthy retention type margarus salicina fries into a pond, and feeding the healthy retention type margarus salicina fries; then putting fish fries of the retention type Malus salicina infected with ichthyophthiriasis, and feeding the fish fries of the retention type Malus salicina; then the salinity of the pond is adjusted to obtain cured retention type Malus salicina, and the cured retention type Malus salicina is the ichthyophthiriasis immune fry of the retention type Malus salicina. The retention type ichthyophthiriasis immune offspring seed of the Malus salicina obtains an antibody for resisting ichthyophthiriasis infection, so that the offspring seed cannot be infected with ichthyophthiriasis.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture vaccines, and in particular relates to a method for preparing an Ichthyophthirius spp. vaccine for stranded Masson salmon. Background Art

[0002] The stranded masu salmon (Oncorhynchusmasou) belongs to the Salmoniformes, Salmonidae, and Masu salmon genus. It is also called cherry trout, cherry salmon, mountain trout, etc. It is a precious economic fish in my country. The fish has the advantages of graceful body, elegant and bright colors, tender and fresh meat, fast growth rate, and strong disease resistance, and is deeply loved by the masses. Under the condition that the breeding cost is almost the same, the economic benefit of this fish is 5 times that of rainbow trout. The current market price is far higher than that of rainbow trout, and the general selling price per kilogram can reach more than 100 yuan. The fish roe of the stranded masu salmon is even more precious food, and the selling price per kilogram can reach more than 5,000 yuan. It can be seen that the stranded masu salmon has a unique market advantage. It not only provides a high-quality breeding variety for the adjustment of fish structure, but also will have a positive impact and promote the development of regional fishery economy. In recent years, the seed breeding of stranded masu salmon has made a breakthrough. The weight of commercial fish in the two-year breeding of adult fish can generally reach 1000-1500 grams per tail.

[0003] However, since the stranded salmon is a cold-water fish, its optimal living water temperature is 10-20℃, and the optimal growth water temperature is 15-18℃. It is very susceptible to Ichthyophthirius multifiliis disease under normal breeding conditions.

[0004] Ichthyophthirius is a disease caused by Ichthyophthirius gracilis invading the skin and gills of fish in large numbers. Sick fish become restless, swim in groups around the edge of the pond, and constantly rub against other objects or jump out of the water. The fish become thin and black, the gills become congested, and they have difficulty breathing. Soon, they die in large numbers. The pathogen's optimal temperature for reproduction is 15-25℃, so it is very easy to infect stranded salmon and cause large-scale deaths of fish, sometimes even all of them. Ichthyophthirius gracilis is extremely difficult to cure and is extremely harmful when breeding stranded salmon.

[0005] Therefore, according to the physiological characteristics and living habits of the stranded type salmon, combined with the physiological characteristics and living habits of Ichthyophthirius scoparia, producing seedlings of the stranded type salmon that are immune to Ichthyophthirius scoparia is an effective measure to prevent farmed stranded type salmon from being infected with Ichthyophthirius scoparia. Summary of the invention

[0006] The invention aims to solve the technical problem that the stranded type Masson salmon is very easy to be infected with Ichthyophthirius avium disease under normal breeding conditions, resulting in the death of a large number of fish or all of them, and provides a method for preparing an Ichthyophthirius avium disease-free vaccine seed of the stranded type Masson salmon.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows.

[0008] The present invention provides a method for preparing a vaccine seed of Ichthyophthirius spp. for a retained type mackerel salmon, comprising the following steps:

[0009] Step 1: disinfect, fill and oxygenate several ponds;

[0010] Step 2, putting healthy fry of the stranded type masu salmon into each pond, raising them for a period of time, then putting fry of the stranded type masu salmon infected with Ichthyophthirius avium into each pond, and then raising them until all the fry of the healthy stranded type masu salmon are infected with Ichthyophthirius avium;

[0011] The number of the fry of the stranded type salmon infected with Ichthyophthirius scoparia is 0.8%-1.2% of the number of the fry of the healthy stranded type salmon;

[0012] Step 3, adding raw salt to each pond until the salinity is 0.4wt%-0.6wt%, increasing the salinity to 0.9wt%-1.1wt% on the second day, and increasing the salinity to 1.4wt%-1.6wt% on the third day;

[0013] Step 4, maintaining the salinity at 1.4wt%-1.6wt% and raising the fish fry of the stranded type salmon infected with Ichthyophthirius avium until all the fish fry are cured, thereby obtaining the Ichthyophthirius avium vaccine-free strain of the stranded type salmon.

[0014] Preferably, in step 1, the pond is disinfected with a trichloroisocyanuric acid solution and oxygenated with an aerator.

[0015] Preferably, in step 1, the water injection depth is 0.5-1.0 meter.

[0016] Preferably, in step 2, the volume of the pond is 15m 3 According to the calculation, the number of the healthy retained type masu salmon fry is 45,000 to 50,000.

[0017] Preferably, in step 2, the body lengths of the healthy fry of the stranded salmon and the fry of the stranded salmon infected with Ichthyophthirius scoparia are independently 6-8 cm.

[0018] Preferably, in step 2, after 3 days of rearing, fry of stranded type salmon infected with Ichthyophthirius gracilis are placed in each pond.

[0019] Preferably, in step 2, the fry of the stranded type salmon infected with Ichthyophthirius scoparia is 1.0% of the fry of the healthy stranded type salmon.

[0020] Preferably, in step 3, raw salt is added to the pond to a salinity of 0.5 wt %, the salinity is increased to 1.0 wt % on the second day, and the salinity is increased to 1.5 wt % on the third day.

[0021] Preferably, in step 4, the salinity is maintained at 1.4wt%-1.6wt% for 10 days.

[0022] Preferably, the method further comprises step 5, transferring the cured stranded type Salmon to a culture pond for culture, so as to obtain Ichthyophthirius spp. vaccine-free strains of stranded type Salmon.

[0023] The principle of the present invention is:

[0024] 1. Studies have shown that the retentive salmon can survive in water with a salinity of 2wt% for more than 15 days when the salinity is gradually increased.

[0025] 2. The growth cycle of Ichthyophthirius punctatus is divided into three main stages: cyst stage, predator stage and trophozoite stage.

[0026] Cyst stage: After the adult Ichthyophthirius spp. leaves the fish body, it will form a cyst to completely wrap itself up, and generally slowly sink to the bottom of the fish pond. Inside the cyst, the worm will divide, and after 8-9 binary fissions, 512-1024 merozoites will be formed. When the merozoites develop into individuals with swimming and infectious abilities, they will break out of the cyst and enter the next stage.

[0027] Predatory stage: The Ichthyophthirius punctatus in the predatory stage can swim in the water for about 4 days. During this period, it will look for a host fish and attach to its body surface. Then, within 5 minutes, it will break through the defense of the fish's epidermal cells, drill into the epithelial cells and parasitize, entering the trophozoite stage.

[0028] Trophozoite stage: After invading the fish's epidermis, Ichthyophthirius spp. begins to use the fish's internal cells as nutrition to support its own development. The development time is about 7 days at around 20°C. It will not fall off the fish body until it matures, re-forms a cyst, and enters the next development cycle.

[0029] The reason why Ichthyophthirius is difficult to treat is that when Ichthyophthirius divides in the cyst, the drug cannot enter the cyst. It is only sensitive to the drug when it swims to find a host. When Ichthyophthirius parasitizes under the fish skin, it is difficult for the drug to penetrate due to the barrier of the fish skin. Therefore, in order to completely cure Ichthyophthirius, the diseased fish must be exposed to at least one Ichthyophthirius life cycle in water with a drug concentration that can effectively kill Ichthyophthirius. When the water temperature is 15-25℃, the life cycle of Ichthyophthirius is 4 days. At low temperatures such as below 7℃, the life cycle of Ichthyophthirius takes more than 5 weeks. At high temperatures such as above 30℃, Ichthyophthirius cannot develop.

[0030] Studies have shown that Ichthyophthirius punctatus cannot survive when the water temperature is 15-25°C and the salinity of the water is higher than 0.5wt%.

[0031] 3. Studies have shown that stranded salmon that have been infected with Ichthyophthirius avium will not be infected with Ichthyophthirius avium again.

[0032] Therefore, based on the characteristics that the stranded type salmon, as a salmonid fish, has a wide adaptability range and fast adaptability to the salinity of the culture water body, combined with the fact that the freshwater Ichthyophthirius cannot tolerate high salinity and that fish will produce antibodies after recovering from the infection with Ichthyophthirius and will not be infected with Ichthyophthirius again, the present invention first artificially infects the stranded type salmon with Ichthyophthirius disease, and then cultures the stranded type salmon infected with Ichthyophthirius disease in a water body with high salinity that it can tolerate (while Ichthyophthirius cannot tolerate this salinity), so that the stranded type salmon fry can obtain antibodies to resist Ichthyophthirius infection while curing the Ichthyophthirius disease, and will never be infected with Ichthyophthirius disease again.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The method for preparing the Ichthyophthirius spp. vaccine-free seed of the stranded type salmon can enable the stranded type salmon fry to obtain antibodies that resist Ichthyophthirius spp. infection, and the stranded type salmon that has been infected with Ichthyophthirius spp. will not be infected with Ichthyophthirius spp. again, thus completely avoiding the problem that the stranded type salmon is very likely to be infected with Ichthyophthirius spp. during the breeding process, thereby damaging the breeding yield of the stranded type salmon. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0036] Figure 1 The figure shows the survival of the test fish in the infection experiment continued for 20 days in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to further understand the present invention, preferred embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0038] The method for preparing the Ichthyophthirius spp. vaccine-free seed of the retained type Masson salmon of the present invention comprises the following steps:

[0039] Step 1: disinfect, fill and oxygenate several ponds;

[0040] Step 2, putting healthy fry of the stranded type masu salmon into each pond, and after raising them for a period of time, putting fry of the stranded type masu salmon infected with Ichthyophthirius avium into each pond, and then raising them until all the fry of the healthy stranded type masu salmon are infected with Ichthyophthirius avium and the fry begin to gather together and are unwilling to eat;

[0041] Among them, the number of fry of stranded salmon infected with Ichthyophthirius was 0.8%-1.2% of the number of fry of healthy stranded salmon;

[0042] Step 3, adding raw salt to each pond until the salinity is 0.4wt%-0.6wt%, increasing the salinity to 0.9wt%-1.1wt% on the second day, and increasing the salinity to 1.4wt%-1.6wt% on the third day, at which time the symptoms of Ichthyophthirius punctatus begin to significantly alleviate;

[0043] Step 4, maintaining the salinity at 1.4wt%-1.6wt% and raising the fish until all the fish fry infected with Ichthyophthirius avium are cured, at which time antibodies have been produced in the bodies of the cured fish fry, thereby obtaining Ichthyophthirius avium vaccine-free strains of fish fry.

[0044] In some embodiments, the pond can be a single pond for breeding, or multiple ponds can be used for breeding at the same time. The pond can be a greenhouse cement pond or a glass pond. The pond is preferably 5 meters long, 2 meters wide, and 1.5 meters high, which is convenient for operation and management, but is not limited thereto.

[0045] In some embodiments, 20 mg / L trichloroisocyanuric acid solution is used for disinfection, and the pond is filled with water 3 days after disinfection to a depth of 0.5-1.0 meters. Each pond is equipped with a 0.2 kW microporous aerator for oxygenation.

[0046] In some embodiments, the volume of the pond is 15m 3 It is estimated that the number of healthy retained salmon fry released into each pond is 45,000 to 50,000.

[0047] In some embodiments, the body lengths of healthy stranded salmon fry and stranded salmon fry infected with Ichthyophthirius avium are independently 6-8 cm; after 3 days of breeding, stranded salmon fry infected with Ichthyophthirius avium are put into each pond; the stranded salmon fry infected with Ichthyophthirius avium is 1.0% of the healthy stranded salmon fry; under normal circumstances, all the fry in the pond will be infected with Ichthyophthirius avium within 3 days.

[0048] In some embodiments, raw salt is added to the pond to a salinity of 0.5 wt %, the salinity is increased to 1.0 wt % on the second day, and the salinity is increased to 1.5 wt % on the third day.

[0049] In some embodiments, the salinity is maintained at 1.4 wt%-1.6 wt% for 10 days, and all the fry of the stranded type salmon infected with Ichthyophthirius scoparia are cured.

[0050] In some embodiments, the method further includes step 5, transferring the cured stranded type masu salmon fry into a normal breeding pond for intensive cultivation, so that the fry can recover to normal as soon as possible and improve their immune ability, and then they can be sold or continued to be raised. The cured stranded type masu salmon fry already has antibodies to Ichthyophthirius spp. and will not be infected with Ichthyophthirius spp. again.

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0052] In the following examples, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, devices, instruments, equipment, etc. used in the following examples can be obtained from commercial sources.

[0053] Example 1

[0054] From August to October 2024, the fry of stranded salmon were immunized against Ichthyophthirius microcarpa at the Changchun Aquatic Products Quality and Safety Testing Center, with good results.

[0055] 1. Breeding conditions

[0056] The fish were temporarily kept in a greenhouse glass tank, the length × width × height of which was 1.5 m × 0.8 m × 0.8 m. After strict disinfection, the glass tank was filled with water to a depth of 0.5 m.

[0057] 2. Preparation

[0058] The glass tank has a matching filtration and oxygenation system.

[0059] 3. Release seedlings

[0060] After the glass tank was filled with water for 3 days, 3,000 healthy and well-proportioned 7 cm long fry of the resident type salmon were placed in it and then raised.

[0061] 4. Ichthyophthirius infection

[0062] After 3 days of feeding, 30 fry of the stranded type of salmon infected with Ichthyophthirius quinquefasciatus with a body length of 7 cm and a relatively uniform size were put into the water.

[0063] 5. Kill Ichthyophthirius chinensis

[0064] After 3 days, it was found that all the healthy fry of the retained type salmon in the glass tank were infected with Ichthyophthirius spp., and the fry began to gather together and refused to eat. Then raw salt was added to the culture pond to make the salinity of the water in the culture pond 0.5wt%. The salinity was increased to 1.0wt% on the second day, and to 1.5wt% on the third day. After 3 days, the symptoms of Ichthyophthirius spp. began to be significantly alleviated.

[0065] 6. Feeding of fry

[0066] The fish were then kept in a culture pond with a salinity of 1.5 wt% for 10 days until the Ichthyophthirius spp. disease was completely cured. The cured fish fry of the retained type salmon were then transferred to a freshwater fish tank for further breeding.

[0067] 7. Serum antibody test

[0068] At 1, 2, 3, 4, 8 and 16 weeks after the fry of the stranded type salmon were cured, blood was collected from the immunized group and the control group, and serum was separated. The immunized group, i.e., the fry of the stranded type salmon cured as mentioned above, and the control group, i.e., the fry of healthy stranded type salmon that had not been infected with Ichthyophthirius microtyphus, were tested for serum antibodies, respectively.

[0069] First, the collected serum was diluted twice with freezing saline, 100 μL of the sample to be tested was added to each 96-well plate, and then 100 μL of Ichthyophthirius punctatus predator solution (containing 30-40 predators) was dropped into each well. The plate was placed in a 23°C environment and observed for 4 hours. The highest dilution factor of the serum in the well plate where all the insects were immobilized and killed was the titer of the serum antibody to be tested.

[0070] The test results are shown in Table 1. It can be seen from Table 1 that the immune serum produced by the immunized group can immobilize the isolated Ichthyophthirius larvae for up to 8 weeks, while the serum collected from the control group cannot immobilize the isolated Ichthyophthirius larvae.

[0071] Table 1 Agglutination titer of serum immune-immobilizing antibodies in the immunized group and the control group at different time periods

[0072]

[0073] In Table 1, "-" indicates that the test serum failed to completely stop the added predators within 4 hours, and "+" indicates that the test serum could completely stop the added predators within 4 hours.

[0074] 8. Continued infection experiment and immune protection determination

[0075] After the cured stranded type machu salmon fry were cultured for one month, they were placed in a fish tank with a length × width × height = 0.6 m × 0.25 m × 0.5 m, with 100 fish in each fish tank. A total of 6 fish tanks were set as immunization groups. The fish tanks were numbered A1, A2, A3, A4, A5, and A6, and each fish tank had a filtration and oxygenation system.

[0076] At the same time, the fry of the stranded type of Masson salmon that were not infected with Ichthyophthirius multifiliis were also placed in a fish tank of length × width × height = 0.6m × 0.25m × 0.5m, with 100 fish in each fish tank. A total of 6 fish tanks were set up as the control group. The fish tanks were numbered B1, B2, B3, B4, B5, and B6. Each fish tank had a filtration and oxygenation system.

[0077] After the above fry were temporarily kept for 3 days, 5 fry of the stranded type of Masu salmon infected with Ichthyophthirius were placed in each fish tank. After 5 days, the fry in the 6 fish tanks of group A were not infected with Ichthyophthirius and ate normally. The fry in the 6 fish tanks of group B were all infected with Ichthyophthirius, and the fry did not eat and were in a dying state. The statistical results are shown in Table 2.

[0078] Table 2 Statistical results of infection experiments

[0079] Group A 1 2 3 4 5 6 Infect Not infected Not infected Not infected Not infected Not infected Not infected Group B 1 2 3 4 5 6 Infect Infect Infect Infect Infect Infect Infect

[0080] From the above statistical results, it can be seen that the stranded salmon in the 6 fish tanks of Group A that had been immunized were not infected with Ichthyophthirius microcarpa; the stranded salmon in the 6 fish tanks of Group B that had not been immunized were all infected with Ichthyophthirius microcarpa.

[0081] After the infection experiment started, the average survival of group A and group B was recorded for 20 consecutive days. Figure 1 The fish in group A did not get sick, but the fish in group B got sick to varying degrees and died one after another. The survival of the test fish within 20 days of the infection experiment was as follows Figure 1 As shown. Calculation shows that the immunized group obtained a protection rate of 100%. Therefore, it can be considered that the present invention can make the fry of the retained type masu salmon 100% immune to Ichthyophthirius spp.

[0082] Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the embodiments. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A method for preparing a vaccine against Ichthyophthirius spp. of a stranded type salmon, characterized in that: The following steps are involved: Step 1: disinfect, fill and oxygenate several ponds; Step 2, putting healthy fry of the stranded type masu salmon into each pond, raising them for a period of time, then putting fry of the stranded type masu salmon infected with Ichthyophthirius avium into each pond, and then raising them until all the fry of the healthy stranded type masu salmon are infected with Ichthyophthirius avium; The number of the fry of the stranded type salmon infected with Ichthyophthirius scoparia is 0.8%-1.2% of the number of the fry of the healthy stranded type salmon; Step 3, adding raw salt to each pond until the salinity is 0.4wt%-0.6wt%, increasing the salinity to 0.9wt%-1.1wt% on the second day, and increasing the salinity to 1.4wt%-1.6wt% on the third day; Step 4, maintaining the salinity at 1.4wt%-1.6wt% and raising the fish fry of the stranded type salmon infected with Ichthyophthirius avium until all the fish fry are cured, thereby obtaining the Ichthyophthirius avium vaccine-free strain of the stranded type salmon.

2. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type masu salmon according to claim 1, characterized in that: In step 1, the pond is disinfected with trichloroisocyanuric acid solution and oxygenated with an aerator.

3. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 1, the water injection depth is 0.5-1.0 meter.

4. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 2, the volume of the pond is 15m 3 According to the calculation, the number of the healthy retained type masu salmon fry is 45,000 to 50,000.

5. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 2, the body lengths of the healthy fry of the stranded type salmon and the fry of the stranded type salmon infected with Ichthyophthirius scoparia are independently 6-8 cm.

6. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 2, after 3 days of rearing, fry of stranded type salmon infected with Ichthyophthirius gracilis were released into each pond.

7. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 2, the number of the fry of the stranded type salmon infected with Ichthyophthirius scoparia is 1.0% of the number of the fry of the healthy stranded type salmon.

8. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type Salmon according to claim 1, characterized in that: In step 3, raw salt is added to the pond to a salinity of 0.5 wt %, and the salinity is increased to 1.0 wt % on the second day, and the salinity is increased to 1.5 wt % on the third day.

9. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type maso salmon according to claim 1, characterized in that: In step 4, the salinity is maintained at 1.4 wt%-1.6 wt% for 10 days.

10. The method for preparing the Ichthyophthirius spp. vaccine for the stranded type Salmon according to claim 1, characterized in that: The method also includes step 5, transferring the cured stranded type Salmon to a culture pond for culture, so as to obtain the Ichthyophthirius vaccine-free strain of stranded type Salmon.

Images