Preparation method and application of rimorella anatipestifer and escherichia coli bivalent multivalent inactivated vaccine

Abstract

The invention discloses a preparation method of rimosella anatipestifer and escherichia coli bivalent multivalent inactivated vaccine, and relates to the field of preparation of canine anatipestifer vaccine.The preparation method comprises the steps that a CpG immune synergist and a nano slow-release carrier suspension are mixed to obtain a composite water-based adjuvant; adding the purified and inactivated antigen concentrated solution of rimorella anatipestifer and O78 type escherichia coli into a composite water-based adjuvant, and carrying out high-pressure homogenization to obtain a vaccine semi-finished product; standing and defoaming the vaccine semi-finished product to obtain the vaccine; the preparation method of the CpG immune synergist comprises the following steps: grafting CpG oligonucleotide onto sodium alginate; wherein the preparation of the nano sustained-release carrier suspension comprises the step of preparing nano sustained-release microspheres from polylactic acid and chitosan, and the animal injection site inoculated with the vaccine has no adverse reactions such as obvious swelling and induration, and is fast to absorb.

Description

Technical Field

[0001] This invention relates to the field of duck disease vaccine preparation, specifically to a method for preparing a bivalent inactivated vaccine containing duck disease bacillus and Escherichia coli, and its application. Background Technology

[0002] Anal lemuriasis and Escherichia coli O78 infection are major infectious diseases affecting the healthy development of waterfowl farming. Epidemiological monitoring data shows that the serotypes of anal lemuriasis are becoming more diverse, with RA2, RA6, and RA7 becoming the dominant strains in some parts of China. This poses higher requirements for comprehensive disease control. To achieve broad coverage and effective protection against the currently prevalent strains, developing a combined vaccine capable of preventing multiple pathogens with a single injection is of significant practical importance. Meanwhile, in the field of vaccine adjuvant technology, developing novel adjuvants to further enhance the immunogenicity and safety of vaccines is an important direction for technological development in this field. This invention aims to provide a safe and effective broad-spectrum combined vaccine by isolating, identifying, and applying the currently dominant prevalent strains, combined with CpG immunostimulants and nano-sustained-release carrier technology. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for preparing a bivalent inactivated vaccine containing duck plague bacillus and Escherichia coli, and its application.

[0004] To achieve the above objectives, this invention provides a method for preparing a bivalent inactivated vaccine against duck plague bacilli and Escherichia coli, comprising the following steps: CpG immunostimulant and nano-sustained-release carrier suspension were mixed to obtain a composite aqueous adjuvant; A purified and inactivated antigen concentrate containing at least two or more serotypes of Riemerella anatipestifer and Escherichia coli O78 is added to the composite aqueous adjuvant and homogenized under high pressure to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to remove bubbles, thus obtaining the vaccine; The preparation of the CpG immune enhancer includes grafting CpG oligonucleotides onto sodium alginate; The preparation of the nano-sustaining carrier suspension includes preparing polylactic acid and chitosan into nano-sustaining microspheres.

[0005] Furthermore, each dose of the vaccine contains 10-40 μg of CpG immunostimulant and a total antigen concentration of 6 x 10⁻⁶. 9 Up to 10x10 9 CFU.

[0006] Furthermore, the pressure of the high-pressure homogenization is 800 Bar.

[0007] Furthermore, the preparation method of the nano-sustained-release carrier suspension includes the following steps: Chitosan was dissolved in acetic acid solution, and polylactic acid was dissolved in dichloromethane. Polylactic acid solution is added dropwise to chitosan solution and subjected to high-speed shearing to form an emulsion; The emulsion was ultrasonically dispersed using a probe and then evaporated under reduced pressure to obtain a colloidal solution containing nano-slow-release microspheres. The colloidal solution was centrifuged, washed, and resuspended to obtain the nano-sustained-release carrier suspension.

[0008] Furthermore, the mass ratio of chitosan to polylactic acid is 6:4; the mass ratio of chitosan to 2% acetic acid solution is 6:100; and the mass ratio of polylactic acid to dichloromethane is 4:50.

[0009] Furthermore, the preparation method of the CpG immune enhancer includes the following steps: Sodium alginate was dissolved in PBS buffer, and EDC and NHS were added for activation in the dark. Amino-modified CpG oligonucleotides were added to an activated sodium alginate solution and subjected to a isothermal shaking reaction. The reaction product was subjected to gradient dialysis and then freeze-dried to obtain CpG immune enhancer powder.

[0010] Furthermore, the mass ratio of sodium alginate, CpG oligonucleotide, EDC and NHS is 5:0.05:1:0.6.

[0011] Furthermore, the serotypes of the duck plague bacillus include at least RA2, RA6 and RA7.

[0012] A bivalent inactivated vaccine against Rimodia dysenteriae and Escherichia coli prepared by the preparation method described above.

[0013] Application of a bivalent inactivated vaccine against duck plague bacillus and Escherichia coli in the prevention of diseases caused by duck plague bacillus and Escherichia coli type O78.

[0014] Compared with the prior art, the beneficial effects of the present invention are: significantly enhanced immune effect and stronger protection. The present invention prepares a composite aqueous adjuvant by combining CpG immune enhancer and nano-sustained-release carrier. This adjuvant has a synergistic effect, which not only solves the problem of insufficient immune response intensity or short duration of action of single adjuvant, but also shows that the vaccine prepared by the present invention can stimulate a high level of antibody response, and its antibody titer is significantly higher than that of traditional oil adjuvant vaccines, and can provide a challenge protection rate of more than 90%.

[0015] Safety is greatly improved and side effects are significantly reduced. This invention uses a compound aqueous adjuvant to replace the traditional mineral white oil adjuvant. Safety tests have confirmed that animals vaccinated with the vaccine of this invention do not show obvious adverse reactions such as swelling or induration at the injection site, and absorption is rapid.

[0016] This invention uses currently prevalent duck plague bacillus strains RA2, RA6, and RA7 as antigens, combined with O78 type Escherichia coli, to effectively solve the problem of insufficient cross-protection of existing vaccines, achieving highly efficient protection against multiple major pathogens with a single immunization, resulting in a more comprehensive epidemic prevention effect. Attached Figure Description

[0017] Figure 1 The flowchart is as follows: This invention is prepared by [the present invention]. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 See Figure 1 This embodiment provides a method for preparing a bivalent inactivated vaccine against duck plague bacilli and Escherichia coli, comprising the following steps: Preparation of polyvalent antigens for *Rimonella anatipestifer* and *Escherichia coli* antigens: Pathological samples were collected from the liver and brain tissues of ducks that had recently exhibited typical infectious serositis symptoms. The samples were isolated and cultured using standard bacteriological methods, and slide agglutination tests were performed. Serotyping of the strains was conducted using diagnostic serum to screen for representative dominant *Rimonella anatipestifer* strains RA2, RA6, and RA7. Simultaneously, *Escherichia coli* strain O78 was screened and identified. Strain isolation and purification were performed using standard bacteriological methods: aseptically sampled pathological tissues were enriched in BHI broth, then streaked onto bovine serum agar plates and incubated at 37°C (5% CO2) for 24-48 hours. For *Rimonella anatipestifer*, single colonies with smooth, moist surfaces, regular edges, and a grayish-white, dew-like appearance were selected. For *Escherichia coli* O78, single red, round, smooth colonies were selected on MacConkey agar plates. The strain identification criteria are as follows: Morphological identification: The purified strain was subjected to Gram staining and microscopic observation; *Rhizobium anatipestifer* should be a Gram-negative, short bacillus or slender filaments with blunt ends; *Escherichia coli* O78 is a Gram-negative short bacillus; Biochemical characterization: Based on relevant veterinary pharmacopoeia standards, a series of biochemical reactions were conducted, including oxidase, catalase, motility, VP test, and indole assay. The typical characteristics of Riemerella anatipestifer are oxidase positivity, catalase positivity, and non-fermentation of sugars. Serological identification: Slide agglutination test was performed using polyvalent and monovalent diagnostic sera; pure culture and diagnostic serum were mixed on a clean glass slide, and the appearance of visible agglutination clumps was considered positive, thus determining the sera type as RA2, RA6, RA7 or O78. Molecular biological confirmation: To ensure the accuracy of strain identification, strain DNA was extracted and finally confirmed by polymerase chain reaction (PCR); amplification was performed using species-specific primers for *Remus dysenteriae* anatipestifer, serotype-specific primers for RA2, RA6, and RA7, and serotype-specific primers for *Escherichia coli* O78; PCR products were subjected to agarose gel electrophoresis, and those with band sizes consistent with the expected fragment size were confirmed as the target strain.

[0020] To further ensure the selected strains possess excellent immunogenicity and pathogenicity, the immunoprotective efficacy of the candidate strains of *Rimonella anatipestifer* identified as RA2, RA6, and RA7 was evaluated. Each candidate strain was prepared into an inactivated bacterial solution and used to immunize 14-day-old healthy Cherry Valley ducks. A challenge protection test was conducted 21 days post-immunization. The median lethal dose (LD50) in the ducklings was determined. To evaluate the pathogenicity of each strain, the Reed-Muench method was used for pathogenicity assessment. The calculation formula is as follows:

[0021] in, ; Meanwhile, the protection rate of each strain of inactivated vaccine against virulent challenge was recorded, and the calculation formula is as follows:

[0022] Ultimately, the choice The strains with the lowest values, i.e. the strongest pathogenicity and the highest immune protection rate, are selected as the dominant circulating strains of RA2, RA6, and RA7 for subsequent vaccine preparation.

[0023] The identified and qualified *Remus aedes* strains RA2, RA6, and RA7, along with *Escherichia coli* strain O78, were separately fermented. By optimizing the culture medium composition and fermentation parameters, the viable cell count of each strain reached 1.2 x 10⁻⁶ after cultivation. 10For strains with a concentration of CFU / ml or higher, the fermentation broth of each strain was added to a formaldehyde solution with a final concentration of 0.2% and inactivated at 37°C for 24 hours. After confirming complete inactivation, the inactivated RA2, RA6, and RA7 strains of *Rimonis agalactiae* and O78 strains of *Escherichia coli* were mixed in equal amounts according to their titers to prepare a concentrated bivalent inactivated antigen solution of *Rimonis agalactiae* and *Escherichia coli*.

[0024] The preparation of CpG immunostimulant involved dissolving sodium alginate in PBS buffer, adding EDC and NHS for light-protected activation, adding amino-modified CpG oligonucleotides to the activated sodium alginate solution, and performing a isothermal shaking reaction. The reaction product was then subjected to gradient dialysis and freeze-dried to obtain CpG immunostimulant powder. The mass ratio of sodium alginate, CpG oligonucleotides, EDC, and NHS was 5:0.05:1:0.6.

[0025] In this process, EDC acts as a carboxyl activator, reacting with the carboxyl group (-COOH) on the sodium alginate molecular chain to form an active but unstable O-acyl isourea intermediate. The addition of NHS (N-hydroxysuccinimide) rapidly reacts with this intermediate to generate a relatively stable and non-hydrolyzable NHS active ester. This activated sodium alginate then undergoes a nucleophilic substitution reaction with an amino-modified CpG oligonucleotide (CpG-NH2) to form a stable amide bond (-CO-NH-), thereby covalently grafting the CpG oligonucleotide onto the sodium alginate backbone. The purpose of gradient dialysis is to utilize molecular weight differences to efficiently remove unreacted EDC, NHS, free CpG molecules, and other reaction byproducts, purifying the CpG-sodium alginate conjugate. To evaluate grafting efficiency, the absorbance of the coupling agent solution at 260 nm can be measured by ultraviolet spectrophotometry. The CpG concentration of the grafted agent can be calculated based on the standard curve, and the grafting rate can be calculated using the following formula:

[0026] in, Grafting rate; The concentration of grafted CpG oligonucleotides was determined by spectrophotometry. This represents the total volume of the coupling solution; The mass of sodium alginate initially fed into the feed; Fourier transform infrared spectroscopy (FTIR) analysis confirmed the formation of new amide bonds, further demonstrating the success of the grafting.

[0027] The preparation of the nano-sustained-release carrier suspension involves dissolving chitosan in acetic acid solution and polylactic acid in dichloromethane. The polylactic acid solution is then added dropwise to the chitosan solution, followed by high-speed shearing to form an emulsion. The emulsion is then ultrasonically dispersed using a probe and subjected to reduced-pressure evaporation to obtain a colloidal solution containing nano-sustained-release microspheres. The colloidal solution is centrifuged, washed, and resuspended to obtain the nano-sustained-release carrier suspension. The mass ratio of chitosan to polylactic acid is 6:4; the mass ratio of chitosan to 2% acetic acid solution is 6:100; and the mass ratio of polylactic acid to dichloromethane is 4:50.

[0028] The preparation of the bivalent multivalent inactivated vaccine involves mixing a prepared CpG immunostimulant and a prepared nano-sustained-release carrier suspension to obtain a composite aqueous adjuvant. A concentrated solution of bivalent multivalent inactivated antigens of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) is added to the composite aqueous adjuvant, followed by high-pressure homogenization to obtain a vaccine semi-finished product. The high-pressure homogenization pressure is 800 Bar. The vaccine semi-finished product is then allowed to stand to defoam, yielding the final vaccine. Each dose of the final vaccine contains 20 μg of CpG immunostimulant and a total antigen concentration of 8 x 10⁻⁶. 9 CFU, in which the ratio of Anatidae RA2, RA6, RA7 and Escherichia coli O78 strains in the total antigen is 1:1:1:1.

[0029] Example 2 The preparation method of this embodiment is exactly the same as that described in Example 1, including antigen preparation, CpG immunostimulant preparation, and nano-sustained-release carrier suspension preparation. The difference lies in the preparation of the bivalent multivalent inactivated vaccine, which is as follows: The prepared CpG immunostimulant and the prepared nano-sustained-release carrier suspension are mixed to obtain a composite aqueous adjuvant. The prepared bivalent multivalent inactivated antigen concentrate of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) is added to the composite aqueous adjuvant and homogenized under high pressure (800 Bar) to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to defoam, and the vaccine is obtained. The final prepared vaccine contains 10 μg of CpG immunostimulant and a total antigen concentration of 8 x 10⁻⁶ per dose. 9 CFU, in which the ratio of Anatidae RA2, RA6, RA7 and Escherichia coli O78 strains in the total antigen is 1:1:1:1.

[0030] Example 3 The preparation method of this embodiment is exactly the same as that described in Example 1, including antigen preparation, CpG immunostimulant preparation, and nano-sustained-release carrier suspension preparation. The difference lies in the preparation of the bivalent multivalent inactivated vaccine, which is as follows: The prepared CpG immunostimulant and the prepared nano-sustained-release carrier suspension are mixed to obtain a composite aqueous adjuvant. The prepared bivalent multivalent inactivated antigen concentrate of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) is added to the composite aqueous adjuvant and homogenized under high pressure (800 Bar) to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to defoam, and the vaccine is obtained. The final prepared vaccine contains 40 μg of CpG immunostimulant and a total antigen concentration of 8 x 10⁻⁶ per dose. 9 CFU, in which the ratio of Anatidae RA2, RA6, RA7 and Escherichia coli O78 strains in the total antigen is 1:1:1:1.

[0031] Example 4 The preparation method of this embodiment, including antigen preparation, CpG immunostimulant preparation, and nano-sustained-release carrier suspension preparation, is exactly the same as that described in Example 1. The difference lies in the preparation of the bivalent multivalent inactivated vaccine, which is as follows: The prepared CpG immunostimulant and the prepared nano-sustained-release carrier suspension are mixed to obtain a composite aqueous adjuvant. The prepared bivalent multivalent inactivated antigen concentrate of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) is added to the composite aqueous adjuvant and homogenized under high pressure (800 Bar) to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to defoam, and the vaccine is obtained. The final prepared vaccine contains 20 μg of CpG immunostimulant and a total antigen concentration of 6 x 10⁻⁶ per dose. 9 CFU, in which the ratio of Anatidae RA2, RA6, RA7 and Escherichia coli O78 strains in the total antigen is 1:1:1:1.

[0032] Example 5 The preparation method of this embodiment, including antigen preparation, CpG immunostimulant preparation, and nano-sustained-release carrier suspension preparation, is exactly the same as that described in Example 1. The difference lies in the preparation of the bivalent multivalent inactivated vaccine, which is as follows: The prepared CpG immunostimulant and the prepared nano-sustained-release carrier suspension are mixed to obtain a composite aqueous adjuvant. The prepared bivalent multivalent inactivated antigen concentrate of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) is added to the composite aqueous adjuvant and homogenized under high pressure (800 Bar) to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to defoam, and the vaccine is obtained. The final prepared vaccine contains 20 μg of CpG immunostimulant and a total antigen concentration of 10 x 10⁻⁶ per dose. 9 CFU, in which the ratio of Anatidae RA2, RA6, RA7 and Escherichia coli O78 strains in the total antigen is 1:1:1:1.

[0033] Comparative Example 1 This comparative example aims to test the effect of the absence of CpG immunostimulant. Its adjuvant component only contains a nano-suspension of sustained-release carrier. Its antigen is the same as that of Riemerella anatipestifer (including RA2, RA6, and RA7 types) and Escherichia coli (O78 type) bivalent inactivated antigen concentrate in Example 1. Its preparation method is exactly the same as that of Example 1, except that CpG immunostimulant is not added in the step of preparing the compound adjuvant. All other steps and parameters are exactly the same as those in Example 1.

[0034] Comparative Example 2 This comparative example aims to test the effect of lacking the nano-release carrier and high-pressure homogenization step. The antigen used is the same bivalent multivalent inactivated antigen concentrate as in Example 1. The preparation method is as follows: the same amount of multivalent antigen concentrate as in Example 1 and CpG immunostimulant (20 μg / dose) are directly mixed in PBS buffer without adding nano-release carrier suspension and without high-pressure homogenization.

[0035] Comparative Example 3 This comparative example is intended to compare with traditional oil-adjuvanted vaccines. The antigen used is the same bivalent multivalent inactivated antigen concentrate of *Rhizobium anatipestifer* (including RA2, RA6, and RA7 types) and *Escherichia coli* (O78 type) as in Example 1. This antigen concentrate is emulsified with traditional mineral white oil adjuvant to prepare a water-in-oil inactivated vaccine. The preparation process refers to the existing conventional oil-adjuvanted vaccine production process.

[0036] In Examples 1-5 and Comparative Examples 1-3, medical-grade chitosan was obtained from Qingdao Bohai Biotechnology Co., Ltd., with a degree of deacetylation >95%; polylactic acid (PLA) was obtained from Shenzhen Guanghua Weiye Co., Ltd., with an intrinsic viscosity of 1.6-2.4 dL / g; sodium alginate was obtained from Sinopharm Chemical Reagent Co., Ltd.; amino-modified CpG oligonucleotides were custom-synthesized by Shanghai Sangon Biotech Co., Ltd., purified by HPLC, and their sequences were designed according to this scheme; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; all other products were commercially available analytical grade products.

[0037] The bivalent inactivated vaccines against Riemerella anatipestifer and Escherichia coli prepared in Examples 1-5 and Comparative Examples 1-3 were tested accordingly. Healthy 14-day-old Cherry Valley ducks, weighing approximately 60-80g, were selected as test animals, ensuring they had no history of infection with the relevant pathogens. Each vaccine was prepared according to the corresponding method. The test animals were randomly divided into 8 groups of 30 animals each (20 for efficacy testing and 10 for safety testing). Immunization was performed by intramuscular injection of 0.5 mL of the corresponding vaccine per animal into the chest. A saline control group was included. Safety was observed by recording local and systemic reactions for 72 consecutive hours post-immunization. Local reactions were quantified and scored according to the swelling diameter: Grade 0 (<0.5cm), Grade 1 (0.5-1.5cm), and Grade 2 (>1.5cm). Immunopotency testing was conducted on day 28 post-immunization, collecting serum to detect HI antibody titers. The remaining animals were then challenged with a strong virus (using a 10x LD50). 50 The dominant circulating strains of *Rhizobium anatipestifer* RA2, RA6, and RA7 were challenged with a mixed bacterial solution of *Escherichia coli* O78. After challenge, the bacteria were observed for 14 consecutive days. Those that survived and had no clinical symptoms were considered protected, and the protection rate was calculated.

[0038] The test results are shown below: (1) Immunoefficacy test: The vaccines prepared in Examples 1-5 and Comparative Examples 1-3 were tested for immunoefficacy. The antibody titer (HI titer) was detected 4 weeks after immunization, and a virulent challenge experiment was conducted. The challenge protection rate was recorded. The test results are shown in Table 1.

[0039] Table 1

[0040] As shown in Table 1, the vaccines prepared according to this protocol can all provide effective protection for animals. The vaccines in Examples 1, 2, 3, and 5 can all elicit high levels of antibody responses and provide a challenge protection rate of over 90%, indicating that when the total antigen concentration is not less than 8 x 10⁻⁶, the antibody response is effective. 9When CFU / dose is used, CpG immunostimulant can effectively enhance immunity within the range of 10-40 μg / dose. In Example 4, compared to Example 1, the total antigen concentration was reduced to 6 x 10⁻⁶. 9 When CFU / dose was administered, the protection rate against challenge decreased to 85%, indicating that a sufficient amount of antigen is fundamental to ensuring immunization efficacy. Compared with the comparative examples, the antibody titer and protection rate of Example 1 were significantly better than those of Comparative Examples 1 and 2. Comparative Example 1 lacked CpG immune enhancer, resulting in insufficient immune response strength; Comparative Example 2 lacked nano-sustained-release carrier, which may have caused the antigen to be cleared too quickly, failing to form effective long-term immunity; Comparative Example 3 used a traditional oil adjuvant, which had a higher protection rate, but its antibody titer was lower than that of Examples 1, 3, and 5, indicating that the composite aqueous adjuvant of this scheme has certain advantages in stimulating humoral immunity. (2) Safety test: The safety of the vaccines prepared in Example 1 and Comparative Examples 1-3 was tested. After the test animals were injected with the vaccine, they were observed continuously for 72 hours and the local reaction at the injection site was recorded. The test results are shown in Table 2. Table 2

[0041] Table 2 shows that the vaccine of Example 1 prepared according to this scheme, with a compound aqueous adjuvant, was rapidly absorbed by animals after injection. No visible swelling or induration was observed at the injection site during the observation period, indicating good animal welfare. Comparative Examples 1 and 2, which did not use oil adjuvants, also showed good safety. However, Comparative Example 3, using a traditional mineral white oil adjuvant, showed significant redness and induration at the injection site within 24-72 hours after injection. Autopsy revealed incompletely absorbed oil droplets, causing a strong stress response in the animals. These results indicate that the vaccine prepared by the method described in claim 1 can significantly improve vaccine safety while ensuring immunization efficacy and reducing stress and adverse reactions caused by adjuvants. This is of great significance for maintaining the production performance and animal welfare of farmed animals.

[0042] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing a bivalent inactivated vaccine against duck plague bacilli and Escherichia coli, characterized in that, Includes the following steps: CpG immunostimulant and nano-sustained-release carrier suspension were mixed to obtain a composite aqueous adjuvant; A purified and inactivated antigen concentrate containing at least two or more serotypes of Riemerella anatipestifer and Escherichia coli O78 is added to the composite aqueous adjuvant and homogenized under high pressure to obtain a vaccine semi-finished product. The vaccine semi-finished product is allowed to stand to remove bubbles, thus obtaining the vaccine; The preparation of the CpG immune enhancer includes grafting CpG oligonucleotides onto sodium alginate; The preparation of the nano-sustaining carrier suspension includes preparing polylactic acid and chitosan into nano-sustaining microspheres.

2. The preparation method according to claim 1, characterized in that, Each dose of the vaccine contains 10-40 μg of CpG immunostimulant and a total antigen concentration of 6 x 10⁻⁶ μg. 9 Up to 10x10 9 CFU.

3. The preparation method according to claim 1, characterized in that, The pressure of the high-pressure homogenizer is 800 Bar.

4. The preparation method according to claim 1, characterized in that, The preparation method of the nano-sustained-release carrier suspension includes the following steps: Chitosan was dissolved in acetic acid solution, and polylactic acid was dissolved in dichloromethane. Polylactic acid solution is added dropwise to chitosan solution and subjected to high-speed shearing to form an emulsion; The emulsion was ultrasonically dispersed using a probe and then evaporated under reduced pressure to obtain a colloidal solution containing nano-slow-release microspheres. The colloidal solution was centrifuged, washed, and resuspended to obtain the nano-slow-release carrier suspension.

5. The preparation method according to claim 4, characterized in that, The mass ratio of chitosan to polylactic acid is 6:4; the mass ratio of chitosan to 2% acetic acid solution is 6:100; and the mass ratio of polylactic acid to dichloromethane is 4:

50.

6. The preparation method according to claim 1, characterized in that, The preparation method of the CpG immune enhancer includes the following steps: Sodium alginate was dissolved in PBS buffer, and EDC and NHS were added for activation in the dark. Amino-modified CpG oligonucleotides were added to an activated sodium alginate solution and subjected to a constant-temperature oscillation reaction. The reaction product was then subjected to gradient dialysis and freeze-dried to obtain CpG immune enhancer powder.

7. The preparation method according to claim 6, characterized in that, The mass ratio of sodium alginate, CpG oligonucleotide, EDC and NHS is 5:0.05:1:0.

6.

8. The preparation method according to claim 1, characterized in that, The serotypes of *Rimorrhageella anatipestifer* include at least RA2, RA6, and RA7.

9. A bivalent inactivated vaccine against duck plague bacilli and Escherichia coli prepared by the preparation method according to any one of claims 1-8.

10. The use of the bivalent inactivated vaccine of Riemerella anatipestifer and Escherichia coli as described in claim 8 in the prevention of diseases caused by Riemerella anatipestifer and Escherichia coli type O78.

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