Turtle vaccines, methods of making and using the same

Abstract

The present application relates to a kind of turtle vaccines and its preparation method and application, the immunogen of the turtle vaccine is prepared from the inactivated strain of Vibrio parahaemolyticus H0711 And / or the inactivated strain of Vibrio harveyi TL0816, the preservation number of the Vibrio parahaemolyticus H0711 It is CCTCC No: M 20221681, the preservation number of the Vibrio harveyi TL0816 It is CCTCC No: M 20221682.The strain Vibrio parahaemolyticus H0711 And Vibrio harveyi TL0816 with strong protective power to turtle can be prepared into inactivated vaccine and applied to green turtle to obtain immunity protection to bacteria, especially prepared into suitable bivalent vaccine, relative protection rate reaches 75%, can be used for preventing and treating turtle bacterial infection, which is of great significance to improve the immune ability of green turtle to specific pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a turtle vaccine and a preparation method and application thereof. BACKGROUND

[0002] Turtles are "living fossils" left over from the dinosaur era, and have great economic, scientific research, ornamental, cultural and ecological value. Among them, the green turtle (scientific name: Chelonia mydas) is a kind of turtle of the Cheloniidae family, which has been listed in Appendix I of the Washington Convention, the endangered species of the International Union for Conservation of Nature (IUCN), and the first-level national protected wildlife list of China. In recent years, due to human fishing and ecological environment destruction, the number of green turtles has decreased significantly, so it is particularly important to protect the green turtles.

[0003] With the maturation of the artificial breeding technology of green turtles, the disease problem also hinders the smooth progress of the conservation work. There are a large number of marine microorganisms in seawater, such as bacteria, fungi, parasites and parasitic organisms, etc. Due to the poor water quality, the decline of individual immunity, and the inadaptability of food, etc., all of which can lead to the outbreak of green turtle diseases. Since the green turtle is a national first-class protected animal, there are few reports on its common pathogenic bacteria and susceptible pathogenic bacteria, and it is difficult to carry out prevention and treatment work. Based on the fact that the green turtle must eventually return to the ocean, we do research on enhancing the disease resistance of the green turtle, and the vaccine developed for pathogenic bacteria is more effective.

[0004] The green turtle mainly feeds on seaweed and algae, so there is a great weakness in the food chain. Whether in the artificial breeding stage or in the marine life stage, bacterial infection is a common phenomenon. It may be sick due to accidentally eating plastic bags and other garbage, or it may be injured by sharp objects such as stones during swimming, or it may be attacked by natural enemies. There are a large number of bacteria in the ocean, especially Vibrio, so how to effectively resist the fatal influence of multiple pathogenic Vibrio on turtles has great significance for the protection of inactivated vaccine. SUMMARY

[0005] Based on this, the purpose of the present application is to provide a turtle vaccine and a preparation method and application thereof.

[0006] The first aspect of the present application is to provide a turtle vaccine, which has good immune protection for green turtles.

[0007] A turtle vaccine, the immunogen of which is prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and / or an inactivated strain of Vibrio harveyi TL0816, Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 of the present application were deposited with the China Center for Type Culture Collection (CCTCC) on October 28, 2022, and the deposit numbers are CCTCC No: M 20221681 for Vibrio parahaemolyticus H0711 and CCTCC No: M 20221682 for Vibrio harveyi TL0816.

[0008] In some embodiments, the turtle is a green turtle.

[0009] In some embodiments, the turtle vaccine is prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and an inactivated strain of Vibrio harveyi TL0816.

[0010] In some embodiments, the inactivated strain of Vibrio parahaemolyticus H0711 and the inactivated strain of Vibrio harveyi TL0816 are used in a ratio of 1-3:1-3, preferably 0.9-1.1:0.9-1.1, and further preferably 1:1.

[0011] In some embodiments, a vaccine adjuvant is further included.

[0012] In some preferred embodiments, the vaccine adjuvant is an aluminum salt adjuvant.

[0013] In some preferred embodiments, the inactivated strain of Vibrio parahaemolyticus H0711 and / or the inactivated strain of Vibrio harveyi TL0816 have a bacterial solution concentration of 1×10 8 cfu / mL to 2×10 9 cfu / mL, and more preferably 5×10 8 cfu / mL to 1×10 9 cfu / mL, respectively.

[0014] The second aspect of the present application provides a preparation method of the above-mentioned turtle vaccine.

[0015] The preparation method of the above-mentioned turtle vaccine comprises the following steps:

[0016] inactivating the cultured Vibrio parahaemolyticus H0711 to obtain an H0711 inactivated bacterial solution;

[0017] inactivating the cultured Vibrio harveyi TL0816 to obtain a TL0816 inactivated bacterial solution;

[0018] Mix the H0711 inactivated bacteria solution and the TL0816 inactivated bacteria solution.

[0019] In some embodiments, the inactivation is: adding each quantitative volume concentration of 0.18%-0.22% final concentration of formalin in Vibrio parahaemolyticus H0711 bacteria solution and / or Vibrio harveyi TL0816 bacteria solution, placing in a 28-32℃ constant temperature incubator, and placing for 22-26h for inactivation, i.e. formalin inactivated bacteria solution.

[0020] The third aspect of the present application is to provide the above-mentioned turtle vaccine for use in the preparation of a drug for preventing and treating turtle bacterial infection, preferably, the bacteria is Vibrio parahaemolyticus and / or Vibrio harveyi.

[0021] In the research of the present application, from a large number of bacteria isolated from sick green sea turtles, through artificial infection test of Chinese grass turtles, the strains Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 with strong protection to turtles were obtained, and were biologically preserved (preservation numbers are CCTCC No: M 20221681 and CCTCC No: M 20221682), which can be prepared into inactivated vaccines and applied to green sea turtles to obtain immunity protection against bacteria, especially prepared into suitable double vaccines, which have much better immunity against pathogenic bacterial infection than single vaccine, and the relative protection rate reaches 75%, which has important significance for improving the immunity of green sea turtles against specific pathogenic bacteria, and can be used for preventing and treating turtle bacterial infection. Moreover, the inactivated vaccine is also economical and inexpensive in preparation method, and is convenient for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Clinical symptoms of artificially infected dead grass turtles, wherein 1 and 2 are Vibrio harveyi TL0816 bacteria, and 3 and 4 are Vibrio parahaemolyticus H0711.

[0023] Figure 2 HE staining diagram of liver tissue sections of healthy and sick grass turtles, wherein A is HE (100X) of liver tissue sections of healthy turtles, B is HE (400X) of liver tissue sections of healthy turtles, C is HE (100X) of liver tissue sections of sick turtles infected with H0711, D is HE (400X) of liver tissue sections of sick turtles infected with H0711, E is HE (100X) of liver tissue sections of sick turtles infected with TL0816, and F is HE (400X) of liver tissue sections of sick turtles infected with TL0816.

[0024] Figure 3HE staining of lung tissue sections of healthy and sick grass turtles, wherein A is HE staining of lung tissue sections of healthy turtles (100X), B is HE staining of lung tissue sections of healthy turtles (400X), C is HE staining of lung tissue sections of sick turtles infected with H0711 (100X), D is HE staining of lung tissue sections of sick turtles infected with H0711 (400X), E is HE staining of lung tissue sections of sick turtles infected with TL0816 (100X), and F is HE staining of lung tissue sections of sick turtles infected with TL0816 (400X).

[0025] Figure 4 HE staining of intestinal tissue sections of healthy and sick grass turtles, wherein A is HE staining of intestinal tissue sections of healthy turtles (100X), B is HE staining of intestinal tissue sections of healthy turtles (400X), C is HE staining of intestinal tissue sections of sick turtles infected with H0711 (100X), D is HE staining of intestinal tissue sections of sick turtles infected with H0711 (400X), E is HE staining of intestinal tissue sections of sick turtles infected with TL0816 (100X), and F is HE staining of intestinal tissue sections of sick turtles infected with TL0816 (400X).

[0026] Figure 5 HE staining of kidney tissue sections (healthy and sick) results, wherein A is HE staining of kidney tissue sections of healthy turtles (100X), B is HE staining of kidney tissue sections of healthy turtles (400X), C is HE staining of kidney tissue sections of sick turtles infected with H0711 (100X), D is HE staining of kidney tissue sections of sick turtles infected with H0711 (400X), E is HE staining of kidney tissue sections of sick turtles infected with TL0816 (100X), and F is HE staining of kidney tissue sections of sick turtles infected with TL0816 (400X).

[0027] Figure 6 Morphology on TCBS plate (left) and Gram staining (right), wherein A is H0711 and B is TL0816.

[0028] Figure 7 16S rRNA amplification electrophoresis results of strains H0711 and TL0816, wherein M is a 2000 bp DNA marker, - is a negative control, and 1 and 2 correspond to the amplification bands of strains H0711 and TL0816, respectively.

[0029] Figure 8 Heat shock protein 60 gene amplification electrophoresis results of strains H0711 and TL0816, wherein M is a 2000 bp DNA marker, - is a negative control, and 1 and 2 correspond to strains H0711 and TL0816, respectively.

[0030] Figure 9is a schematic diagram of direct counting method under microscope, wherein A is a blood cell counting plate, B is a small grid volume (1 / 400 x 0.1) mm 3 , each of the black areas (the number of counts is set as A1 / A2 / A3 / A4 / A5) has a small grid, the total number of small grids is 16 x 5, and the minimum volume is (1 / 400) x 0.1 x 0.001 mL.

[0031] Figure 10 is a schematic diagram of significant analysis results of antibody titer.

[0032] Figure 11 is a schematic diagram of survival curve results of mixed bacteria challenge.

[0033] Figure 12 is a schematic diagram of survival curve results of H0711 bacteria challenge.

[0034] Figure 13 is a schematic diagram of survival curve results of TL0816 bacteria challenge. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0036] The experimental methods in the following examples not specifically noted are generally carried out according to the conventional conditions, for example, Green and Sambrook, eds., Molecular Cloning: A Laboratory Manual, 4th edition, published in 2013, or according to the conditions suggested by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used in the present application includes any and all combinations of one or more related listed items.

[0038] Monovalent vaccine only contains a single antigen component, which can only prevent one infectious disease or one type of pathogen infection. Combination vaccine refers to containing two or more live, inactivated organisms or purified antigens, which can be used to prevent multiple diseases. In fact, both have advantages and disadvantages. Although the application rate of monovalent vaccine is low, the protection effect of monovalent vaccine against a certain pathogen is better than that of combination vaccine. The advantage of combination vaccine is to reduce the number of inoculations, and at the same time, it can prevent multiple diseases and has good applicability. However, the process of combination vaccine is more complex, whether there is competition inhibition between combination vaccines, whether it is more likely to cause stress response of inoculated individuals, how to combine and other factors need to be considered.

[0039] In general, under the premise of safe and effective vaccine, combination vaccine can reduce the number of vaccine injections, prevent more types of diseases at the same time, improve the vaccination rate, reduce the stress of multiple immunization on individuals, reduce the cost of vaccine transportation, storage and inoculation, reduce the inoculation and management cost, and also reduce the dosage of preservatives and adjuvants and other agents in vaccine production, reduce the adverse reactions of vaccine, and thus the effective combination vaccine has more advantages.

[0040] The present application isolates and identifies 40 strains from sick turtles. Through isolation, purification and identification of the strains, the determination of artificial infection of Chinese grass turtle LD 50 , pathological section of artificially infected dead Chinese grass turtle and pathological section of sick and dead green sea turtle, two green sea turtle-derived Vibrio H0711 and TL0816 are obtained, it is found that the two strains of bacteria have high pathogenicity to the selected attack model Chinese grass turtle, and can be prepared into inactivated bivalent vaccine with good protection rate. In some embodiments, the immune dose, immune cycle, immune protection rate and serum antibody titer after immunization of the inactivated bivalent vaccine are evaluated.

[0041] The Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 of the present application were deposited with the China Center for Type Culture Collection (CCTCC) in Wuhan, China on October 28, 2022, and the deposit accession numbers are CCTCC No: M 20221681 and CCTCC No: M 20221682, respectively.

[0042] In some embodiments, the obtained virulent strains of Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 are inactivated to prepare a bivalent inactivated vaccine. Through grouping tests, the highest protection rate of 75% and the highest antibody titer of the aluminum hydroxide adjuvant high-dose group are obtained after mixed bacteria challenge. At the same time, the protection rate of the bivalent vaccine to single bacteria challenge can reach more than 50%, and it has good effect on grass turtles, and can be applied to green sea turtles. The screening of the strains described in the application has strict procedures, the preparation of the inactivated vaccine is simple, the operability is high, the steps are simple, the repeatability is high, and it has good specificity.

[0043] The application will be further described in detail below in combination with specific embodiments.

[0044] Example 1

[0045] We conducted several epidemiological investigations on the artificial breeding environment and sick individuals in Guangdong Huidong Sea Turtle National Nature Reserve, and found that bacterial diseases were the most common. During 2019-2021, we isolated 40 strains from the surface and body of sick green sea turtles in the reserve, and screened the virulence of the 40 strains. See Table 1 for details.

[0046] (1) Strain purification and expansion

[0047] The isolation medium is mainly blood plate and TCBS agar (both purchased from Guangdong Huan Kai Microbial Technology Co., Ltd.), and the blood plate is a finished medium. The TCBS agar medium is prepared by dissolving the powder according to the instructions, boiling for less than 2 min, and pouring the plate for standby use. The expansion medium of the strain is LB (tryptone 1 g / 100 mL, yeast powder 0.5 g / 100 mL, sodium chloride 2 g / 100 mL, 121℃ for 15 min), and the solid medium is inoculated and cultured at 30℃ for about 20 h. The liquid medium is inoculated and cultured at 30℃ for 20 h with constant temperature shaking at 200 rpm.

[0048] (2) 16S rRNA identification of strains

[0049] The 40 strains obtained from the epidemiological investigation were purified and cultured, and bacterial 16S rRNA identification was performed to determine the bacterial species. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID NO. 1) and 1492R (5'-TACGACTTAACCCCAATCGC-3' SEQ ID NO. 2) were used for gene amplification. The reaction system was 2x Taq PCR Master Mix 25 μL, 2 μL of each of the upstream and downstream primers, 1 μL of DNA template, and 20 μL of ddH2O. The reaction program was 95℃ for 5 min, 94℃ for 30 s, 56℃ for 30 s, 72℃ for 90 s, 35 cycles, and 72℃ for 7 min.

[0050] (3) Plate colony counting method (for live bacteria counting in challenge experiment)

[0051] 1. Number, mark the concentration of the plate and centrifuge tube respectively.

[0052] 2. Dilute the sample, dilute the sample with challenge bacteria appropriately. Take the sample, accurately pipette 10 -6 , 10 -7 , 10 -8 Dilute 1 ml of bacterial solution each time, and put 200 uL into a sterile plate.

[0053] 3. Pour the plate, pour 15 ml of medium melted and cooled to about 45°C into the above plate as soon as possible, and mix well by rotating. 4. Count, take out the culture plate after 24 h of culture at 30°C, calculate the average number of colonies in three plates of the same dilution, and then calculate the number of live bacteria per milliliter of the original sample according to the formula.

[0054] 5. Data processing, generally select the plates with an average number of colonies between 30 and 300 at the same dilution. For example, multiply the dilution factor by the average number of colonies between 30 and 300 at one dilution.

[0055] (3) Isolation of strain virulence primary screening

[0056] Through plate colony counting method, obtain the original bacterial solution concentration of 40 strains after expansion, 10000 rpm, 2 min, remove the supernatant, suspend the bacteria with physiological saline, adjust the bacterial solution to 10 8 cfu / mL, inject 200 uL into the abdominal cavity of each turtle, count the death within 14 days, and preliminarily screen the strains with strong virulence.

[0057] Table 2. Bacterial isolation and identification from green sea turtles and death rate statistics of artificial infection of Chinese grass turtles with strains

[0058]

[0059]

[0060] Four, virulence secondary screening of bacteria from green sea turtles

[0061] Through virulence primary screening, 9 strains with strong virulence (mortality rate greater than 50%) were obtained from 40 strains. To further screen strong strains, we challenged Chinese grass turtles with the 9 strains with strong virulence in the primary screening and counted the death to obtain strains with higher virulence and eliminate strains with lower virulence. Through purification and expansion, 9 strains of bacterial solution were obtained, the original bacterial solution concentration was determined by plate live bacteria counting method, and the original bacterial solution was diluted to 5×10 8As the initial concentration of the two-screen attack, the gradient concentration of each bacterial solution was obtained by 10-fold gradient dilution: 5 x 10 8 , 5 x 10 7 , 5 x 10 6 , 5 x 10 5 , 5 x 10 4 , 5 x 10 3 . The control group and 9 experimental groups used 20 healthy grass turtles, all weighing 32 ± 2 g, 200 uL intraperitoneal injection, placed in glass jars for normal feeding, water temperature 26 ± 2℃, survival statistics for 14 days. Please refer to Tables 3-12 for detailed attack statistics.

[0062] Table 3. T2019H0711 attack statistics table (referred to as H0711 strain)

[0063]

[0064] Table 4. T2020TL0816 attack statistics table (referred to as TL0816 strain)

[0065]

[0066] Table 5. T20201117TL1 attack statistics table

[0067]

[0068]

[0069] Table 6. T20200420GQ4 attack statistics table

[0070]

[0071] Table 7. T20210322H5 attack statistics table

[0072]

[0073] Table 8. T20210515#4HG1 attack statistics table

[0074]

[0075]

[0076] Table 9. T20210515#24HG attack statistics table

[0077]

[0078] Table 10. T20210818Q4 attack statistics table

[0079]

[0080] Table 11. Statistics on the Infection Situation of T20210818#23

[0081]

[0082]

[0083] Example 2

[0084] After further screening, Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 were selected in this invention, and the LD50 of highly virulent strains was determined (calculated using SPSS software).

[0085] LD50% 50 LD50 refers to the drug dose that can cause death in half of the test animals, usually expressed as the logarithm of the lethal dose. This invention directly uses SPSS software to calculate LD50 using a probability-weighted regression method (Bliss method). 50 The method is simple, fast, intuitive and convenient.

[0086] Taking Vibrio parahaemolyticus strain H0711 as an example, variables were set and input data was entered. Analyze---Regression—Probit (probability unit). "Challenge dose" was selected in the "Covariate" column; "Number of deaths" was selected in the "Response frequency" column; "Total number of experimental animals" was selected in the "Summary of observations" column; in the "Transformation" column, "Logarithm base is 10, other options remain at default." The resulting regression equation is: Probit = --4.660 + 0.758X. The residuals of the probability table for experimental and expected values ​​at different doses follow a normal distribution. Standardized residual = Mean of residuals / Standard deviation of residuals. The probability that the standardized residual of the experimental point falls outside the normal distribution interval is ≤0.05.

[0087] The estimated value corresponding to a probability of 0.5 can be found in the 95% confidence interval table; this is the LD. 50 Dosage 3.52 × 10 6 cfu / mL, that is, a bacterial concentration of 3.52 × 10⁻⁶. 6 A concentration of cfu / mL and an attack of 0.2 mL of the virus caused death in half of the grass turtles. The LD50 of strain TL0816 was obtained using the same method. 50 Dosage 5.11×10 5 cfu / mL. Please refer to Table 12 for the SPSS calculation results of the LD50 of the two highly virulent strains.

[0088] Table 12. LD50 of highly virulent strains 50 Calculation results (SPSS software)

[0089] Strains Strain No. LD 50 dose (cfu / mL) Vibrio parahaemolyticus H0711 H0711 3.52 x 10 6 ]]> Vibrio harveyi TL0816 TL0816 5.11 x 10 5 ]]>

[0090] Example 3

[0091] Analysis of pathological sections of Chinese grass turtles infected with Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816.

[0092] Through artificial infection experiments by initial screening of virulence, we found that the clinical symptoms of Chinese grass turtles infected with Vibrio harveyi and Vibrio parahaemolyticus were consistent with those of the green sea turtles that died of the disease: liver enlargement and hemorrhage, intestinal swelling and hyperemia, lung tissue atrophy, and kidney enlargement and hyperemia. We promptly cut liver, lung, intestinal, and kidney tissues from the grass turtles that were dying and fixed them in 10% formalin, and we also took samples from healthy grass turtles as a control group, which were used to prepare paraffin sections for pathological analysis.

[0093] The clinical symptoms of the dead grass turtles infected artificially are shown in Figure 1 .

[0094] 1. Comparison and analysis of tissue sections of grass turtles that died of the disease and healthy grass turtles (liver, intestine, lung, kidney)

[0095] 1.1 HE staining of liver tissue sections (healthy and diseased), the results of which are shown in Figure 2 .

[0096] As can be seen from Figure 2 , the liver tissue sections of the diseased turtles showed disordered arrangement of hepatocytes, vacuolization of hepatocytes, and a small amount of lymphocyte infiltration; there was a large amount of hemosiderin deposition. This suggests liver hemorrhage and inflammation.

[0097] 1.2 The results of HE staining of lung tissue sections (healthy and diseased) are shown in Figure 3 .

[0098] As can be seen from Figure 3 , the alveolar septum of the diseased turtles was broken, and the alveoli were atrophic; large cystic cavities were visible in the lung tissue, which may have been caused by the proliferation of abnormal tissue in the alveoli due to acute infection. This suggests that the lung nodules may have been caused by inflammation, accompanied by symptoms of lung atrophy. Figure 4 .

[0099] As can be seen from Figure 4 , both showed villous mucosal shedding and submucosal layer damage; the serous layer was thin, and the muscle layer was damaged to varying degrees. This suggests that the intestinal mucosa is thin, swollen, or has enteritis.

[0100] 1.4 The results of HE staining of kidney tissue sections (healthy and diseased) are shown in Figure 5 .

[0101] As can be seen fromFigure 5 In the healthy state, the glomerular vascular loop is thin and clear. The number of endothelial cells and mesangial cells is normal. The surrounding renal tubules are also normal; after the onset, the renal tubule lumen is blocked, the renal tubule is enlarged, the lumen is enlarged, and the glomerular capillary is proliferated, indicating that there is obvious nephritis.

[0102] Example 4 Vibrio culture

[0103] 1. Strain purification, expansion and Gram staining (culture method is the same as above)

[0104] 10 uL of the preserved H0711 and TL0816 bacteria was taken and placed on a TCBS plate, and cultured at 30°C for 24 h. Single colonies on the TCBS plate were picked into LB medium for expansion. At the same time, single colonies were picked for Gram staining. The Vibrio parahaemolyticus H0711 colony on the TCBS plate was round, with neat edges, moist, slightly turbid, translucent, green color, and a diameter of 2-4 mm; Gram staining was negative bacilli. The Vibrio harveyi TL0816 colony on the TCBS plate was round, with neat edges, moist, translucent, yellow color, and a diameter of 2-4 mm; Gram staining was negative bacilli.

[0105] 2. Strain 16S rRNA identification

[0106] Universal primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID NO. 1), 1492R (5'-TACGACTTAACCCCAATCGC-3' SEQ ID NO. 2) Reaction system: 2 x TaqPCR Master Mix 25 uL, 2 uL of upstream and downstream primers, 1 uL of DNA template, 20 uL of ddH2O; Reaction program: 95°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 7 min. The amplified product was sequenced to determine.

[0107] Note: The figure shows the 16S rRNA amplification electrophoretic bands of strains H0711 and TL0816, M is a 2000 bp DNA marker, - is a negative control, 1 and 2 correspond to the amplification bands of strains H0711 and TL0816, respectively.

[0108] 2.1 H0711 strain 16S rRNA sequencing results:

[0109] TGCAAGTCGAGCGGAAACGAGTTATCAGAACCTTCGGGGAACGATAACGGCGTCGAGC

[0110] GGCGGACGGGTGAGTAATGCCTAGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAA

[0111] CGATGGCTAATACCGCATGATGCCTACGGGCCAAAGAGGGGGACCTTCGGGCCTCTCGC

[0112] GTCAGGATATGCCTAGGTGGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGAC

[0113] GATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGA

[0114] CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCA

[0115] TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCGTGAGGAAGGT

[0116] AGTGTAGTTAATAGCTGCATTATTTGACGTTAGCGACAGAAGAAGCACCGGCTAACTCC

[0117] GTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAA

[0118] AGCGCATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAATT

[0119] GCATTTGAAACTGGCAGACTAGAGTACTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGG

[0120] TGAAATGCGTAGAGATCTGAAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAGATA

[0121] CTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC

[0122] GCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACG

[0123] CGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGG

[0124] GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACC

[0125] TACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGA

[0126] CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAAC

[0127] GAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTAATGTCGGGAACTCCAGGGAGACTGC

[0128] CGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTA

[0129] GGGCTACACACGTGCTACAATGGCGCATACAGAGGGCAGCCAACTTGCGAAAGTGAGC

[0130] GAATCCCAAAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTC

[0131] GGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACA

[0132] CACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGGGGACGCTTACCAC (SEQ ID NO. 3)

[0133] 2.2 Sequencing results of 16S rRNA of strain TL0816:

[0134] TAAGCGTCCTCCCGAAGGTTAAACTACCTACTTCTTTTGCAGCCCACTCCCATGGTGTGA

[0135] CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACT

[0136] AGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTT

[0137] TTGGGATTCGCTCACTCTCGCAAGTTGGCCGCCCTCTGTATGCGCCATTGTAGCACGTGT

[0138] GTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTAT

[0139] CACCGGCAGTCTCCCTGGAGTTCCCACCCGAAGTGCTGGCAAACAAGGATAAGGGTTG

[0140] CGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGC

[0141] ACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAA

[0142] GAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGG

[0143] GCCCCCGTCAATTCATTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACTTA

[0144] ACGCGTTAGCTCCGAAAGCCACGGCTCAAGGCCACAACCTCCAAGTAGACATCGTTTAC

[0145] GGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCATCTGAGTGTC

[0146] AGTATCTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTTCAGATCTCTACGCATTTCA

[0147] CCGCTACACCTGAAATTCTACCCCCCTCTACAGTACTCTAGTCTGCCAGTTTCAAATGCTA

[0148] TTCCGAGGTTGAGCCCCGGGCTTTCACATCTGACTTAACAAACCACCTGCATGCGCTTTA

[0149] CGCCCAGTAATTCCGATTAACGCTCGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAG

[0150] TTAGCCGGTGCTTCTTCTGTCGCTAACGTCAAATAATGCAGCTATTAACTACACTACCTTC

[0151] CTCACGACTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGC

[0152] ATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACC

[0153] GTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAGGGATCGTCGCCTTGG

[0154] TGAGCCATTACCTCACCAACTAGCTAATCCCACCTAGGCATATCCTGACGCGAGAGGCCC

[0155] GAAGGTCCCCCTCTTTGACCCGTAGGTATTATGCGGTATTAGCCATCGTTTCCAATGGTTA

[0156] TCCCCCACATCAGGGCAATTTCCTAGGCATTACTCACCCGTCCGCCGCTCGACGCCGTTATCGTTCCCCGAAGGTTCAGATAACTCGTTTCCGCTCGACTTGC (SEQ ID NO. 4).

[0157] 3. Amplification of HSP60 (Heat Shock Protein-60) gene of strain H0711

[0158] Primer: HSP60-F (5'-ACAACAGCAACGGTACTAGC-3' SEQ ID NO. 5), HSP60-R (5'-CAACTTTCACGATGCCAC-3' SEQ ID NO. 6),

[0159] Reaction system: 2 x TaqPCR Master Mix 25 μL, 2 μL of upstream and downstream primers, 1 μL of DNA template, 20 μL of ddH2O; reaction program: 95 °C for 5 min; 94 °C for 30 s, 56 °C for 30 s, 72 °C for 90 s, 35 cycles; 72 °C for 7 min. The amplified product was sequenced to determine.

[0160] 3.1 Sequencing results of H0711 strain HSP60 amplified fragment:

[0161] CGCAAGCAATCGTAAATGAAGGTCTAAAAGCAGTTGCAGCGGGTATGAACCCAATGGAT

[0162] CTTAAGCGCGGTATCGACAAAGCTGTTGCAGCGGCAGTAGAGCAACTAAAAGAGCTTTC

[0163] TGTTGAGTGTAACGACACCAAAGCAATCGCACAGGTTGGTACTATCTCTGCGAACTCTG

[0164] ACGCAAGCGTAGGTAACATCATTGCTGAAGCAATGGAACGCGTTGGCCGCGACGGTGTT

[0165] ATCACTGTTGAAGAAGGTCAGGCTCTACAAGACGAGCTAGACGTAGTAGAAGGTATGCA

[0166] GTTCGACCGCGGTTACCTATCTCCTTACTTCATCAACAACCAAGAAGCGGGCAGCGTTG

[0167] AGCTAGAAAACCCATTCATCCTTCTAGTTGATAAGAAGATCTCAAACATTCGTGAGCTTC

[0168] TACCAACTCTAGAAGCAGTAGCAAAAGCATCTCGTCCACTGCTAATCATCGCAGAAGAC

[0169] GTAGAAGGCGAAGCACTAGCGACATTGGTTGTGAACAACATGCGTGTCTGGGAAAA (SEQ ID NO. 7)

[0170] 3.2 TL0816 strain HSP60 amplified fragment sequencing results:

[0171] AACTTTTCCACGAGCCACGCATGTTGTTCACAACAAGTGTTGCTAGTGCTTCACCTTCTA

[0172] CGTCTTCAGCGATGATAAGTAGTGGGCGAGATGCTTTTGCTACTGCTTCTAGTGCTGGAA

[0173] GAAGTTCACGGATGTTCGATACTTTCTTATCGATCAGAAGGATGAATGGGTTTTCTAGAT

[0174] CAACAGAACCTGCTTCTTGGTTGTTGATGAAGTAAGGAGATAGGTAACCGCGGTCGAAC

[0175] TGCATACCTTCAACTACATCTAGCTCATCTTGTAGTGCTTGACCTTCTTCAACAGTGATAA

[0176] CACCGTCGCGACCAACTTTTTCCATTGCTTCAGCAATGATGTTACCTACGCTTGAGTCAG

[0177] AGTTCGCAGAGATAGTACCAACCTGTGCGATTGCTTTGGTGTCGTTACACTCAACAGATA

[0178] GCTCTTTTAGTTGCTCAACTGCTGCGATAACTGCTTTGTCGATACCGCGCTTAAGGTCCA

[0179] TTGGGTTCATACCCGCTGCAACTGCTTTTAGACCTTCATTTACGATTGCTTGCGCTAG (SEQ ID NO. 8).

[0180] 4 Biochemical identification of strains

[0181] Table 4-1 Biochemical identification results of H0711 and TL0816 strains

[0182]

[0183]

[0184] After two rounds of screening, two strains of Vibrio (numbered H0711, TL0816) isolated from diseased green sea turtles were obtained, and their strong virulence to healthy grass turtles was demonstrated by artificial infection virulence test. They were inoculated in LB liquid (2% NaCl) medium, placed in a 30°C constant temperature incubator at 200 rpm for 20h, then centrifuged at 10000 rpm for 2 min, washed with sterile physiological saline for 3 times, and then the bacterial cells were collected.

[0185] 5 Direct counting method under microscope (for inactivated bacteria immune counting)

[0186] 5.1 Observe the concentration of the bacteria suspension to be tested, and dilute it appropriately with sterile physiological saline (usually dilute it by 100 times), so that the number of bacteria per small grid can be counted.

[0187] 5.2 Take a clean blood cell counting plate, and first cover it with a cover glass on the counting area.

[0188] 5.3 Shake the original bacterial suspension thoroughly, dilute it to the desired concentration, and then use a pipette to take 8uL. After placing the cover glass, add the bacterial solution dropwise in the gap, and the bacterial solution will enter the counting plate by capillary action.

[0189] 5.4 Let it stand for 3-5 minutes to allow the bacteria to settle on the counting plate and not to drift with the liquid. Place the blood cell counting plate on the stage of the microscope and clamp it firmly. First find the counting area under low magnification, then switch to high magnification to observe and count.

[0190] 5.5 The central counting area is composed of 25 middle squares. In addition to the four middle squares mentioned above, the number of bacteria in the central middle square (i.e. 25x16 small squares) also needs to be counted. In order to ensure the accuracy of the count, avoid repeated counting and missed counting;

[0191] 5.6 When counting, the statistics of cells settled on the grid lines should have uniform provisions. If the bacteria are located on the double lines of the large square, count the upper line and do not count the lower line, count the left line and do not count the right line when counting, so as to reduce the error. That is, the cells located on the upper line and the left line of the grid are counted into the grid, and the cells on the lower line and the right line of the grid are counted into the corresponding grid according to the provisions.

[0192] 5.7 Each sample is counted 2-3 times (the value of each count should not differ too much, otherwise it should be re-operated), and the number of bacteria contained in each mL of bacterial suspension is calculated according to the formula.

[0193] 5.8 After counting, remove the cover glass and rinse the blood cell counting plate with water. Do not use hard objects to wash or wipe, so as not to damage the grid scale. After washing, dry and store in the box.

[0194] 5.9 Calculation formula: [(A1+A2+A3+A4+A5) / (5x16)] / [(1 / 400)x0.1x0.001]x dilution factor (cfu / mL).

[0195] The two strains of bacteria in the application are identified by 16S rRNA and hsp60 gene sequences. The sequencing results are subjected to BLAST comparison in NCBI. After comparison, the 16S rRNA and hsp60 gene sequences of H0711 strain have 100% and 99% homology with the 16S RNA and hsp60 gene sequences of Vibrio parahaemolyticus in the NCBI database, respectively; and combined with the biochemical identification results, it is comprehensively determined as Vibrio parahaemolyticus. The 16S rRNA and hsp60 gene sequences of TL0816 strain have 100% and 99.63% homology with the 16S RNA and hsp60 gene sequences of Vibrio harveyi in the NCBI database, respectively, and combined with the biochemical identification results, it is comprehensively determined as Vibrio harveyi.

[0196] The Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 have been preserved in the China Center for Type Culture Collection on October 28, 2022. The preservation number of the Vibrio parahaemolyticus H0711 is CCTCC No: M 20221681, and the preservation number of the Vibrio harveyi TL0816 is CCTCC No: M 20221682.

[0197] Example 5, preparation of immunogen

[0198] The calculated bacterial liquid concentration is adjusted to 109 After the concentration of the two strains of bacteria H0711 and TL0816 reached 5 x 107cfu / mL, the two strains of bacteria were mixed at a ratio of 1:1 to prepare a mixed bacteria solution as an immunogen for inoculating the turtles and a reactogen for determining the agglutination titer of the immune serum. The experimental group was divided into two groups according to whether an adjuvant was used: an aluminum adjuvant group (aluminum adjuvant: bacteria solution = 1:3) and an adjuvant-free group. The immunization dose was divided into three groups: high dose, medium dose, and low dose. Thus, there were seven groups: ① aluminum adjuvant-high dose, ② aluminum adjuvant-medium dose, ③ aluminum adjuvant-low dose; ④ adjuvant-free-high dose, ⑤ adjuvant-free-medium dose, ⑥ adjuvant-free-low dose, and ⑦ a control group, with 20 turtles in each group. 9 The medium dose was 5 x 107cfu / mL, the low dose was 10 8 cfu / mL, and the high dose was 10 8 cfu / mL.

[0199] In the prepared two strains of bacteria (H0711 and TL0816) solution, 0.2% formalin was added to each to prepare a final concentration of 0.2% formalin, and the solution was placed in a constant temperature incubator at 30.0°C for 24 hours for inactivation. Then, 100 μL of the inactivated bacteria solution was taken and spread on TCBS plates and LB plates, which were placed in a constant temperature incubator at 30.0°C for 24 hours. No bacterial colonies grew on the culture medium, indicating that the bacteria had been completely inactivated and could be used as an inactivated vaccine (reactogen).

[0200] Example 6 Safety test of inactivated vaccine

[0201] To ensure that the inactivated vaccine was safe and non-toxic for the turtles used in the experiment, the two strains of bacteria H0711 and TL0816 were inactivated and then mixed at a ratio of 1:1 to prepare a solution with a concentration of 10 10 cfu / mL. Three groups of turtles of different sizes were used for the safety test: large turtles, each weighing about 37 g; medium turtles, each weighing about 32 g; and small turtles, each weighing about 28 g. Each group had 20 turtles, and 100 uL of the inactivated bacteria was injected intramuscularly. The turtles were observed for safety for two weeks. A control group was injected with sterile saline.

[0202] Table 9-1. Statistics of 14-day safety test of high-concentration inactivated vaccine

[0203]

[0204]

[0205] Analysis: The experimental results showed that the use of high-concentration 10 10The survival rate of different sizes of grass turtles immunized with the inactivated dual bacterial vaccine was high, reaching 90% or more. The preliminary speculation of the death of the grass turtles was that they died of stress. Overall, the vaccine was highly safe.

[0206] Example 7 Immunization

[0207] Healthy grass turtles with a weight of about 35 g were injected intramuscularly with 100 uL of inactivated dual bacterial vaccine, and 80 grass turtles were inoculated with each strain of inactivated bacterial vaccine. The control group was injected with 100 uL of sterile saline. The inoculated grass turtles were normally fed, and the grass turtles were fed with artificial compound feed once a day. During the feeding period, the water temperature in the aquarium was controlled at 24-28°C. After 10 days of feeding after the first immunization with the inactivated dual bacterial vaccine, the second immunization was performed, and the dosage was the same. The feeding time was 20 days.

[0208] Blood sampling and serum separation

[0209] Six grass turtles were randomly taken from each test and control group in the aquarium, and blood was collected by the method of subvertebral vein. About 0.8-1 mL of blood was collected and placed in a 2 mL centrifuge tube. The tube was placed horizontally at room temperature for more than 2 hours, centrifuged at 4000 g for 20 min, and the immune serum was separated. The serum was stored at -80°C for later use.

[0210] Immune protection rate statistics: for the second immunization (30 days) end, the challenge strain concentration was 2 times the LD 50 of the two strains (H0711, TL0816), and after mixing at a ratio of 1:1, each test turtle was injected with 200 uL into the abdominal cavity, and normally fed for 30 days. The survival rate was calculated, and the immune protection rate was calculated.

[0211] Determination of serum antibody titer

[0212] 1. In the first row and first column of the 96-well plate V-shaped plate (90°), 50 microliters of normal saline was added to each well, and 50 microliters was added to each well in the other wells.

[0213] 2. Add 20 microliters of grass turtle serum to the first well and mix well. Take out 50 microliters from the first well and add it to the second well, and mix well.

[0214] 3. Take out 50 microliters from the second well and add it to the third well, and mix well. Repeat the same process for the other wells up to the 11th well. Discard 50 microliters from the 11th well, and do not add to the 12th well.

[0215] 4. Add 50 microliters of inactivated bacteria solution (1 x 10 8 cfu / mL, 0.2% formalin inactivation) to wells 1-12. Shake for 1 minute, then place in a 30°C incubator for 2 hours, and then place in a 4°C refrigerator overnight. Observe the results the next day.

[0216] 5. Observe under low magnification and record the reciprocal of the highest dilution of agglutinated precipitate as the antibody titer. The highest dilution that shows agglutination is taken as the agglutination titer, and the antibody titer is recorded as the titer above the one marked (+).

[0217] 6. Calculate the geometric mean titer of the antibody. The geometric mean (G) is the nth root of the product of all n observations. It is often used to describe the central location of data with a few large extreme values ​​or data that are multiples or approximately multiples of each other.

[0218] 7. Perform one-way ANOVA on antibody titers using SPSS. Step 1: Open the software and click "Analyze – Compare Means – One-way ANOVA". Step 2: In the "One-way ANOVA" window, place "Antibody Titer" in the "Dependent Variable List" box and "Reactor" in the "Factor" box. Step 3: In the "One-way ANOVA" window, click the "Post-hoc Comparisons" option. The "One-way ANOVA: Post-hoc Multiple Comparisons" window will appear. Under "Assuming Homogeneity of Variance", check the "Bonfrenny" method. Under "Not Assuming Homogeneity of Variance", check the "Tammani" method. Then click "Continue". Step 4: In the "One-way ANOVA" window, click "Options". The "One-way ANOVA: Options" window will appear. Check "Descriptive", "Homogeneity of Variance Test", and "Welch". Then click "Continue".

[0219] Table 13-1 Results of serum agglutination antibody titers in experimental and control groups

[0220]

[0221] Please see the results of the antibody titer significance analysis. Figure 10 The immunogens were H0711:TL0816 = 1:1, with a high dose of 10 for each bacterium. 9 cfu / mL, medium dose 5×10 8 cfu / mL, low dose 10 8 cfu / mL.

[0222] from Figure 10As can be seen, antibody titer significance analysis showed that the antibody titers of the experimental groups were higher than those of the control group, and the high-dose adjuvant group was significantly higher than the other groups (P<0.5); the antibody geometric mean titer calculation results showed that the highest was the high-dose adjuvant immunization group, which was 1:1:114.04, followed by the medium-dose adjuvant group, which was 1:50.80, and the control group was 1:5.04. The experimental results showed that the agglutination antibody titer of the serum of the Chinese grass turtle was significantly higher than that of the control group after the second immunization for 30 days. This shows that the inactivated vaccine can produce common antibodies in the turtle body, which can effectively protect the grass turtle from the invasion of the bacterial disease.

[0223] Example 8 Relative protection rate of bivalent vaccine

[0224] Vaccine relative protection rate = (1-immune turtle mortality rate / control group turtle mortality rate) x 100%

[0225] The 6 immunization groups and the control group were subjected to challenge test using the following 4 different strain combinations. Challenge group 1 was challenged with 2LD 50 dose (1:1) mixed with 200 uL. Challenge group 2 was injected with 2LD 50 concentration H0711 single bacteria uL, challenge group 3 was injected with 2LD 50 concentration TL0816 single bacteria 200 uL.

[0226] Table 15. Relative protection rate statistics table of mixed challenge

[0227] Grouping Number of formal challenge Number of 30d death 30d mortality 30d protection rate Control group 20 16 80.00％ - Adjuvant high dose immunization group 20 4 20.00％ 75.00％ Adjuvant medium dose immunization group 20 8 40.00％ 50.00％ Adjuvant low dose immunization group 20 10 50.00％ 37.50％ Non-adjuvant high dose immunization group 20 5 25.00％ 68.75％ Non-adjuvant medium dose immunization group 20 7 35.00％ 56.25％ Non-adjuvant low dose immunization group 20 9 45.00％ 43.75％

[0228] The description of the survival curve of mixed bacteria challenge can be seen in Figure 11 .

[0229] Table 16. Relative protection rate statistics table of H0711 single bacteria challenge

[0230] H0711 single strain challenge Number of formal challenge Number of 30d death 30d mortality 30d protection rate Control group 20 17 85.00％ - Adjuvant high dose immunization group 20 7 35.00％ 58.82％ Adjuvant medium dose immunization group 20 7 35.00％ 58.82％ Adjuvant low dose immunization group 20 11 55.00％ 35.29％ Non-adjuvant high dose immunization group 20 9 45.00％ 47.06％ Non-adjuvant medium dose immunization group 20 10 50.00％ 41.18％ Non-adjuvant low dose immunization group 20 10 50.00％ 41.18％

[0231] The description of the survival curve of H0711 bacteria challenge can be seen in Figure 12 .

[0232] Table 17. Relative protection rate statistics table of TL0816 single bacteria challenge

[0233] TL0816 single strain challenge Number of formal challenge Number of 30d death 30d mortality 30d protection rate Control group 20 16 80.00％ - Adjuvant high dose immunization group 20 7 35.00％ 56.25％ Adjuvant medium dose immunization group 20 9 45.00％ 43.75％ Adjuvant low dose immunization group 20 10 50.00％ 37.50％ Non-adjuvant high dose immunization group 20 9 45.00％ 43.75％ Non-adjuvant medium dose immunization group 20 9 45.00％ 43.75％ Non-adjuvant low dose immunization group 20 11 55.00％ 31.25％

[0234] The above tables 15-17, and Figure 12-Figure 13As can be seen from the survival curves of the grass turtles immunized and then attacked one month later, the survival rates of the immunized groups were higher than those of the control groups. Among them, the protection rate of the grass turtles in the high-dose adjuvant immunization group was the highest, reaching 75% after being attacked by mixed bacteria, followed by the high-dose non-adjuvant immunization group, with a protection rate of 68.75%; the protection rates of the single-bacteria attack were all above 50%, and the protection rates after high-dose immunization were higher than those after low-dose immunization, indicating that high-dose immunization could stimulate individuals to produce more antibodies and improve the disease resistance.

[0235] The above-described embodiments only express several embodiments of the present application, which are described in detail, but should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A vaccine for turtles, wherein, Its immunogen was prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and an inactivated strain of Vibrio harveyi TL0816, wherein Vibrio parahaemolyticus ( Vibrio parahemolyticus The preservation number of H0711 is CCTCC No: M 20221681, and the Vibrio harveyi ( Vibrio harveyi The preservation number of TL0816 is CCTCC No: M 20221682; the ratio of the inactivated strain of Vibrio parahaemolyticus H0711 to the inactivated strain of Vibrio harveyi TL0816 is 0.9-1.1:0.9-1.1, and the bacterial concentrations of the inactivated strains of Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 are 1×10⁻⁶. 8 cfu / mL to 2×10 9 cfu / mL, the turtle in question is a green sea turtle.

2. The vaccine for turtles according to claim 1, wherein, The inactivated strain of Vibrio parahemolyticus H0711 and the inactivated strain of Vibrio harveyi TL0816 are used in a ratio of 1:

1.

3. The vaccine for turtles according to any one of claims 1-2, wherein, A vaccine adjuvant is also included.

4. The vaccine for turtles according to claim 3, wherein, The vaccine adjuvant is an aluminum salt adjuvant.

5. The vaccine for turtles according to claim 1, wherein, The bacterial liquid concentration of the inactivated strain of Vibrio parahaemolyticus H0711 and / or the inactivated strain of Vibrio harveyi TL0816 is 5×10 8 cfu / mL to 1×10 9 cfu / mL.

6. A method of preparing the vaccine of any one of claims 1-5, wherein, The method comprises the following steps: The cultured Vibrio parahemolyticus H0711 is inactivated to obtain an H0711 inactivated bacteria solution; The cultured Vibrio harveyi TL0816 is inactivated to obtain a TL0816 inactivated bacteria solution; The H0711 inactivated bacteria solution and the TL0816 inactivated bacteria solution are mixed.

7. Use of the turtle vaccine of any one of claims 1-5 in the preparation of a medicament for preventing Vibrio parahemolyticus and / or Vibrio harveyi infection in turtles.

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