Bovine pasteurella multocida (group A and group B) and mannheimia haemolytica bivalent trivalent inactivated vaccine and preparation method thereof

Abstract

The invention relates to the technical field of veterinary drug preparation, in particular to a bovine pasteurella multocida (group A and group B) and mannheimia haemolytica bivalent trivalent inactivated vaccine and a preparation method thereof. The inactivated vaccine comprises an adjuvant and antigens, wherein the antigens are a bovine pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine haemolytic mannheimia A1 type BMh / NM2018 strain. According to the bovine pasteurellosis bivalent trivalent inactivated vaccine, multiple technical breakthroughs are realized, the bovine pasteurellosis bivalent trivalent inactivated vaccine which is good in safety, high in immune efficacy and controllable in quality is finally successfully developed, irrelevant impurities and endotoxin are removed by adopting a novel concentration process, and the bovine pasteurellosis bivalent trivalent inactivated vaccine has the characteristics of prevention of three diseases by one injection, low immune dose, high safety and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of veterinary medicine preparation, and more particularly to a bovine Pasteurella multocida (group A and group B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine and a preparation method thereof. BACKGROUND

[0002] Bovine Pasteurella multocida disease and Mannheimia haemolytica disease are caused by Pasteurella multocida (Pm) and Mannheimia haemolytica (Mh) respectively, and Pm and Mh belong to the Pasteurellaceae family and are conditional pathogenic bacteria residing in the upper respiratory tract (especially the tonsillar crypt) of healthy livestock. In the presence of stress factors such as long-distance transportation, early weaning or poor ventilation, the resistance of cattle decreases significantly, thereby inducing bovine fibrinous pneumonia, hemorrhagic septicemia (HS) and injury-related major diseases. Pm and Mh cause infection to other cattle through secretions, excretions, droplets and the like, which brings great losses to the cattle industry. Pm and Mh are both gram-negative coccobacilli, 0.2-2.0 μm, and show bipolar staining with Wright's staining. Pm is divided into five types (A, B, D, E and F) according to the capsular serotype (16 types according to the bacterial serotype), and Mh is divided into 12 serotypes (Al, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16 and A17).

[0003] The bovine Pasteurella multocida vaccine in the prior art is only directed against capsular type B bacteria, and cannot provide protection against capsular type A bacteria, which brings great difficulties to the prevention and control of bovine Pasteurella multocida disease. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art, and for this purpose, an aspect of the present application aims to provide a bovine Pasteurella multocida (group A, group B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine, the inactivated vaccine comprising an adjuvant and an antigen, wherein the antigen is a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica A1 type BMh / NM2018 strain; the antigen is a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica A1 type BMh / NM2018 strain, which were deposited on November 27, 2018 at the China General Microbiological Culture Collection Center, and the classification name is bovine Mannheimia haemolytica, and the accession number is CGMCC No.16895.

[0005] Preferably, the bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, the bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and the bovine Mannheimia haemolytica A1 type BMh / NM2018 strain are all in the range of 1×10 9 ~1×10 10 CFU / ml; the volume ratio of the adjuvant and the antigen is 1:1~1:6.

[0006] Preferably, the adjuvant is an aluminum hydroxide adjuvant.

[0007] Preferably, the effective content of the antigen is not less than 1×10 9 ~1×10 10 CFU / ml.

[0008] Another aspect of the present application aims to provide a preparation method of a bovine Pasteurella multocida (group A, group B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine, and the specific steps of the preparation method are as follows:

[0009] S1: respectively ferment and cultivate a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica BMh / NM2018 strain to obtain three kinds of fermentation bacterial liquids;

[0010] S2: concentrating and purifying the three kinds of fermentation bacterial liquids of S1, inactivating to obtain three kinds of inactivated antigen bacterial liquids;

[0011] S3: mixing the three kinds of inactivated antigen bacterial liquids of S2 with an aluminum hydroxide adjuvant and uniformly mixing to prepare an inactivated vaccine.

[0012] Preferably, the fermentation culture in S1 is that the Pasteurella multocida capsular group A BPmA / XJ2016 strain is inoculated into Martin's broth medium at an inoculum of 1% to 2% (v / v), the Pasteurella multocida capsular group B BPmB / NM2016 strain is inoculated into Martin's broth medium at an inoculum of 1% to 2% (v / v), and the Mannheimia haemolytica A1 type BMh / NM2018 strain is inoculated into BHI medium with sheep lysed blood at an inoculum of 1% to 2% (v / v), the culture temperature is 37±1℃, the rotation speed is 100-150 r / min, the culture time is 10-16 h, and the fermentation broth is obtained.

[0013] Preferably, the fermentation culture in S1 is divided into primary seed propagation and secondary seed propagation; the freeze-dried spores of the Pasteurella multocida capsular group A BPmA / XJ2016 strain or the Pasteurella multocida capsular group B BPmB / NM2016 strain are inoculated into Martin's broth medium, and the freeze-dried spores of the Mannheimia haemolytica A1 type BMh / NM2018 strain are inoculated into BHI medium with sheep lysed blood, the culture temperature is 37±1℃, the rotation speed is 100-150 r / min, the culture time is 10-16 h, then streak inoculation is performed on Martin's plate, the culture temperature is 37±1℃, the culture time is 10-16 h, typical colonies are selected under a microscope, and Martin's slant is inoculated with several branches, the culture temperature is 37±1℃, and the culture time is 10-16 h, serving as the primary seed; the secondary seed propagation is that the primary seed of the Pasteurella multocida capsular group A BPmA / XJ2016 strain or the Pasteurella multocida capsular group B BPmB / NM2016 strain is inoculated into Martin's broth medium, and the primary seed of the Mannheimia haemolytica A1 type BMh / NM2018 strain is inoculated into BHI medium with sheep lysed blood, the culture temperature is 37±1℃, the rotation speed is 100-150 r / min, and the culture time is 10-16 h, serving as the secondary seed.

[0014] Preferably, the fermentation broth in S1 is that the secondary seeds of the Pasteurella multocida capsular group A BPmA / XJ2016 strain, the Pasteurella multocida capsular group B BPmB / NM2016 strain, and the Mannheimia haemolytica A1 type BMh / NM2018 strain are inoculated into the culture medium at an inoculum of 1% to 2% (v / v), the culture temperature is 37±1℃, the rotation speed is 100-150 r / min, and the culture time is 10-16 h.

[0015] Preferably, the fermentation broth in S2 is concentrated and purified by centrifugation at a rotation speed of 12500-14000 r / min and a temperature of 4±1℃ for 15-25 min, then the bacterial precipitate is collected and resuspended with sterile PBS at pH 7.2-7.4 to prepare concentrated bacterial liquid.

[0016] Preferably, the inactivation in S2 is adding formaldehyde solution at 0.1%-0.15% (v / v) of the concentrated bacteria solution, inactivating at 37±1℃ for 8-10 hours to prepare the inactivated antigen bacteria solution.

[0017] The present application has the following beneficial effects:

[0018] The present application adopts the bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and bovine Mannheimia haemolytica A1 type BMh / NM2018 strain with good immunogenicity, respectively inoculates suitable culture medium for culture, and the fermentation bacteria collection time is early, so that the phenomenon of a large number of dead bacteria due to long fermentation time and reaching the growth cycle is avoided, and the number of live bacteria is large, the culture product is inactivated by formaldehyde solution, the safety of the combined vaccine is high, and the antigen is prepared after concentration and purification, and the three kinds of antigens are mixed according to a certain proportion and then added into aluminum hydroxide adjuvant to be mixed to prepare the vaccine. The vaccine is used for preventing bovine Pasteurella multocida disease caused by bovine Pasteurella multocida capsular group A and B and bovine Mannheimia haemolytica disease caused by Mannheimia haemolytica A1 type.

[0019] The present application realizes a plurality of technical breakthroughs, and finally successfully develops the bovine Pasteurella multocida disease double combination three-valent inactivated vaccine with good safety, high immunization efficacy and controllable quality. The present application adopts a new type of concentration process to remove irrelevant impurities and endotoxins, and has the characteristics of preventing two diseases with one needle, long immunization protection period and high safety.

[0020] The bovine Pasteurella multocida disease double combination three-valent inactivated vaccine prepared by the present application is not only the updated version of bovine hemorrhagic septicemia vaccine, but also fills the blank of prevention and control of bovine Pasteurella multocida capsular type A disease in China, provides an effective tool for the comprehensive prevention and control of bovine Pasteurella multocida disease in China, and provides strong support for the improvement of the bovine disease prevention and control system in China.

[0021] Additional aspects and advantages of the present application will become apparent from the following description, or will be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the experiment, combined with the following drawings, in which:

[0023] Figure 1 is the average body temperature change curve of 2-6 month-old cattle in experiment three after single-dose vaccination of the vaccine;

[0024] Figure 2 is the average body temperature change curve of 2-6 month-old cattle in experiment three after single-dose repeated vaccination of the vaccine;

[0025] Figure 3 is the average body temperature change curve of 2-6 month-old cattle in experiment three after super-dose vaccination of the vaccine;

[0026] Figure 4 is the average body temperature change curve of 8-18 month-old cattle after single dose vaccination of the present application experiment three;

[0027] Figure 5 is the average body temperature change curve of 8-18 month-old cattle after single dose vaccination of the present application experiment three;

[0028] Figure 6 is the average body temperature change curve of 8-18 month-old cattle after single dose vaccination of the present application experiment three. DETAILED DESCRIPTION

[0029] In order to enable the above-mentioned objects, features and advantages of the present application to be more clearly understood, the following further describes the present application with reference to the accompanying drawings and specific experiments. It should be noted that the experiments and features in the experiments of the present application can be combined with each other without conflict.

[0030] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can also be implemented in other ways different from the description, therefore, the scope of protection of the present application is not limited by the specific experiments disclosed below.

[0031] Experiment 1 Strain source

[0032] From 2015 to 2016, suspected bovine Pasteurella multocida infection samples collected from Xinjiang, Inner Mongolia, Shandong, Henan, Hebei, Shanxi, Ningxia, Jiangsu and Hubei cattle farms were inoculated on Martin's broth and blood plate after processing, and the pathogenic bacteria were cloned and purified on Martin's broth and blood plate for 2 generations. Through strain morphology, microscopic examination, Pasteurella multocida multiple capsule PCR typing system identification and mouse virulence test, a strain of bovine Pasteurella multocida was preliminarily screened out. Further culture characteristics, 16S sequencing, serological characteristics (slide agglutination test, Pasteurella multocida multiple capsule PCR typing system identification), biochemical identification, animal pathogenicity test and immunogenicity test of the strain were carried out, and finally it was confirmed as bovine Pasteurella multocida capsular group A, named BPmA / XJ2016 strain.

[0033] A strain of bovine Pasteurella multocida was preliminarily screened out through the strain morphology, microscopic examination, multiple capsular PCR typing system identification of Pasteurella multocida, and rabbit virulence test after the tissue samples of a dead cow due to septicemia in a cattle farm in Inner Mongolia in 2016 were treated and inoculated on Martin's meat broth and blood plate. The strain was further confirmed as bovine Pasteurella multocida capsular group B, named BPmB / NM2016 strain, through further culture characteristics, 16S sequencing, serological characteristics (slide agglutination test, multiple capsular PCR typing system identification of Pasteurella multocida), biochemical identification, animal pathogenicity test, and immunogenicity test.

[0034] A strain of bovine Mannheimia haemolytica was preliminarily screened out through the strain morphology, staining microscopic examination, PCR identification, and guinea pig virulence test after the suspected bovine Mannheimia haemolytica infection samples collected from Inner Mongolia, Shanxi, Hebei, Shandong, and Henan were treated and inoculated on brain-heart infusion agar and blood plate. The strain was further confirmed as bovine Mannheimia haemolytica A1 type, named BMh / NM2018 strain, through further culture characteristics, PCR identification, 16S sequencing, PCR serotype identification, biochemical identification, animal pathogenicity test, and immunogenicity test.

[0035] Experiment 2: Preparation of vaccine

[0036] The present experiment aims to conduct a fermenter preparation of bovine Pasteurella multocida (group A and group B) and Mannheimia haemolytica bivalent inactivated vaccine process test, further verify the optimized process parameters, and at the same time prepare test samples for product safety, potency, shelf life, and immune duration tests, so as to comprehensively evaluate the quality and performance of bovine Pasteurella multocida (group A and group B) and Mannheimia haemolytica bivalent inactivated vaccine.

[0037] 1. Materials

[0038] 1.1 Main equipment: 100L fermenter (Shanghai Ritek Medical Equipment Co., Ltd.), centrifuge (Hunan Xiangyi).

[0039] 1.2 Production strains: bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain F5, bovine Pasteurella multocida capsular group B BPmB / NM2016 strain F5, and bovine Mannheimia haemolytica BMh / NM2018 strain F5, all identified, stored, and supplied by the project team.

[0040] 1.3 Test strains: BPmA / XJ2016 strain JY2018001, BPmB / NM2016 strain JY2018001, BMh / NM2018 strain JY2019001, all identified, preserved and supplied by the project team.

[0041] 1.4 Culture medium: Brain-Heart Infusion Liquid Medium (BHI), purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd.; Martin's broth medium, thioethanolate fluid medium (TG), tyrosine agar medium (GA), trypticase soy broth agar medium (TSA), trypticase soy broth liquid medium (TSB), all purchased from Zhonghai Biological Technology Co., Ltd.

[0042] 1.5 Concentration system: VERSAflux filtration system (GE Company), 0.1 um hollow fiber column (GE Company).

[0043] 1.6 Other reagents: aluminum hydroxide gel, purchased from Inner Mongolia Mengheli Biological Co., Ltd.

[0044] 1.7 Test animals: 2-3 month-old healthy susceptible cattle, purchased from Inner Mongolia designated cattle farm; 18-22 g body weight mice, 1.5-2 kg body weight healthy susceptible rabbits, 300-350 g body weight guinea pigs, purchased from the designated test animal base.

[0045] 2 Method

[0046] 2.1 Seed preparation for production

[0047] 2.1.1 Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain

[0048] 2.1.1.1 Primary seed propagation and identification: The bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain F5 freeze-dried seed was inoculated into 5 ml of Martin's broth medium containing 0.1% sheep lysed blood and incubated at 36-37°C with 120 r / min shaking for 12-16 hours. It was then streaked onto Martin's broth agar plates containing 0.1% sheep lysed blood and 4% newborn calf serum and incubated at 36-37°C for 12-16 hours. Three to five typical colonies were selected under low-power microscopy at a 45-degree refraction and inoculated into several Martin's broth agar slants containing 10% sheep lysed blood, which were incubated at 36-37°C for 12-16 hours as the primary seed.

[0049] 2.1.1.2 Secondary seed propagation and identification: One primary seed was inoculated into 200 ml of Martin's broth medium containing 0.1% sheep lysed blood and incubated at 36-37°C with 120 r / min shaking for 12-16 hours. A sample was taken for purity test.

[0050] 2.1.2 Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain

[0051] 2.1.2.1 Primary seed propagation and identification: the bovine Pasteurella multocida capsular group B BPmB / NM2016 strain freeze-dried seed F5 was inoculated into 5 ml of Martin's broth medium containing 0.1% sheep lysed blood, and was cultured at 36-37°C with 120 r / min shaking for 18-22 hours. The culture was streaked onto Martin's broth agar plates containing 0.1% sheep lysed blood and 4% newborn calf serum, and was cultured at 36-37°C for 18-22 hours. Three to five typical colonies were selected under low-power microscopy at 45 degrees of refraction, and were streaked onto several Martin's broth agar slants containing 10% sheep defibrinated blood, and were cultured at 36-37°C for 18-22 hours as the primary seed.

[0052] 2.1.2.2 Secondary seed propagation: one primary seed was inoculated into 200 ml of Martin's broth medium containing 0.1% sheep lysed blood, and was cultured at 36-37°C with 120 r / min shaking for 18-22 hours. The sample was taken for purity test.

[0053] 2.1.3 Bovine Mannheimia haemolytica BMh / NM2018 strain

[0054] 2.1.3.1 Primary seed propagation and identification: the bovine Mannheimia haemolytica BMh / NM2018 strain freeze-dried seed F5 was inoculated into 10 ml of BHI medium containing 0.1% sheep lysed blood, and was cultured at 36-37°C with 120 r / min shaking for 10-16 hours. The culture was streaked onto BHI agar plates containing 0.1% sheep lysed blood and 4% newborn calf serum, and was cultured at 36-37°C for 10-16 hours. Three to five typical colonies were selected under low-power microscopy at 45 degrees of refraction, and were streaked onto several BHI agar slants containing 10% sheep defibrinated blood, and were cultured at 36-37°C for 10-16 hours as the primary seed.

[0055] 2.1.3.2 Secondary seed propagation: one primary seed was inoculated into 200 ml of BHI medium containing 0.1% sheep lysed blood, and was cultured at 36-37°C with 120 r / min shaking for 6-10 hours. The sample was taken for purity test.

[0056] 2.2 Culture medium for vaccine production

[0057] 2.2.1 Martin's broth medium was prepared according to the instruction for the bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain and group B BPmB / NM2016 strain, and after sterilization, 0.1% (v / v) of sheep lysed blood was added to the culture medium.

[0058] 2.2.2 Mannheimia haemolytica BMh / NM2018 strain was cultured in brain-heart infusion broth (BHI) prepared according to the formula in the specification, sterilized, and then 0.1% (v / v) of the volume of the medium was added with sheep lysed whole blood.

[0059] 2.3 Preparation of the vaccine bacterial solution

[0060] 2.3.1 Mannheimia haemolytica capsular group A BPmA / XJ2016 strain

[0061] 2.3.1.1 Bacterial solution culture: inoculate the secondary seed according to 1%-2% of the volume of the culture medium, and add an appropriate amount of antifoam agent, and set the stirring speed and dissolved oxygen in association, set the dissolved oxygen (DO) to 15%, and the pH value to 7.20±0.20, and culture at 37.0±0.2°C for 6-8 hours, harvest the bacterial solution, and take samples for purity test and viable bacterial count.

[0062] 2.3.1.2 Purity test: according to the current Chinese Veterinary Pharmacopoeia, inoculate 2 tubes of TG, 2 tubes of GA, and 2 tubes of Martin's broth agar slant, each with 0.2 ml, 1 tube is cultured at 35-37°C, and 1 tube is cultured at 23-25°C, and observe for 3-5 days, and it should be pure.

[0063] 2.3.1.3 Viable bacterial count: according to the current Chinese Veterinary Pharmacopoeia, use Martin's broth agar plates containing 0.1% lysed blood and 4% newborn calf serum for viable bacterial count, and the viable bacterial count should be ≥1×10 10 CFU / ml.

[0064] 2.3.1.4 Inactivation: add 0.1%-0.15% of the volume of the cultured bacterial solution with formaldehyde solution, and inactivate at 37°C for 10 hours. Take samples, inoculate 2 tubes of TG, 2 tubes of TSA, and 2 tubes of Martin's broth medium, each with 0.2 ml, and culture at 35-37°C for 7 days, and it should be sterile growth.

[0065] 2.3.1.5 Concentration and purification: the inactivated bacterial solution is concentrated 10 times by 0.1 μm hollow fiber column, the concentrated bacterial solution is centrifuged at 10000 r / min for 30 min, the bacterial slurry precipitate is resuspended with an equal volume of sterile PBS solution, and the process is repeated 2 times, after the last centrifugation, according to the results of the viable bacterial count of the bacterial solution, the bacterial slurry precipitate is resuspended with an appropriate amount of sterile PBS solution, and the hollow fiber concentrated bacterial solution is concentrated 1-3 times again, and the concentration and purification recovery rate is calculated as 60%. The number of inactivated bacteria (CFU / ml) in the concentrated antigen = (the number of bacteria before inactivation × the volume of the bacterial solution before concentration × 60%) / the volume after concentration and purification.

[0066] 2.3.2 Mannheimia haemolytica capsular group B BPmB / NM2016 strain

[0067] 2.3.2.1 Bacterial culture: Inoculate secondary seed at 1% to 2% of the culture medium volume, add an appropriate amount of defoamer, and set the stirring speed and dissolved oxygen to 15%, pH 7.20±0.20, and culture at 37.0±0.2℃ for 6 to 8 hours. Harvest the bacterial culture and take samples for purity testing and viable cell count.

[0068] 2.3.2.2 Purity test: According to the current Chinese Veterinary Pharmacopoeia, inoculate 0.2 ml each of TG tubes, GA slant and Martin's broth agar slant. Incubate one tube at 35-37℃ and the other at 23-25℃. Observe for 3-5 days. It should be pure.

[0069] 2.3.2.3 Viable Bacterial Count: Viable bacterial counts were performed using Martin's broth agar plates containing 0.1% lysed blood and 4% newborn calf serum, according to the current Chinese Veterinary Pharmacopoeia. The viable bacterial count should be ≥1×10⁻⁶. 10 CFU / ml.

[0070] 2.3.2.4 Inactivation: Add formaldehyde solution at 0.1%–0.15% of the bacterial culture volume and inactivate at 37°C for 10 hours. Take samples and inoculate 0.2 ml each into two vials of TG, TSA, and Martin's broth medium. Incubate at 35–37°C for 7 days. No bacterial growth should be observed.

[0071] 2.3.2.5 Concentration and Purification: The inactivated bacterial solution was concentrated 10-fold using a 0.1 μm hollow fiber column. The concentrated bacterial solution was collected and centrifuged at 6000 r / min for 30 min. The bacterial sludge precipitate was collected and resuspended with an equal volume of sterile PBS. This process was repeated twice. After the last centrifugation, based on the viable cell count, the bacterial sludge precipitate was resuspended with an appropriate amount of sterile PBS, and the bacterial solution concentrated by the hollow fiber column was concentrated again by 1-3 times. The concentration and purification recovery rate was calculated as 85%. The number of inactivated bacteria (CFU / ml) in the concentrated antigen = (number of bacteria before inactivation × volume of bacterial solution before concentration × 85%) / volume after concentration and purification.

[0072] 2.3.3 Bovine hemolytic Mansula BMh / NM2018 strain

[0073] 2.3.3.1 Bacterial culture: Inoculate secondary seed at 1% to 2% of the culture medium volume, add an appropriate amount of defoamer, and set the stirring speed and dissolved oxygen to 15% and pH 7.20±0.20. Incubate at 37.0±0.2℃ for 6 to 10 hours, harvest the bacterial culture, and take samples for purity testing and viable cell count.

[0074] 2.3.3.2 Purity test: According to the current Chinese Veterinary Pharmacopoeia, inoculate 0.2 ml each of TG tubes, GA slant and Martin's broth agar slant. Incubate one tube at 35-37℃ and the other at 23-25℃. Observe for 3-5 days. It should be pure.

[0075] 2.3.3.3 Viable count: According to the current "Chinese Veterinary Pharmacopoeia", use BHI agar plates containing 0.1% sheep lysed blood and 4% newborn calf serum to count viable bacteria. The viable count should be ≥2x10 9 CFU / ml.

[0076] 2.3.3.4 Inactivation: Add 0.1%-0.15% formaldehyde solution based on the volume of bacterial solution, and inactivate at 37°C for 8 hours. Take samples and inoculate 2 branches of TG, TSA and BHI medium, each 0.2 ml, and cultivate at 35-37°C for 7 days. There should be no sterile growth.

[0077] 2.3.3.5 Concentration and purification: Inactivate the bacterial solution with 0.1 μm hollow fiber column to concentrate 10 times. Collect the concentrated bacterial solution, centrifuge at 6000 r / min for 30 min, collect the bacterial slurry precipitate, resuspend with an equal volume of sterile PBS solution, repeat 2 times, and after the last centrifugation, according to the viable count of the bacterial solution, resuspend the bacterial slurry precipitate with an appropriate amount of sterile PBS solution, and concentrate the hollow fiber concentrated bacterial solution again by 1-3 times. The concentration and purification recovery rate is calculated as 85%. The number of inactivated bacteria (CFU / ml) in the concentrated antigen = (number of bacteria before inactivation x volume of bacterial solution before concentration x 85%) / volume after concentration and purification.

[0078] 2.4 Semi-finished product inspection: According to the current "Chinese Veterinary Pharmacopoeia", perform sterile test and meet the requirements.

[0079] 2.5 Vaccine preparation: According to the total amount of the vaccine to be prepared and the number of inactivated bacteria contained in each concentrated and purified antigen solution, the number of inactivated B. bovis capsular group A BPmA / XJ2016 strain, B. bovis capsular group B BPmB / NM2016 strain and M. haemolytica BMh / NM2018 strain in each dose (2 ml) of vaccine should be 3.0x10 10 CFU, 1.5x10 10 CFU, 3.0x10 10 CFU, respectively. Calculate the required amount (volume) of each type of concentrated and purified antigen solution, mix the required 3 types of concentrated and purified antigen solutions, and supplement the volume of the mixed antigen solution with sterile PBS or physiological saline to 80% of the total amount of the vaccine to be prepared, adjust the pH to 6.8-7.0, and then prepare according to the antigen (v) : aluminum hydroxide gel (w) = 4:1.

[0080] 2.6 Sub-packaging: After stirring evenly, sub-packaging, sealing, and labeling.

[0081] 3 Finished product inspection

[0082] 3.1 Physical properties: After standing, the upper layer is a clear liquid, the lower layer is a grayish white or yellowish precipitate, and after shaking, it is a uniform suspension.

[0083] 3.2 Sterility test: according to the current "Chinese Veterinary Pharmacopoeia".

[0084] 3.3 Loading inspection: according to the current "Chinese Veterinary Pharmacopoeia".

[0085] 4 Results

[0086] 4.1 Bacterial solution production: According to the methods of 2.1, 2.2 and 2.3, nine batches of bacterial solutions PmA2020001, PmA2020002, PmA2020003, PmB2020001, PmB2020002, PmB2020003, M.h2020001, M.h2020002 and M.h2020003 were prepared. The purity test of each batch of secondary seed met the requirements, and the purity test and viable bacterial count of the bacterial solution met the requirements. The results are shown in Table 1 below.

[0087] Table 1. Summary of bacterial solution production

[0088]

[0089] 4.2 Inactivated bacterial solution test results: MPmA2020001, MPmA2020002, MPmA2020003, MPmB2020001, MPmB2020002, MPmB2020003 batches of inactivated bacterial solution were inactivated at 37℃ for 10 hours; MM.h2020001, MM.h2020002, MM.h2020003 batches of inactivated bacterial solution were inactivated at 37℃ for 8 hours. The inactivation test results met the requirements, and the results are shown in Table 2 below.

[0090] Table 2. Inactivated bacterial solution test results

[0091]

[0092]

[0093] Note: "-" indicates sterile growth.

[0094] 4.3 Concentrated antigen test results: The nine batches of inactivated antigens were concentrated 10 times by 0.1 μm hollow fiber column, and then centrifuged and concentrated, with a total concentration ratio of 15 times. The sterility test results met the requirements, and the results are shown in Table 3 below.

[0095] Table 3. Centrifugation and concentrated antigen test results

[0096]

[0097] Note: "-" indicates sterile growth

[0098] 4.4 Preparation and dispensing of inactivated vaccine: 3 batches of bovine Pasteurella multocida (group A, group B) and Mannheimia haemolytica dual inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) were prepared and dispensed according to the method 2.5, with batch numbers S2020001, S2020002, and S2020003, respectively. The results are shown in Table 4 below.

[0099] Table 4. Vaccine preparation and dispensing

[0100]

[0101] 4.5 Product testing

[0102] 4.5.1 The physical properties, fill weight, formaldehyde residue, and sterility test results of the 3 batches of inactivated vaccine all met the requirements. The results are shown in Table 5.

[0103] Table 5. Summary of the physical properties, fill weight, formaldehyde residue, and sterility test results of the 3 batches of inactivated vaccine

[0104]

[0105] Note: “—” indicates no growth

[0106] Safety test of vaccine

[0107] The prepared 3 batches of bovine Pasteurella multocida (group A, group B) and Mannheimia haemolytica dual inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) were subjected to safety test on three non-target animals (rabbits, mice, and guinea pigs), and single-dose vaccination, single-dose repetition, and super-dose vaccination safety research on target animals of different ages were conducted. The results showed that there was no necrosis at the injection site of experimental animals, and all animals were healthy and alive after 10 days of observation. The immune cows had normal mental state, no obvious food reduction, no injection site infection and necrosis, and the body temperature results during the post-immunization observation period were all within the normal range, and no obvious clinical abnormal reactions were observed. The body weight of the target animals vaccinated with the super-dose of one batch of test vaccine showed no significant difference compared with the control group. This indicated that the vaccine was safe for both target animals and non-target animals.

[0108] 1 Materials

[0109] 1.1 Vaccine: 3 batches of bovine Pasteurella multocida (group A, group B) and Mannheimia haemolytica dual inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) with batch numbers S2020001, S2020002, and S2020003 were provided by the project team.

[0110] 1.2 Test animals: 40 healthy susceptible cattle aged 2-6 months and 8-18 months, from designated cattle farms in Inner Mongolia; 18-22 g mice, 1.5-2 kg healthy susceptible rabbits, and 300-350 g guinea pigs, purchased from designated test animal bases.

[0111] 2 Method

[0112] 2.1 Non-target animal safety test: 3 batches of vaccines S2020001, S2020002, and S2020003 were respectively injected subcutaneously into the neck and back of 10 mice weighing 18-22 g at 0.2 ml per mouse, 10 healthy susceptible rabbits weighing 1.5-2 kg at 2.0 ml per rabbit, and 10 guinea pigs weighing 300-350 g at 1.0 ml per guinea pig, and observed for 10 days.

[0113] 2.2 Target animal safety test

[0114] 2.2.1 Single-dose inoculation safety test: 20 healthy susceptible cattle aged 2-6 months and 8-18 months were selected and inoculated with 5 cattle each of S2020001, S2020002, and S2020003 batches of vaccines subcutaneously at 2.0 ml per head, and another 5 cattle of different ages were used as control cattle and injected with the same dose of PBS. The average of the body temperature 2 days before inoculation and the body temperature before inoculation on the day of inoculation was used as the basal body temperature, and the body temperature of the test animals was determined at the same point after feeding every day for 14 days after inoculation. The test animals were also observed for whether there was pus or necrosis at the injection site and adverse reactions in the whole body after inoculation.

[0115] 2.2.2 Single-dose repeated inoculation safety test: the cattle used in the single-dose inoculation safety test were used again, and were inoculated with the corresponding batch of vaccine or PBS 14 days later subcutaneously at 2.0 ml per head. The average of the body temperature 2 days before inoculation and the body temperature before inoculation on the day of inoculation was used as the basal body temperature, and the body temperature of the test animals was determined at the same point after feeding every day for 14 days after inoculation. The test animals were also observed for their reactions, whether there was pus or necrosis at the injection site, and adverse reactions in the whole body after inoculation.

[0116] 2.2.3 Overdose safety test: 20 healthy susceptible 2-6 month and 8-18 month old calves were selected and vaccinated with S2020001, S2020002 and S2020003 respectively, 5 calves for each vaccine, 4.0 ml / each (2 doses) were injected subcutaneously. Another 5 calves of different ages were used as control and injected with the same dose of PBS. The average of the body temperature 2 days before vaccination and the body temperature before vaccination on the day of vaccination were used as the basal body temperature. The body temperature of the test animals was measured at the same time every day for 14 days after vaccination. The reaction of the test animals after vaccination, whether there was pus or necrosis at the injection site, and whether there was any adverse reaction in the whole body were observed. The weight changes of the 5 2-6 month old healthy susceptible calves vaccinated with S2020001 and the 5 control calves were recorded before and 14 days after vaccination to test the effect of the vaccine on the weight gain of the calves.

[0117] 3 Results

[0118] 3.1 Non-target animal safety test: 3 batches of vaccines S2020001, S2020002 and S2020003 prepared in the laboratory were injected into laboratory animals mice, rabbits and guinea pigs respectively, and continuously observed for 10 days. No necrosis or other adverse reactions were observed at the injection site. The results are shown in Table 6 below.

[0119] Table 6. Safety test results of 3 batches of vaccines on laboratory animals

[0120]

[0121] 3.2 Safety test on 2-6 month old calves

[0122] 3.2.1 Single dose safety test: After single dose vaccination, the immune calves were observed for 14 days. The spirit and appetite of the immune calves were normal. The body temperature of individual calves increased slightly after vaccination, which was not more than 1.5°C compared with the basal body temperature. No pus, necrosis or other adverse reactions caused by vaccination were observed at the injection site, indicating that the vaccine was safe for 2-6 month old calves when vaccinated in single dose. The results are shown in Tables 7-9 and Figure 1 .

[0123] Table 7. Safety test results of 3 batches of vaccines on 2-6 month old calves vaccinated in single dose

[0124] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 2.0ml No abscess and necrosis were seen 0

[0125] Table 8. Body temperature results of 3 batches of vaccines on 2-6 month old beef calves vaccinated in single dose

[0126]

[0127] Table 9. Average body temperature of 2-6 month old calves vaccinated with 3 batches of vaccines in single dose

[0128]

[0129]

[0130] 3.2.2 Single dose repeated vaccination safety test: After single dose repeated vaccination, the immune cattle were observed for 14 days. No abnormality was found in spirit and appetite. The body temperature of individual cattle increased slightly after immunization. The body temperature of 1 cattle was more than 1.5℃ compared with the basic body temperature, but less than 2 temperature times. The body temperature of the rest of the cattle was not more than 1.5℃ compared with the basic body temperature. No abscess and necrosis was found at the injection site, and no other adverse reactions caused by injection of vaccine were found. It was indicated that the vaccine was safe for 2-6 month old calves after single dose repeated vaccination, and the results were shown in Tables 10-12 and Figure 2 .

[0131] Table 10. Safety test results of 3 batches of vaccine for 2-6 month old cattle after single dose repeated vaccination

[0132] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe

[0133] Table 11. Animal body temperature results of 3 batches of vaccine for 2-6 month old cattle after single dose repeated vaccination

[0134]

[0135] Table 12. Average body temperature of animals in safety test of 3 batches of vaccine for 2-6 month old cattle after single dose repeated vaccination

[0136]

[0137] 3.2.3 Overdose vaccination safety test: After 2 times overdose vaccination, the immune cattle were observed for 14 days. No abnormality was found in spirit and appetite. The body temperature of individual cattle increased slightly after immunization, and was not more than 1.5℃ compared with the basic body temperature. No abscess and necrosis was found at the injection site, and no other adverse reactions caused by injection of vaccine were found. There was no significant difference in weight gain between the immune group and the control group, which indicated that the vaccine was safe for 2-6 month old calves after overdose vaccination, and the results were shown in Tables 13-16 and Figure 3 .

[0138] Table 13. Safety test results of 3 batches of vaccine for 2-6 month old cattle after overdose vaccination

[0139] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe

[0140] Table 14. Animal body temperature results of 3 batches of vaccine for 2-6 month old cattle after overdose vaccination

[0141]

[0142] Table 15. Average body temperature of animals in safety test of 3 batches of vaccines over-dosed in 2-6 month-old cattle

[0143]

[0144] Table 16. Body weight gain results of S2020001 batch of vaccine over-dosed in 2-6 month-old cattle

[0145]

[0146] Note: The body weight of the control group is the average of 5 cattle.

[0147] 3. Safety test of 3.38-18 month-old cattle

[0148] 3.3.1 Single dose vaccination safety test: After single dose vaccination, the immune cattle were observed for 14 days, and no abnormalities were found in their spirit and appetite. The body temperature of individual cattle increased slightly after vaccination, and the increase was not more than 1.5°C compared with the basic body temperature. No abscess or necrosis was found at the injection site, and no other adverse reactions caused by vaccination were found. This indicates that the vaccine is safe for 8-18 month-old cattle after single dose vaccination. The results are shown in Tables 17-19 and Figure 4 .

[0149] Table 17. Results of safety test of 3 batches of vaccines single-dosed in 8-18 month-old cattle

[0150] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe

[0151] Table 18. Body temperature results of safety test of 3 batches of vaccines single-dosed in 8-18 month-old cattle

[0152]

[0153]

[0154] Table 19. Average body temperature of animals in safety test of 3 batches of vaccines single-dosed in 8-18 month-old cattle

[0155]

[0156] 3.3.2 Single dose repeated vaccination safety test: After single dose repeated vaccination, the immune cattle were observed for 14 days, and no abnormalities were found in their spirit and appetite. The body temperature of individual cattle increased slightly after vaccination, and the increase was not more than 1.5°C compared with the basic body temperature. No abscess or necrosis was found at the injection site, and no other adverse reactions caused by vaccination were found. This indicates that the vaccine is safe for 8-18 month-old cattle after single dose repeated vaccination. The specific results are shown in Tables 20-22 and Figure 5 .

[0157] Table 20. Results of safety test of 3 batches of vaccines single-dosed repeatedly in 8-18 month-old cattle

[0158] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 2.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 2.0ml No abscess and necrosis were seen 0

[0159] Table 21. Body temperature results of safety test animals of 3 batches of vaccine single dose repeated vaccination of 8-18 month old cattle

[0160]

[0161]

[0162] Table 22. Average body temperature results of safety test animals of 3 batches of vaccine single dose repeated vaccination of 8-18 month old cattle

[0163]

[0164] 3.2.3 Overdose vaccination safety test: After the overdosage vaccination of the vaccine, the immune cattle were continuously observed for 14 days, and no abnormalities were found in the spirit and appetite of the immune cattle. The body temperature of individual cattle increased slightly after immunization, and the increase was not more than 1.5°C compared with the basal body temperature. No abscess and necrosis was found at the injection site, and no other adverse reactions caused by injection of the vaccine were found, indicating that the vaccine was safe for 8-18 month old cattle after overdosage vaccination. The results are shown in Tables 23-25 and Figure 6 .

[0165] Table 23. Safety test results of 3 batches of vaccine overdosage vaccination of 8-18 month old cattle

[0166] Vaccine lot Number of animals (heads) Single immunization dose Injection site Clinical abnormality (heads) Result determination S2020001 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020002 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe S2020003 5 4.0ml No abscess and necrosis were seen 0 5 / 5 safe Control group 5 4.0ml No abscess and necrosis were seen 0

[0167] Table 24. Body temperature results of safety test animals of 3 batches of vaccine overdosage vaccination of 8-18 month old cattle

[0168]

[0169]

[0170] Table 25. Average body temperature of safety test animals of 3 batches of vaccine overdosage vaccination of 8-18 month old cattle

[0171]

[0172] 4 Conclusion

[0173] 4.1 The 3 batches of bovine Pasteurella multocida (group A, group B), Hemolytic Mannheimia haemolytica bivalent inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) prepared in the laboratory were not found to be abnormal after overdosage vaccination of related non-target animals (mice, rabbits, guinea pigs), indicating that the vaccine was safe for target animals (mice, rabbits, guinea pigs).

[0174] 4.2 Single dose and single dose repeated, over-dose vaccination of 2-6 month-old calves and 8-18 month-old cattle, immune cattle are healthy and normal, no clinical adverse reactions such as loss of appetite, depression, death, etc., indicating that the vaccine is safe for different age target animals.

[0175] 4.3 Over-dose vaccination has no significant effect on the weight gain of calves, indicating that the vaccine can be safely used for fattening calves.

[0176] Experiment 4 Vaccine potency test in animals

[0177] Three batches of vaccines S2020001, S2020002, S2020003 were respectively immunized with 4-6 month-old healthy susceptible cattle, and 21 days after the first immunization, the second immunization was carried out by the same route and dose. 14 days after the second immunization, BPmA / XJ2016 strain, BPmB / NM2016 strain and BMh / NM2018 strain were challenged. The results showed that the potency of the three batches of vaccines was in line with the requirements after the animal potency test

[0178] 1 Test materials

[0179] 1.1 Vaccine: Bovine Pasteurella multocida (group A, group B), Haemophilus somnus bivalent inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) S2020001, S2020002, S2020003.

[0180] 1.2 Test strains: Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain JY2018001, Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain JY2018001, Bovine Haemophilus somnus BMh / NM2018 strain JY2019001, provided by the project team.

[0181] 1.3 Test animals: 4-6 month-old healthy susceptible cattle, purchased from designated cattle farms.

[0182] 2 Methods

[0183] 2.1 Animal preparation: 60 healthy susceptible cattle of 4-6 months old were selected.

[0184] 2.2 Immunization: Randomly take 1 bottle of vaccine S2020001, S2020002, S2020003, shake well, then subcutaneously inject 15 heads of each batch of vaccine in the neck, 2.0 ml per head, 21 days after the first immunization, the second immunization, the second immunization method and dose are the same as the first immunization, the remaining 15 heads are used as control cattle.

[0185] 2.3 Challenge and determination

[0186] 2.3.1 Bovine Pasteurella multocida capsularis group A strain BPmA / XJ2016: 14 days after the second immunization, 5 cattle each of the S2020001, S2020002, and S2020003 immunizations, along with 5 control cattle, were intratracheally injected with 5 ml of BPmA / XJ2016 bacterial suspension per head (containing 1×10⁶ live bacteria). 8 ~1×10 10 CFU), observed for 10 days, temperature monitored for 7 days, then autopsy performed.

[0187] 2.3.1.1 Criteria for determining the onset of disease: The challenged cattle exhibit one or more clinical symptoms such as depression, loss of appetite, runny nose, and cough, or a body temperature of 40°C or higher (lasting for one day); necropsy 10 days after challenge reveals obvious purulent or solid lesions in the apical, cardiac, or diaphragmatic lobes of the lungs, with extensive abscesses and significant fibrinous exudation in severe cases, and bacterial isolation and PCR identification of the lesion tissue confirming positive for Pasteurella multocida capsulatum group A; and the lung lesion rate reaches 5% (5% or higher) according to the lung lesion rate statistical method in 2.3.1.2.

[0188] 2.3.1.2 Statistics on the incidence of lung lesions

[0189] 2.3.1.2.1 Visual lung lesion rate: The proportion of lesion area in each of the seven lung lobes was visually assessed to evaluate the overall area of ​​the lobe. A lobe with no lesions was recorded as having a lesion rate of 0%. A lobe with a lesion rate greater than 0% but less than or equal to 25% was recorded as having a lesion rate of 25%. A lobe with a lesion rate greater than 25% but less than or equal to 50% was recorded as having a lesion rate of 50%. A lobe with a lesion rate greater than 50% but less than or equal to 75% was recorded as having a lesion rate of 75%. A lobe with a lesion rate greater than 75% was recorded as having a lesion rate of 100%. For cattle that died within 3 days of infection (with necropsy findings and molecular biological diagnosis confirming death was not due to external causes), the lesion rate of each lung lobe was recorded as 100%.

[0190] The lesion rate of each lung lobe was multiplied by the percentage of each lung lobe's mass relative to the total lung mass in Table 26 to obtain the lesion rate of that lung lobe relative to the total lung mass. The sum of the lesion rates of the seven lung lobes was then used to obtain the lung tissue lesion rate. The lung tissue lesion rate of cattle that died within 3 days after the challenge (after necropsy and molecular biological diagnosis to determine that the death was not due to external causes) was 100%.

[0191] Table 26. Percentage of lung lobe mass to total lung mass

[0192] Lobe name Percentage of total lung Left apical lobe 5％ Left cardiac lobe 6％ Left diaphragmatic lobe 32％ Right apical lobe 11％ Right cardiac lobe 7％ Right diaphragmatic lobe 35％ Accessory lobe 4％ Total 100％

[0193] 2.3.1.2.2 Lung tissue lesion rate: The lung tissue lesion rate is calculated according to the following formula.

[0194] Rate of lung tissue lesion = rate of lesion in left apical lobe x 5% + rate of lesion in left cardiac lobe x 6% + rate of lesion in left diaphragmatic lobe x 32% + rate of lesion in right apical lobe x 11% + rate of lesion in right cardiac lobe x 7% + rate of lesion in right diaphragmatic lobe x 35% + rate of lesion in accessory lobe x 4%

[0195] 2.3.1.3 Challenge protection criteria

[0196] The rate of lung tissue lesion of the immune group and the challenge control group was counted, and according to 2.3.1.1 and 2.3.1.2, at least 4 / 5 of the challenge control group were sick, and the lung tissue lesion reduction rate of the immune group was calculated according to the following formula: it should not be less than 60% to be qualified.

[0197]

[0198] 2.3.1.4 Collect the obviously lesioned tissues, and detect and isolate the pathogenic bacteria in the tissues, and identify the suspected colonies isolated by PCR.

[0199] 2.3.2 On the 14th day after the second immunization of bovine Pasteurella multocida capsular group B BPmB / NM2016 strain, 5 heads of S2020001, S2020002 and S2020003 immune cattle were injected with 2 ml of BPmB / NM2016 strain bacterial liquid (containing 1 x 10 6 ~ 1 x 10 8 CFU) per head, together with 5 heads of control cattle, and observed for 10 days. The feeding and mental state of the immune group and the control group were observed daily, and the number of surviving and dead cattle was counted after 10 days of challenge.

[0200] 2.3.2.1 Challenge protection criteria

[0201] 2.3.2.1.1 Within 10 days after challenge, the spirit and appetite are normal.

[0202] 2.3.2.1.2 If the spirit or appetite is reduced after challenge, the duration should not exceed 7 days.

[0203] If conditions 2.3.2.1.1 or 2.3.2.1.2 are met, it is judged as challenge protection.

[0204] 2.3.2.2 Result determination

[0205] 2.3.2.2.1 At least 4 heads of the 5 heads of control cattle should be dead;

[0206] 2.3.2.2.2 At least 4 heads of the 5 heads of immune cattle should be protected.

[0207] If 2.3.2.2.1-2.3.2.2.2 are met, it is determined that the vaccine bovine Pasteurella multocida capsular group B BPmB / NM2016 strain is qualified for efficacy test in cattle.

[0208] 2.3.3. 2 Lung lesion rate statistics: the method is the same as 2.3.1.2. The lung tissue lesion rate of the cattle died within 5 days after challenge (non-external cause of death identified by post-mortem examination and molecular biology diagnosis) is 100%. 8 10 CFU), observed for 10 days, temperature measured for 7 days, and post-mortem examination performed.

[0209] 2.3.3.1 Incidence determination criteria: the challenged cattle showed one or more clinical symptoms such as depression, loss of appetite, runny nose, cough, or body temperature reached 40°C (lasted for one temperature) and above within 5 days after challenge; post-mortem examination showed lung hemorrhage, consolidation, and purulent lesions, lung and pericardium or thoracic cavity adhesion, and the lesion tissues were positive for M. haemolytica by bacterial isolation and PCR identification; the lung tissue lesion rate reached 20% (20% and above) according to the lung lesion rate statistical method of 2.3.1.2.

[0210] 2.3.3.2 Lung lesion rate statistics: the method is the same as 2.3.1.2. The lung tissue lesion rate of the cattle died within 5 days after challenge (non-external cause of death identified by post-mortem examination and molecular biology diagnosis) is 100%.

[0211] 2.3.3.3 Challenge protection determination criteria

[0212] The lung tissue lesion rate of the immune group of cattle and the challenged control group of cattle was calculated, and at least 4 / 5 of the challenged control group of cattle were determined to be ill according to 2.3.1.1 and 2.3.1.2, and the lung tissue lesion reduction rate of the immune group of cattle should not be less than 60% to be determined to be qualified according to the following formula.

[0213]

[0214] 2.3.3.4 Collect the tissues with obvious lesions for pathogen detection and isolation in the tissues, and perform PCR identification on the suspected colonies isolated.

[0215] 3 Results

[0216] 3.1 The efficacy test results of M. bovis capsular group A BPmA / XJ2016 strain in cattle: observed for 10 days, 4 / 5 of the challenged control group of cattle had body temperature increased to 40°C or above, and the results are shown in Table 27. Post-mortem examination results showed that 4 / 5 of the challenged control group of cattle had lung lesion rate of 5% or above, and 4 / 5 of the control group of cattle were ill. The average lung tissue lesion reduction rates of the immune S2020001, S2020002, and S2020003 groups of cattle relative to the control group of cattle were 77.7%, 81.0%, and 74.4%, respectively, and the results are shown in Table 28.

[0217] ​Table 27. Body temperature results of cattle challenged with BPmA / XJ2016 strain

[0218]

[0219] Note: "1 day" in "body temperature after challenge" refers to the body temperature measured on the 2nd day after challenge, and so on. The temperature was measured continuously for 7 days.

[0220] Table 28. Statistical results of lung lesion rate of cattle challenged with BPmA / XJ2016 strain

[0221]

[0222] Note: "a" indicates that the pathogen PCR identification is positive for B. multocida capsular group A, and "-" indicates that it is not related.

[0223] 3.2 Potency test results of B. multocida capsular group B BPmB / NM2016 strain in cattle: observed for 10 days, 5 / 5 control group cattle died, and the challenge protection rates of S2020001, S2020002, S2020003 immune groups were 100% (5 / 5), 80% (4 / 5), and 100% (5 / 5), respectively. The results are shown in Table 29 below.

[0224] Table 29. Results of cattle challenged with BPmB / NM2016 strain

[0225]

[0226]

[0227] 3.3 Potency test results of B. manillense BMh / NM2018 strain in cattle: observed for 10 days, 5 / 5 control group cattle had body temperature increased by more than 40°C, and 1 control cattle died on the 3rd day after challenge. The results are shown in Table 30 below. The results of necropsy showed that the lung lesion rate of 5 / 5 control group cattle was more than 20%, and 5 / 5 control group cattle were sick. The average lesion reduction rate of lung tissue of S2020001, S2020002, and S2020003 immune groups relative to the control group was 70.3%, 72.6%, and 72.1%, respectively. The results are shown in Table 31 below.

[0228] Table 30. Body temperature results of cattle challenged with BMh / NM2018 strain

[0229]

[0230] Note: "1 day" in "body temperature after challenge" refers to the body temperature measured on the 2nd day after challenge, and so on. The temperature was measured continuously for 7 days. "-" indicates death.

[0231] Table 31. Statistical results of lung lesion rate of cattle challenged with BMh / NM2018 strain

[0232]

[0233] Note: "a" means the pathogenic PCR identification is positive for Mannheimia haemolytica, and "-" means not involved.

[0234] 4 Conclusion

[0235] 4.1 The 3 batches of vaccines S2020001, S2020002 and S2020003 were used for potency test in cattle, and the results of challenge test of BPmA / XJ2016 strain, BPmB / NM2016 strain and BMh / NM2018 strain were in line with the requirements.

[0236] 4.2 The results of potency test in animals of the 3 batches of prepared vaccines met the requirements of quality standards. Further, it was proved that the cattle Pasteurella multocida (group A, group B) and Mannheimia haemolytica dual inactivated vaccine (BPmA / XJ2016 strain + BPmB / NM2016 strain + BMh / NM2018 strain) using experimental animal substitution potency test qualified vaccine, the potency test in animals was qualified, and the substitution potency test method and the potency test in animals had good correlation.

[0237] Experiment 5 Substitution animal potency test of vaccine

[0238] 3 batches of vaccines were subjected to substitution animal potency test

[0239] 1 Material

[0240] Test strains: BPmA / XJ2016 strain JY2018001, BPmB / NM2016 strain JY2018001, BMh / NM2018 strain JY2019001, 3 batches of vaccines S2020001, S2020002 and S2020003.

[0241] 2 Method

[0242] 2.1 Cattle Pasteurella multocida capsular group A BPmA / XJ2016 strain: 10 mice with a body weight of 18-22 g were injected intramuscularly with 0.1 ml of vaccine per mouse, and 14 days after the first immunization, the same route and dose were used for the second immunization. Seven days after the second immunization, 10 mice in the control group were subcutaneously injected with 0.1 ml of BPmA / XJ2016 strain bacterial solution per mouse (containing 2 x 10 4 CFU), and observed for 7 days.

[0243] 2.2 Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain: 5 healthy susceptible rabbits with body weight of 1.5-2 kg were injected intramuscularly with 0.5 ml of vaccine per rabbit, and 21 days later, together with 5 rabbits in the control group, they were injected intramuscularly with 1 ml of BPmB / NM2016 bacterial solution per rabbit (containing 1-4 CFU of live bacteria), and observed for 8 days.

[0244] 2.3 Bovine Mannheimia haemolytica BMh / NM2018 strain: 5 healthy susceptible guinea pigs with body weight of 300-350 g were injected intramuscularly with 0.4 ml of vaccine per pig, and 14 days after the first injection, they were given a second injection by the same route and dose. Seven days after the second injection, together with 5 guinea pigs in the control group, they were injected intraperitoneally with 1 ml of BMh / NM2018 bacterial solution per pig (containing 2 x 10 8 CFU of live bacteria), and observed for 7 days.

[0245] 3 Results

[0246] 3.1 Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain efficacy test results: 3 batches of inactivated vaccine were used to immunize mice, and 7 days after the second injection, together with the control group, they were injected subcutaneously with 0.1 ml of BPmA / XJ2016 bacterial solution per mouse (containing 2 x 10 4 CFU of live bacteria), and observed for 7 days. The control group of 10 mice all died, S2022001, S2022002, and S2022003 protected 10, 9, and 10 mice respectively, and the results all met the standard. The results are shown in Table 32.

[0247] Table 32. Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain mouse efficacy test results

[0248]

[0249] 3.5.3.2 Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain efficacy test results: 3 batches of inactivated vaccine were used to immunize rabbits, and 21 days later, together with the control group, they were injected intramuscularly with 1 ml of BPmB / NM2016 bacterial solution per rabbit (containing 1-4 CFU of live bacteria), and the control group of 5 rabbits all died, S2022001, S2022002, and S2022003 protected 4, 5, and 5 rabbits respectively, and the results all met the standard. The results are shown in Table 33.

[0250] Table 33. Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain rabbit efficacy test results

[0251]

[0252] 3.5.3.3. Results of efficacy test of Mannheimia haemolytica BMh / NM2018 strain on guinea pigs: 3 batches of inactivated vaccine were used to immunize guinea pigs, 7 days after the second immunization, together with the control group, 1 ml of BMh / NM2018 strain bacterial solution (containing 2 x 10 8 CFU) was injected intraperitoneally, 4 / 5 in the control group died, S2022001, S2022002, S2022003 protected 4, 5, and 5 respectively, the results meet the standard, the results are as follows in Table 34.

[0253] Table 34. Results of efficacy test of Mannheimia haemolytica BMh / NM2018 strain on guinea pigs

[0254]

[0255] Establishment of indirect hemagglutination test for antibodies between vaccines

[0256] 1. Materials and equipment: Martin's broth, BHI medium, Pasteurella multocida capsular group A BPmA / XJ2016 strain, Pasteurella multocida capsular group B BPmB / NM2016 strain, Mannheimia haemolytica BMh / NM2018 strain, formaldehyde solution, tannic acid, glutaraldehyde, sheep ghost red blood cells, positive serum of Pasteurella multocida capsular group A BPmA / XJ2016 strain (serum antibody titer is 1:64) and negative serum (serum antibody titer is ≤1:2) (self-made), positive serum of Pasteurella multocida capsular group B BPmB / NM2016 strain (serum antibody titer is 1:64) and negative serum (serum antibody titer is ≤1:2), positive serum of Mannheimia haemolytica BMh / NM2018 strain (serum antibody titer is 1:64) and negative serum (serum antibody titer is ≤1:2), ultrasonic disrupter (Ningbo Xinzhi), 0.04M PBS (pH 7.2-7.4), biological safety cabinet, KDC-1044L large-capacity low-speed centrifuge, pipette, reaction plate (V-shaped 110°), etc.

[0257] 2. Operation method

[0258] 2.1 Preparation of sensitizing antigen

[0259] 2.1.1. Bovine Pasteurella multocida Group A BPmA / XJ2016 strain: One vial of F5 generation freeze-dried bacteria was inoculated into 5 ml of Martin broth liquid medium and incubated at 36-37°C and 120 r / min for 12-16 hours. Then, it was inoculated onto a Martin agar plate containing 4% of newborn bovine serum and 0.1% of lysed blood and incubated at 36-37°C for 12-16 hours. The bacterial lawn on the surface of the plate was washed with 0.04M PBS solution at pH 7.2, and the collected bacterial solution was mixed to form a suspension. Then, 0.15% formaldehyde solution was added to the bacterial suspension, and the mixture was inactivated at 37°C for 16 hours, ultrasonically cracked in an ice bath for 30 min at 200W, and stored at 2-8°C.

[0260] 2.1.2. Bovine Pasteurella multocida Group B BPmB / NM2016 strain: One vial of F5 generation freeze-dried bacteria was inoculated into 5 ml of Martin broth liquid medium and incubated at 36-37°C and 120 r / min for 18-22 hours. Then, it was inoculated onto a Martin agar plate containing 4% of newborn bovine serum and 0.1% of lysed blood and incubated at 36-37°C for 18-22 hours. The bacterial lawn on the surface of the plate was washed with 0.04M PBS solution at pH 7.2, and the collected bacterial solution was mixed to form a suspension. Then, 0.15% formaldehyde solution was added to the bacterial suspension, and the mixture was inactivated at 37°C for 16 hours, ultrasonically cracked in an ice bath for 30 min at 200W, and stored at 2-8°C.

[0261] 2.1.3. Bovine Mannheimia haemolytica BMh / NM2018 strain: One vial of F5 generation freeze-dried bacteria was inoculated into 5 ml of BHI medium and incubated at 36-37°C and 120 r / min for 10-16 hours. Then, it was inoculated onto a BHI agar plate containing 4% of newborn bovine serum and 0.1% of lysed blood and incubated at 36-37°C for 10-16 hours. The bacterial lawn on the surface of the plate was washed with 0.04M PBS solution at pH 7.2, and the collected bacterial solution was mixed to form a suspension. Then, 0.15% formaldehyde solution was added to the bacterial suspension, and the mixture was inactivated at 37°C for 16 hours, ultrasonically cracked in an ice bath for 30 min at 200W, and stored at 2-8°C.

[0262] 2.2. Glutaraldehyde-treated sheep red blood cells: Freshly collected male sheep blood (containing 50% Al's solution) was washed with PBS solution at 2000 r / min for 10 min for 5 times, and then centrifuged at 2000 r / min for 30 min in the last time. The final product was a 5% red blood cell suspension prepared with PBS solution. 2.5% glutaraldehyde stored at 2-8°C was mixed with the 5% red blood cell suspension at a ratio of 1:5 (v / v) in a shaking bed at room temperature at 100 r / min for 2 hours. The mixture was washed with PBS solution at 2000 r / min for 2 min for 5 times and then with sterilized deionized water at 2000 r / min for 2 min for 5 times to obtain a 5% glutaraldehyde-treated red blood cell suspension.

[0263] 2.3 Tannic acidization: Mix the prepared 1:20000 tannic acid solution with 5% aldehyde-treated red blood cell suspension in equal volume, 37°C water bath for 20 min, shake once in 5 min, wash with PBS liquid 2000 r / min for 2 min for 3 times, prepare 5% aldehyde-treated-tannic acidized red blood cell suspension, and store at 2-8°C. For antigen sensitization.

[0264] 2.4 Antigen sensitization unit determination

[0265] 2.4.1 Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain: Dilute the antigen with PBS liquid by 25 times, 50 times, 75 times, 100 times, and 200 times respectively, mix the diluted antigen with 5% aldehyde-treated-tannic acidized red blood cells in a volume ratio of 1:1, and place in a 37°C water bath for 45 min, shake once in 5 min; wash twice with PBS liquid containing 1% healthy rabbit serum (diluent). Prepare 2% antigen-sensitized red blood cell suspension with PBS liquid containing 1% healthy rabbit serum, react with immune serum, and take the antigen with the highest titer as 1 sensitization unit, and the antigen-sensitized red blood cells have no self-coagulation or rupture phenomenon.

[0266] 2.4.2 Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain: Dilute the antigen with PBS liquid by 90 times, 100 times, 110 times, 120 times, and 130 times respectively, mix the diluted antigen with 5% aldehyde-treated-tannic acidized red blood cells in a volume ratio of 1:1, and place in a 37°C water bath for 45 min, shake once in 5 min; wash twice with PBS liquid containing 1% healthy rabbit serum (diluent). Prepare 2% antigen-sensitized red blood cell suspension with PBS liquid containing 1% healthy rabbit serum, react with immune serum, and take the antigen with the highest titer as 1 sensitization unit, and the antigen-sensitized red blood cells have no self-coagulation or rupture phenomenon.

[0267] 2.4.3 Bovine Mannheimia haemolytica BMh / NM2018 strain: Dilute the antigen with PBS liquid by 50 times, 60 times, 70 times, 80 times, and 90 times respectively, mix the diluted antigen with 5% aldehyde-treated-tannic acidized red blood cells in a volume ratio of 1:1, and place in a 37°C water bath for 45 min, shake once in 5 min; wash twice with PBS liquid containing 1% healthy rabbit serum (diluent). Prepare 2% antigen-sensitized red blood cell suspension with PBS liquid containing 1% healthy rabbit serum, react with immune serum, and take the antigen with the highest titer as 1 sensitization unit, and the antigen-sensitized red blood cells have no self-coagulation or rupture phenomenon.

[0268] 2.5 Antigen-sensitized red blood cell preparation

[0269] Mix 1 part of diluted antigen (containing 1-2 sensitizing units) with 1 part of 5% aldehyde-tanned red blood cell suspension, mix well, and incubate at 37°C for 45 min. Wash twice with PBS containing 1% healthy rabbit serum (diluent), and prepare a 2% antigen-sensitized red blood cell suspension with PBS containing 1% healthy rabbit serum.

[0270] 2.6 Antibody detection and determination

[0271] 2.6.1 Antibody detection

[0272] 2.6.1.1 Label: Use a marker pen to mark the sample identification on the left side of the 96-well "V" type 110° micro-reaction plate, including positive serum control, negative serum control, and antigen control.

[0273] 2.6.1.2 Add diluent: Add 0.05 ml of PBS containing 1% healthy rabbit serum to each well using a pipette.

[0274] 2.6.1.3 Serum dilution: Use a micropipette to take 0.05 ml of the serum to be tested and add it to the first well of the 96-well "V" type micro-reaction plate marked in advance, then use the micropipette to perform a 2-fold serial dilution to the 6th well (dilution 2 6 times); dilute the positive serum to the 12th well (dilution 2 12 times); dilute the negative serum to the 6th well (dilution 2 6 times).

[0275] 2.6.1.4 Add antigen-sensitized red blood cells: Use a micropipette to take 0.05 ml of 2% antigen-sensitized red blood cells after shaking, and add it to each well, with 4 wells as antigen control.

[0276] 2.6.1.5 Shake and observe: After adding the sample, mix gently, stand at room temperature for 45-60 min, observe and record the results.

[0277] 2.6.2 Determination

[0278] 2.6.2.1 Determination criteria: The highest dilution with (++) hemagglutination reaction is taken as the final antibody titer of the serum. The intensity of hemagglutination reaction is shown as follows.

[0279] ++++ Red blood cells form a large area of clumps at the bottom of the well.

[0280] +++ Red blood cells form a thick layer of agglutination at the bottom of the well, with curled edges or jagged shape.

[0281] ++ Red blood cells form a thin layer of uniform agglutination at the bottom of the well, with an area larger than the above two, and agglutination above "++" is positive for red blood cell agglutination.

[0282] + Red blood cells do not completely settle at the bottom of the well, with a small amount of agglutination around.

[0283] ± Red blood cells sink to the bottom of the well, but the periphery is not smooth or the center is blank.

[0284] - Red blood cells are dotted at the bottom of the well, and the periphery is smooth.

[0285] 2.6.2.2 Control standard

[0286] 2.6.2.2.1 Positive serum control: The positive serum antibody titer of Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and Bovine Mannheimia haemolytica BMh / NM2018 is 1:32-1:128, and the positive serum control is established.

[0287] 2.6.2.2.2 Negative serum control: The negative serum antibody titer of Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and Bovine Mannheimia haemolytica BMh / NM2018 is not higher than 1:2, and the negative serum control is established.

[0288] 2.6.2.2.3 Antigen sensitized red blood cell control: There is no agglutination phenomenon of antigen sensitized red blood cells, and the antigen sensitized red blood cell control is established.

[0289] 2.6.2.3 Result determination

[0290] 2.6.2.3.1 Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain serum antibody titer: The positive serum antibody titer of Bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain is 1:32-1:128; the negative serum antibody titer is not higher than 1:2; and there is no agglutination phenomenon of antigen sensitized red blood cells. The serum antibody titer of the detected serum is higher than 1:8, which is determined to be positive; the serum antibody titer of the detected serum is not higher than 1:8, which is determined to be negative.

[0291] 2.6.2.3.2 Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain serum antibody titer: The positive serum antibody titer of Bovine Pasteurella multocida capsular group B BPmB / NM2016 strain is 1:32-1:128; the negative serum antibody titer is not higher than 1:2; and there is no agglutination phenomenon of antigen sensitized red blood cells. The serum antibody titer of the detected serum is higher than 1:4, which is determined to be positive; the serum antibody titer of the detected serum is not higher than 1:4, which is determined to be negative.

[0292] 2.6.2.3.3 Serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain: The serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain reached 1:32-1:128; the negative serum antibody titer was not higher than 1:2; and there was no agglutination phenomenon of antigen sensitized red blood cells. The serum antibody titer higher than 1:8 was determined as positive; and the serum antibody titer not higher than 1:8 was determined as negative.

[0293] 3 Results

[0294] 3.1 Relationship between serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain and protection against challenge The results of observation on the 10th day showed that the lung lesion rate of 5 / 5 control group of cattle reached more than 5%, and 4 / 5 of the control group of cattle were sick. The average lung lesion reduction rates of S2020001, S2020002 and S2020003 groups of cattle relative to the control group of cattle were 77.7%, 81.0% and 74.4%, respectively. When the antibody level was greater than 6, all could be protected, and the results were as shown in Table 35.

[0295] Table 35. Statistical results of relationship between serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain and protection against challenge

[0296]

[0297]

[0298] 3.2 Relationship between serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain and protection against challenge The results of observation on the 10th day showed that the lung lesion rate of 5 / 5 control group of cattle reached more than 5%, and 4 / 5 of the control group of cattle were sick. The average lung lesion reduction rates of S2020001, S2020002 and S2020003 groups of cattle relative to the control group of cattle were 77.7%, 81.0% and 74.4%, respectively. When the antibody level was greater than 6, all could be protected, and the results were as shown in Table 35.

[0299] Table 36. Statistical results of relationship between serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain and protection against challenge

[0300]

[0301] 3.3 Relationship between serum antibody titer of Mannheimia haemolytica BMh / NM2018 strain and protection against challenge The results of observation on the 10th day showed that the lung lesion rate of 5 / 5 control group of cattle reached more than 5%, and 4 / 5 of the control group of cattle were sick. The average lung lesion reduction rates of S2020001, S2020002 and S2020003 groups of cattle relative to the control group of cattle were 77.7%, 81.0% and 74.4%, respectively. When the antibody level was greater than 6, all could be protected, and the results were as shown in Table 35.

[0302] Table 37. Serum antibody titers of bovine M. haemolytica BMh / NM2018 strain and statistical results of challenge protection

[0303]

[0304] The above merely describes preferred experiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can also have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine, characterized in that: The inactivated vaccine comprises an adjuvant and an antigen, wherein the antigen is a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica A1 type BMh / NM2018 strain; the antigen is a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica A1 type BMh / NM2018 strain, which were deposited on November 27, 2018 at the China General Microbiological Culture Collection Center, and the classification and naming is bovine Mannheimia haemolytica, and the preservation number is CGMCC No. 16895.

2. A Bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine as claimed in claim 1, wherein: The said bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and bovine Mannheimia haemolytica type A1 BMh / NM2018 strain are all at 1×10 9 ~1×10 10 CFU / ml; the volume ratio of the adjuvant and the antigen is 1:1~1:

6.

3. A Bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine according to claim 1 or 2, characterized in that: The adjuvant is an aluminum hydroxide gel adjuvant.

4. The bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine according to any one of claims 1 to 3, characterized in that, The effective content of the antigen is not less than 1 x 10 9 ~1 x 10 10 CFU / ml.

5. The method for preparing a bivalent trivalent inactivated vaccine of Pasteurella multocida (group A and group B) and hemolytic Mansoni according to claim 1, characterized in that: The preparation method comprises the following specific steps: S1: respectively fermenting and culturing a bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain, a bovine Pasteurella multocida capsular group B BPmB / NM2016 strain and a bovine Mannheimia haemolytica BMh / NM2018 strain to obtain three kinds of fermentation bacterial liquids; S2: concentrating and purifying the three kinds of fermentation bacterial liquids in S1, inactivating to obtain three kinds of inactivated antigen bacterial liquids; S3: mixing the three kinds of inactivated antigen bacterial liquids in S2 with an aluminum hydroxide gel adjuvant to prepare an inactivated vaccine.

6. A process for the preparation of a Bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine as claimed in claim 5, wherein the process comprises the steps of: In the fermentation culture in S1, the bovine Pasteurella multocida capsular group A BPmA / XJ2016 strain is inoculated into Martin's meat medium at an inoculation amount of 1% to 2% (v / v), the bovine Pasteurella multocida capsular group B BPmB / NM2016 strain is inoculated into Martin's meat medium at an inoculation amount of 1% to 2% (v / v), and the bovine Mannheimia haemolytica BMh / NM2018 strain is inoculated into BHI culture medium containing sheep lysed blood at an inoculation amount of 1% to 2% (v / v), the culture temperature is 37±1℃, the rotation speed is 100 to 150 r / min, the culture time is 10 to 16 h, and the fermentation bacterial liquid is obtained.

7. A process for the preparation of a Bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine as claimed in claim 6, characterized in that: The fermentation culture in S1 is divided into primary seed propagation and secondary seed propagation; the primary seed propagation is that the freeze-dried bacterial strain of Bovine Pasteurella Multocida Capsular Group A BPmA / XJ2016 or Bovine Pasteurella Multocida Capsular Group B BPmB / NM2016 is inoculated into Martin's broth medium, the freeze-dried bacterial strain of Mannheimia Haemolytica BMh / NM2018 is inoculated into BHI medium of sheep lysed blood, the culture temperature is 37±1℃, the rotation speed is 100-150r / min, and the culture time is 10-16h, then streak inoculation is carried out on Martin plate, the culture temperature is 37±1℃, and the culture time is 10-16h, typical colonies are selected under a microscope, and Martin slant is inoculated with several branches, the culture temperature is 37±1℃, and the culture time is 10-16h, which is used as the primary seed; the secondary seed propagation is that the primary seed of the Bovine Pasteurella Multocida Capsular Group A BPmA / XJ2016 or Bovine Pasteurella Multocida Capsular Group B BPmB / NM2016 is inoculated into Martin's broth medium, the primary seed of Mannheimia Haemolytica BMh / NM2018 is inoculated into BHI medium of sheep lysed blood, the culture temperature is 37±1℃, the rotation speed is 100-150r / min, and the culture time is 10-16h, which is used as the secondary seed.

8. A process for the preparation of a Bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine as claimed in claim 7, characterized in that: The fermentation bacterial liquid in S1 is that the secondary seeds of Bovine Pasteurella Multocida Capsular Group A BPmA / XJ2016, Bovine Pasteurella Multocida Capsular Group B BPmB / NM2016 and Mannheimia Haemolytica A1 BMh / NM2018 are inoculated into culture medium at an inoculation amount of 1%-2% (v / v), the culture temperature is 37±1℃, the rotation speed is 100-150r / min, and the culture time is 10-16h.

9. A process for the preparation of a bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine according to any one of claims 5 to 8, characterized in that: The concentrated and purified fermentation bacterial liquid in S2 is prepared by centrifugation at a rotation speed of 12500-14000r / min and a temperature of 4±1℃ for 15-25min, then the bacterial precipitate is collected and resuspended with sterile PBS at pH 7.2-7.4 to prepare the concentrated bacterial liquid.

10. A process for the preparation of a bovine Pasteurella multocida (A, B) and Mannheimia haemolytica bivalent trivalent inactivated vaccine as claimed in any one of claims 5 to 9, characterized in that: The inactivation in S2 is that formaldehyde solution is added at an amount of 0.1%-0.15% (v / v) of the concentrated bacterial liquid, and the inactivation is carried out at 37±1℃ for 8-10h to prepare the inactivated antigen bacterial liquid.

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