A system, immunization method and use for fish immunization

Abstract

The present invention belongs to the biological field and discloses a system for fish immunization, comprising a device for generating flowing water and an immunization area; the flowing water generated by the device flows through the immunization area; and the water contains a vaccine. The system can generate flowing water containing the vaccine, which can improve the immunization effect. At the same time, the present invention also discloses an optimized immunization method suitable for fish and the use of the system.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a system, an immunization method and a use for fish immunization. Background Art

[0002] Grouper refers to fish animals belonging to the genus Epinephelus of the family Serranidae in the order Perciformes. They grow fast, have tender meat, are rich in nutrition, and have high economic benefits from breeding. In my country, they are mainly farmed in the southern coastal areas. With the continuous expansion of the scale of breeding in recent years, the occurrence of diseases has become more frequent and serious, which has had a serious impact on the development of the grouper breeding industry and caused huge economic losses. Grouper frog iridovirus disease is one of the main viral diseases in the artificial breeding of grouper, and grouper Vibrio harveyi is one of the important bacterial pathogens. Grouper frog iridovirus and grouper Vibrio harveyi can often be detected in breeding areas where diseases break out. Therefore, the main cause of infectious disease outbreaks in grouper is the co-infection caused by grouper frog iridovirus or grouper Vibrio harveyi.

[0003] Vaccines are an effective means of preventing and controlling infectious diseases in aquaculture. There are many types of vaccines reported for grouper, but most of them are injected. The preparation method is to use oil or aluminum gel as an adjuvant to prepare the vaccine and then immunize, which can obtain a very high immune protection rate. However, this immunization method requires a lot of labor, which also makes the acceptance rate of injectable vaccines not high. Summary of the invention

[0004] The main purpose of the present invention is to provide a system for fish immunization; the system can generate flowing water containing vaccines, which can improve the immunization effect.

[0005] At the same time, the invention also discloses an optimized immunization method suitable for fish and the use of the system.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A system for fish immunization comprises a device for generating flowing water and an immunization area; the flowing water generated by the device flows through the immunization area; and the water contains a vaccine.

[0008] In the above-mentioned system for fish immunization, the vaccine is an inactivated vaccine of grouper frog iridovirus and / or an inactivated vaccine of grouper Vibrio harveyi.

[0009] In the above-mentioned system for fish immunization, the water body also contains an adjuvant, and the adjuvant is MontanideIMS 1312 VG vaccine adjuvant.

[0010] In the above-mentioned system for fish immunization, the vaccine and the adjuvant are configured into a vaccine solution and added into the water body; 0.5L to 5L of the vaccine solution is added into every 45 to 50L of the water body; the vaccine solution is composed of a virus solution containing an inactivated vaccine of grouper frog iridovirus, a bacterial solution containing an inactivated vaccine of grouper Vibrio harveyi, and an adjuvant.

[0011] In the above-mentioned system for fish immunization, the volume ratio of the virus solution containing the grouper frog iris virus inactivated vaccine, the bacterial solution containing the grouper Vibrio harveyi inactivated vaccine, and the adjuvant is 20-30:20-30:40-60.

[0012] In the above-mentioned system for fish immunization, the flow rate of the water body is 6-20L / min / 0.16m 2 Flow cross-sectional area.

[0013] The 6-20 L / min / 0.16 m 2 The flow cross-sectional area refers to the cross-sectional area perpendicular to the flow direction, which is 0.16m 2 In the flow space, water flows at a speed of 6 to 20 L / min, which can have a better immune effect.

[0014] In the above-mentioned system for fish immunization, the device includes a water tank, a net cage, and a pump; the pump is used to make the water in the water tank flow; the net cage is used to place the fry to be immunized; the net cage is placed in the water tank and allows the flowing water to pass through.

[0015] In the above-mentioned system for fish immunization, the pump is placed outside or inside the water tank;

[0016] If the pump is placed outside the water tank, the water tank and the pump form a water circulation system, which is used to circulate the water externally;

[0017] If the pump is placed in the water tank, the pump is used to circulate the water in the water tank.

[0018] Meanwhile, the invention also discloses a method for fish immunization, which includes placing the fish to be immunized in a flowing water body containing a vaccine for immunization.

[0019] Finally, the present invention also discloses the use of the system as described above for immunizing fish.

[0020] One of the above technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0021] Compared with the simple immersion immunization method, the system of the present invention can generate flowing water containing vaccines, and using the water to immunize fish fry can effectively improve the immunization effect and enhance the protective power of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the structure of the flowing water immunization device of Example 2. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0024] The red-spotted grouper spleen (EAGS) cell line was established by the inventors of the present invention (X. Huang, Y. Huang, J. Sun, X. Han, Q. Qin, Characterization of two grouper Epinephelus akaara cell lines: Application to studies of Singapore grouper iridovirus (SGIV) propagation and virus–host interaction, Aquaculture (2009).), and the grouper Vibrio harveyi was isolated, identified and preserved by the inventors of the present invention, and the classification name is: Vibrio harveyi GD25, the preservation number is CCTCC NO: M 2023630, and it is preserved in the China Center for Type Culture Collection, the preservation date is April 26, 2023, and the preservation address is: Wuhan University, Wuhan, China. Leibovitz's L-15 medium was purchased from Gibco.

[0025] Example 1 Preparation of Grouper Bivalent Inactivated Immersion Vaccine

[0026] 1. Preparation and Inactivation of Grouper Frog Iridescent Virus (SGIV)

[0027] 1.1 Virus reproduction

[0028] When the red-spotted grouper spleen (EAGS) cells grew into a good monolayer, they were replaced with Leibovitz's L-15 culture medium containing 2% fetal bovine serum. Then, the grouper frog iridovirus (SGIV) virus solution was inoculated into the EAGS cells at a certain ratio and cultured at 28°C. When the cytopathic effect (CPE) was higher than 90%, the virus solution was repeatedly frozen and thawed three times below -20°C to harvest the virus solution.

[0029] 1.2 Virus inactivation

[0030] The propagated SGIV virus solution was repeatedly frozen and thawed three times, and then formaldehyde solution was added at a ratio of 0.1% (v / v) and placed in a 4°C refrigerator for inactivation for 96 hours.

[0031] 2. Preparation and Inactivation of Vibrio harveyi (VH) from Grouper

[0032] 2.1 Bacterial reproduction

[0033] Take the seed liquid of Vibrio harveyi from grouper and inoculate 2% NaCl LB liquid culture medium (10 g / L peptone, 5 g / L yeast extract powder, 30 g / L sodium chloride) at a ratio of 1:100 (v / v).

[0034] 2.2 Bacterial inactivation

[0035] The culture of Vibrio harveyi in grouper was inactivated by adding formaldehyde solution at a ratio of 0.2% (v / v) and incubating at 28°C and 50 r / min for 24 hours.

[0036] 3. Preparation of Grouper Bivalent Inactivated Immersion Vaccine

[0037] The inactivated SGIV virus solution was centrifuged at 3000r / min to remove the precipitate; the inactivated Vibrio harveyi solution of grouper was centrifuged at 7000r / min to collect the precipitate, and resuspended with an appropriate amount of PBS; then the inactivated supernatant virus solution (containing 10 8.0 TCID 50 Virus): Inactivated PBS resuspended bacteria (8.3×10 per ml before inactivation) 8 CFU bacteria): IMS 1312 VG was mixed at a ratio of 25:25:50 (v / v) at 200 r / min for half an hour to prepare a grouper bivalent inactivated immersion vaccine.

[0038] Example 2 Flowing water immunization of grouper bivalent inactivated immersion vaccine

[0039] 1. Experimental methods

[0040] 3,000 healthy pearl groupers (SGIV nucleic acid test was negative) with a size of 2 to 3 cm per tail were collected from the fishery workshop and divided into 6 groups, with 500 fish in each group. Five groups were immunized groups and one group was a control group. After stopping feeding for 12 hours, they were immunized using a flowing water immunization device.

[0041] The structure of the flowing water immune device is as follows Figure 1 As shown;

[0042] It includes a submersible pump 1, a water flow pipe 2, and a square cylinder 3. The square cylinder has four hole screens.

[0043] The dimensions of the square tank are 0.5m×0.4m×0.4m (length*width*height); the rated power of the submersible pump is 6W, and it can pump 10L of water in 1min.

[0044] The experimental groups were divided into groups A to E; groups A to D were immunized groups; group E was the control group; and the remaining group was the blank group;

[0045] The immunization method for group A was as follows: 50L of aquaculture water was added to a square tank, and 5L of grouper bivalent inactivated immersion vaccine was poured in, 500 healthy groupers were poured into a four-hole sieve, and then the whole sieve was placed in an immunization device for running water immunization for 2 minutes;

[0046] The immunization method for group B was as follows: 50L of aquaculture water was added to the immunization device, 5L of grouper bivalent inactivated immersion vaccine was poured in, 500 healthy groupers were poured into a four-hole sieve, and then the whole sieve was placed in the immunization device for running water immunization for 30 seconds;

[0047] The immunization method of group C was as follows: 50L of aquaculture water was added to the immunization device, 0.5L of the grouper bivalent inactivated immersion vaccine was poured in, 500 healthy groupers were poured into a four-hole sieve, and then the whole sieve was placed in the immunization device for running water immunization for 10 minutes;

[0048] The immunization method of group D was as follows: 50L of aquaculture water was added to the immunization device, 0.5L of the grouper bivalent inactivated immersion vaccine was poured in, 500 healthy groupers were poured into a four-hole sieve, and then the whole sieve was placed in the immunization device for running water immunization for 3 minutes;

[0049] The immunization method for the E group immunization group was as follows: 50 L of aquaculture water was added to the immunization device, 1.25 L of inactivated SGIV virus and 1.25 L of inactivated Vibrio harveyi were poured in, 500 healthy groupers were poured into a four-sided hole sieve, and then the entire sieve was placed in the immunization device for running water immunization for 2 minutes.

[0050] The immunization method of the blank control group was as follows: 50 L of aquaculture water was added to the immunization device, 500 healthy groupers were poured into a four-hole sieve, and then the entire sieve was placed in the immunization device and run water for 2 minutes.

[0051] After immunization, the fish in each group were returned to the mesh screen in the cement pool and raised in groups. (Aeration pumps were used to provide oxygen during the immunization process of each group)

[0052] After 14 days of immunization, 80 fish were randomly selected from each group and transported back to the laboratory. After 7 days of temporary storage, the virus attack evaluation was performed: 30 fish were randomly selected from each group and injected intraperitoneally with grouper frog iridovirus. 5.0 TCID 50 / fish, and were raised in circulating water at 28-30℃. The fish were observed for 10 consecutive days, and the mortality of each group was recorded and the immune protection rate was calculated. Thirty fish were randomly selected from each group and injected intraperitoneally with Vibrio harveyi, 3×10 5 CFU / tail, reared in circulating water at 28-30℃, observed continuously for 10 days, recorded the mortality of each group of fish, and calculated the immune protection rate; the immune protection rate formula is as follows:

[0053] Immune protection rate = ×100%.

[0054] Comparison Example 1 Immersion Immunization of Grouper Bivalent Inactivated Immersion Vaccine

[0055] 1. Experimental methods

[0056] 1,500 healthy pearl groupers (SGIV nucleic acid test was negative) with a size of 2 to 3 cm per tail were collected from the fishery workshop and divided into three groups, with 500 in each group. After stopping feeding for 12 hours, they were immunized by immersion using a conventional immersion immunization device.

[0057] The immunization method for the F group was as follows: take a square tank, add 50L of aquaculture water, and pour in 5L of the grouper bivalent inactivated immersion vaccine, stir evenly, add an air stone and use an aerator pump to oxygenate, pour 500 healthy groupers into the net frame, and then put the whole frame into the square tank for immersion immunization for 2 minutes;

[0058] The immunization method for the G group was as follows: take a square tank, add 50L of aquaculture water, and pour in 0.5L of the grouper bivalent inactivated immersion vaccine, stir evenly and add air stone for oxygenation, pour 500 healthy groupers into the net frame, and then put the whole frame into the square tank for immersion immunization for 10 minutes;

[0059] The immunization method for the H group immunization group is as follows: take a square tank, add 50L of breeding water, and pour in 1.25L of inactivated SGIV virus and 1.25L of inactivated Vibrio harveyi, stir evenly, add air stone and use an aerator to oxygenate, pour 500 healthy groupers into the net frame, and then put the whole frame into the square tank and soak for 2 minutes for immunization.

[0060] After immunization, the fish in each group were returned to the mesh screen in the cement pool and raised in groups.

[0061] After 14 days of immunization, 80 fish were randomly selected from each group and transported back to the laboratory. After 7 days of temporary storage, the virus attack evaluation was performed. 30 fish were randomly selected from each group and injected intraperitoneally with grouper frog iridovirus. 5.0 TCID 50 / fish, bred in 28℃ circulating water, and observed for 10 consecutive days. The mortality of each group of fish was recorded and the immune protection rate was calculated. Thirty fish from each group were randomly selected and injected intraperitoneally with Vibrio harveyi, 3×10 5CFU / tail, reared in circulating water at 28℃, observed continuously for 10 days, recorded the mortality of each group of fish, and calculated the immune protection rate; the immune protection rate formula is as follows:

[0062] Immune protection rate = ×100%.

[0063] Results Statistics:

[0064] Judging from the test results, the immunization effect of using the traditional immersion immunization method, whether it is high-concentration immersion or low-concentration long-term immersion, is obviously limited. However, using the flowing water immunization method with a certain water flow rate, whether it is high-concentration flowing water immunization or low-concentration flowing water immunization, can obviously speed up the speed of vaccine entering the fish body and significantly improve the immune protection rate.

[0065] In the absence of adjuvant, conventional immersion immunization with high concentration of SGIV antigen also provides certain protection, and using running water immunization instead of conventional immersion immunization can also improve its protection rate, which shows that running water immunization is not limited to IMS 1312 adjuvant. However, conventional immersion immunization with high concentration of Vibrio harveyi antigen and running water immunization with high concentration of Vibrio harveyi did not provide obvious protection.

[0066] The IMS 1312 adjuvant was used to prepare a two-in-one inactivated immersion vaccine for grouper. Regardless of immersion immunization or flowing water immunization, it provided protection against grouper frog iridovirus and grouper Vibrio harveyi. Therefore, the IMS 1312 adjuvant can also be used to prevent and control other viral or bacterial pathogens of grouper.

[0067] The challenge results of grouper frog iridovirus and grouper Vibrio harveyi are shown in Tables 1 and 2.

[0068] Table 1 Immune protection results after infection with grouper frog iridovirus

[0069]

[0070] Table 2 Immune protection rate of grouper after challenge with Vibrio harveyi

[0071]

[0072] Result analysis:

[0073] 1. As shown in Table 1 above, compared with Group A, Group B and Group F, the 30s immune effect is better than the 2min immune effect of Group F, and the advantage is very obvious;

[0074] 2. As shown in Table 1 above, compared with Group C, Group D and Group G, the 3-minute immune effect is better than the 10-minute immune effect of Group G, and the advantage is very obvious;

[0075] 3. By comparing Group A and Group E, and Group F and Group H, it can be seen that IMS 1312 VG is a very important factor whether in static water or in flowing water.

[0076] Example 3 Effect of flow rate on flowing water immunization of grouper dual inactivated immersion vaccine

[0077] 1. Experimental methods

[0078] 2000 healthy pearl grouper with a size of 2-3 cm / tail (SGIV nucleic acid test was negative) were collected from the fishery workshop and divided into 4 groups, 500 in each group, 3 groups were immunized groups and 1 group was the control group. After stopping feeding for 12 hours, they were immunized using a flowing water immunization device.

[0079] Group 1: The size of the square tank is (0.5m×0.4m×0.4m); the rated power of the submersible pump is 3W, which can pump about 6L of water in 1min. 50L of breeding water is added to the immunization device, and 0.5L of grouper bivalent inactivated immersion vaccine is poured in. 500 healthy groupers are poured into a four-sided hole sieve, and then the whole sieve is placed in the immunization device for running water immunization for 3 minutes.

[0080] Group 2: The size of the square tank is (0.5m×0.4m×0.4m); the rated power of the submersible pump is 6W, which can pump about 10L of water in 1min. 50L of breeding water is added to the immunization device, and 0.5L of grouper bivalent inactivated immersion vaccine is poured in. 500 healthy groupers are poured into a four-sided hole sieve, and then the whole sieve is placed in the immunization device for running water immunization for 3 minutes.

[0081] Group 3: The size of the square tank is (0.5m×0.4m×0.4m); the rated power of the submersible pump is 15W, which can pump about 20L of water in 1min. 50L of breeding water is added to the immunization device, and 0.5L of grouper bivalent inactivated immersion vaccine is poured in. 500 healthy groupers are poured into a four-sided hole sieve, and then the whole sieve is placed in the immunization device for running water immunization for 3 minutes.

[0082] Group 4: The size of the square tank is (0.5m×0.4m×0.4m); the rated power of the submersible pump is 6W, which can pump about 10L of water in 1min. 50L of breeding water is added to the immunization device, and 500 healthy groupers are poured into a sieve with holes on all four sides. Then the whole sieve is placed in the immunization device for running water immunization for 3 minutes.

[0083] After immunization, the fish in each group were returned to the mesh screen in the cement pool and raised in groups.

[0084] After 14 days of immunization, 80 fish were randomly selected from each group and transported back to the laboratory. After 7 days of temporary storage, the virus attack evaluation was performed. 30 fish were randomly selected from each group and injected intraperitoneally with grouper frog iridovirus. 5.0 TCID 50 / fish, bred in 28℃ circulating water, and observed for 10 consecutive days. The mortality of each group of fish was recorded and the immune protection rate was calculated. Thirty fish from each group were randomly selected and injected intraperitoneally with Vibrio harveyi, 3×10 5 CFU / tail, reared in circulating water at 28℃, observed continuously for 10 days, recorded the mortality of each group of fish, and calculated the immune protection rate; the immune protection rate formula is as follows:

[0085] Immune protection rate = ×100%.

[0086] Results Statistics:

[0087] The challenge results of grouper frog iridovirus and grouper Vibrio harveyi are shown in Tables 3 and 4.

[0088] Table 3 Immune protection results after infection with grouper frog iridovirus

[0089] Group Group 1 Group 2 Group 3 Group 4 (Control) Flow rate 6L / min 10L / min 20L / min 10L / min mortality rate 6 / 30 7 / 30 10 / 30 23 / 30 Immune protection rate 73.91% 69.57% 56.52% -

[0090] Table 4 Immune protection rate of grouper after challenge with Vibrio harveyi

[0091] Group Group 1 Group 2 Group 3 Group 4 (Control) Flow rate 6L / min 10L / min 20L / min 10L / min mortality rate 4 / 30 4 / 30 9 / 30 21 / 30 Immune protection rate 80.95% 80.95% 57.14% -

[0092] When grouper is immunized with the grouper bivalent inactivated immersion vaccine at flow rates of 6L / min and 10L / min, a higher immune protection rate can be obtained, while the immune protection rate obtained at a flow rate of 20L / min is somewhat lower, indicating that it is necessary to control the slow water flow when using flowing water for immunization, and turbulent water flow cannot be used. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A system for fish immunization, characterized in that: The invention comprises a device for generating flowing water and an immunization area; the flowing water generated by the device flows through the immunization area; the water contains a vaccine and an adjuvant, and the adjuvant is a Montanide IMS 1312 VG vaccine adjuvant; The vaccine and adjuvant are configured into a vaccine solution, and added to the water body; 0.5L to 5L of the vaccine solution is added to every 45 to 50L of the water body; the vaccine solution is composed of a virus solution containing an inactivated vaccine of grouper frog iridovirus, a bacterial solution containing an inactivated vaccine of grouper Vibrio harveyi, and an adjuvant; the volume ratio of the virus solution containing an inactivated vaccine of grouper frog iridovirus, the bacterial solution containing an inactivated vaccine of grouper Vibrio harveyi, and the adjuvant is 20 to 30:20 to 30:40 to 60, and the flow rate of the water body is 6 to 20L / min / 0.16m 2 Flow cross-sectional area.

2. The system for fish immunization according to claim 1, characterized in that: The device comprises a water tank, a net cage and a pump; the pump is used to make the water in the water tank flow; the net cage is used to place the fry to be immunized; the net cage is placed in the water tank and allows the flowing water to pass through.

3. The system for fish immunization according to claim 2, characterized in that: The pump is placed outside or inside the water tank; If the pump is placed outside the water tank, the water tank and the pump form a water circulation system, which is used to circulate the water externally; If the pump is placed in the water tank, the pump is used to circulate the water in the water tank.

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