A trivalent inactivated vaccine for avian Escherichia coli disease and preparation method thereof

Abstract

The present invention provides a trivalent inactivated vaccine for avian colibacillosis and a preparation method thereof. The avian colibacillosis composition of the present invention includes three serotypes of Escherichia coli, namely O145, O78 and O111; the O145 serotype Escherichia coli is Escherichia coli JX164; the O78 serotype Escherichia coli is Escherichia coli JX119; the O111 serotype Escherichia coli is Escherichia coli JX033; the avian colibacillosis composition can be used to prepare a trivalent inactivated vaccine of O111, O78 and O145; the trivalent inactivated vaccine of avian colibacillosis of the present invention has no obvious adverse reactions in each group of ducks within 7 days after immunization, and the survival rate reaches 100% at 7 days after immunization; compared with similar commercial vaccines, the two trivalent inactivated vaccines provided by the present invention have better immune protection effects on infection of two serotypes of Escherichia coli, namely O78 and O111, than the competing product group.

Description

Technical Field

[0001] The invention relates to the technical field of inactivated vaccines, and in particular to a trivalent inactivated vaccine for avian Escherichia coli disease and a preparation method thereof. Background Art

[0002] Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens in the poultry industry. Due to its high morbidity and mortality, the poultry industry suffers significant economic losses. When poultry is infected with APEC, it can cause sepsis and fibrinous inflammation of multiple organs, such as pericarditis, air sacculitis, perihepatitis and peritonitis. APEC is also a potential zoonosis pathogen, with the risk of infecting humans through the environment, feeding links or consumption of animal-derived foods, threatening human public health safety.

[0003] Prevention and treatment strategies for APEC infection usually include vaccine prevention, antibiotics, probiotics, phages and various new therapies, such as innate immune stimulants and small molecule antimicrobial peptides. Clinical practice shows that except for vaccines that can provide protection against infection with the same serotype and the definite efficacy of antibiotics, the effectiveness of other prevention and control measures is still uncertain in clinical practice. However, due to the widespread prevalence of drug-resistant strains, the diversity of APEC serotypes and the poor cross-protection ability of E. coli vaccines against different serotypes, the prevention and control of APEC remains a difficult problem in poultry farming.

[0004] At present, only the APEC quadrivalent propolis adjuvant inactivated vaccine (O78, O111, O2, O5) has been approved for marketing and used in veterinary clinics in China. However, many studies have shown that other serotypes are widely prevalent in different regions of my country, such as O1, O18, O92, O93, O145, O109, O36, etc. However, there are currently few studies on the development of APEC vaccines for other serotypes. On the other hand, although propolis adjuvanted vaccines have the advantages of rapid absorption and low cost, propolis adjuvanted inactivated vaccines are difficult to provide a high protection rate. Therefore, it is of great significance to explore adjuvant types with high protection, high safety and low side effects for the dominant serotype strains prevalent in clinical practice, and to develop APEC multivalent vaccines. Summary of the invention

[0005] The invention provides a trivalent inactivated vaccine for avian Escherichia coli disease and a preparation method thereof, so as to solve the defects in the prior art.

[0006] In a first aspect, the present invention provides an avian Escherichia coli composition, comprising three serotypes of Escherichia coli, namely O145, O78 and O111;

[0007] Among them, the O145 serotype Escherichia coli is Escherichia coli JX164, with a deposit number of CCTCC NO: M 20231988;

[0008] O78 serotype Escherichia coli, which is Escherichia coli JX119, with a deposit number of CCTCC NO: M 20231989;

[0009] The O111 serotype Escherichia coli is Escherichia coli JX033, and its deposit number is CCTCC NO: M 20231990.

[0010] In a second aspect, the present invention also provides a use of the avian Escherichia coli composition in the preparation of a trivalent inactivated vaccine for avian Escherichia coli disease.

[0011] In a third aspect, the present invention also provides a trivalent inactivated vaccine for avian Escherichia coli disease, comprising the avian Escherichia coli composition.

[0012] Preferably, in the trivalent inactivated vaccine for avian Escherichia coli disease, the antigen content of each inactivated Escherichia coli strain is ≥ 6.0×10 9 CFU / mL.

[0013] Preferably, the trivalent inactivated vaccine for avian Escherichia coli disease further comprises an adjuvant.

[0014] Preferably, the trivalent inactivated vaccine for avian Escherichia coli disease is prepared with aluminum gel adjuvant or MONTANIDE GELP PR adjuvant.

[0015] Preferably, in the trivalent inactivated vaccine for avian Escherichia coli disease, the volume fraction of the adjuvant in the trivalent inactivated vaccine for avian Escherichia coli disease is 20-30%.

[0016] In a fourth aspect, the present invention further provides a method for preparing the trivalent inactivated vaccine for avian Escherichia coli disease, comprising the following steps:

[0017] Preparation of inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring culture and performing inactivation treatment;

[0018] Preparation of vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the trivalent inactivated vaccine for avian Escherichia coli disease.

[0019] Preferably, in the preparation method of the trivalent inactivated vaccine for avian Escherichia coli disease, the transfer culture conditions are as follows: the bacterial solution of the secondary seed is transferred to a shaking bottle at a ratio of 1: (30-70), and the bacteria are fermented at 170-190 r / min in a shaking shaker at 35-38°C. After fermentation for 3-4 hours, the OD is measured every 0.5-0.6 hours. 600 , when OD 600 Stop fermentation when the plateau phase is reached, centrifuge at 7000-9000 r / min for 2-4 min at 3-5°C in a high-speed refrigerated centrifuge, discard the supernatant and collect the bacteria, resuspend the bacteria in physiological saline, take 90-110 μL of the bacterial solution for multiple dilution, and calculate the viable bacteria concentration of the resuspended bacterial solution by counting the viable bacteria.

[0020] Preferably, in the method for preparing the trivalent inactivated vaccine for avian Escherichia coli disease, the inactivation treatment is specifically as follows: adding a 0.3-0.7% formaldehyde solution by volume to the bacterial solution, and inactivating the solution at 35-38°C in a shaking incubator at 80-120 r / min for 40-60 hours.

[0021] The trivalent inactivated vaccine for avian Escherichia coli disease and the preparation method thereof of the present invention have the following beneficial effects compared with the prior art:

[0022] The poultry Escherichia coli composition of the present invention comprises three serotypes of Escherichia coli, namely, O145, O78 and O111; wherein, the O145 serotype Escherichia coli is Escherichia coli (Escherichia coli) JX164, and the preservation number is CCTCC NO: M 20231988; the O78 serotype Escherichia coli is Escherichia coli (Escherichia coli) JX119, and the preservation number is CCTCC NO: M 20231989; the O111 serotype Escherichia coli is Escherichia coli (Escherichia coli) JX033, and the preservation number is CCTCC NO: M 20231990, the avian Escherichia coli composition can be used to prepare a trivalent inactivated vaccine of O111, O78 and O145; the trivalent inactivated vaccine of avian Escherichia coli disease of the present invention has no obvious adverse reactions in each group of ducks within 7 days after immunization, and the survival rate 7 days after immunization reaches 100%; compared with similar commercial vaccines, the two trivalent inactivated vaccines provided by the present invention have better immune protection effects against infection with two serotypes of Escherichia coli, O78 and O111, than those of the competing product group. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The figures are the statistical results of the serotype types and their corresponding strain numbers in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The following are described in detail. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any quoted numbers (fractions or integers) within the indicated range.

[0027] The present invention provides an avian Escherichia coli composition, comprising three serotypes of Escherichia coli, namely O145, O78 and O111;

[0028] Among them, the O145 serotype Escherichia coli is Escherichia coli JX164, with a deposit number of CCTCC NO: M 20231988;

[0029] O78 serotype Escherichia coli, which is Escherichia coli JX119, with a deposit number of CCTCC NO: M 20231989;

[0030] The O111 serotype Escherichia coli is Escherichia coli JX033, and its deposit number is CCTCC NO: M 20231990.

[0031] The Escherichia coli JX164 of the present invention is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M20231988, and the deposit date is October 23, 2023; the Escherichia coli JX119 is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M 20231989, and the deposit date is October 23, 2023; the Escherichia coli JX033 is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M 20231990, and the deposit date is October 23, 2023.

[0032] Based on the same inventive concept, the present invention also provides an application of the above-mentioned avian Escherichia coli composition in the preparation of a trivalent inactivated vaccine for avian Escherichia coli disease.

[0033] Based on the same inventive concept, the present invention also provides a trivalent inactivated vaccine for avian Escherichia coli disease, comprising the above-mentioned avian Escherichia coli composition.

[0034] In some embodiments, the antigen content of each inactivated Escherichia coli strain in the trivalent inactivated vaccine for avian Escherichia coli is ≥ 6.0×10 9 CFU / mL.

[0035] In some embodiments, the trivalent inactivated vaccine for avian Escherichia coli disease further comprises an adjuvant.

[0036] In some embodiments, the adjuvant is aluminum gel adjuvant or MONTANIDE GEL P PR adjuvant. Specifically, the aluminum gel adjuvant is aluminum hydroxide gel adjuvant.

[0037] In some embodiments, the volume fraction of the adjuvant in the trivalent inactivated vaccine for avian colibacillosis is 20-30%, that is, the volume ratio of the Escherichia coli strain to the adjuvant in the trivalent inactivated vaccine for avian colibacillosis is (80-70):(20-30).

[0038] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned trivalent inactivated vaccine for avian Escherichia coli disease, comprising the following steps:

[0039] S1. Preparing an inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring the culture and performing inactivation treatment;

[0040] S2. Prepare the vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the trivalent inactivated vaccine for avian Escherichia coli disease.

[0041] In some embodiments, the transfer culture conditions are: the bacterial solution of the secondary seed is transferred to the shaking bottle at a ratio of 1: (30-70), and the bacteria are fermented at 170-190 r / min in a shaking shaker at 35-38°C. After fermentation for 3-4 hours, the OD is measured every 0.5-0.6 hours. 600 , when OD 600 Stop fermentation when the plateau phase is reached, centrifuge at 7000-9000 r / min for 2-4 min at 3-5°C in a high-speed refrigerated centrifuge, discard the supernatant and collect the bacteria, resuspend the bacteria in physiological saline, take 90-110 μL of the bacterial solution for multiple dilution, and calculate the viable bacteria concentration of the resuspended bacterial solution by counting the viable bacteria.

[0042] In some embodiments, the inactivation treatment is specifically as follows: adding a 0.3-0.7% formaldehyde solution by volume to the bacterial solution, and inactivating the bacterial solution at 35-38° C. in a shaking incubator at 80-120 r / min for 40-60 h.

[0043] The following further describes the trivalent inactivated vaccine of avian Escherichia coli disease of the present application and its preparation method with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] In the following examples, aluminum hydroxide gel adjuvant: aluminum hydroxide is a crystalline aluminum hydroxide, which is positively charged at physiological pH (pI=11) and suitable for adsorbing negatively charged acidic proteins (such as albumin). It is a commercial product purchased; MONTANIDE TM GEL P PR adjuvant is a newly developed polymer adjuvant, which is a high molecular weight polypropylene and is a purchased commercial product; competing vaccine: Propolis inactivated Escherichia coli vaccine (EC24 strain + EC30 strain + EC45 strain + EC50 strain), used to prevent Escherichia coli disease in chickens caused by serotypes O78, O111, O2, and O5 of Escherichia coli, and produced by Shandong Huahong Bioengineering Co., Ltd.

[0045] Example 1: Collection and screening of vaccine strains

[0046] (1) Isolation and identification of duck-derived Escherichia coli

[0047] Experimental methods: Suspected avian Escherichia coli disease materials were collected from poultry farms in different regions of China between April 2020 and December 2022, mainly including extraintestinal tissues (brain, heart, liver, lung) and fluid samples (pericardial effusion, semen, eggs). These dead animals showed different types of pathological changes of avian Escherichia coli infection, such as perihepatitis, pericarditis, air sacculitis, salpingitis, and peritonitis. The inventors cut the tissue block, soaked it in a 75% ethanol solution for 1 minute to kill pathogenic microorganisms on the surface of the liver; placed the tissue block in a sterile EP tube and washed it twice with 1.5 mL of sterile saline; spread the ground tissue block and the aseptically collected liquid sample on a MacConkey plate, and cultured it at 37°C for 24 hours; picked a single colony with a dark pink center, round, flat, neat edges, smooth surface, and moistness, and streaked it on an LB plate, and cultured it at 37°C for 24 hours; resuspended the bacteria with sterile saline, and stored one portion of the bacterial suspension at -20°C with 25% glycerol, and the other portion was sent to Shanghai Shenggong Biotechnology Co., Ltd. for whole genome sequencing.

[0048] Experimental results: The sequencing results were subjected to nucleotide BLAST analysis in NCBI, and a total of 163 strains of Escherichia coli were identified.

[0049] (2) Determination of dominant serotype and analysis of strain virulence genes

[0050] The whole genome sequencing results of 163 Escherichia coli strains were uploaded one by one using the SerotypeFinder 2.0 (https: / / cge.food.dtu.dk / services / SerotypeFinder / ) online database. Serotype comparison analysis was performed on the wzx gene and wzy gene to determine the dominant serotype of APEC. The whole genome sequencing results of the dominant serotype strains were uploaded using the VirulenceFinder 2.0 (https: / / cge.food.dtu.dk / services / VirulenceFinder / ) online database for virulence gene prediction analysis.

[0051] Experimental results: Figure 1 As shown in Figure 1, among the 163 APEC strains, 145 strains completed serotyping, and the dominant serotypes were O145 (16.6%), O78 (9.2%), and O111 (6.7%). As shown in Table 1, the predicted number of virulence genes of strains with serotypes of O145, O78, and O111 was statistically analyzed.

[0052] Table 1 - Statistics of the predicted number of virulence genes

[0053]

[0054] (3) Animal pathogenicity experiments

[0055] According to the dominant serotype and the number of virulence genes, 6 strains numbered APEC-33 (O111), APEC-34 (O111), APEC-111 (O78), APEC-119 (O78), APEC-40 (O145), and APEC-164 (O145) were selected as vaccine candidate strains, and the pathogenicity test for ducklings was carried out. 240 healthy female ducklings aged 14 days were selected and randomly divided into 24 groups, with 10 ducklings in each group. Four challenge concentrations were set for each vaccine candidate strain, and the number of surviving ducklings in each group after 7 days of infection was counted. The results are shown in Table 3.

[0056] Experimental results: As shown in Table 3, the six selected vaccine candidate strains are all highly pathogenic strains. APEC-33 (O111), APEC-119 (O78), and APEC-164 (O145) strains with strong infection virulence were selected as vaccine preparation strains, and were named Escherichia coli JX033, Escherichia coli JX119, and Escherichia coli JX164, respectively. At the same time, they were sent to the China Center for Type Culture Collection for preservation. The preservation address is: Wuhan University, Wuhan, China, and the preservation numbers are CCTCC M 20231990, CCTCC M20231989, and CCTCC M 20231988, respectively.

[0057] Table 3 - Pathogenicity test results

[0058]

[0059]

[0060] Example 2: Preparation of trivalent inactivated vaccine of duck-derived Escherichia coli O111, O78 and O145

[0061] (1) Preparation of bacterial solution

[0062] Take the frozen avian pathogenic Escherichia coli JX033, Escherichia coli JX119, and Escherichia coli JX164 strains and streak them on the plates, and place them in an incubator at 37°C for overnight culture to recover. The next day, single colonies with neat colony edges and shiny, moist, and smooth surfaces were selected for primary and secondary seed propagation in LB culture medium. The secondary seed culture was transferred to a shake flask at a ratio of 1:50 (i.e., a volume ratio of 2%), and fermented in a 37°C shaking incubator at 180 r / min. After fermentation for 3 hours, the OD was measured every 0.5 hours. 600 , when OD 600 Stop fermentation when reaching the plateau phase; centrifuge at 4°C, 8000r / min for 3min in a high-speed refrigerated centrifuge, discard the supernatant and collect the bacteria, first resuspend the bacteria in an appropriate volume of physiological saline. Take 100μl of bacterial solution for each serotype strain and dilute it 10 times, and calculate the viable bacterial concentration by counting the viable bacteria.

[0063] (2) Seedling production

[0064] Trivalent vaccine (containing 20% ​​volume of MONTANIDE TM GEL P PR adjuvant): According to the live bacterial count results, the concentration of the three serotype bacterial solutions was adjusted to 2.25×10 10 CFU / mL. Take 16mL of the three serotype bacterial solutions of the adjusted concentrations and mix them in equal proportions (a total of 48mL), add 0.5% formaldehyde solution to the total volume, and inactivate the bacterial solution in a 37℃ shaker at 100r / min for 48h; dip a sterile cotton swab into the inactivated bacterial solution and apply it to the LB plate for sterility testing. Place the sterile immunogen solution in a stirrer at 200rpm and quickly add 12mL of MONTANIDE TM GEL P PR adjuvant (added within 2 seconds), stirred at 200 rpm for 10 min, final antigen content: 6×10 9 CFU / mL. Place the prepared vaccine at 4℃ for 12h to observe whether it is evenly distributed and store it at 2-8℃.

[0065] Trivalent vaccine (containing 30% volume aluminum hydroxide gel adjuvant): According to the live bacteria count results, the concentration of the three serotypes was adjusted to 2.57×10 10CFU / mL. Take 14mL of the three serotype bacterial solutions of adjusted concentrations and mix them in equal proportions (a total of 42mL), add 0.5% formaldehyde solution to the total volume, and inactivate the bacterial solution in a 37℃ shaker at 100r / min for 48h; dip a sterile cotton swab in the inactivated bacterial solution and apply it to the LB plate for sterility testing. Shake the unopened aluminum hydroxide gel adjuvant vigorously to resuspend it, drop 18mL of aluminum hydroxide into the immunogen solution, and add 0.01% thimerosal at a final concentration (mass concentration). Pipette up and down several times to mix to ensure the correct absorption of the antigen by the alum adjuvant, incubate for 5-10min, and the final antigen content is 6×10 9 CFU / mL. Place the prepared vaccine at 4℃ for 12h to observe whether it is evenly distributed and store it at 2-8℃.

[0066] (4) Safety verification of trivalent inactivated duck Escherichia coli O111, O78, and O145 vaccines

[0067] Sixty healthy Mallard ducks aged 7 days were randomly divided into 6 groups, with 10 ducks in each group. All the ducks were warmed in a 41°C water bath for 30 minutes before vaccination. Vaccine safety was verified at 1, 2, and 3 times the designed immunization dose. The results are shown in Table 4.

[0068] Table 4 - Safety verification results of inactivated vaccines

[0069]

[0070]

[0071] As can be seen from Table 4, no obvious adverse reactions occurred in the ducks of each group within 7 days after immunization, and the survival rate reached 100% 7 days after immunization. This shows that the two prepared trivalent inactivated vaccines have good safety.

[0072] Example 3: Experiment on the immune protection of duck-derived Escherichia coli O111, O78, and O145 trivalent inactivated vaccine

[0073] As shown in Table 5, 170 healthy Mallard ducks aged 7 days were selected and randomly divided into 17 groups, with 10 ducks in each group, including experimental group, competitive product control group, adjuvant control group and blank control group. The body weight before immunization was weighed after grouping. The immunization site was subcutaneous in the neck. The immunization dose for the experimental group and the competitive product control group was 0.3 ml per duck, and the immune antigen content was 1.8×10 9 CFU / feather. 21 days after immunization, the same strains were used for challenge. The challenge dose of Escherichia coli JX033 was 1.28×10 10 CFU / feather, the challenge dose of Escherichia coli JX119 was 1.56×10 10CFU / feather, the challenge dose of Escherichia coli JX164 was 2.50×10 10 CFU / feather. At the same time, the weight of each group of ducks was weighed 21 days after immunization. Comprehensively evaluate the immune protection effect of the vaccine and the effect of vaccine immunization on animal weight gain.

[0074] Table 5 - Results of inactivated vaccine immune protection experiments

[0075]

[0076]

[0077]

[0078] As can be seen from Table 5, both inactivated vaccines with two adjuvants have good protective effects; MONTANIDE TM The protective effect of the vaccine prepared with GELP PR adjuvant is better than that prepared with aluminum hydroxide gel adjuvant; compared with similar commercial vaccines, the two trivalent inactivated vaccines provided by the present invention have better immune protection against O78 and O111 serotypes of Escherichia coli infection than the competing products; in terms of animal weight gain, compared with the blank control, there is no significant difference in the average daily weight gain after immunization with the vaccine provided by the present invention, which proves that vaccine immunization has no effect on the daily weight gain of animals. TM The GEL PPR adjuvant trivalent inactivated vaccine has excellent immune protection, good safety and low side effects, and has broad application prospects and practical value.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An avian Escherichia coli composition, characterized in that Including three serotypes of Escherichia coli: O145, O78 and O111; Among them, the O145 serotype Escherichia coli is Escherichia coli JX164, with a deposit number of CCTCC NO: M 20231988; O78 serotype Escherichia coli, which is Escherichia coli JX119, with a deposit number of CCTCC NO: M 20231989; The O111 serotype Escherichia coli is Escherichia coli JX033, and its deposit number is CCTCC NO: M 20231990.

2. Use of the avian Escherichia coli composition as claimed in claim 1 in the preparation of a trivalent inactivated vaccine for avian Escherichia coli disease.

3. A trivalent inactivated vaccine for avian Escherichia coli disease, characterized in that: The invention comprises the avian Escherichia coli composition as claimed in claim 1.

4. The trivalent inactivated vaccine for avian Escherichia coli disease according to claim 3, characterized in that: In the trivalent inactivated vaccine for avian Escherichia coli disease, the antigen content of each inactivated Escherichia coli strain is ≥ 6.0×10 9 CFU / mL.

5. The trivalent inactivated vaccine for avian Escherichia coli disease according to claim 3, characterized in that: Adjuvants are also included.

6. The trivalent inactivated vaccine for avian Escherichia coli disease according to claim 5, characterized in that The adjuvant is aluminum gel adjuvant or MONTANIDE GEL P PR adjuvant.

7. The trivalent inactivated vaccine for avian Escherichia coli disease according to claim 5, characterized in that: The volume fraction of the adjuvant in the trivalent inactivated vaccine for avian Escherichia coli disease is 20-30%.

8. A method for preparing a trivalent inactivated vaccine for avian Escherichia coli disease as claimed in any one of claims 3 to 7, characterized in that: The steps include: Preparation of inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring culture and performing inactivation treatment; Preparation of vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the trivalent inactivated vaccine for avian Escherichia coli disease.

9. The method for preparing a trivalent inactivated vaccine for avian Escherichia coli disease as claimed in claim 8, characterized in that: The transfer culture conditions are as follows: the bacterial solution of the secondary seed is transferred to the shaking bottle at a ratio of 1: (30-70), and the bacteria are fermented at 170-190 r / min in a shaking shaker at 35-38°C. After fermentation for 3-4 hours, the OD is measured every 0.5-0.6 hours. 600 , when OD 600 Stop fermentation when the plateau phase is reached, centrifuge at 7000-9000 r / min for 2-4 min at 3-5°C in a high-speed refrigerated centrifuge, discard the supernatant and collect the bacteria, resuspend the bacteria in physiological saline, take 90-110 μL of the bacterial solution for multiple dilution, and calculate the viable bacteria concentration of the resuspended bacterial solution by counting the viable bacteria.

10. The method for preparing the trivalent inactivated vaccine for avian Escherichia coli disease according to claim 9, characterized in that: The inactivation treatment is specifically as follows: adding a formaldehyde solution with a volume ratio of 0.3-0.7% to the bacterial solution, and inactivating the bacterial solution at 35-38° C. in a shaking incubator at 80-120 r / min for 40-60 hours.

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