CN115944720A - Turtle vaccine and preparation method and application thereof - Google Patents

Abstract

The invention relates to a turtle vaccine and a preparation method and application thereof, wherein the immunogen of the turtle vaccine is prepared from an inactivated strain of vibrio parahaemolyticus H0711 and/or an inactivated strain of vibrio harveyi TL0816, and the preservation number of the vibrio parahaemolyticus H0711 is CCTCC No: m20221681, and the preservation number of the Vibrio harveyi TL0816 is CCTCC No: m20221682. The invention obtains the strains of vibrio parahaemolyticus H0711 and vibrio harveyi TL0816 with strong protection to the tortoise, can prepare the strains into inactivated vaccine to be applied to green tortoise to obtain the immunity protection to the bacteria, especially prepare the proper bivalent vaccine, the relative protection rate reaches 75 percent, can be used for preventing and treating the tortoise bacterial infection, and has important significance for improving the immunity of the green tortoise to the specific pathogenic bacteria.

Description

Turtle vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a turtle vaccine and a preparation method and application thereof.

Background

The sea turtle is an 'activated stone' left by the dinosaur age, and has great economic, scientific, ornamental, cultural and ecological values. Among them, a tortoise of the genus chelonian of the family cheloniaceae, the name of green sea turtle (Chelonia mydas), is listed at first in appendix I bibliography of the "Washington convention", endangered species of the world nature protection alliance (IUCN), and bibliography of wild animals of the national emphasis protection of China. In recent years, the number of green sea turtles is greatly reduced due to artificial fishing and ecological environment destruction, so that the maintenance of green sea turtles is particularly important.

With the maturity of the artificial propagation technology of the green sea turtle, the smooth progress of the conservation work is also hindered by the disease problem. A large amount of marine microorganisms, such as bacteria, fungi, parasites, parasitic organisms and the like exist in seawater, and the marine microorganisms cause the outbreak of the green sea turtle diseases due to poor water quality, the reduction of the immunity of individuals, food inadaptability and the like. Since the green sea turtle is a national first-class protected animal, common pathogenic bacteria and susceptible pathogenic bacteria are reported, and the development of prevention and treatment work is difficult. Based on the fact that the green sea turtle finally needs to return to the sea, the research on enhancing the disease resistance of the green sea turtle is carried out, and the vaccine developed aiming at pathogenic bacteria is more effective.

The green sea turtles mainly feed on seaweed, algae and the like, so that great weakness exists in the food chain, and bacterial infection is a common phenomenon no matter in the artificial breeding stage or the marine life stage. The food is eaten by mistake, so that the food is ill, or is scratched by sharp objects such as stones in the swimming process, and can be injured by natural enemy attacks and the like. A large amount of bacteria exist in the sea, especially vibrio is taken as the main bacteria, so that the research on how to effectively resist the fatal influence of various pathogenic vibrios on the turtle has great significance on the protection effect of the inactivated vaccine.

Disclosure of Invention

Based on the above, the invention aims to provide a turtle vaccine and a preparation method and application thereof.

In a first aspect of the invention, a turtle vaccine is provided, which has good immune protection against green turtles.

A sea turtle vaccine is prepared by an inactivated strain of Vibrio parahaemolyticus H0711 and/or an inactivated strain of Vibrio harveyi TL0816, wherein the Vibrio parahaemolyticus (Vibrio parahaemolyticus) H0711 and Vibrio harveyi TL0816 are preserved in China center for type culture collection (CCTCC; address: wuhan, wuhan university; zip code: 430072) at 28 th 10 th 2022, and the preservation number of the Vibrio parahaemolyticus H0711 is CCTCC No: m20221681, the preservation number of the Vibrio harveyi TL0816 is CCTCC No: m20221682.

In some of these embodiments, the turtle is a green turtle.

In some of these examples, the turtle vaccine is prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and an inactivated strain of Vibrio harveyi TL0816.

In some of the examples, the inactivated strain of vibrio parahaemolyticus H0711 and the inactivated strain of vibrio harveyi TL0816 are used in the following ratio: 1-3:1-3, preferably 0.9-1.1:0.9 to 1.1, more preferably 1:1.

in some of these embodiments, a vaccine adjuvant is also included.

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

In some preferred embodiments, the bacteria liquid concentration of the inactivated strain of Vibrio parahaemolyticus H0711 and/or the inactivated strain of Vibrio harveyi TL0816 is 1 × 10 ⁸ cfu/mL to 2X 10 ⁹ cfu/mL, more preferably 5X 10 ⁸ cfu/mL to 1X 10 ⁹ cfu/mL。

In a second aspect of the present invention, a method for preparing the above-mentioned turtle vaccine is provided.

The preparation method of the sea turtle vaccine comprises the following steps:

inactivating the cultured vibrio parahaemolyticus H0711 to obtain H0711 inactivated bacteria liquid;

inactivating the cultured Vibrio harveyi TL0816 to obtain TL0816 inactivated bacterial liquid;

h0711 inactivated bacteria solution and TL0816 inactivated bacteria solution are mixed.

In some of these embodiments, the inactivation is: formalin with the volume concentration of 0.18-0.22% is quantitatively added into vibrio parahaemolyticus H0711 bacterial liquid and/or vibrio harveyi TL0816 bacterial liquid, and the formalin sterilized bacterial liquid is obtained by placing the formalin in a constant-temperature incubator with the temperature of 28-32 ℃ for 22-26H for inactivation.

In a third aspect of the present invention, there is provided the use of the above-mentioned turtle vaccine in the preparation of a medicament for the prevention and treatment of bacterial infection in turtles, preferably, the bacteria are vibrio parahaemolyticus and/or vibrio harveyi.

In the research, from a plurality of bacteria separated from the onset green sea turtle, through the artificial infection test of the Chinese grass turtle, the strains of vibrio parahaemolyticus H0711 and vibrio harveyi TL0816 with strong protection to the sea turtle are obtained, and biological preservation (preservation numbers are CCTCC No: M20221681 and CCTCC No: M20221682) is carried out, so that the inactivated vaccine can be prepared to be applied to the green sea turtle to obtain the immunity protection to the bacteria, particularly to prepare a proper bivalent vaccine, the immunity protection to the sea turtle for preventing the infection of pathogenic bacteria is far better than that of a single bacterium, the relative protection rate reaches 75%, the method has important significance for improving the immunity of the green sea turtle to specific pathogenic bacteria, and the method can be used for preventing and treating the bacterial infection of the sea turtle. And the inactivated vaccine is economical and cheap in preparation method and convenient for large-scale production.

Drawings

FIG. 1 clinical symptoms of artificially infected killed scarab, wherein 1 and 2 are Vibrio harveyi TL0816 bacteria, and 3 and 4 are Vibrio parahaemolyticus H0711.

FIG. 2 is a schematic diagram showing the staining of liver tissue slices HE of healthy and diseased turtles, wherein A is the liver tissue slice HE (100X) of healthy turtles, B is the liver tissue slice HE (400X) of healthy turtles, C is the liver tissue slice HE (100X) of diseased turtles infected with H0711, D is the liver tissue slice HE (400X) of diseased turtles infected with H0711, E is the liver tissue slice HE (100X) of diseased turtles infected with TL0816, and F is the liver tissue slice HE (400X) of diseased turtles infected with TL0816.

FIG. 3 is the staining of lung tissue sections HE of healthy and diseased turtles, wherein A is the lung tissue section HE (100X) of healthy turtles, B is the lung tissue section HE (400X) of healthy turtles, C is the lung tissue section HE (100X) of diseased turtles infected with H0711, D is the lung tissue section HE (400X) of diseased turtles infected with H0711, E is the lung tissue section HE (100X) of diseased turtles infected with TL0816, and F is the lung tissue section HE (400X) of diseased turtles infected with TL0816.

Figure 4 intestinal tissue section HE staining of healthy and diseased turtles: a is healthy tortoise intestinal tissue slice HE (100X), B is healthy tortoise intestinal tissue slice HE (400X), C is diseased tortoise intestinal tissue slice HE (100X) infected with H0711, D is diseased tortoise intestinal tissue slice HE (400X) infected with H0711, E is diseased tortoise intestinal tissue slice HE (100X) infected with TL0816, and F is diseased tortoise intestinal tissue slice HE (400X) infected with TL0816.

FIG. 5 is a graph showing the results of staining (healthy versus diseased) kidney tissue sections HE, where A is healthy turtle kidney tissue section HE (100X), B is healthy turtle kidney tissue section HE (400X), C is diseased turtle kidney tissue section HE (100X) infected with H0711, D is diseased turtle kidney tissue section HE (400X) infected with H0711, E is diseased turtle kidney tissue section HE (100X) infected with TL0816, and F is diseased turtle kidney tissue section HE (400X) infected with TL0816.

FIG. 6 shows morphology (left panel) and gram stain (right panel) on TCBS plates, where A is H0711 and B is TL0816.

FIG. 7 is a diagram showing the results of 16SrRNA amplification electrophoresis of strains H0711 and TL0816, wherein M is 2000bpDNAmarker and-1 and 2 are negative controls corresponding to the amplified bands of strains H0711 and TL0816, respectively.

FIG. 8 is a schematic diagram showing the results of heat shock protein 60 gene amplification electrophoresis of strains H0711 and TL0816, wherein M is 2000bpDNAmarker and-is a negative control, and 1 and 2 correspond to strains H0711 and TL0816, respectively.

FIG. 9 is a schematic view of direct counting under a microscope, in which A is a blood cell counting plate and B is a cell having a volume of 1/400X 0.1 mm ³ In the black regions shown, the number of counts (the number of counts is set to A1/A2/A3/A4/A5) is small, the total number of small squares is 16X 5, and the minimum volume is (1/400). Times.0.1X 0.001mL.

Fig. 10 is a graph showing the results of antibody titer significance analysis.

FIG. 11 is a graph showing the results of the survival curves of mixed bacteria for combating poison.

FIG. 12 is a graph showing the result of the H0711 strain challenge survival curve of the present invention.

FIG. 13 is a diagram showing the result of the TL0816 bacteria challenge survival curve of the present invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures not specifying specific conditions in the following examples are generally published in 2013 according to conventional conditions, for example, molecular Cloning: A Laboratory Manual, fourth edition, master code of Green and Sambrook, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Vaccines that contain a single antigenic component per unit dose often prevent infection by only one infectious disease or one type of pathogen. Combination vaccines are defined as vaccines containing two or more live, inactivated organisms or purified antigens that can be used to prevent a variety of diseases. The two have advantages and disadvantages, the monovalent vaccine has low utilization rate, but has better protection effect than combined immunity aiming at a certain pathogen, the combined vaccine has the advantages of reducing inoculation times, preventing a plurality of diseases, and having good broad-spectrum applicability, but the process of combining the vaccine is more complicated, whether competitive inhibition exists among the combined vaccines, whether stress response of inoculated individuals is caused more easily, and how to combine and other factors need to be considered.

In general, the combined vaccine can reduce the injection frequency of the vaccine and prevent more diseases on the premise that the vaccine is safe and effective. The vaccination combination vaccine can improve the vaccination rate, reduce the stress of individual caused by multiple times of immunization, reduce the cost of vaccine transportation, storage and vaccination, reduce the vaccination and management cost, reduce the dosage of preservatives, adjuvants and the like which are necessary in vaccine production, reduce the adverse reaction of vaccine and the like, thereby having more advantages in effective vaccine combination.

The invention separates and identifies 40 strains from diseased sea turtle, separates, purifies and identifies the strainsThe same includes artificially infected Chinese tortoise LD ₅₀ The determination of the method comprises the steps of obtaining two strains of vibrio H0711 and TL0816 derived from green sea turtles through researches on pathological sections of artificially infected and dead Chinese turtles, researching pathological sections of diseased and dead green sea turtles and the like, finding that the two strains have high pathogenicity on selected attack models of Chinese turtles and can be prepared into inactivated bigeminy seedlings with good protection rate, and in some embodiments, evaluating the immune dose, immune cycle, immune protection rate and serum antibody titer after immunization of the inactivated bigeminy seedlings.

The Vibrio parahaemolyticus (Vibrio parahaemolyticus) H0711 and the Vibrio harveyi TL0816 of the invention are preserved in China center for type culture Collection (CCTCC for short; address: wuhan, wuhan university; zip code: 430072) at 28 months 10 and 2022, and the preservation numbers are CCTCC Nos: m20221681 and CCTCC No: m20221682.

In some embodiments, the obtained virulent strains of Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816 are inactivated to prepare bivalent inactivated vaccines. Through grouping tests, the alumina gel adjuvant high-dose group with the highest protection rate of 75% after mixed bacteria challenge and the highest antibody titer is obtained. Meanwhile, the combined seedling has the protection rate of more than 50 percent for single bacterium attack, has good effect on the grass turtles and can be applied to the green sea turtles. The screening of the strain has strict procedures, the inactivated vaccine is easy to prepare, the operability is high, the steps are simple, the repeatability is high, and the pertinence is better.

The present invention will be described in further detail with reference to specific examples.

Example 1

The bacterial diseases are found to be the most common by carrying out a plurality of epidemiological surveys on artificial breeding environments and diseased individuals in the national natural protection zone of the sea turtle in the east of Guangdong province. During 2019-2021, 40 strains are sequentially and jointly separated from the body surface and the body of the diseased green sea turtle in the protected area, and virulence screening is carried out on the 40 strains, and the details are shown in a table 1.

(1) Purifying and expanding culture of bacterial strain

The separation culture medium is mainly blood plate and TCBS agar (both culture media are purchased from Guangdong Huaqiao microbiological science and technology Co., ltd.), the blood plate is the finished product culture medium, the TCBS agar culture medium is prepared by weighing powder according to the instruction, dissolving, boiling for less than 2min, and pouring the plate for later use. The amplification culture medium of the strain is LB (tryptone 1g/100mL, yeast powder 0.5g/100mL, sodium chloride 2g/100mL,121 ℃ for 15 min), the solid culture medium is inoculated and then cultured at 30 ℃ for about 20h, the liquid culture medium is inoculated and then cultured at 30 ℃ for 20h and 200rpm under constant temperature shaking.

(2) Identification of Strain 16SrRNA

Purifying and culturing 40 strains obtained by epidemiological investigation, identifying the bacteria 16SrRNA, and determining the bacterial species 16SrRNA by adopting a universal primer: 27F (5 '-AGAGAGTTTGATCCTGGCTCAG-3' SEQ ID NO. 1) and 1492R (5 '-TACGACTTACCCCAATCGC-3' SEQ ID NO. 2) were subjected to gene amplification. Reaction system: 2 XTaqPCR Master Mix 25. Mu.L, upstream and downstream primers 2. Mu.L each, DNA template 1. Mu.L, ddH ₂ O20 mu L; reaction procedure: 5min at 95 ℃; 30s at 94 ℃, 30s at 56 ℃, 90s at 72 ℃ and 35 cycles; 7min at 72 ℃.

(3) Plate colony counting method (viable count for challenge experiment)

1. Numbering, and marking the concentration of the plate and the centrifuge tube respectively.

2. Diluting the sample, and properly diluting the sample with the challenge bacteria. Sampling, accurately sucking 10 with a pipettor ^-6 、10 ^-7 、10 ^-8 Each 1ml of diluted bacterial solution was placed in 200uL sterile plates.

3. Pouring the plate, pouring 15ml of the culture medium which is melted and cooled to about 45 ℃ into the plate as soon as possible, and uniformly mixing by rotation. 4. Counting, taking out the culture dish for counting after culturing for 24h at 30 ℃, calculating the average number of bacterial colonies in three plates with the same dilution degree, and calculating the number of viable bacteria in each milliliter of the original sample according to a formula.

5. Data processing, generally take the same dilution of the average colony number between 30 and 300 plate. For example, one dilution, the average number of colonies between 30 and 300, is reported multiplied by the dilution factor.

(3) Initial screening for separating virulence of strain

Passing through dull-stereotyped bacteriaPerforming colony counting method to obtain original bacterial liquid concentration of 40 strains after culture, 10000rpm,2min, removing supernatant, suspending the strain with physiological saline, and adjusting bacterial liquid to 10 ⁸ cfu/mL, 200uL of each tortoise is injected into the abdominal cavity, the death condition in 14 days is counted, and a strain with stronger toxicity is primarily screened.

TABLE 2 isolation and identification of bacteria from Tortoise Green and initial screening mortality statistics of artificially infected Chinese grass turtle by strain

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4. Green sea turtle source bacterium virulence secondary sieve

9 strains with stronger toxicity (the death rate is more than 50 percent) are obtained from 40 strains through initial toxicity screening. In order to further screen virulent strains, the stronger strains are screened for 9 strains for the first time, the Chinese turtles are attacked again, the death condition is counted, the strains with higher toxicity are obtained, and the strains with lower toxicity are eliminated. Purifying and culturing to obtain 9 strains of bacterial liquid, determining the concentration of original bacterial liquid by flat viable count method, and diluting the original bacterial liquid to 5 × 10 with normal saline ⁸ As the initial concentration of the secondary screening for counteracting the poison, the gradient concentration of each bacterial liquid is obtained by 10 times of gradient dilution: 5X 10 ⁸ 、5×10 ⁷ 、5×10 ⁶ 、5×10 ⁵ 、5×10 ⁴ 、5×10 ³ . 20 healthy turtles are used in the control group and 9 experimental groups, the average weight is 32 +/-2 g, 200uL of the turtles are injected into the abdominal cavity and placed in a glass jar to be normally raised, the water temperature is 26 +/-2 ℃, and the survival condition in 14 days is counted. Please refer to tables 3-12 for the statistics of the toxicity attack.

TABLE 3 statistical table of toxicity challenge situation (H0711 strain for short)

TABLE 4 T2020TL0816 attacking situation statistical Table (TL 0816 strain for short)

TABLE 5 T20201117TL1 statistical table of the situation of attacking

TABLE 6 T20200420GQ4 statistics table for toxicity attacking situation

TABLE 7 T20210322H5 statistics of toxicity attacking conditions

TABLE 8 statistics of the toxicity attacking conditions of T20210515#4HG1

TABLE 9 T20210515#24HG statistics table for toxic substance counteracting

TABLE 10 T20210818Q4 attacking situation statistical table

TABLE 11 T20210818#23 statistical table of the toxicity attacking situation

Example 2

Through further screening, the invention selects Vibrio parahaemolyticus H0711 and Vibrio harveyi TL0816, and carries out virulent strain LD50 determination (SPSS software calculation).

Half lethal dose (Lethal dose 50%, LD) ₅₀ ): refers to the dose of drug that causes half of the test animal to die, usually expressed as a logarithmic value for the lethal dose of the drug. The invention directly uses SPSS software to calculate LD by probability unit weighted regression method (Bliss method) ₅₀ The method has the characteristics of simplicity, rapidness, intuition and convenience.

Setting variables by taking a strain Vibrio parahaemolyticus H0711 as an example, inputting data Analyze- - -Regression- - -Probit (probability unit) "attacking dose" and selecting into a "covariate" column; "death count" is selected in the "response frequency" column; the total number of experimental animals is selected into an observation value summary column; in the "convert" column, the "log base is selected to be 10, and others remain default. The regression equation for the option results is: probit = -4.660+0.758X. The probability that the standard residuals of the experimental points of the experimental values and the expected value probability tables of the residuals of different doses fall outside the normal distribution interval according to the normal distribution standard residuals = the mean value of the residuals/the standard deviation of the residuals is less than or equal to 0.05.

Finding the estimated value corresponding to the probability 0.5 in the 95% confidence interval table is the LD ₅₀ Dosage 3.52X 10 ⁶ cfu/mL, i.e., the concentration of the bacterial liquid is 3.52X 10 ⁶ cfu/mL and 0.2mL of counteracting toxic substance can kill half of the turtles. To be combined withLD of the strain TL0816 obtained by the method ₅₀ Dosage is 5.11X 10 ⁵ cfu/mL. The results of SPSS calculation of LD50 from 2 virulent strains are shown in Table 12.

TABLE 12 virulent strain LD ₅₀ Calculation results (SPSS software)

Bacterial strains	Strain numbering	LD 50 Dose (cfu/mL)
			Vibrio parahaemolyticus H0711	H0711	3.52×10 6
Vibrio harveyi TL0816	TL0816	5.11×10 5

Example 3

Analysis of pathological tissue sections of Chinese turtles infected by vibrio parahaemolyticus H0711 and vibrio harveyi TL0816.

Through artificial infection experiments of toxicity preliminary screening, the clinical symptoms of Chinese turtles infected with vibrio harveyi and vibrio parahaemolyticus and the symptoms of the ill dead green turtles are consistent: swelling and bleeding of liver, swelling and congestion of intestinal tract, collapse of lung tissue, swelling and congestion of kidney. The liver, lung, intestinal tract and kidney tissues of the diseased morinda citrifolia turtles are cut in time and fixed in 10% formalin, and meanwhile, the healthy turtles sampled by the user are used as a control group and are all used for preparing paraffin sections for pathological analysis.

Clinical symptoms of artificially infected dead turtles are shown in figure 1.

1. Contrast analysis of the tissue slices of the diseased and dead scarabs and the tissue slices of the healthy scarabs (liver, intestine, lung and kidney)

1.1 HE staining of liver tissue sections (healthy and diseased), see FIG. 2 for results.

As can be seen from FIG. 2, the liver tissue section of the diseased tortoise shows the disorganization of the hepatocytes, vacuolation of the hepatocytes, and a small amount of lymphocyte infiltration; there is more ferrohemoglobin deposited. Indicating liver bleeding with inflammation.

1.2 results of HE staining (health and morbidity) of lung tissue sections are shown in FIG. 3.

As can be seen in fig. 3, the alveolar space of the diseased tortoise is ruptured and the alveoli are atrophied; the lung tissue is seen as a large sac, and it is possible that acute infection causes heterogeneous tissue proliferation in the alveoli. It is suggested that the pulmonary nodule hyperplasia, possibly caused by inflammation, is accompanied by symptoms of lung atrophy. 1.3 results of HE staining (healthy and diseased) of intestinal tissue sections are shown in FIG. 4.

As can be seen from fig. 4, both of them are seen with intestinal villus mucosa detachment and submucosal lesion; the serosal layer becomes thin and the muscular layer is damaged to different degrees. Indicating thinning, swelling or enteritis of the intestinal mucosa.

1.4 results of HE staining (health and morbidity) of renal tissue sections are shown in FIG. 5.

As can be seen in FIG. 5, in healthy state, the glomerular vascular loop is thin and clear. Endothelial and mesangial cell numbers were normal. The surrounding renal tubules are also normal; after the disease, the renal tubule lumen is blocked, the renal tubules are enlarged, the lumen is enlarged, and capillary vessels in glomerulus are proliferated, which indicates that the nephritis is obvious.

EXAMPLE 4 culture of Vibrio

1 Strain purification, propagation and gram staining (cultivation methods same as above)

10uL of the strain H0711 and TL0816 is sucked and placed on a TCBS plate, and is subjected to streak culture by sterile inoculation, and is subjected to inversion culture at the constant temperature of 30 ℃ for 24 hours. And (4) picking a single colony on the TCBS to an LB culture medium for propagation. At the same time, single colonies were picked for gram staining. H0711 vibrio parahaemolyticus is circular on a TCBS flat plate, neat, wet, slightly turbid, semitransparent and green colony is formed, and the diameter is 2-4mm; gram stain is negative bacilli. The colony of TL0816 Vibrio harveyi on the TCBS plate is circular, the edge is neat, the colony is moist, translucent, yellow colony, the diameter is 2-4mm; gram-staining was negative bacilli.

2 identification of 16S rRNA of strain

The general primer is as follows: 27F (5 '-AGAGAGTTTGATCCTGGCTCAG-3' SEQ ID NO. 1), 1492R (5 '-TACGACTAACCCCCAATCGC-3' SEQ ID NO. 2) reaction system: 2 XTaqPCR Master Mix 25. Mu.L, upstream and downstream primers 2. Mu.L each, DNA template 1. Mu.L, ddH2O 20. Mu.L; reaction procedure: 5min at 95 ℃; 30s at 94 ℃, 30s at 56 ℃, 90s at 72 ℃ and 35 cycles; 7min at 72 ℃. The amplification product was determined by sequencing.

Note: the 16S rRNA amplified electrophoretic bands of strains H0711 and TL0816 are shown, M is 2000bpDNAmarker, and-is a negative control, and 1 and 2 correspond to the amplified bands of strains H0711 and TL0816, respectively.

2.1h0711 strain 16SrRNA sequencing results:

TGCAAGTCGAGCGGAAACGAGTTATCAGAACCTTCGGGGAACGATAACGGCGTCGAGC

GGCGGACGGGTGAGTAATGCCTAGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAA

CGATGGCTAATACCGCATGATGCCTACGGGCCAAAGAGGGGGACCTTCGGGCCTCTCGC

GTCAGGATATGCCTAGGTGGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGAC

GATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGA

CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCA

TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCGTGAGGAAGGT

AGTGTAGTTAATAGCTGCATTATTTGACGTTAGCGACAGAAGAAGCACCGGCTAACTCC

GTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAA

AGCGCATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAATT

GCATTTGAAACTGGCAGACTAGAGTACTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGG

TGAAATGCGTAGAGATCTGAAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAGATA

CTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC

GCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACG

CGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGG

GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACC

TACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGA

CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAAC

GAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTAATGTCGGGAACTCCAGGGAGACTGC

CGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTA

GGGCTACACACGTGCTACAATGGCGCATACAGAGGGCAGCCAACTTGCGAAAGTGAGC

GAATCCCAAAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTC

GGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACA

CACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGGGGACGCTTACCAC(SEQ ID NO.3)

2.2tl0816 strain 16SrRNA sequencing results:

TAAGCGTCCTCCCGAAGGTTAAACTACCTACTTCTTTTGCAGCCCACTCCCATGGTGTGA

CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACT

AGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTT

TTGGGATTCGCTCACTCTCGCAAGTTGGCCGCCCTCTGTATGCGCCATTGTAGCACGTGT

GTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTAT

CACCGGCAGTCTCCCTGGAGTTCCCACCCGAAGTGCTGGCAAACAAGGATAAGGGTTG

CGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGC

ACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGGATGTCAA

GAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGG

GCCCCCGTCAATTCATTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCTACTTA

ACGCGTTAGCTCCGAAAGCCACGGCTCAAGGCCACAACCTCCAAGTAGACATCGTTTAC

GGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCATCTGAGTGTC

AGTATCTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTTCAGATCTCTACGCATTTCA

CCGCTACACCTGAAATTCTACCCCCCTCTACAGTACTCTAGTCTGCCAGTTTCAAATGCTA

TTCCGAGGTTGAGCCCCGGGCTTTCACATCTGACTTAACAAACCACCTGCATGCGCTTTA

CGCCCAGTAATTCCGATTAACGCTCGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAG

TTAGCCGGTGCTTCTTCTGTCGCTAACGTCAAATAATGCAGCTATTAACTACACTACCTTC

CTCACGACTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGC

ATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACC

GTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAGGGATCGTCGCCTTGG

TGAGCCATTACCTCACCAACTAGCTAATCCCACCTAGGCATATCCTGACGCGAGAGGCCC

GAAGGTCCCCCTCTTTGACCCGTAGGTATTATGCGGTATTAGCCATCGTTTCCAATGGTTA

TCCCCCACATCAGGGCAATTTCCTAGGCATTACTCACCCGTCCGCCGCTCGACGCCGTTATCGTTCCCCGAAGGTTCAGATAACTCGTTTCCGCTCGACTTGC(SEQ ID NO.4)。

3 bacterial strain HSP60 (heat shock protein-60) gene amplification

Primer: HSP60-F (5 '-ACAACACGCAACGTACTAGC-3' SEQ ID NO. 5), HSP60-R (5 '-CAACTTTCACGATGCCAC-3' SEQ ID NO. 6),

reaction system: 2 XTaqPCR Master Mix 25. Mu.L, upstream and downstream primers 2. Mu.L each, DNA template 1. Mu.L, ddH ₂ O20 mu L; reaction procedure: 5min at 95 ℃; 30s at 94 ℃, 30s at 56 ℃, 90s at 72 ℃ and 35 cycles; 7min at 72 ℃. The amplification products were determined by sequencing.

3.1H0711 strain HSP60 amplified fragment sequencing result:

CGCAAGCAATCGTAAATGAAGGTCTAAAAGCAGTTGCAGCGGGTATGAACCCAATGGAT

CTTAAGCGCGGTATCGACAAAGCTGTTGCAGCGGCAGTAGAGCAACTAAAAGAGCTTTC

TGTTGAGTGTAACGACACCAAAGCAATCGCACAGGTTGGTACTATCTCTGCGAACTCTG

ACGCAAGCGTAGGTAACATCATTGCTGAAGCAATGGAACGCGTTGGCCGCGACGGTGTT

ATCACTGTTGAAGAAGGTCAGGCTCTACAAGACGAGCTAGACGTAGTAGAAGGTATGCA

GTTCGACCGCGGTTACCTATCTCCTTACTTCATCAACAACCAAGAAGCGGGCAGCGTTG

AGCTAGAAAACCCATTCATCCTTCTAGTTGATAAGAAGATCTCAAACATTCGTGAGCTTC

TACCAACTCTAGAAGCAGTAGCAAAAGCATCTCGTCCACTGCTAATCATCGCAGAAGAC

GTAGAAGGCGAAGCACTAGCGACATTGGTTGTGAACAACATGCGTGTCTGGGAAAA(SEQ ID NO.7)

3.2TL0816 strain HSP60 amplified fragment sequencing results:

AACTTTTCCACGAGCCACGCATGTTGTTCACAACAAGTGTTGCTAGTGCTTCACCTTCTA

CGTCTTCAGCGATGATAAGTAGTGGGCGAGATGCTTTTGCTACTGCTTCTAGTGCTGGAA

GAAGTTCACGGATGTTCGATACTTTCTTATCGATCAGAAGGATGAATGGGTTTTCTAGAT

CAACAGAACCTGCTTCTTGGTTGTTGATGAAGTAAGGAGATAGGTAACCGCGGTCGAAC

TGCATACCTTCAACTACATCTAGCTCATCTTGTAGTGCTTGACCTTCTTCAACAGTGATAA

CACCGTCGCGACCAACTTTTTCCATTGCTTCAGCAATGATGTTACCTACGCTTGAGTCAG

AGTTCGCAGAGATAGTACCAACCTGTGCGATTGCTTTGGTGTCGTTACACTCAACAGATA

GCTCTTTTAGTTGCTCAACTGCTGCGATAACTGCTTTGTCGATACCGCGCTTAAGGTCCA

TTGGGTTCATACCCGCTGCAACTGCTTTTAGACCTTCATTTACGATTGCTTGCGCTAG(SEQ ID NO.8)。

4 Biochemical identification of strains

TABLE 4 Biochemical identification of 1H0711 and TL0816 strains

After two previous rounds of screening, two strains of Vibrio (accession No. H0711, TL 0816) isolated from diseased green turtle were separately inoculated into LB liquid (2% NaCl) medium for 20H in 30 ℃ constant temperature shaking incubator at 200rpm, centrifuged at 10000rpm,2min, washed with sterilized normal saline 3 times, and collected.

5 direct counting method under microscope (for viable bacteria immune counting)

5.1 observing the concentration of the suspension of the bacteria to be detected, adding sterile normal saline for proper dilution (generally 100 times of dilution), and taking the bacteria number of each cell as the degree;

5.2 taking one clean blood counting chamber, and covering a cover glass on the counting area;

5.3 thoroughly shaking the original bacterial suspension, diluting the gradient concentration, sucking 8uL by using a liquid transfer device, dropwise adding bacterial liquid into a gap after a cover glass is placed, and allowing the bacterial liquid to enter a counting plate by means of siphon.

And 5.4 standing for 3-5min to make the bacteria settle on the counting plate and not drift with the liquid. The blood cell counting plate is placed on an object stage of a microscope and clamped stably, and after a counting area is found under a low-power lens, the high-power lens is converted for observation and counting.

5.5 the central counting area is a counting area composed of 25 middle squares, and the bacteria number of the central 1 middle squares (namely 25 multiplied by 16 small squares) is counted besides the four middle squares. In order to ensure the accuracy of counting, repeated counting and missing recording are avoided;

5.6 there should be a uniform specification for the statistics of the cells settled on the grid lines at the time of counting. If the thalli are positioned on double lines of the large square grids, the upper line is counted, the lower line is counted, and the left line is counted, the right line is counted, so that errors are reduced. That is, the cells on the upper line and the left line of the grid are counted in the grid, and the cells on the lower line and the right line of the grid are counted in the corresponding grid according to the regulations.

5.7 repeat counting for 2-3 times (the value should not be too different each time, otherwise, the operation should be repeated), and calculating the number of bacteria contained in each mL of bacterial suspension according to the formula.

5.8 after counting, taking down the cover glass, washing the blood counting chamber with water, and washing or wiping with hard objects to avoid damaging the scales of the grids. After cleaning, the mixture is automatically dried and put into a box for storage.

5.9 calculation formula: [ (A1 + A2+ A3+ A4+ A5)/(5X 16) ]/[ (1/400). Times.0.1X 0.001 ]. Times.dilution factor (cfu/mL).

The two strains are identified by adopting the gene sequences of the strains 16SrRNA and hsp60, the sequencing result is subjected to BLAST comparison at NCBI, and after comparison, the homology between the gene sequences of the H0711 strain 16SrRNA and hsp60 and the gene sequences of Vibrio parahaemolyticus (Vibrio parahaemolyticus) 16SRNA and hsp60 in NCBI database is 100 percent and 99 percent respectively; and combining the biochemical identification result to comprehensively judge that the gene sequences of the 16SrRNA and the hsp60 of the TL0816 strain are 100 percent and 99.63 percent of homology with the gene sequences of the 16SRNA and the hsp60 of the NCBI database Vibrio harveyi (Vibrio harveyi) respectively, and combining the biochemical identification result to comprehensively judge that the gene sequences of the Vibrio harveyi (Vibrio harveyi) are 100 percent and 99.63 percent.

The Vibrio parahaemolyticus (Vibrio parahaemolyticus) H0711 and Vibrio harveyi (Vibrio harveyi) TL0816 have been preserved in China Center for Type Culture Collection (CCTCC) at 28 months 10 and 2022, and the preservation number of the Vibrio parahaemolyticus H0711 is CCTCC No: m20221681, and the preservation number of the Vibrio harveyi TL0816 is CCTCC No: m20221682.

Example 5 preparation of immunogen

Adjusting the concentration of the calculated bacteria liquid to 10 by adopting sterilized normal saline ⁹ After cfu/mL, the 2 strains H0711 and TL0816 were mixed at a ratio of 1:1 preparing a mixed bacterial liquid as immunogen (immunogen) for inoculating the scarab and reaction source (reactogen) for measuring the agglutination titer of immune serum. Experimental groups two groups were set up with or without adjuvant: aluminum adjuvant group (aluminum adjuvant: bacterial suspension = 1)Adjuvant group, three groups were set up on the immunization dose: high, medium and low doses. If so, there are the following 7 groups: (1) aluminum adjuvant-high dose, (2) aluminum adjuvant-medium dose, (3) aluminum adjuvant-low dose; (4) no adjuvant-high dose, (5) no adjuvant-medium dose, (6) no adjuvant-low dose, and (7) control group of 20 turtles per group. Wherein: high dose 10 ⁹ cfu/mL, medium dose 5X 10 ⁸ cfu/mL, low dose 10 ⁸ cfu/mL。

And quantitatively adding 0.2 percent of formalin with final concentration into the prepared vibrio (H0711 and TL 0816) bacterial solutions, placing the two bacterial solutions in a constant-temperature incubator at 30.0 ℃, and placing the two bacterial solutions for 24 hours for inactivation to obtain the formalin inactivated bacterial solution. Taking 100 mu L of the inactivated bacteria liquid of 3 groups, respectively coating the inactivated bacteria liquid on a TCBS (trichloroacetic acid) plate and an LB (lysogeny broth) plate, placing the plates in a constant-temperature incubator at 30.0 ℃, observing after placing for 24h, and growing sterile colonies on a culture medium, namely proving that the bacteria are completely inactivated and can be used as inactivated bacterins (reaction sources).

Example 6 inactivated vaccine safety test

In order to ensure that the turtles for the inactivated vaccine injection experiment are safe, nontoxic and reliable, 2 strains of bacteria H0711 and TL0816 are inactivated according to the ratio of 1:1 mixing them uniformly and adjusting the concentration to 10 ¹⁰ cfu/mL. Safety tests were performed using 3 groups of turtles of different specifications: the weight of large-size scarab turtles is about 37g, the weight of middle-size scarab turtles is about 32g, the weight of small-size scarab turtles is about 28g, 20 scarab turtles in each group are injected into 100uL muscle of inactivated bacteria, and the scarab turtles are bred for 2 weeks to observe whether the scarabs are safe or not. While the control group was injected with sterile saline.

TABLE 9-1 statistics of high concentration inactivated vaccine 14d safety test

And (3) analysis: the experimental results show that a high concentration of 10 is used ¹⁰ cfu/mL inactivated dual-bacterium is used for immunizing turtles with different specifications,the survival rate is higher and reaches 90 percent or above. Death of turtles is initially presumed to be fatal by stress. The vaccine has high safety in the whole.

Example 7 immunization

For healthy turtles with the average weight of about 35g, 100uL of inactivated bigeminal vaccine is injected into muscles, 80 turtles are inoculated to the inactivated vaccine of each strain, and 100uL of sterile normal saline is injected into a control group. And (4) normally feeding the inoculated turtles, and feeding the turtles with the artificial compound feed once a day. During the raising period, the water temperature in the aquarium is controlled between 24-28 deg.c. After the first immunization inactivated bigeminy vaccine is bred for 10 days, the second immunization is carried out, the dose of the second immunization is the same, and the breeding time is 20 days.

Blood collection and serum separation

Randomly fishing 6 turtles from aquarium tanks of each test group and control group, collecting blood by using a vertebral inferior vein method, collecting about 0.8-1mL of blood, placing the blood in a 2mL centrifuge tube, placing the blood in a flat state at room temperature for more than 2h, 4000g, centrifuging for 20min, and separating immune serum. Storing at-80 deg.C for use.

And (3) immune protection rate statistics: after the secondary immunization (30 d) is finished, the challenge strain concentration is 2 times LD of the two strains (H0711 and TL 0816) ₅₀ ,1:1, injecting 200uL of the abdominal cavity of each test tortoise, normally feeding for 30d, counting the survival rate, and calculating the immune protection rate.

Determination of serum antibody titer

1. 50 microliters of normal saline is respectively added into the first grid in the first row in a V-shaped plate (90 degrees) of a 96-pore plate, and 50 microliters of normal saline is respectively added into other pores;

2. adding 20 microliters of scarab serum into the first hole, mixing uniformly, taking out 50 microliters of scarab serum from the first hole, adding into the second hole, and mixing uniformly;

3. then taking 50 microliters of the mixture out of the second hole, adding the mixture into the third hole, uniformly mixing, adding the mixture into the 11 holes in the same manner, taking 50 microliters of the mixture out of the 11 hole, and discarding the mixture, wherein the 50 microliter of the mixture is not added into the 12 hole;

4. adding inactivated bacteria solution (1X 10) into 1-12 holes ⁸ cfu/mL, inactivated with 0.2% formalin) 50. Mu.L, shaking for 1 minute, placing in a 30 ℃ incubator for 2 hours, placing in a4 ℃ refrigerator overnight,observing the result on the next day;

5. the reciprocal of the highest dilution of the agglutinated precipitate was recorded as the antibody titer upon low power lens observation. The one with the highest dilution and showing agglutination is taken as the agglutination titer, and the antibody titer is recorded as the last titer of the mark (+);

6. calculating the geometric mean titer (G) of the antibody, and the n-th power of the product of all n observation data, and is commonly used for describing the positive bias distribution with few large extreme values or the concentration position of data with multiple relation or approximate multiple relation among the observation values.

7. Single factor analysis of variance was performed on antibody titers using SPSS, step1: opening the software, and sequentially clicking 'analysis-comparison mean-one-way ANOVA'; step2: in the one-way analysis of variance window, the antibody titer is placed in the dependent variable list box and the reactogens are placed in the factor box. Step3: one-way ANOVA "click in Window" compare after the fact "option appears: after the fact, multiple comparison is carried out on a window, a method of 'Ponfereny' is selected under the column of 'assumed homogeneity of variance', a method of 'Tamkini' is selected under the column of 'un-assumed homogeneity of variance', and then 'continue' is clicked. Step4: click on "option" in the "one-way analysis of variance" window, a "one-way ANOVA: the option "window, check" descriptive "and" homogeneity of variance test "and" welch ", and then click" continue ".

TABLE 13-1 measurement results of serum agglutination antibody titer of experimental group and control group

The results of the antibody titer significance analysis are shown in fig. 10. Wherein the immunogen is H0711: TL0816=1:1, high dose of 10 per bacterium ⁹ cfu/mL, medium dose 5X 10 ⁸ cfu/mL, low dose 10 ⁸ cfu/mL。

As can be seen from fig. 10, the antibody titer significance analysis showed that the antibody titers in the experimental groups were all higher than those in the control group, wherein the adjuvant high dose group was significantly higher than those in the other groups (P < 0.5); the calculation result of the geometric mean titer of the antibody shows that the adjuvant high-dose immunization group is the highest and is 1:1:114.04, followed by the adjuvant medium dose group, were all 1:50.80, wherein the control group is 1:5.04. the experimental result shows that the agglutination antibody titer of the serum of the Chinese grass turtles is obviously higher than that of the control group after 30 days of two-time immunization. This indicates that the inactivated vaccine produces common antibody in the tortoise body, and can effectively protect the tortoise from the invasion of the bacterial diseases.

Example 8 Dual relative protection Rate

Relative vaccine protection = (1-mortality of protected turtle/mortality of control group turtle) × 100%

The 6 immunization groups and the control group were subjected to challenge test using the following combination of 4 different strains. Challenge group 1 challenge dose was 2LD for each of two strains H0711 and TL0816 ₅₀ Dose (1. 2 groups of toxin counteracting injection 2LD ₅₀ H0711 concentration of single bacterium uL, and 3 groups of counteracting toxic substance injected 2LD ₅₀ The concentration TL0816 is 200uL of single bacterium.

TABLE 15 statistical table of relative protection rates of mixed attack

Grouping	Number of official offensive	Number of deaths of 30d	Death rate of 30 days	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％
Adjuvant-free high dose immunization group	20	5	25.00％	68.75％
					Adjuvant-free medium dose immunization groups	20	7	35.00％	56.25％
Adjuvant-free low dose immunization group	20	9	45.00％	43.75％

The mixed bacteria challenge survival curve is described in fig. 11.

TABLE 16.H0711 statistics table for relative protection rate of single bacterium attack

H0711 single bacterium detoxification	Amount of official poison attack	Number of deaths of 30d	30d mortality rate	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％
Adjuvant-free high dose immunization group	20	9	45.00％	47.06％
					Adjuvant-free medium dose immunization group	20	10	50.00％	41.18％
Adjuvant-free low dose immunization group	20	10	50.00％	41.18％

The H0711 bacteria challenge survival curve is depicted in FIG. 12.

TABLE 17 TL0816 statistical table of relative protection rate of single bacterium attack

TL0816 single bacterium toxin counteracting	Number of official offensive	Number of deaths of 30d	Death rate of 30 days	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％
Adjuvant-free high dose immunization group	20	9	45.00％	43.75％
					Adjuvant-free medium dose immunization groups	20	9	45.00％	43.75％
Adjuvant-free low dose immunization group	20	11	55.00％	31.25％

As can be seen from tables 15 to 17, and fig. 12 to 13, the survival rate of the immunized group after the second immunization was higher than that of the control group in the survival curve one month after challenge. Wherein the protective rate of the adjuvant high-dose immune group turtles after using mixed bacteria to detoxify is the highest and is 75 percent, and the protective rate of the adjuvant-free high-dose immune group is 68.75 percent; the protection rate of single bacterium to attack the poison can reach more than 50 percent, and after high-dose immunization, the protection rate to attack the poison is higher than that of low-dose attack, which shows that after high-dose immunization, individuals can be stimulated to generate more antibodies, and the disease resistance is improved.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A turtle vaccine, wherein the immunogen is prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and/or an inactivated strain of Vibrio harveyi TL0816, and the preservation number of the Vibrio parahaemolyticus (Vibrio parahaemolyticus) H0711 is CCTCC No: m20221681, and the preservation number of the Vibrio harveyi (Vibrio harveyi) TL0816 is CCTCC No: m20221682.

2. The turtle vaccine of claim 1, wherein the turtle is a green turtle.

3. The turtle vaccine of claim 1, wherein the immunogen is prepared from an inactivated strain of Vibrio parahaemolyticus H0711 and an inactivated strain of Vibrio harveyi TL0816.

4. The turtle vaccine according to claim 1, wherein the ratio of the inactivated strain of vibrio parahaemolyticus H0711 to the inactivated strain of vibrio harveyi TL0816 is 1-3:1-3, preferably 0.9-1.1:0.9-1.1, more preferably 1:1.

5. the turtle vaccine according to any one of claims 1-4, further comprising a vaccine adjuvant.

6. The turtle vaccine of claim 5, wherein the vaccine adjuvant is an aluminum salt adjuvant.

7. The sea turtle vaccine according to any one of claims 1-4, wherein the bacterial liquid concentration of the inactivated strain of Vibrio parahaemolyticus H0711 and/or the inactivated strain of Vibrio harveyi TL0816 is 1 x 10 ⁸ cfu/mL to 2X 10 ⁹ cfu/mL。

8. The turtle vaccine according to claim 7, wherein the bacteria solution concentration of the inactivated strain of Vibrio parahaemolyticus H0711 and/or the inactivated strain of Vibrio harveyi TL0816 is 5 x 10 ⁸ cfu/mL to 1X 10 ⁹ cfu/mL。

9. A method for preparing the turtle vaccine of claims 1-8, comprising the steps of:

inactivating the cultured vibrio parahaemolyticus H0711 to obtain H0711 inactivated bacteria liquid;

inactivating the cultured Vibrio harveyi TL0816 to obtain TL0816 inactivated bacterial liquid;

h0711 inactivated bacteria solution and TL0816 inactivated bacteria solution are mixed.

10. Use of the turtle vaccine of claims 1-8 for the preparation of a medicament for the prevention and treatment of infection by vibrio parahaemolyticus and/or vibrio harveyi in turtles.

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