Carp mixed bacteria inactivated vaccine as well as preparation method and application thereof
By isolating and inactivateing a variety of pathogenic bacteria from carp and preparing mixed bacterial inactivated vaccines, the problem of preventing and treating bacterial diseases in carp is solved, and the effect of significantly improving the immune protection rate and breeding survival rate of carp is achieved.
Patent Information
- Application Number
- CN202411561318.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-04
- Publication Date
- 2025-02-14
AI Technical Summary
Bacterial diseases of carp are often outbreaks and prevalent. The existing prevention and treatment methods have hidden dangers of drug residues and bacterial resistance, and there is a lack of effective mixed bacterial inactivated vaccines.
Aeromonas Vickers, Aeromonas hydrophila, Citrobacterium Freunde and Shigmomonasoid were isolated from Henan carp, and mixed bacterial inactivated vaccine was prepared after formalin, and Inoculation of healthy carp by intraperitoneal injection.
After 20 days of vaccination, the immune protection rate of carp reaches 40% to 83%, which significantly improves the defense ability of carp against multiple bacterial infections. Compared with a single bacterial component vaccine, the protection range is wider and the breeding survival rate is improved .
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Figure CN119424623A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and specifically relates to the field of fish breeding vaccines, and in particular to a carp mixed bacterial inactivated vaccine and a preparation method and application thereof. Background Art
[0002] Carp (Cyprinuscarpio) is a high-quality species in aquaculture. It belongs to the Cypriniformes, Cyprinidae, and Cyprinidae. It has thick meat, tender and delicious, rich in nutrients, rich in essential amino acids and trace elements for the human body, and has a beautiful shape of a spindle-like body, which is widely loved by consumers. In recent years, with the continuous expansion of aquaculture scale and the continuous increase in aquaculture density, bacterial diseases of carp often break out, causing huge economic losses to farmers and seriously affecting the healthy development of the carp industry. At present, the main prevention and control methods for bacterial diseases of carp are external disinfectants and oral antibiotics, but these two methods have hidden dangers such as drug residues and bacteria are prone to drug resistance. Therefore, it is urgent to develop effective vaccines against common pathogens of carp.
[0003] Inactivated vaccine is a relatively safe and effective method in the prevention and treatment of aquatic diseases. Its principle is to maintain the immunogenicity of the microorganism while inactivating the microorganism by physical or chemical methods, and the animal body can produce specific immunity to the microorganism after vaccination. According to the preliminary investigation of this laboratory, cases caused by mixed infection of multiple pathogenic bacteria often occur in carp in Henan Province, but the research on mixed bacterial inactivated vaccine of carp has not been reported so far. Aeromonas veronii, Aeromonas hydrophila, Citrobacter freundii and Plesiomonas shigelloides separated from the sick carp in the aquaculture base of Henan Normal University are made into formalin mixed inactivated vaccine, which is used to immunize healthy carp and detect the immune protection effect of the vaccine, aiming to more effectively solve the problem of mixed infection of carp bacteria often encountered in practice, and provide theoretical basis for the application of mixed inactivated vaccine in bacterial disease prevention and treatment of cultured fish such as carp. There is no relevant report in this regard at present. Summary of the invention
[0004] The technical problem solved by the invention is to provide a carp mixed bacterial inactivated vaccine and a preparation method and application thereof.
[0005] The technical solution used to achieve the purpose of the present invention is: a mixed bacterial inactivated vaccine for carp, which comprises the following inactivated antigen composition: inactivated liquid of Aeromonas hydrophila, inactivated liquid of Aeromonas veronii, inactivated liquid of Citrobacter freundii and inactivated liquid of Pleistomonas shigellae.
[0006] Preferably, the volume ratio of the Aeromonas hydrophila inactivation liquid, the Aeromonas vernix inactivation liquid, the Citrobacter freundii inactivation liquid and the Shigella-like Pleistomonas inactivation liquid is 1:1:1:1, wherein the concentration of the Aeromonas hydrophila inactivation liquid is 1×10 9 CFU / mL, the concentration of Aeromonas victoriae inactivated solution is 1×10 9 CFU / mL, the concentration of the inactivated solution of Citrobacter freundii was 1×10 9 CFU / mL, the concentration of Shigella-like inactivated solution is 1×10 9 CFU / mL.
[0007] The present invention also provides a method for preparing a mixed bacterial inactivated vaccine for carp, the specific steps of which are as follows:
[0008] (1) Aeromonas hydrophila, Aeromonas vermifuge, Citrobacter freundii and Plesiomonas shigellae were inoculated on LB solid medium respectively and cultured at 28°C for 16 to 24 hours;
[0009] (2) Pick out single colonies of the four bacteria and place them in LB liquid culture medium, and culture them at 28°C and 180 rpm for 24 to 48 h;
[0010] (3) After the bacterial cells were expanded and cultured, they were centrifuged at 4°C and 5000 rpm for 10-15 min to collect the bacterial precipitate, and then washed three times with sterile saline. Finally, the cells were resuspended in PBS and the OD value of the bacterial solution was adjusted. 600 to 1.0 (the bacterial solution concentration is 1.0×10 9 CFU / mL), formalin was added to the bacterial solution to a final volume concentration of 1%, and the solution was inactivated at 25°C for 24 hours, during which the bacterial solution was shaken once every 3 to 4 hours;
[0011] (4) Take 100 μL of the inactivated bacterial solution and spread it on LB solid culture medium, culture it at 28°C for 24 h, and after detecting that there are no live bacteria, centrifuge the inactivated bacterial solution at 4°C and 5000 rpm for 10-15 min to collect the bacterial precipitate, wash it three times with sterile saline, and finally resuspend the bacteria in PBS. Adjust the concentration of the inactivated vaccine to 1×10 9 CFU / mL;
[0012] (5) The four kinds of inactivated bacterial vaccines were mixed in a volume ratio of 1:1:1:1 to obtain a mixed carp bacterial inactivated vaccine, which was then packaged and stored in a 4°C refrigerator for later use.
[0013] The carp mixed bacterial inactivated vaccine of the invention is used as a vaccine for preventing and / or treating carp bacterial infection.
[0014] Preferably, the mixed inactivated bacterial vaccine is used to immunize carp by intraperitoneal injection.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] (1) The present invention separates bacteria from diseased carp and prepares a vaccine, wherein a mixed bacterial inactivation of Aeromonas hydrophila, Aeromonas vernix, Citrobacter freundii and Pleistomonas shigellae is used. The four bacteria are common pathogens in aquaculture. After 20 days of inoculation of healthy carp with the inactivated vaccine, the immune protection rate reaches 40% to 83%, and the immune protection effect is significant, indicating that inoculation with the mixed bacterial inactivated vaccine enables carp to form a stronger defense against the co-infection of the four bacteria. Compared with a single bacterial component vaccine, the protection range is wider and the survival rate of aquaculture can be further improved.
[0017] (2) The antigen components (i.e., four inactivated bacteria) of the mixed bacterial inactivated vaccine of the present invention can induce a more comprehensive humoral immunity in carp, increase the activity of disease-resistant related enzymes in carp and induce a higher level of antibody production. The use of the mixed bacterial inactivated vaccine also has the advantages of reducing the number of immunizations, saving immunization costs, and reducing the damage caused to carp by multiple vaccinations. The present invention shows that the mixed bacterial inactivated vaccine has a good development prospect in the research and practice of carp vaccines, and has a high application value in actual production. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The changes of acid phosphatase activity in carp at different time periods after injection of mixed inactivated vaccine are shown in Table 1. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there is a significant difference (P < 0.05); when marked with **, there is a very significant difference (P < 0.01).
[0019] Figure 2 The changes of alkaline phosphatase activity in carp at different time periods after injection of mixed inactivated vaccine are shown in Table 1. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there is a significant difference (P < 0.05); when marked with **, there is a very significant difference (P < 0.01).
[0020] Figure 3 The results are shown as mean ± standard deviation (n = 4). When marked with *, there is a significant difference (P < 0.05); when marked with **, there is a very significant difference (P < 0.01).
[0021] Figure 4Figure 3 Changes in the levels of anti-Citrobacter freundii antibodies in carp serum at different time periods after injection of mixed inactivated vaccine. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there are significant differences (P < 0.05); when marked with **, there are extremely significant differences (P < 0.01).
[0022] Figure 5 Figure 3 Changes in anti-Shigella-like antibodies in carp serum at different time periods after injection of mixed inactivated vaccine. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there is a significant difference (P < 0.05); when marked with **, there is a very significant difference (P < 0.01).
[0023] Figure 6 Figure 3 Changes in the levels of anti-Aeromonas hydrophila antibodies in carp serum at different time periods after injection of mixed inactivated vaccine. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there are significant differences (P < 0.05); when marked with **, there are extremely significant differences (P < 0.01).
[0024] Figure 7 Figure 3 Changes in the levels of anti-Aeromonas veronii antibodies in carp serum at different time periods after injection of mixed inactivated vaccine. The results are expressed as mean ± standard deviation (n = 4). When marked with *, there are significant differences (P < 0.05); when marked with **, there are extremely significant differences (P < 0.01). DETAILED DESCRIPTION
[0025] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.
[0026] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.
[0027] Example 1
[0028] Preparation of mixed inactivated bacterial vaccine for carp
[0029] 1. Experimental Materials
[0030] Healthy carp were purchased from a farm in Xinxiang City, Henan Province, weighing about (15±3)g. They were disinfected with 2.5wt% saline before stocking. The active, uninjured, and uniform individuals were selected and placed in the aquaculture barrels of Henan Normal University Aquaculture Base. After 30 days of quarantine observation and random sampling, the carp were confirmed to be normal and then used for artificial infection experiments. The water temperature was (28±2)℃ during the entire experiment.
[0031] The strains used in the mixed vaccine preparation and artificial infection test were Aeromonas hydrophila, Aeromonas vernix, Citrobacter freundii, and Pleistomonas shigellae, all of which were isolated from diseased carp in the aquaculture base of Henan Normal University and stored in a -80°C refrigerator after identification. Before the experiment, the four strains were again subjected to physiological and biochemical identification and 16S rRNA molecular biological identification for verification. The GenBank gene sequence numbers of the 16S rRNA of the four strains were PQ524494, PQ524498, PQ524499, and PQ524500, respectively. Formalin was purchased from Chengdu Cologne Chemicals Co., Ltd.
[0032] 2. Vaccine Preparation
[0033] Aeromonas hydrophila, Aeromonas vernix, Citrobacter freundii and Oleomonas shigellae, which were refrigerated in a refrigerator, were picked out with an inoculation needle and inoculated on solid culture medium respectively, and cultured at 28°C for 16 to 24 hours; single colonies of the four bacteria were picked out and placed in LB liquid culture medium respectively, and cultured at 28°C and 180rpm for 24 to 48 hours; after the bacteria were expanded and cultured, they were centrifuged at 4°C and 5000rpm for 10 to 15 minutes to collect the bacterial precipitate, and washed 3 times with sterile saline, and finally the bacteria were resuspended in phosphate buffered saline (PBS) to adjust the bacterial solution OD 600 to 1.0 (the bacterial solution concentration is 1.0×10 9 CFU / mL), formalin was added to the bacterial solution to a final volume concentration of 1%, and the solution was inactivated at 25°C for 24 hours, during which the bacterial solution was shaken once every 3 to 4 hours; 100 μL of the inactivated bacterial solution was spread on LB solid culture medium, and cultured at 28°C for 24 hours. After detecting that there were no live bacteria, the inactivated bacterial solution was centrifuged at 4°C and 5000 rpm for 10 to 15 minutes to collect the bacterial precipitate, which was washed three times with sterile saline, and finally the bacteria were resuspended with PBS, and the concentration of the inactivated vaccine was adjusted to 1×10 9 CFU / mL; Mix the inactivated vaccines of the four bacteria in a volume ratio of 1:1:1:1, divide them into portions and store them in a 4°C refrigerator for later use.
[0034] 3. Safety test
[0035] 60 healthy carps were divided into three groups: experimental group, control group and blank group, with 20 carps in each group. The experimental group was injected with 0.1 mL / tail of mixed inactivated vaccine, the control group was injected with the same amount of PBS, and the blank group was not treated. After injection, they were observed for 14 days, during which they were fed conventionally, and the feeding and activity of the carps were observed every day.
[0036] 4. Protection rate test
[0037] 4.1 Immunization injection test
[0038] 200 healthy carps were randomly divided into 5 groups, each with 40 carps (20 carps for control and 20 carps for immunization), and carps were injected intraperitoneally, with mixed inactivated vaccine and PBS (0.1 mL / carp) injected into each immunization group and control group, respectively. After immunization injection, each group of test fish was routinely raised.
[0039] 4.2 Artificial infection test
[0040] The test strains Aeromonas hydrophila, Aeromonas vernix, Citrobacter freundii and Plesiomonas shigellae were inoculated into LB liquid culture medium respectively. After culturing at 28°C for 24-48 hours, the culture medium was washed off with sterile saline under sterile conditions and resuspended with PBS to prepare bacterial suspension. The concentrations of the four bacteria were adjusted to about 1×10 8 CFU / mL, and take part of the four bacterial suspensions in a volume ratio of 1:1:1:1 to mix evenly to make a mixed bacterial suspension, and keep them separately with each bacterial suspension for use. Artificial infection test was carried out on the carp in the test group 20 days after immunization injection (0.1mL / tail of bacterial suspension was injected intraperitoneally). The immunization treatment groups in the five groups were artificially infected with Aeromonas hydrophila, Aeromonas vernix, Citrobacter freundii, Shigella-like Oligomonas and mixed bacterial liquid, and the control group was injected with PBS (0.1mL / tail of intraperitoneal injection). After artificial infection injection, the carp in each group were routinely fed and observed for 15 consecutive days, and the incidence and death of the test fish were recorded every day. The vaccine protection rate was calculated according to the following formula.
[0041] RPS = (1-mortality rate of immunized group / mortality rate of control group) × 100%.
[0042] 4.3 Determination of blood biochemical indicators
[0043] The changes of acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) in the blood of carp were detected. All evaluation indicators were measured according to the corresponding kit operation instructions of Nanjing Jiancheng Bioengineering Institute.
[0044] 4.4 Blood antibody content determination
[0045] The serum antibody content was determined by enzyme-linked immunosorbent assay (ELISA). Carbonate buffer was used as the coating solution, and the inactivated bacterial solution was coated at 4°C overnight; 5wt% BSA was used as the optimal blocking solution, and the solution was blocked at 37°C for 2h; carp serum was diluted 200 times with PBS, and 100μL was added to the ELISA plate, and incubated at 37°C for 2h; the primary antibody (mouse anti-carp IgM antibody) was diluted at a volume ratio of 1:1000, and incubated at 37°C for 2h; the horseradish peroxidase-labeled secondary antibody (sheep anti-mouse IgG) was diluted at a volume ratio of 1:10000, and incubated at 37°C for 2h; TMB color development was performed at 37°C for 5min; 2M H2SO4 was added to terminate the color development, and the plate was incubated at OD 450 Read the numbers.
[0046] Example 2
[0047] Application of mixed bacterial inactivated vaccine for carp
[0048] 1. Vaccine safety
[0049] No bacterial growth was observed after the inactivated vaccine coated on the solid culture medium was cultured at 28°C for 48 hours. After the inactivated vaccine and PBS were injected into the carp, the feeding and activities of the carp in each group were normal during the 15-day observation period, and there was no visible disease or death. This shows that the mixed inactivated vaccine prepared by the present invention has reached the complete inactivation requirement and is safe for carp.
[0050] 2. Vaccine protection rate
[0051] As shown in Table 1, the survival rate of the immunized carp was improved compared with that of the control group, among which the immune protection effect against Shigella-like Pleistomonas was the best, with a relative immune protection rate of 83%. After 20 days of immunization, the mortality rate (85%) of the control group that was not vaccinated with the mixed inactivated vaccine in the Aeromonas vernix group was higher than that of the immunized test group (30%), and the protection rate was 64.7%; in the Aeromonas hydrophila group, the mortality rate of the control group (85%) was higher than that of the test group (45%), and the protection rate was 47%; in the Shigella-like Pleistomonas group, the mortality rate of the control group (60%) was higher than that of the test group (10%), and the protection rate was 83%; in the Citrobacter freundii group, the mortality rate of the control group (100%) was also higher than that of the test group (60%), and the protection rate was 40%. In the mixed infection group, the mortality rate of the control group (100%) was also higher than that of the test group (35%), and the protection rate was 65%. The autopsy of the dying individuals revealed that the intestines were filled with a large amount of gas, the eyes, lower jaw, base of the pectoral fins, anus were red, and there was ascites. The disease was caused by an artificial infection experiment, and the infection symptoms were basically consistent with those of naturally sick carp at the aquatic base of Henan Normal University.
[0052] Table 1 Test results of immune protection rate of mixed inactivated vaccine against various bacteria
[0053]
[0054] 3. Blood biochemical index test results
[0055] from Figure 1 , 2 , 3 It can be seen that compared with the control group, the blood ACP activity, AKP activity, and SOD activity of carp in the immunized group were significantly increased. Among them, the acid phosphatase ACP activity of the immunized group was significantly higher than that of the control group from the 3rd day, and it was still the case when it was detected on the 35th day; the alkaline phosphatase AKP activity of the immunized group was significantly higher than that of the control group from the 3rd day, and reached 26 Jin units / 100mL on the 35th day; the superoxide dismutase SOD activity of the immunized group was significantly higher than that of the control group from the 3rd day, and continued until the 21st day.
[0056] 4. Blood antibody content test results
[0057] from Figure 4 , 5 , 6, and 7 show that compared with the control group, the antibody content of the immunized group was significantly increased. For the antibody content produced by Citrobacter freundii, the antibody content produced by the immunized group reached the highest level on the 14th day and was significantly higher than that of the control group, and continued until the 21st day, after which the antibody content began to decline; for the antibody content produced by Shigella-like Pleistomonas, the overall trend of first increasing and then decreasing was shown, and it reached the highest level on the 21st day, and was significantly higher than the control group from the 7th day, and continued to be significantly higher than the control group until the 35th day, indicating that the inactivated vaccine has a better immune effect on Shigella-like Pleistomonas and a longer immune time; for Aeromonas hydrophila and Aeromonas veronii, it was significantly higher than the control group from the 3rd day and continued until the 35th day.
[0058] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.
Claims
1. A mixed bacterial inactivated vaccine for carp, characterized in that The vaccine comprises the following inactivated antigen composition: inactivated liquid of Aeromonas hydrophila, inactivated liquid of Aeromonas veronii, inactivated liquid of Citrobacter freundii and inactivated liquid of Pleistomonas shigellae.
2. The carp mixed bacterial inactivated vaccine according to claim 1, characterized in that: The volume ratio of the Aeromonas hydrophila inactivation liquid, the Aeromonas versii inactivation liquid, the Citrobacter freundii inactivation liquid and the Shigella-like Pleistomonas inactivation liquid is 1:1:1:1, wherein the concentration of the Aeromonas hydrophila inactivation liquid is 1×10 9 CFU / mL, the concentration of Aeromonas victoriae inactivated solution is 1×10 9 CFU / mL, the concentration of the inactivated solution of Citrobacter freundii was 1×10 9 CFU / mL, the concentration of Shigella-like inactivated solution is 1×10 9 CFU / mL.
3. A method for preparing the carp mixed bacterial inactivated vaccine according to claim 1 or 2, characterized in that The specific steps are as follows: (1) Aeromonas hydrophila, Aeromonas vermifuge, Citrobacter freundii and Plesiomonas shigellae were inoculated on LB solid medium respectively and cultured at 28°C for 16-24 h; (2) Pick single colonies of the four bacteria and place them in LB liquid culture medium. Culture them at 28°C and 180 rpm for 24 to 48 hours. (3) After the cells were expanded and cultured, they were centrifuged at 4°C and 5000 rpm for 10-15 min to collect the cell pellet, and then washed three times with sterile saline. Finally, the cells were resuspended in PBS and the OD value of the cell solution was adjusted. 600 To 1.0, add formalin to the bacterial solution to a final volume concentration of 1%, inactivate at 25 °C for 24 h, and shake the bacterial solution every 3 to 4 h; (4) Take 100 μL of the inactivated bacterial solution and spread it on LB solid culture medium, incubate at 28 °C for 24 h, and after testing for the absence of live bacteria, centrifuge the inactivated bacterial solution at 4 °C and 5000 rpm for 10-15 min to collect the bacterial precipitate, wash it three times with sterile saline, and finally resuspend the bacteria in PBS. Adjust the inactivated vaccine concentration to 1×10 9 CFU / mL; (5) Mix the four kinds of inactivated bacterial vaccines in a volume ratio of 1:1:1:1 to obtain a mixed carp bacterial inactivated vaccine, then divide the mixture into smaller packages and store it in a 4°C refrigerator for later use.
4. The carp mixed bacterial inactivated vaccine according to claim 1 or 2 is used as a vaccine for preventing and / or treating carp bacterial infection.
5. The use according to claim 4, characterized in that: The mixed inactivated bacterial vaccine is used to immunize carp by intraperitoneal injection.