CN109675024B - Combined vaccine for bacterial septicemia and red skin disease of grass carp and preparation method thereof - Google Patents
Combined vaccine for bacterial septicemia and red skin disease of grass carp and preparation method thereof Download PDFInfo
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
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- A61K39/02—Bacterial antigens
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention discloses a grass carp bacterial septicemia and red skin disease combined vaccine and a preparation method thereof. After grass carp is immunized, the expression of immune related genes of IgM, IL-1 beta, IFN, Mx, C3 and TLR3 in spleen and head kidney tissues of a fish body can be stimulated, the vaccine can generate a relative immune protection rate of 80-90% for the grass carp, and the relative protection rate can still keep 50% 1 year after immunization; the method is simple and convenient to operate, safe and effective, can prevent two diseases through one-time immunization, not only reduces the use of antibiotics and chemical drugs, but also reduces pollution and damage to the external environments such as the culture environment, the ecological environment and the like, and guarantees the food safety and the water ecological safety of the grass carp.
Description
Technical Field
The invention belongs to the field of fish culture, and particularly relates to a combined vaccine for grass carp bacterial septicemia and red skin disease and a preparation method thereof.
Background
Grass carp is one of the main breeding varieties in Guangdong province, the annual output reaches more than 400 million tons, but the annual disease loss is more than 20 hundred million yuan. Septicemia and red skin disease of grass carp are serious bacterial diseases harmful to grass carp, and have wide epidemic range and high death rate. The grass carp bacterial septicemia is caused by aeromonas hydrophila infection, is a high-risk disease of grass carps in recent years, has fierce attack, causes death of a large number of fishes in a short time and is fulminant in epidemic, once the attack is difficult to control, the attack is long in season and wide in epidemic range, and grass carp main culture areas all occur to different degrees. The grass carp red skin disease troubles vast grass carp farmers in China for a long time, the pathogen is pseudomonas aeruginosa, the epidemic area is wide and can be seen all the year round, if the treatment is not carried out in time, the higher death rate can be caused, and the appearance quality of the grass carp is seriously influenced. Therefore, the bacterial disease of the grass carps becomes a barrier for the sustainable development of grass carp breeding in China.
For the prevention and control of grass carp bacterial diseases, a tissue slurry vaccine or a single vaccine immunization mode is mostly adopted, but the satisfactory effect cannot be achieved. Therefore, in order to solve the problem, the development of a vaccine capable of preventing bacterial sepsis and red skin disease of grass carp in combination is an urgent solution.
Disclosure of Invention
The invention aims to provide an efficient combined prevention and treatment method for grass carp bacterial septicemia and red skin disease.
The technical scheme adopted by the invention is as follows:
a vaccine for treating bacterial septicemia and red skin disease of grass carp is prepared from beta-propiolactone, inactivated aeromonas hydrophila liquid and inactivated pseudomonas aeruginosa liquid.
Furthermore, the grass carp bacterial septicemia and red skin disease vaccine also contains an adjuvant; preferably, the adjuvant is a water-in-oil-in-water adjuvant.
The preparation method of the grass carp bacterial septicemia and red skin disease vaccine comprises the following steps:
(1) respectively carrying out amplification culture on aeromonas hydrophila and pseudomonas aeruginosa;
(2) respectively adding beta-propiolactone into aeromonas hydrophila liquid and pseudomonas aeruginosa liquid obtained by amplification culture, inactivating, and carrying out water bath for 2-3 h at 37-40 ℃;
(3) and mixing the aeromonas hydrophila liquid, the pseudomonas aeruginosa liquid and an adjuvant to obtain the inactivated bivalent vaccine.
Further, the preparation method of the grass carp bacterial septicemia and erythroderma vaccine comprises the step (2) that the concentration of the aeromonas hydrophila bacterial liquid after the amplification culture is 7.5 multiplied by 108～9×108CFU mL-1The concentration of the pseudomonas aeruginosa bacterial liquid is 4.2 multiplied by 108～6×108CFU mL-1(ii) a The volume concentration of the beta-propiolactone in the step (2) is 0.1-0.5%.
Further, the preparation method of the grass carp bacterial septicemia and red skin disease vaccine comprises the step (2) of inactivating for 72-80 hours at 4-5 ℃.
Further, the preparation method of the grass carp bacterial septicemia and red skin disease vaccine comprises the following steps of (3) mixing aeromonas hydrophila liquid, pseudomonas aeruginosa liquid and adjuvant according to the volume ratio of 1-2: 1-2: 1 to 4.
Further, the preparation method of the grass carp bacterial septicemia and red skin disease vaccine is characterized in that the adjuvant is a water-in-oil-in-water adjuvant.
The application of the vaccine in preparing a reagent for treating grass carp bacterial septicemia and red skin disease.
The vaccine is applied to the preparation of a reagent for improving the grass carp immunity gene expression.
Further, the immune genes are IgM, IL-1 beta, IFN, Mx, C3 and TLR 3.
The invention has the beneficial effects that:
according to the invention, the inactivating agent beta-propiolactone is added according to the epidemic strains screened at the earlier stage, so that the method is safe and efficient, and can effectively control the occurrence of grass carp bacterial septicemia and red skin disease; the invention can stimulate the expression of IgM, IL-1 beta, IFN, Mx, C3 and TLR3 immune related genes in spleen and head kidney tissues of a fish body, and can generate 80-90% relative immune protection rate to grass carp, and the relative protection rate can still reach 50% 1 year after immunization; the invention has simple operation, safety and effectiveness, can prevent two diseases by one-time immunization, not only reduces the use of antibiotics and chemical drugs, but also reduces the damage to the breeding environment, and ensures the food safety and the water ecological safety.
Drawings
FIGS. 1A-L are schematic diagrams of IgM and spleen tissues IgM of grass carp head and kidney respectively injected with the bivalent inactivated vaccine of the present invention; grass carp head kidney IL-1 beta, spleen tissue IL-1 beta; grass carp head kidney IFN, spleen tissue IFN; grass carp head kidney Mx protein, spleen tissue Mx protein; grass carp head kidney C3, spleen tissue C3; influence of expression level of grass carp head kidney TLR3 and spleen tissue TLR 3.
FIG. 2 shows the effective period of the inactivated bivalent vaccine of the present invention.
Detailed Description
Example 1 vaccine preparation
(1) Aeromonas hydrophila (GA201 strain) and pseudomonas aeruginosa (JP802 strain) preserved at-70 ℃ are respectively inoculated into nutrient agar culture media, incubated at the constant temperature of 28 ℃ for 24h, and single colonies are respectively picked and inoculated into nutrient broth culture media and cultured at 28 ℃ overnight (18 h).
(2) Absorbing 100 mu L of each of the aeromonas hydrophila and pseudomonas aeruginosa bacteria liquid cultured overnight in the step (1), respectively inoculating into the nutrient broth culture medium again, performing amplification culture at 28 ℃, 180 revolutions per minute and shaking for 18-20 hours in a shaking table, and determining the concentration of the aeromonas hydrophila to be 7.5 multiplied by 10 by the concentration of the aeromonas hydrophila by using a turbidimetric method8CFU mL-1Pseudomonas aeruginosa of 4.2X 108CFU mL-1。
(3) Respectively adding beta-propiolactone with final concentration of 0.1% (V/V) into the amplified aeromonas hydrophila solution and the pseudomonas aeruginosa solution, inactivating for 72h at 4 ℃, and then carrying out water bath for 2h at 37 ℃ to hydrolyze the inactivator. And inoculating a small amount of the bacterial liquid obtained in the previous step into a nutrient agar culture medium, culturing at 28 ℃ for 72h for inactivation test, and generating a sterile colony to indicate that the bacterial liquid is completely inactivated and can be used for subsequent seedling preparation.
(4) Emulsifying and stirring the inactivated aeromonas hydrophila liquid, the inactivated pseudomonas aeruginosa liquid and a water-in-oil-in-water adjuvant MONTANIDETMISA 201VG according to the proportion of 1:1:2 to form the inactivated bivalent vaccine, and storing the inactivated aeromonas hydrophila liquid, the inactivated pseudomonas aeruginosa liquid and the water-in-oil-in-water adjuvant MONTANIDETMISA 201VG at the temperature of 4 ℃ for later use.
Wherein, the nutrient broth culture medium formula is (per liter): 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride, and final pH of 7.4 +/-0.2. The nutrient agar culture medium formula is as follows (per liter): 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride and 15.0g of agar, and the final pH value is 7.4 +/-0.2.
Example 2 vaccine preparation
(1) Aeromonas hydrophila (GA201 strain) and pseudomonas aeruginosa (JP802 strain) preserved at-70 ℃ are respectively inoculated into nutrient agar culture media, incubated at the constant temperature of 28 ℃ for 24h, and single colonies are respectively picked and inoculated into nutrient broth culture media and cultured at the temperature of 28 ℃ overnight (24 h).
(2) Absorbing 90 mu L of each of the aeromonas hydrophila and pseudomonas aeruginosa bacteria liquid cultured overnight in the step (1), respectively inoculating into the nutrient broth culture medium again, carrying out amplification culture at 28 ℃, 180 r/min, shaking for 18-20 h by a shaking table, and determining the concentration of the aeromonas hydrophila to be 9 x 10 by a turbidimetry8CFU mL-1The pseudomonas aeruginosa is 6 multiplied by 108CFU mL-1。
(3) Respectively adding beta-propiolactone with final concentration of 0.5% (V/V) into the amplified aeromonas hydrophila solution and the pseudomonas aeruginosa solution, inactivating for 80h at 5 ℃, and hydrolyzing the inactivator in water bath for 3h at 40 ℃. And inoculating a small amount of the bacterial liquid obtained in the previous step into a nutrient agar culture medium, culturing at 28 ℃ for 72h for inactivation test, and generating a sterile colony to indicate that the bacterial liquid is completely inactivated and can be used for subsequent seedling preparation.
(4) Emulsifying and stirring the inactivated aeromonas hydrophila liquid, the inactivated pseudomonas aeruginosa liquid and a water-in-oil-in-water adjuvant MONTANETMISA 201VG according to the proportion of 2:2:4 to form the inactivated bivalent vaccine, and storing the inactivated aeromonas hydrophila liquid, the inactivated pseudomonas aeruginosa liquid and the water-in-oil-in-water adjuvant MONTANETMISA 201VG at the temperature of 4 ℃ for later use.
Wherein, the nutrient broth culture medium formula is (per liter): 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride, and final pH of 7.4 +/-0.2. The nutrient agar culture medium formula is as follows (per liter): 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride and 15.0g of agar, and the final pH value is 7.4 +/-0.2.
The inactivated vaccine prepared was further tested for efficacy as follows.
Safety of aeromonas hydrophila septicemia and pseudomonas aeruginosa erythroderma bigeminal white oil inactivated vaccine to grass carp
The method comprises the following steps: grass carp (8-10 cm in body length and 12-15g in body weight) is purchased from a certain culture farm in the area of the golden wine in Guangzhou city, and is free from insects through microscopic examination, and bacteria are separated to find that common strains such as aeromonas hydrophila and pseudomonas aeruginosa and the like do not exist. After one week of temporary rearing, the healthy grass carp 120 tails with good state and no body surface damage are selected and randomly divided into 6 groups, namely 3 groups of vaccine injection group and blank control group, and 20 tails of each group. Wherein, the vaccine injection group adopts 2 times of dosage, namely 0.4mL per tail, the inactivated bivalent vaccine prepared in the embodiment 1 is injected into the abdominal cavity, and the blank control group is not treated at all. During the test, the water temperature was controlled at (28. + -. 2) ° C, and the observation was continued for 14 days.
As a result: the health condition and the number of dead individuals of the tested fish are observed 3 times in the morning, in the middle and at the evening every day, during the whole test period, no dead fish exists in the vaccine injection group and the blank control group, the fish is healthy and alive and normal in food intake, the survival rate is 100 percent, and the condition that the health condition of the grass carp is not influenced by injecting the bivalent vaccine with double doses is shown.
Expression of immune related genes in head kidney and spleen tissues of grass carp after injection of aeromonas hydrophila septicemia and pseudomonas aeruginosa erythroderma bigeminal white oil inactivated vaccine
The method comprises the following steps: each grass carp is injected with 0.2mL of the bivalent vaccine prepared in the example 1 in an abdominal cavity, 30 grass carps are injected into each group, 3 parallel groups are arranged at the same time, and the same amount of blank healthy grass carps are taken as a control. Taking spleen and head kidney tissues of immunized grass carps and control grass carp groups 0, 6h, 12h, 1d, 3d, 5d, 7d, 14d and 21d after immunization respectively, extracting RNA of the spleen and the head kidney tissues respectively, carrying out reverse transcription to obtain cDNA, taking the cDNA as a template, selecting beta-actin as an internal reference gene, and carrying out real-time fluorescent quantitative PCR (qRT-PCR) detection on mRNA of 6 immune related genes including Interferon (IFN), immunoglobulin M (IgM), complement 3(C3), interleukin 1 beta (IL-1 beta), Mx protein (Mx) and Toll-like receptor 3(TLR3) by a relative expression quantity determination method. Wherein the qRT-PCR amplification system is shown in Table 1, and the primer sequence is shown in Table 2.
TABLE 1 real-time fluorescent quantitative PCR amplification System
TABLE 2 primer sequences for real-time fluorescent quantitative PCR
The qRT-PCR reaction amplification program is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 30s for 40 cycles.
As a result: during the whole experiment, the expression quantity of mRNA of 6 immune related genes (shown in figures 1A-L) shows the trend of increasing and then reducing in head kidney and spleen tissues of grass carp, but the peak value is high and low, and the appearance time is different. Wherein IgM, IL-1 beta, IFN and TLR3 are expressed in head and kidney at high level (see FIG. 1A, FIG. 1C, FIG. 1E and FIG. 1K).
The relative expression level of the IgM gene peaked 168h after immunization in both the head kidney and spleen tissues, the expression level in the head kidney was 80 times higher than that in the control group, the expression level in the spleen was 50 times higher than that in the control group, and the expression level in the head kidney was higher than that in the spleen as a whole.
IL-1 beta is a proinflammatory cytokine with immune regulation and control, and has the potential of initiating inflammation, promoting immunological activity and the like and enhancing immune response. The relative expression level of IL-1 beta gene in spleen reached a peak value 24h after immunization, which was 28 times that of the control group, and reached a peak value 72h in the head and kidney, which was 300 times that of the control group, which was much higher than the expression level in spleen.
IFN is a glycoprotein which is produced rapidly and in large quantities when the body is stimulated by pathogenic invasion, exerts an immunomodulatory effect and reaches its peak value within hours after infection. The grass carp IFN is mainly produced by lymphocytes in immune tissues such as head, kidney and the like, and has the functions of promoting phagocytosis and sterilization of macrophages, regulating lymphocyte transformation and the like. In the experiment, the relative expression quantity of IFN in the spleen of the grass carp reaches a peak value 24 hours after immunization, and is 12 times of that of a control group; the peak value in the head kidney is reached at 72h, which is more than 150 times of that in the control group, and the expression level is far higher than that in the spleen.
Mx proteins are a class of gtpase-induced substances produced by type I IFN. In the experiment, the relative expression quantity of the gene in the spleen of the grass carp reaches a peak value 120h after immunization, and is 18 times of that of a control group; peak at 168h in the head and kidney tissue, 25-fold higher than the control group.
The complement system is very complex in composition and is an important pivotal point between innate immunity and adaptive immunity. The C3 molecule, one of the intrinsic components, is the core of the complement system, and is involved in a variety of immune regulation and response reactions, and when bacteria, polysaccharides, antigen-antibody complexes activate any one of the complement pathways, the C3 molecule is activated through a cascade of reactions. In the experiment, the relative expression of the C3 gene reaches a peak value 120h after immunization in spleen and head kidney tissues of the grass carp, and the expression in the head kidney is 4.5 times higher than that in a control group; the expression level in spleen was 12 times higher than that in control group, but higher than that in head kidney.
Toll-like receptors (TLRs) activate downstream immune factors by a unique MyD88 independent way, and induce the expression and secretion of type I interferons (alpha-IFN and beta-IFN), thereby achieving the purpose of inhibiting virus replication. In the experiment, the relative expression of the TLR3 gene in spleen and head kidney tissues is higher and reaches a peak value 120h after immunization, the expression in the head kidney is 130 times higher than that in a control group, and the expression in the spleen is 100 times higher than that in the control group.
Effectiveness of injection of bigeminal inactivated vaccine on prevention of bacterial septicemia and red skin disease of grass carp
The method comprises the following steps: 150 healthy grass carps are selected and randomly divided into 5 groups, wherein two groups are vaccine immunization groups, the other 3 groups are blank control groups, and each group contains 30 fish. The vaccine immunization group was administered a dose of 0.2mL of the bivalent vaccine per tail of the abdominal cavity. After the immunization for 21d, selecting 1 vaccine immunization group and 1 blank control group of grass carp abdominal cavity injection aeromonas hydrophila virulent strain liquid; and (3) selecting grass carp abdominal cavities of another 1 vaccine immunization group and another 1 blank control group to inject freshly cultured pseudomonas aeruginosa virulent strain liquid.
The toxin counteracting dosage is selected to ensure that the minimum viable bacteria amount of the blank healthy grass carp which can die 100 percent, namely the aeromonas hydrophila is 0.6 multiplied by 107CFU/tail, Pseudomonas aeruginosa at 1 × 108CFU/tail. And (5) observing survival conditions of the fish bodies every day after using the bacterial liquid to counteract the toxin, recording the number of diseases and deaths, and continuously observing for 14 d. Different times were chosen for 3 independent replicates. Each group of the experiment was set up with 3 parallel experiments.
The relative immunoprotection rate (RPS%) was calculated with reference to the following formula:
RPS ═ (1-mortality in the immune group/mortality in the control group) × 100%
As a result: 3 times of effectiveness tests show that the effective protection rates of the aeromonas hydrophila vaccine components in the bivalent inactivated vaccine are respectively 90%, 100% and 95%, and the effective protection rates of the pseudomonas aeruginosa vaccine components are respectively 80%, 90% and 95%.
Immune protection rate of vaccine
The method comprises the following steps: 3500 tail healthy grass carps are prepared, wherein 1500 tails are injected intraperitoneally to immunize the aeromonas hydrophila and pseudomonas aeruginosa bigeminal white oil inactivated vaccine (immunization group) prepared in the invention example 1, and 2000 tails are used as a control group for standby. After immunizing vaccine for 21d, beginning to attack virus for the first time, and then attacking the virus for 1 time every 30d, and respectively taking fish of 100 immune groups and fish of 100 control groups every time, and grouping, namely immunizing 2 groups of fish, and 50 fishes in each group; control 2 groups, 50 tails each. And placing the mixture in an aquarium for temporary culture for 3d, and then performing a challenge experiment. 1 group of immune fish and 1 group of control fish are injected with aeromonas hydrophila virulent strain bacterial liquid, and the other 1 group of immune fish and the other 1 group of control fish are injected with pseudomonas aeruginosa virulent strain bacterial liquid.
The dose of the drug is the same as that in example 4, i.e., the dose of Aeromonas hydrophila is 0.6X 107CFU/tail, Pseudomonas aeruginosa at 1 × 108CFU/tail. The experiment lasts for 1 year, once every month, and the effective protection rate of each toxin attacking is shown in figure 2, wherein the total time of the experiment is 12 times.
As a result: the relative immune protection rate of the two vaccine components (inactivated, namely aeromonas hydrophila vaccine and inactivated pseudomonas aeruginosa vaccine) is maintained between 60% and 90% in the 1 st month to 6 th month after the intraperitoneal injection of the vaccine to the grass carp; the relative immune protection rate is reduced to 50 to 60 percent from the 6 th to the 12 th months. The vaccine prepared by the invention can immunize grass carp in abdominal cavity for 6 months, has good immune protection effect on infection of aeromonas hydrophila virulent strain liquid and pseudomonas aeruginosa virulent strain liquid, and can only play a part of protection effect after 6 months.
The beta-propiolactone screened by the invention is used as the inactivator of the bivalent vaccine, and the immune protection rate is improved by 20-30% compared with other inactivators.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhujiang aquatic products institute of China aquatic science research institute of Guangzhou Promu biological products GmbH
<120> grass carp bacterial septicemia and erythroderma combined vaccine and preparation method thereof
<130>
<160> 14
<170> PatentIn version 3.5
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Claims (4)
1. A grass carp bacterial septicemia and red skin disease vaccine is characterized in that the vaccine consists of an adjuvant, inactivated aeromonas hydrophila liquid and inactivated pseudomonas aeruginosa liquid; the adjuvant is a water-in-oil-in-water adjuvant; the concentration of the aeromonas hydrophila bacterial liquid is 7.5 multiplied by 108～ 9×108 CFU mL-1The concentration of the pseudomonas aeruginosa bacterial liquid is 4.2 multiplied by 108～6×108 CFU mL-1(ii) a The adjuvant is MONTANIDETMISA 201VG, and the inactivated aeromonas hydrophila liquidThe volume ratio of the pseudomonas aeruginosa liquid to the adjuvant is 1:1: 2.
2. The method for preparing the vaccine for grass carp bacterial septicemia and red skin disease of claim 1, comprising the following steps:
(1) respectively carrying out amplification culture on aeromonas hydrophila and pseudomonas aeruginosa;
(2) respectively adding beta-propiolactone into aeromonas hydrophila liquid and pseudomonas aeruginosa liquid obtained by amplification culture, inactivating, and carrying out water bath for 2-3 h at 37-40 ℃;
(3) mixing the aeromonas hydrophila liquid, the pseudomonas aeruginosa liquid and the adjuvant obtained in the previous step according to the proportion of 1:1:2 to obtain an inactivated bivalent vaccine;
the specific inactivation condition in the step (2) is inactivation for 72-80 h at 4-5 ℃.
3. The method for preparing the vaccine for bacterial septicemia and red skin disease of grass carp as claimed in claim 2, wherein the concentration of the aeromonas hydrophila bacterial liquid after the expanded culture in the step (2) is 7.5 x 108～ 9×108 CFU mL-1The concentration of the pseudomonas aeruginosa bacterial liquid is 4.2 multiplied by 108～6×108 CFU mL-1(ii) a The volume concentration of the beta-propiolactone in the step (2) is 0.1-0.5%.
4. The use of the vaccine of claim 1 in the preparation of an agent for increasing expression of genes for enhancing immunity in grass carp, wherein the genes for enhancing immunity are IgM, IL-1 β, IFN, Mx, C3 and TLR 3.
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