CN110840841B

CN110840841B - Turbot oil emulsion vaccine and application thereof

Info

Publication number: CN110840841B
Application number: CN201911058070.1A
Authority: CN
Inventors: 李�杰; 莫照兰; 李贵阳; 阎永伟
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2019-11-01
Filing date: 2019-11-01
Publication date: 2021-07-02
Anticipated expiration: 2039-11-01
Also published as: CN110840841A

Abstract

The invention discloses a turbot oil emulsion vaccine which is prepared by mixing vaccine antigens vibrio anguillarum O1 serotype strain VAM003 and O2 serotype strain VAM007 and then emulsifying the mixture with Marcol 52. The invention also discloses application of the turbot oil emulsion vaccine, wherein the final concentration of the turbot oil emulsion vaccine for immunizing the turbot is 10⁹cells/ml, concentration of injection immunization 10⁴‑10⁸CFU/g. The invention has the advantages that the invention can ensure that the immune turbot obtains higher antibody titer and early immune protection effect; the side effect is extremely small, and the weight of the turbot is not obviously influenced on the production scale.

Description

Turbot oil emulsion vaccine and application thereof

Technical Field

The invention relates to the fields of microbiology and immunology of aquaculture animals, in particular to an oil emulsion vaccine of turbot and application thereof.

Background

Turbot belongs to the class of teleostomia, the family of paralichthys olivaceus, the english name Turbot, commonly known as european flounder, and in china as "Turbot" and "oncothorn-flounder". Native to the European Atlantic sea area, is one of the world recognized high-quality flatfish.

The body is rhombus, the side is flat and high, the body length is 1.3-1.6 times of the body height, both eyes are positioned at the left side of the head, and the body length is up to 1M at most. Bare, no scale, with only the eye side being treated with bony prominences that are smaller than the eye diameter. Big mouth, thin and curved jaw teeth, no canine teeth. Most of the fin-ray branches of dorsal and anal fins. The eye side is brownish and has a small amount of skin prickles; the non-eye side light is white, the dorsal fin and the hip have no hardware and are longer. The male of the turbot reaches sexual maturity in one year, the female reaches sexual maturity in two years, and the natural maturity period is 5-8 months per year. Mature ova can be obtained from the artificially cultured parent fish all the year round under the condition of temperature and light control, and the artificial insemination method is adopted for artificial propagation at present. Is marine benthic fish of Paralichthys of Pleuropodales of Osteichthyes. Adult fish feed on benthic fishes, shellfishes, crustaceans, and the like. Is a deep sea bottom layer fish which is distributed from European Scandinavia to Mediterranean sea and is an important economic fish.

Turbot faces troubles of various diseases in the culture process, including vibrio anguillarum, edwardsiella, aeromonas and the like. Vaccines have been successfully used for disease control in farmed fish, such as Aeromonas salmonicida-Vibrio anguillarum bivalent inactivated vaccine for Atlantic salmon (Salmo salar), Anguilla anguillarum (Anguilla anguillaria), Aeromonas salmonicida-Vibrio anguillarum (Yersinia ruckeri) bivalent inactivated vaccine, Aeromonas salmonicida-Vibrio anguillarum-Vibrio maritima (Vibrio ordalii) -Vibrio coldniasis (Vibrio salmonicida) quadruple inactivated vaccine, and the like.

The adjuvant is an important component for improving the immune protection rate of the vaccine and prolonging the immune protection period, and comprises glucan, astragalus polysaccharide, aluminum, liposome, oil adjuvant and the like. The oil emulsion adjuvant is widely used in commercial fish vaccines, and is prepared into a water-in-oil vaccine by emulsifying and mixing an oil phase and a water phase, so that the slow release of antigens is realized, and the immune protection period of the vaccine is prolonged. Compared with an aqueous vaccine, the oil adjuvant can also enhance the immune response of fish bodies, improve the titer of serum antibodies and enhance the immune protection effect of the vaccine. However, in turbot, various oil emulsion adjuvants commonly used for aquatic products, such as Freund's incomplete adjuvant, MONTANIDETM ISA763A and the like, can cause serious adhesion side effects of turbot viscera, and if the oil emulsion adjuvants are used in combination with vaccines, the oil emulsion adjuvants can seriously affect the growth of the turbot. Because the fish culture period is long, the immune system is low and the like, most of the mature salmon and trout commercial vaccines in the world currently use oil adjuvants to emulsify antigens, but the commercial vaccines of turbots do not have oil emulsion products and are aqueous preparations.

Disclosure of Invention

In order to solve the problem of great side effect when the conventional turbot adopts an oil emulsion vaccine, the turbot oil emulsion vaccine and the application thereof are provided.

The invention is realized by the following technical scheme:

in order to realize the purpose, the invention provides a method for preparing a turbot oil emulsion vaccine, which takes vibrio anguillarum bivalent serotype inactivated vaccine as an example: vaccine antigens, namely, vibrio anguillarum O1 serotype strain VAM003 and O2 serotype strain VAM007 are separated from a research room of molecular pathology and biological disease control of the institute of aquatic products in the yellow sea of China academy of aquatic sciences, the strain VAM003 is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No. 6942; the strain VAM007 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.6944, and is cultured in Tryptone Soy Broth (TSB) at the culture temperature of 25 ℃.

The vaccine is prepared by mixing bacterial suspensions of inactivated VAM003 and VAM007 and emulsifying by MOBIL Marcol 52. Streaking strain VAM003 and strain VAM007 on TSA plate, performing serology and 16S rRNA analysis to determine the correct strain, selecting single colony, inoculating to TSB, culturing overnight, inoculating the overnight cultured bacterial liquid to fresh TSB (containing 3% glucose) according to 1% (v: v), culturing for 24h, and determining OD₅₄₀Then, 37% formalin solution was added to a final concentration of 0.2% formaldehyde, and inactivation was performed at 28 ℃ at 140 r/min. And (4) respectively taking the bacteria liquid inactivated for 12h and 24h for inactivation condition detection, and determining the complete inactivation condition of the bacteria liquid after observing that no bacteria grows. The inactivated bacteria solution was centrifuged at 6000g at 4 ℃ for 10min, and the precipitated bacteria was washed twice with sterile PBS (0.2mol, pH 7.6) and then resuspended in PBS (containing 0.1% formaldehyde). The resulting bacterial suspensions of VAM003 and VAM007 were mixed at a ratio of 1:1(v: v) to prepare a bivalent vaccine, which was stored at 4 ℃. Selecting MOBIL Marcol52 white oil as an oil emulsifier, matching span 80 and Tween 80, and emulsifying according to the proportion of water agent to oil agent to be 4: 6; mixing the bacterial liquid with PBS at a ratio of 4:6 to obtain aqueous vaccine composition with final concentration of 10⁹cells/ml。

Preferably, the mixture ratio of the VAM003 bacterial suspension to the VAM007 bacterial suspension is 1: 1.

Preferably, the mixed bacterial suspension and the MOBIL Marcol52 are emulsified according to the ratio of 4: 6.

Preferably, the vaccination mode of the vaccine is injection immunization.

The invention also provides application of the turbot oil emulsion vaccine, and the vaccine is applied to immunization of turbots.

Preferably, the final concentration of the vaccine is 10⁹cells/ml, concentration of injection immunization 10⁴-10⁸CFU/tail (colony forming units/fish body weight).

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention can ensure that the immune turbot obtains higher antibody titer and early immune protection effect;

(2) the invention has little side effect, and has no obvious influence on the body weight of the turbot on the production scale compared with other common oil adjuvants.

Drawings

FIG. 1 shows the antibody titers against VAM003 and VAM007 in the sera of turbot after immunization;

FIG. 2 is Scoring Scophthalmus maximus visceral adhesion 1 week after immunization;

FIG. 3 is Scophthalmus maximus visceral adhesion score 1 month after immunization;

FIG. 4 shows changes in weight of turbot before and after immunization;

FIG. 5 shows the death of Vibrio anguillarum VAM003 infection 7 days after the immunization of turbot;

FIG. 6 shows the death of Vibrio anguillarum VAM007 infected 7 days after the immunization of turbot;

FIG. 7 shows the death of Vibrio anguillarum VAM003 infection 30 days after the immunization of turbot;

FIG. 8 shows the death of Vibrio anguillarum VAM007 infected 30 days after the immunization of turbot.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that various changes or modifications may be made therein without departing from the scope of the invention; and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1:

first, preparation of experiment

1. Experimental Material

The turbot used in the experiment is purchased from a Shandong tobacco stage-farm, and the weight of the turbot is 62.54g +/-9.75 g. Culturing in a factory cement pond (7m multiplied by 1m), controlling the water temperature to be 16 ℃, and carrying out bait casting and water changing according to a normal management mode: the daily feeding amount of the feed is 1.2-1.5% of the weight, the feed is fed for 2 times every day, and the daily water change amount is 6 pond courses. Observing the conditions of turbot movement, ingestion, body surface and the like 2 weeks before the experiment, randomly taking 30 turbots after determining no abnormality, observing whether the body surface and gills are infected by parasites or not by naked eyes and a microscope, taking liver, spleen and kidney tissues of the fish for bacterial separation, and after determining that the turbot is not infected by bacteria and parasites, using the turbot for subsequent experiments.

2. Vibrio anguillarum divalent inactivated vaccine and control preparation

Streaking strain VAM003 and strain VAM007 on TSA plate, performing serology and 16SrRNA analysis to determine the correct strain, selecting single colony, inoculating to TSB, culturing overnight, inoculating the overnight cultured bacterial liquid to fresh TSB (containing 3% glucose) according to 1% (v: v) inoculum size, culturing for 24h, and determining OD₅₄₀Then, 37% formalin solution was added to a final concentration of 0.2% formaldehyde, and inactivation was performed at 28 ℃ at 140 r/min. And (4) respectively taking the bacteria liquid inactivated for 12h and 24h for inactivation condition detection, and determining the complete inactivation condition of the bacteria liquid after observing that no bacteria grows. The inactivated bacteria solution was centrifuged at 6000g at 4 ℃ for 10min, and the precipitated bacteria was washed twice with sterile PBS (0.2mol, pH 7.6) and then resuspended in PBS (containing 0.1% formaldehyde). The resulting bacterial suspensions of VAM003 and VAM007 were mixed at a ratio of 1:1(v: v) to prepare a bivalent vaccine, which was stored at 4 ℃. Selecting Freund's incomplete adjuvant (sigma) and sibic Montanide^TM ISA 763A、Montanide^TMISA780 and Marcol52 white oil as oil emulsifier, respectively^TMThe adjuvant is emulsified by a water agent and an oil agent according to the proportion of 3:7 according to the specification; freund's incomplete adjuvantAnd Marcol52 is emulsified according to the proportion of water solution and oil solution which is 4: 6; mixing the bacterial liquid with PBS at a ratio of 4:6 to obtain aqueous vaccine group with final concentration of 10⁹cells/ml。

3. Vaccine for immunizing turbot

Selection of 5 groups of vaccines (FIA group, Montanide)^TMISA763A group Montanide^TMISA780 group, Marcol52 group and aqua vaccine group), injecting immune turbot by continuous injector (German Hankasos) according to 0.1mL dose per fish, injecting PBS with equal volume to the control group, each group having 500 tails, marking the injected fish on dorsal fin by plastic labels with different colors, culturing in the same culture pond, normally changing water and feeding.

Second, experimental results

1. Serum antibody titer detection

As shown in FIG. 1, at 7d and 30d after immunization, 5 fish tails were randomly selected from each of the immunized group and the control group, blood was collected from the caudal peduncle vein to prepare serum, and the serum of each fish was mixed in equal amounts, dispensed, and stored at-20 ℃. At a concentration of 10⁹cells/ml of inactivated VAM003 and VAM007 cells were used as antigens, and serum antibody titers were determined by microagglutination.

Selection of 5 groups of vaccines (FIA group, Montanide)^TMISA763A group Montanide^TMISA780 group, Marcol52 group and PBS aqua vaccine group) and PBS, taking turbot serum of an immune group and a control group respectively on the 7 th day and the 30 th day after immunization, and detecting the antibody titer of VAM003 and VAM007 respectively, wherein as shown in figure 1, the antibody titer of each immune group is obviously higher than that of the control group by detecting the obvious antibody level rise of each immune group in one week after immunization, the antibody titer of VAM003 is 1: 80-1: 320, and the antibody titer of FIA and ISA763 immune groups to VAM007 is 1: 80-1: 320. One month after immunization, the titer of the antibody to VAM003 is 1: 320-1: 1280, and the titer of the antibody to VAM007 is 1: 1280-1: 2560.

2. Visceral adhesion detection

As shown in FIG. 2 and FIG. 3, at 7d and 30d after immunization, 30 tails of turbot were respectively taken from each immunization group and a control group for dissection, the adhesion condition of the viscera of turbot was observed and recorded, and the adhesion condition of the viscera after adjuvant immunization was evaluated in grades according to the adhesion evaluation standard (Speilberg screening system).

At 7d and 30d after immunization, 30 tails of turbots are respectively taken from each immunization group and a control group for dissection, and visceral adhesion after adjuvant immunization is graded and evaluated according to adhesion evaluation criteria (Speilberg screening system). As shown in the figure, no adhesion was observed in the control group and the aqua vaccine group at day 7 after immunization, and slight adhesion occurred in the adjuvant Marcol52 immunized group, but neither was scored higher than 2 points; extensive adhesion was observed with the adjuvants FIA, ISA763A and ISA780 groups, scoring 3 points and above at 50%, 70% and 80%. At 30 days after immunization, no adhesion was observed in the control group, only 2 of the aqueous vaccine groups showed slight adhesion, and slight adhesion was observed between the peritoneum and the intestinal tract at the injection site. The Marcol52 vaccine immunization group of the adjuvant mostly only has slight membranous adhesion at the injection site or between the intestinal tract and the peritoneum, 1 adhesion is evaluated as 3 points, and the other 29 adhesions have only slight adhesion and are not higher than 2 points. The vaccine immunization groups of adjuvants FIA, ISA763 and ISA780 have serious adhesion, and the rest of the vaccine immunization groups have a score of 2 points except that 1 part of ISA763A and the score of 3 points above. ISA780 adhesion was the most severe, with the abdominal wall of immunized fish thickened and viscera extensively adhered as a whole.

3. Effect of vaccine on turbot growth Performance

As shown in figure 4, 30 turbots were randomly picked from the culture pond and weighed before immunization, and the average body weight of the fish was calculated. And (3) weighing 30d after immunization of each immune group and the control group respectively, and comparing the influence of vaccine immunity on the growth performance of the turbot.

Before immunization, the average weight of the turbot in the culture pond is 62.54g +/-9.75 g. The body weight of each immunized group and control group was significantly higher than that before immunization (P <0.01) 30 days after immunization. The fish weights of the PBS-injected control group, the aqua vaccine immunization group and the Marcol52 adjuvant vaccine immunization group are 94.98g +/-12.41 g, 92.40g +/-14.60 g and 91.44g +/-11.11 g respectively, and no significant difference exists among the 3 groups. The weight of the turbot injected with FIA, ISA763 and ISA 7803 adjuvant vaccines is 78.86g +/-12.36 g, 76.60g +/-12.84 g and 74.92g +/-11.89 g respectively, no significant difference exists among the 3 groups, but the weight is significantly lower than that of a PBS control group, an aqua vaccine immunization group and a Marcol52 adjuvant vaccine immunization group (P is less than 0.01).

4. Immune protection rate detection

As shown in FIGS. 5-8, at 7d and 30d after immunization, 30 turbots were taken from each immunization group and control group, respectively, and cultured in a 300L circulating water culture tank, and then were subjected to intramuscular injection for detoxification with VAM003 or VAM007, respectively. Each tail injected with 0.1mL 10⁷cfu/ml bacterial solution. After the challenge, the fish disease symptoms were observed, the death number was counted, and the immune protective Ratio (RPS) of the vaccine was calculated according to the following formula, where RPS ═ 1-immune group mortality/control group mortality × 100%.

At 7d and 30d after immunization, collecting 30 tails of turbot from each immunization group and control group, and performing intramuscular injection for counteracting toxic substance with VAM003 or VAM007 respectively, wherein each tail is injected with 0.1mL of 10 mL⁷cfu/ml bacterial solution, and the death of fish after infection was recorded. On the 7 th day after immunization, vibrio anguillarum O1 serotype strain VAM003 is used for infection, a control group injected with PBS is completely dead after infection for 3 days, the number of dead fishes in each immune group is lower than that of the control group, and the immune protection rates are respectively 60% of water-based vaccine, 90% of FIA adjuvant vaccine, 100% of Marcol52 adjuvant vaccine, 96.67% of ISA763A adjuvant vaccine and 96.67% of ISA780 adjuvant group. On the 7 th day after immunization, vibrio anguillarum O2 serotype strain VAM007 is used for infection, 26 dead turbots in a control group injected with PBS have immune protection rates of 43.31 percent of aqua vaccine, 100 percent of FIA adjuvant vaccine, 96.15 percent of Marcol52 adjuvant vaccine, 100 percent of ISA763A adjuvant vaccine and 100 percent of ISA780 adjuvant group respectively.

30 days after immunization, vibrio anguillarum O1 serotype strain VAM003 is used for infection, a control group injected with PBS is completely dead after 3 days of infection, the immune protection rate of the aqua vaccine group and the Marcol52 adjuvant vaccine group is 96.67 percent, and the immune protection rate of the rest 3 vaccine groups is 100 percent; the vibrio anguillarum O2 serotype strain VAM007 is used for infection, 27 turbots dead in a control group injected with PBS have the immune protection rate of 96.67 percent in a Marcol52 adjuvant vaccine group, and the immune protection rates of the rest 4 immune groups are all 100 percent.

Third, summarize

Research results show that the oil emulsifying adjuvant can ensure that the immune turbot can obtain higher antibody titer and early immune protection effect, the side effect of the MOBIL Marcol52 oil adjuvant is obviously lower than that of other 3 oil adjuvants, the weight of the turbot is not obviously influenced on the production scale, and the oil emulsifying adjuvant can be used for emulsifying and preparing the vibrio anguillarum divalent inactivated vaccine.

Claims

1. A turbot oil emulsion vaccine is characterized in that: vaccine antigens, namely, vibrio anguillarum O1 serotype strain VAM003 and O2 serotype strain VAM007 are separated from a research room of molecular pathology and biological disease control of the institute of aquatic products in the yellow sea of China academy of aquatic sciences, wherein the strain VAM003 is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No. 6942; the strain VAM007 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 6944; the vaccine is prepared by mixing VAM003 and VAM007 bacterial suspension and emulsifying the mixture with MOBIL Marcol 52.

2. The turbot oil emulsion vaccine of claim 1, wherein: the VAM003 and VAM007 bacterial suspensions and the MOBIL Marcol52 are emulsified according to the proportion of 4: 6.

3. The turbot oil emulsion vaccine of claim 1, wherein: the vaccination mode of the vaccine is injection immunization.

4. The turbot oil emulsion vaccine of claim 1, wherein: the vaccine is applied to immunizing turbots.

5. The turbot oil emulsion vaccine of claim 1, wherein: the final concentration of the vaccine applied by the vaccine is 10⁹cells/ml, concentration of injection immunization 10⁴-10⁸CFU/g。