Rabbit pasteurella outer membrane vesicle trivalent vaccine and preparation method thereof

Abstract

The invention discloses a rabbit pasteurella outer membrane vesicle trivalent vaccine and a preparation method thereof, and belongs to the technical field of multivalent vaccine preparation. According to the trivalent vaccine, outer membrane vesicle extracts from rabbit A type pasteurella multocida PmA HP-18, rabbit D type pasteurella multocida PmD HP-22 and rabbit F type pasteurella multocida PmF HP-11 are taken as antigens. The outer membrane vesicle trivalent vaccine provided by the invention contains a main outer membrane structure and periplasmic protein, has no life activity and good safety, and can effectively activate an immune system and generate protective immune response. A type pasteurella multocida, D type pasteurella multocida and F type pasteurella multocida can be prevented at the same time, and the three-prevention effect is achieved through one injection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of multivalent vaccine preparation, and particularly relates to a rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine and a preparation method thereof. BACKGROUND

[0002] Rabbit Pasteurella multocida disease is a disease caused by Pasteurella multocida, and is a common and frequently-occurring disease in rabbit breeding. The disease can occur throughout the year, and rabbits of all ages can be infected. Clinically, the disease is mainly characterized by septicemia and hemorrhagic syndrome, and is also known as rabbit hemorrhagic septicemia. It is one of the respiratory infectious diseases that seriously endanger animal and human health. In addition to infectious rhinitis, tracheitis and pneumonia, it can also cause otitis media, conjunctivitis, metritis and subcutaneous abscesses. It is widely prevalent in rabbit populations in China, causes serious economic losses, and is one of the important pathogens that hinder the development of the rabbit industry.

[0003] Pasteurella multocida is a zoonotic pathogenic bacterium that can infect various poultry, livestock and wild animals, and can also infect humans. It mainly causes respiratory diseases, focal infections and hemorrhagic septicemia, and the diseases caused by it include not only rabbit pneumonia hemorrhagic septicemia, but also avian cholera, atrophic rhinitis in pigs, swine pneumonia, bovine hemorrhagic septicemia and human skin necrosis. It seriously threatens animal and human health. According to the different capsules, Pasteurella multocida can be divided into five serotypes, A, B, D, E and F. There is no cross protection between strains of different capsule serotypes. According to the reports, the Pasteurella multocida that infects rabbits has capsule types A, D and F. These three serotypes are prevalent in China, and mixed infection of the three serotypes often occurs. However, at present, there is only a commercial inactivated vaccine against rabbit A-type Pasteurella multocida in China. Therefore, a new trivalent vaccine that can prevent rabbit A-type Pasteurella multocida, rabbit D-type Pasteurella multocida and rabbit F-type Pasteurella multocida is needed to effectively prevent the current epidemic of rabbit Pasteurella multocida disease.

[0004] Outer membrane vesicles (OMVs) are a kind of vesicle-like small molecules containing biologically active substances that exist universally in gram-negative bacteria and even some gram-positive bacteria, and their size is between 20-250 nm. The composition of outer membrane vesicles includes lipopolysaccharide, outer membrane protein, phospholipid, DNA, and periplasmic components wrapped in the outer membrane during formation. Because outer membrane vesicles contain a large number of bacterial antigens and cannot replicate, and can effectively activate the immune system, they are considered to be a very potential subunit vaccine. The outer membrane components contained in OMVs can stimulate the body to produce adaptive immune memory, and the contained LPS can act as a self-adjuvant. Moreover, as a non-replicating vaccine, it has certain safety. Therefore, these factors make OMVs a popular choice for developing non-replicating and efficient vaccines.

[0005] Because the outer membrane vesicles of *Pasteurella multocida* contain endotoxins, appropriate amounts of endotoxin can effectively activate the immune system as adjuvants, but excessive amounts can trigger serious vaccine side effects. Therefore, developing a trivalent outer membrane vesicle vaccine targeting *Pasteurella multocida* serotypes A, D, and F simultaneously not only ensures safety but also effectively controls infections caused by these three serotypes. The development of such a vaccine has significant scientific and practical value for the comprehensive prevention and control of rabbit pasteurellosis. Summary of the Invention

[0006] To address the aforementioned shortcomings in the existing technology, this invention provides a rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine and its preparation method. The serotypes of the strains used cover the Pasteurella multocida serotypes currently prevalent in rabbit populations in my country. The outer membrane vesicle trivalent vaccine prepared by the method of this invention can more effectively and comprehensively protect rabbits against Pasteurella multocida infection, achieving the effect of three protections with one injection.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a trivalent rabbit Pasteurella multocida outer membrane vesicle vaccine, which uses outer membrane vesicle extracts from rabbit Pasteurella multocida type A PmA HP-18, rabbit Pasteurella multocida type D PmD HP-22 and rabbit Pasteurella multocida type F PmF HP-11 as antigens. Rabbit Pasteurella multocida type A ( Pasteurella multocida PmA HP-18 was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M 2023854; Rabbit Pasteurella multocida (D) Pasteurella multocida PmD HP-22 was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M 2023855; Rabbit Pasteurella multocida F ( Pasteurella multocida PmF HP-11 was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M 2023856.

[0008] Furthermore, the volume ratio of the outer membrane vesicle extracts of rabbit Pasteurella multocida PmA HP-18, rabbit Pasteurella multocida PmD HP-22, and rabbit Pasteurella multocida PmF HP-11 was 0.5~1:0.5~1:0.5~1.

[0009] Furthermore, the volume ratio of the outer membrane vesicle extracts of rabbit Pasteurella multocida PmA HP-18, rabbit Pasteurella multocida PmD HP-22, and rabbit Pasteurella multocida PmF HP-11 was 1:1:1.

[0010] Furthermore, it also includes its pharmaceutically acceptable adjuvants.

[0011] Furthermore, the adjuvant is aluminum hydroxide gel, and its volume ratio to the mixed outer membrane vesicle extract is 1:9.

[0012] Furthermore, the preparation method of the outer membrane vesicle extract is as follows: (1) Rabbit Pasteurella multocida PmA HP-18, rabbit Pasteurella multocida PmD HP-22, and rabbit Pasteurella multocida PmF HP-11 were inoculated into modified Martin medium containing 0.4% sheep blood cells for expansion culture to obtain rabbit Pasteurella multocida suspension, rabbit Pasteurella multocida suspension, and rabbit Pasteurella multocida suspension; (2) Take the rabbit type A Pasteurella multocida bacterial suspension, rabbit type D Pasteurella multocida bacterial suspension and rabbit type F Pasteurella multocida bacterial suspension obtained in step (1) respectively and centrifuge to remove the bacterial cells; the supernatant is then purified by centrifugation to obtain the outer membrane vesicle extract.

[0013] Another objective of this invention is to provide a method for preparing the above-mentioned rabbit Pasteurella outer membrane vesicle trivalent vaccine, wherein the outer membrane vesicle extract is mixed in proportion, then mixed with an adjuvant and stored to obtain the rabbit Pasteurella outer membrane vesicle trivalent vaccine.

[0014] Another object of the present invention is to provide a rabbit type A Pasteurella multocida ( Pasteurella multocida PmA HP-18, which was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M2023854; Another object of the present invention is to provide a rabbit D-type Pasteurella multocida ( Pasteurella multocida PmD HP-22 was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M2023855; Another object of the present invention is to provide a rabbit type F Pasteurella multocida ( Pasteurella multocida PmF HP-11 was deposited at the China Center for Type Culture Collection on May 29, 2023, with accession number CCTCC NO: M2023856.

[0015] The beneficial effects of this invention are: 1. The outer membrane vesicle trivalent vaccine provided by the application can simultaneously target multiple serotypes or virulence factors of rabbit Pasteurella multocida, provide broader protection, and reduce the risk of infection by different strains. Moreover, the vaccine preparation method is simple and has strong operability.

[0016] 2. The outer membrane vesicle trivalent vaccine provided by the application does not contain live bacteria, avoids the problem of incomplete inactivation that may occur in traditional inactivated vaccines, and is safer.

[0017] 3. The outer membrane vesicle trivalent vaccine provided by the application can reduce the use of antibiotics in breeding and reduce the risk of drug resistance by preventing rabbit Pasteurella multocida disease.

[0018] 4. The adjuvant provided by the application is a pharmaceutically acceptable aluminum hydroxide gel adjuvant. The outer membrane vesicle antigen of the vaccine contains bacterial endotoxin. Endotoxin has immunogenicity, and animals are prone to stress when vaccinated. The aluminum hydroxide gel adjuvant has strong adsorption capacity and can adsorb endotoxin to achieve a slow-release effect, making the trivalent vaccine safer and more effective in preventing the occurrence of rabbit Pasteurella multocida disease. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 PCR amplification results of Pasteurella multocida specific genes kmt1 Figure 2 Multiple PCR amplification results of Pasteurella multocida specific genes kmt1 Capsular type A hyaD-hyaC , type B bcbD , type D dcbF , type E ecbJ and type F fcbD of positive colonies Figure 3 Subcutaneous injection of rabbits with type A (PmA HP-18), type D (PmD HP-22), and type F (PmF HP-11) Pasteurella multocida live bacteria resulted in lung hemorrhage, necrosis, and fibrous pneumonia in rabbits. Figure 4 is a transmission electron microscope observation of rabbit Pasteurella multocida outer membrane vesicles. Figure 5 is the particle size of rabbit Pasteurella multocida outer membrane vesicles. Figure 6 is an SDS-PAGE diagram of rabbit Pasteurella multocida outer membrane vesicles. DETAILED DESCRIPTION

[0020] ​The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0021] Example 1 Isolation, identification and screening of rabbit type A Pasteurella multocida, type D Pasteurella multocida and type F Pasteurella multocida 1. Sterilely collect the lung and liver samples of respiratory disease dead rabbits, inoculate on modified Martin agar plates containing 0.4% sheep blood cells, incubate at 37°C overnight, pick up smooth, moist, round, translucent, grayish white, neat edge dewdrop-shaped colonies, clone on 0.4% sheep blood cell modified Martin medium agar plates, and incubate at 37°C overnight for pure culture.

[0022] 2. Pick up each pure culture clone of step 1, evenly spread on glass slides, observe bacterial morphology under microscope after Gram staining, and select clones of short bacilli with negative Gram staining and bipolar staining.

[0023] 3. Pick up the pure culture of the clones screened in step 2, dissolve in sterile water, extract nucleic acid as template by boiling method, refer to the standard NY / T 567-2017 of "Rabbit Hemorrhagic Septicemia Diagnosis Technology", and synthesize specific primers for Pasteurella multocida kmt1 (457 bp), five capsular specific primers, capsular type A hyaD-hyaC (1044 bp), type B bcbD (760 bp), type D dcbF (657 bp), type E ecbJ (511 bp) and type F fcbD (851 bp), and use plasmid containing target gene as positive control.

[0024] 4. The PCR reaction system of specific primers kmt1 is 25 μL, 2 × PCR MasterMix 12.5 μL, each 1 μL of upstream and downstream primers, 8.5 μL of ultrapure water, and 2 μL of template. The PCR reaction conditions are: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1 min, 30 cycles; 72 ℃ extension for 10 min. The PCR products are detected by 1.5% agarose gel electrophoresis.

[0025] 5. The multiplex PCR reaction system consists of 50 μL, 25 μL of 2×PCR MasterMix, 0.8 μL each of forward and reverse primers, 27.5 μL of ultrapure water, and 2 μL of template. Follow step 4 for PCR reaction and product electrophoresis.

[0026] 6. All monoclonal antibodies kmt1 All genes were positive, and the target fragment size was 457 bp (e.g., Figure 1 ), there are monoclonal strains with capsular type A. hyaD-hyaC Type D dcbF Type F fcbD Positive ， The target fragment sizes were 1044 bp, 657 bp, and 851 bp, respectively. Positive amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were validated by BLAST alignment in the NCBI database. kmt1 Gene sequence, hyaD-hyaC Gene sequence, bcbF Gene sequence, fcbD The gene sequences of the cloned strains showed a homology of more than 99% with the corresponding sequences of Pasteurella multocida in GenBank. Figure 2 (This information is used for further animal reintroduction experiments.)

[0027] 7. The rabbit Pasteurella multocida type A, rabbit Pasteurella multocida D, and rabbit Pasteurella multocida F strains successfully screened and identified in step 6 were used for animal reintroduction experiments. Single colonies were picked and inoculated into Martin's liquid medium, and cultured at 37℃ and 180 r / min for 8 hours. The viable bacterial count was adjusted to 100 CFU / mL with sterile physiological saline for rabbit challenge. Thirty-five rabbits were selected and divided into three groups of 10 rabbits each. The experimental group received a subcutaneous injection of 1 mL of bacterial solution per rabbit in the neck and back, while the control group received a subcutaneous injection of 1 mL of physiological saline per rabbit. The rabbits were observed for 10 days. Clinical symptoms were observed daily, including mental state, appetite, cough, and nasal discharge. Rabbits that died during the experimental period were necropsies performed to observe lesions in various organs. Lung and liver samples were collected and subjected to bacterial isolation and identification again.

[0028] The results showed that all three strains of Pasteurella multocida isolated in this study (PmA HP-18, PmD HP-22, and PmF HP-11) caused disease and death in all experimental rabbits after artificial infection. Infected rabbits exhibited lethargy, reduced food intake, coughing, and serous or purulent nasal discharge. Necropsy revealed typical fibrinous pneumonia lesions. Figure 3), and the corresponding challenge strain was successfully recovered from the lung samples of the dead rabbits. The above results confirmed that PmA HP-18, PmD HP-22, and PmF HP-11 were pathogenic strains of rabbit Pasteurella multocida and could be used as challenge strains after immunization.

[0029] Among them, rabbit A type Pasteurella multocida (PmA) HP-18, rabbit D type Pasteurella multocida (PmD) HP-22, and rabbit F type Pasteurella multocida (PmF) HP-11 were isolated from the lung samples of the dead rabbits. Pasteurella multocida PmA HP-18 was deposited at the China Center for Type Culture Collection on May 29, 2023, with the accession number CCTCC NO: M 2023854.

[0030] Among them, rabbit A type Pasteurella multocida (PmA) HP-18, rabbit D type Pasteurella multocida (PmD) HP-22, and rabbit F type Pasteurella multocida (PmF) HP-11 were isolated from the lung samples of the dead rabbits. Pasteurella multocida PmD HP-22 was deposited at the China Center for Type Culture Collection on May 29, 2023, with the accession number CCTCC NO: M 2023855.

[0031] Among them, rabbit A type Pasteurella multocida (PmA) HP-18, rabbit D type Pasteurella multocida (PmD) HP-22, and rabbit F type Pasteurella multocida (PmF) HP-11 were isolated from the lung samples of the dead rabbits. Pasteurella multocida PmF HP-11 was deposited at the China Center for Type Culture Collection on May 29, 2023, with the accession number CCTCC NO: M 2023856.

[0032] Example 2 Virulence study of rabbit Pasteurella A, D, and F types The pure cultures of rabbit A type Pasteurella multocida PmA HP-18, rabbit D type Pasteurella multocida PmD HP-22, and rabbit F type Pasteurella multocida PmF HP-11 were inoculated into modified Martin broth containing 0.4% sheep blood cells and incubated at 37°C for 12 hours. After viable cell counting, the bacterial solution was adjusted to contain 5 CFU / mL, 10 CFU / mL, 15 CFU / mL, 20 CFU / mL, and 25 CFU / mL of viable cells. Eight to nine-week-old healthy susceptible rabbits were injected subcutaneously on the nape with each dose, 10 rabbits per group. The rabbits were observed for 7 days, and the morbidity and mortality of each test rabbit were recorded. The results are shown in Table 1. After challenge, the test rabbits showed varying degrees of morbidity and mortality, and the same rabbit Pasteurella multocida strains as the inoculated strains were isolated from the lungs, livers, and spleens of the dead rabbits. The virulence test results showed that the minimum lethal dose (MLD) of A type, D type, and F type strains on the test rabbits was 10 CFU, 20 CFU, and 25 CFU, respectively. Among them, the A type rabbit Pasteurella multocida was the most virulent.

[0033] Table 1 Virulence test results of different capsular type Pasteurella multocida on rabbits

[0034] Example 3 Outer membrane vesicle extraction and biological property identification of rabbit Pasteurella 1. Outer membrane vesicle extraction (1) Bacterial culture 1) The freeze-dried strains of rabbit Pasteurella multocida PmA HP-18, rabbit Pasteurella multocida PmD HP-22 and rabbit Pasteurella multocida PmF HP-11 were inoculated onto modified Martin plates containing 0.4% sheep blood cells and streaked, and incubated at 37°C for 18-24 hours.

[0035] 2) Pick 4-5 single colonies and inoculate them into 20 mL of modified Martin broth containing 0.4% sheep blood cells. Incubate at 37°C and 180 rpm for 18-24 h to obtain seed culture.

[0036] 3) The seed culture was inoculated at a ratio of 1:100 into 2 L of modified Martin broth medium containing 0.4% sheep blood cells and cultured overnight at 37°C until the OD value reached 1.

[0037] (2) Bacterial cell isolation The bacterial cells were removed by centrifugation at 10,000×g for 10 min at 4℃.

[0038] (3) Supernatant filtration The collected supernatant was filtered through a 0.45 μm filter to remove residual bacterial cells. Then, the outer membrane vesicles in the supernatant were collected by ultracentrifugation (40,000 × g, 4 °C, 2 h). The collected outer membrane vesicles were resuspended in DPBS and filtered again through a 0.45 μm filter.

[0039] (4) Purification The outer membrane vesicles were further purified by density gradient centrifugation. Using 10 mM HEPES buffer containing 0.85% NaCl as a diluent, eight layers were prepared from top to bottom with density gradient centrifugation buffer at concentrations ranging from 20% to 45%. The outer membrane vesicles collected in the previous step were added to the top layer of the density gradient centrifugation apparatus, and the mixture was centrifuged overnight at 200,000 × g and 4 °C. The large density gradient layers containing the outer membrane vesicles were collected by ultracentrifugation (40,000 × g, 4 °C, 1 h), washed once with DPBS to remove impurities, and stored at -80 °C for later use.

[0040] 2. Morphological characteristics The morphology, size, and structure of the outer membrane vesicles were observed using transmission electron microscopy. The results showed that the outer membrane vesicles of rabbit type A (PmA HP-18), type D (PmD HP-22), and type F (PmF HP-11) Pasteurella multocida all had spherical vesicle-like structures. Figure 4 ).

[0041] 3. Particle size distribution The particle size distribution of the vesicles was determined by dynamic light scattering (DLS). The particle size results showed that the average particle sizes of the outer membrane vesicles of Pasteurella multocida rabbit type A (PmA HP-18), type D (PmD HP-22), and type F (PmF HP-11) were 101.3 nm, 105.0 nm, and 104.4 nm, respectively. Figure 5 ).

[0042] 4. Protein composition analysis SDS-PAGE analysis was used to identify the protein components in the vesicles. The protein bands were mainly concentrated in the range of 40-70 kDa. Figure 6 . Example 4. Preparation of a trivalent vaccine of outer membrane vesicles of Pasteurella multocida and safety and efficacy evaluation 1. Vaccine preparation The outer membrane vesicle antigens of Pasteurella multocida rabbit type A, type D, and type F harvested in Example 3 were quantified using a protein quantification kit, mixed uniformly at a volume ratio of 1:1:1, and then mixed with aluminum hydroxide adjuvant at a volume ratio of 9:1 to obtain the mixed antigen solution, which was the outer membrane vesicle trivalent vaccine. The vaccine was sealed and stored at 4°C after being divided into aliquots.

[0043] 2. Safety test Twenty 4-5-week-old healthy susceptible rabbits were randomly divided into two groups, with 10 rabbits in the immunization group and 10 rabbits in the control group. The rabbits in the immunization group were injected with the outer membrane vesicle trivalent vaccine of Pasteurella multocida rabbit disease in the leg muscles at a dose of 1 mL per rabbit. The rabbits in the control group were not inoculated. The rabbits were observed for 10 days after inoculation, and the clinical manifestations such as spirit, appetite, and fecal state were observed. All the test rabbits were sacrificed at the end of the test, and the vaccine absorption at the injection site and the pathological changes of the organs of the test rabbits were observed. The test results are shown in Table 2.

[0044] Table 2. Safety results of the vaccine inoculated into 4-5-week-old healthy susceptible rabbits

[0045] The results in Table 2 show that after the rabbits were immunized with the vaccine prepared in the present application, the test rabbits had good mental state, normal appetite, and normal feces, and no adverse reactions were observed. The injection sites of all the test rabbits were well absorbed, and no abnormal lesions were observed in the organs after dissection, which proves that the vaccine has good safety.

[0046] 3. Efficacy test Take 4~5 weeks old healthy susceptible rabbits 60, randomly divided into groups, 30 in the test group, divided into 3 groups, 30 in each group; 30 in the control group, divided into 3 groups, 10 in each group. The immunization group was injected with rabbit pasteurellosis outer membrane vesicle trivalent vaccine into the leg muscle, 0.5 mL / one, and the control group was not inoculated. On the 21st day after immunization, 10 immunized rabbits were taken from each group, together with 10 control rabbits, and 1 MLD of rabbit A, D and F type pasteurella multocida was injected subcutaneously on the neck and back, respectively. Observe for 10 days, record the death of rabbits in the immunization group and the control group, and perform necropsy on the dead rabbits. The test results are shown in Table 3.

[0047] Table 3: Protection results of 3 batches of vaccines against rabbit pasteurella multocida challenge

[0048] The results of Table 3 show that the vaccine prepared by the present application can provide good immune protection after immunizing rabbits, among which 8 / 10 protection can be provided against rabbit pasteurella multocida type A, 9 / 10 protection can be provided against rabbit pasteurella multocida type D, and 9 / 10 protection can be provided against rabbit pasteurella multocida type F.

[0049] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the examples, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A trivalent vaccine of outer membrane vesicles of Pasteurella multocida of rabbits, characterized by, The outer membrane vesicle extract from rabbit A Pasteurella multocida PmA HP-18, rabbit D Pasteurella multocida PmD HP-22 and rabbit F Pasteurella multocida PmF HP-11 is used as an antigen; The rabbit type A multi-drug resistant Pasteurella multocida (PmA) Pasteurella multocida ) PmA HP-18 was preserved in China Center for Type Culture Collection on May 29, 2023, and the preservation number is CCTCC NO: M 2023854; The rabbit D type Pasteurella multocida (PmD HP-22) Pasteurella multocida PmD HP-22 was preserved in China Center for Type Culture Collection on May 29, 2023, and the preservation number is CCTCC NO: M 2023855; The rabbit F-type Pasteurella multocida (PmF) Pasteurella multocida PmF HP-11 was deposited at China Center for Type Culture Collection on May 29, 2023, and the deposit number is CCTCC NO: M 2023856.

2. The rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine according to claim 1, characterized in that, The volume ratio of the outer membrane vesicle extract of rabbit A Pasteurella multocida PmA HP-18, the outer membrane vesicle extract of rabbit D Pasteurella multocida PmD HP-22 and the outer membrane vesicle extract of rabbit F Pasteurella multocida PmF HP-11 is 0.5-1:0.5-1:0.5-1.

3. The rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine according to claim 2, characterized in that, The volume ratio of the outer membrane vesicle extract of rabbit A Pasteurella multocida PmA HP-18, the outer membrane vesicle extract of rabbit D Pasteurella multocida PmD HP-22 and the outer membrane vesicle extract of rabbit F Pasteurella multocida PmF HP-11 is 1:1:

1.

4. The trivalent vaccine of outer-membrane vesicles of Pasteurella multocida according to any one of claims 1 to 3, characterized in that, Also included is a pharmaceutically acceptable adjuvant thereof.

5. The rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine according to claim 4, characterized in that, The adjuvant is aluminum hydroxide gel, and the volume ratio of the amount used to the mixed outer membrane vesicle extract is 1:

9.

6. The trivalent vaccine of outer-membrane vesicles of Pasteurella multocida according to any one of claims 1 to 3, characterized in that, The preparation method of the outer membrane vesicle extract is: (1) Rabbit A Pasteurella multocida PmA HP-18, rabbit D Pasteurella multocida PmD HP-22 and rabbit F Pasteurella multocida PmF HP-11 are inoculated into modified Martin medium containing 0.4% sheep blood cells for expansion culture, to obtain rabbit A Pasteurella multocida bacterial liquid, rabbit D Pasteurella multocida bacterial liquid and rabbit F Pasteurella multocida bacterial liquid; (2) The rabbit A Pasteurella multocida bacterial liquid, the rabbit D Pasteurella multocida bacterial liquid and the rabbit F Pasteurella multocida bacterial liquid obtained in step (1) are centrifuged to remove the bacterial bodies, respectively; The supernatant is then purified by centrifugation to obtain the outer membrane vesicle extract.

7. A method of preparing a trivalent vaccine of outer membrane vesicles of B. rabbit pathogens according to any one of claims 1 to 6, characterized in that, The outer membrane vesicle extracts are mixed in proportion, and then mixed with the adjuvant for storage to prepare the rabbit Pasteurella multocida outer membrane vesicle trivalent vaccine.

8. A rabbit type A Pasteurella multocida (PmA) HP-18 characterized by, Pasteurella multocida ) PmA HP-18, characterized by, It was preserved in the China Center for Type Culture Collection on May 29, 2023, and the preservation number is CCTCC NO: M 2023854.

9. A rabbit type D Pasteurella multocida (PmD) HP-22 characterized by, Pasteurella multocida It was preserved in the China Center for Type Culture Collection on May 29, 2023, and the preservation number is CCTCC NO: M 2023855. ​ 10. A rabbit F-type Pasteurella multocida (PmF) HP-11, characterized in that, Pasteurella multocida PmF HP-11 is a bacterin comprising a whole cell of PmF HP-11, and It was preserved in the China Center for Type Culture Collection on May 29, 2023, and the preservation number is CCTCC NO: M 2023856.

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