Mannheimia haemolytica and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a mannheimia haemolytica strain and application thereof. The mannheimia haemolytica strain is a goat species, the preservation number is CGMCC No: 31259, the preservation date is July 10, 2024, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The invention also discloses an application of the mannheimia haemolytica strain in preparation of a mannheimia haemolytica inactivated vaccine, the mannheimia haemolytica strain HNYJWLT19 of a long cashmere goat has good immunogenicity and can stimulate a goat body to generate a strong immunoreaction, and the inactivated vaccine prepared by inoculating the strain can be used for preparing the inactivated vaccine of the mannheimia haemolytica. The goat can generate an effective antibody aiming at mannheimia haemolytica, so that prevention and control of mannheimia haemolytica diseases are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial separation and application, and specifically relates to a strain of Mannheimia hemolytica and application thereof. Background Art

[0002] Mannheimia haemolytica (MH) is a pathogen that poses a significant threat to the goat farming industry. This pathogen is highly contagious and pathogenic, and the pneumonia it causes is severe, manifested by high fever, cough, and serous and fibrinous inflammation of the chest and pleura. The course of the disease can be acute or chronic, and is often accompanied by a high mortality rate. Mannheimia haemolytica is widely prevalent worldwide, especially in areas where goats are intensively raised. The wide range of its impact and the depth of its harm cannot be ignored. Due to its high contagiousness and pathogenicity, once an outbreak occurs, it may cause serious losses to the goat farming industry. Therefore, the prevention and control of Mannheimia haemolytica is of vital importance.

[0003] Mannheimia haemolytica is the main cause of respiratory diseases in goats, and its negative impact on goat health and production performance is very significant. This pathogen not only directly affects the health and life of sick goats, but also has a negative impact on the health status and production performance of the entire goat population. Mannheimia haemolytica is a prevalent pathogen in countries and regions around the world, especially in areas where goats are raised. Due to its high infectivity and pathogenicity, Mannheimia haemolytica spreads quickly in the flock, and once an outbreak occurs, it may cause huge economic losses to farmers. The growth of sick goats is inhibited, production performance is reduced, and even a large number of deaths may occur, which directly affects the economic benefits of farmers.

[0004] The biological characteristics and drug resistance of Mannheimia haemolytica are important reasons why it is difficult to treat. Mannheimia haemolytica has strong drug resistance and can quickly adapt to and resist the effects of multiple antibiotics. This drug resistance may come from genetic variations in the bacteria themselves, or it may be affected by excessive use of antibiotics. The emergence of drug resistance makes conventional antibiotic treatment ineffective in many cases, increasing the difficulty of treatment. Mannheimia haemolytica has the ability to adapt to the environment quickly, including rapid adaptation to antibiotics. This makes it easy for bacteria to develop drug resistance during treatment, leading to treatment failure.

[0005] In addition, due to the harsh growth conditions of Mannheimia haemolytica, it needs strict nutritional and environmental conditions to reproduce, which makes in vitro culture and research very difficult. The lack of an effective in vitro culture system not only limits the in-depth understanding of the biological characteristics of the pathogen, but also hinders the development of new drugs and vaccines. In addition, due to the large number of serotypes of Mannheimia haemolytica and the lack of cross-immunity between serotypes, the development of vaccines is extremely difficult. Although there are vaccines for some serotypes, they do not cover all serotypes. Even if there are vaccines for related serotypes, there are differences in the protective effects against diseases caused by different strains. This also affects the prevention and control of the disease to a certain extent, causing serious economic losses to my country's sheep industry.

[0006] Based on the above problems, through in-depth research on the biological characteristics, pathogenic mechanism and transmission route of goat hemolytic Mannheimia, we explore the vaccine of Mannheimia strain with better protective effect against goat hemolytic Mannheimia, which is of great significance for preventing and controlling goat hemolytic Mannheimia and ensuring the healthy development of the sheep industry. Summary of the invention

[0007] In view of the defects and problems of poor clinical treatment effect of goat hemolytic Mannheimia at present, the present invention provides a hemolytic Mannheimia strain and its application in vaccine preparation. The vaccine strain has strong virulence and the prepared vaccine has good immunity.

[0008] The solution adopted by the present invention to solve its technical problem is: a strain of hemolytic Mannheimia, the hemolytic Mannheimia is hemolytic Mannheimia HNYJWLT19, its preservation number is CGMCC No: 31259, the preservation date is: July 10, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The above-mentioned hemolytic Mannheimia, the hemolytic Mannheimia HNYJWLT19 is type 3 hemolytic Mannheimia.

[0010] The application of the above-mentioned Mannheimia haemolytica in the preparation of an inactivated vaccine for preventing Mannheimia haemolytica infection.

[0011] In the above application, the inactivated vaccine is prepared from inactivated hemolytic Mannheimia HNYJWLT19 strain and an adjuvant.

[0012] In the above application, the adjuvant is propolis adjuvant.

[0013] In the above application, the concentration of the inactivated strain HNYJWLT19 in the inactivated vaccine is 1-3×10 10 CFU / mL.

[0014] In the above application, the method for preparing the inactivated vaccine comprises the following steps:

[0015] (1) Primary seed propagation: The hemolytic Mannheimia strain HNYJWLT19 was inoculated into a tryptone soy broth medium containing 10% fetal bovine serum and cultured in a 37°C incubator for 1 to 2 days as primary seeds;

[0016] (2) Secondary seed propagation: After the primary seeds have passed the pollution inspection, they are inoculated into a tryptone soy broth medium containing 10% fetal bovine serum and cultured at 37°C for 2 to 3 days for secondary seed propagation. After passing the inspection, they are used as secondary seeds.

[0017] (3) Preparation of bacterial solution: 2% to 4% of the seed volume was inoculated into tryptone soy broth medium containing 10% fetal bovine serum and cultured at 37°C for 3 to 4 days. Samples were taken daily to detect the number of hemolytic Mannheimia. When the growth titer of hemolytic Mannheimia reached 1 to 5 × 10 10 CFU / mL, terminate the culture, perform purity test and store for future use;

[0018] (4) Concentration and inactivation: After passing the test, concentrate the bacterial solution to 50% of the original volume, add formaldehyde to the concentrated bacterial solution to a final concentration of 0.2%, mix thoroughly, and inactivate at 37°C for 12 h;

[0019] (5) Vaccine preparation: After the sterility test and inactivation test are passed, add sterilized propolis ethanol extract to make each milliliter of bacterial solution contain 10 mg of propolis. Shake while adding to make it emulsified. Then divide the mixture into portions to make the hemolytic Mannheimia inactivated vaccine.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The hemolytic Mannheimia HNYJWLT19 screened and isolated by the present invention is from a goat that died of respiratory symptoms of typical pneumonia, and is highly representative. It has a strong pathogenicity to goats and can cause the disease and death of goats.

[0022] 2. The vaccine prepared by using the hemolytic Mannheimia HNYJWLT19 isolated by the present invention is safe and reliable, has good immunogenicity, can stimulate the goat body to produce a strong immune response, has a good protective effect on goat hemolytic Mannheimia disease, and can effectively achieve the prevention and control of the disease.

[0023] 3. The present invention uses natural propolis adjuvant with no toxic side effects as the vaccine adjuvant during vaccine preparation, which is safer and more efficient, and can also promote the rising speed of antibody levels, making the immune effect more lasting.

[0024] 4. The vaccine prepared by the present invention has a 100% immune protection effect on goats, and still has a good protective effect on goats even under the conditions of low immunization dose and high challenge dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the evolutionary tree diagram of the strain HNYJWLT19 of the present invention.

[0026] Figure 2 This is a morphological diagram of the strain HNYJWLT19 of the present invention under a Gram-stained 100× microscope.

[0027] Figure 3 It is the PCR identification result of the strain HNYJWLT19 of the present invention. The marker in the figure is DL 2000bp, and from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; 1 is the amplification result of the positive control of Mannheimia hemolytica using primers MHF and MHR; 2 and 3 are the amplification results of the strain HNYJWLT19 using primers MHF and MHR; 4 is the negative control.

[0028] Figure 4 This is the serotype identification result of the strain HNYJWLT19 of the present invention. The marker in the figure is DL 2000bp, and from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; 1-3 are the amplification results of MhF1F / R, MhF2F / R, and MhF3F / R, respectively.

[0029] Figure 5 This is a distribution map of virulence factors related to pathogenicity in the genome of the strain HNYJWLT19 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1: Isolation and identification of hemolytic Mannheimia

[0032] 1. Sample collection

[0033] The specimen came from a two-month-old goat that died of severe pneumonia and breathing difficulties at a plush goat pastoral farm in Ulatai Township, Hami City, Xinjiang in April 2024.

[0034] 2. Culture medium preparation

[0035] Solid culture medium for hemolytic Mannheimia: PPLO broth powder 21g, agar powder 15g, double distilled water to 1000mL. Sterilize the culture medium at 115℃ for 15min, cool to 50℃, add 10% horse serum, pour into a plate, and store at 4℃ after solidification for later use.

[0036] 5. Isolation and culture

[0037] Disinfect the surface of the collected lung tissue under sterile conditions, use an inoculation needle to take the internal lung tissue and smear it on the surface of the culture medium, and place it in a 37°C incubator for culture. After 24 hours, no other bacteria grow significantly, and tiny pinpoint-shaped colonies can be seen with the naked eye. Pick a single colony and purify it in the culture medium. Gram stain (see Figure 2 ) The morphology of the hemolytic Mannheimia bacteria was observed under an oil microscope and was spherical, named HNYJWLT19.

[0038] 4. Strain identification

[0039] (1) Primer design

[0040] A pair of universal primers were designed based on the 16S rRNA sequence of Mannheimia haemolytica for amplification of Mannheimia haemolytica. The primer sequences are as follows:

[0041] MhF: 5'-AGCCGCGGTAATACGGGGGGT-3' (SEQ ID NO. 1);

[0042] MhR: 5'-GAAGTAGATAGCTTAACCGA-3' (SEQ ID NO. 2).

[0043] The size of the amplified fragment was 240 bp.

[0044] (2) PCR identification

[0045] The strain HNYJWLT19 was inoculated into liquid culture medium and cultured in a 37°C incubator for 24 hours. The bacteria were collected and the DNA of the strain HNYJWLT19 was extracted according to the operation steps of the DNA extraction kit. The concentration was determined by spectrophotometer to be 100 μg / mL, and PCR identification was performed using MhF and MhR amplification. At the same time, the hemolytic Mannheimia standard strain was set as a positive control.

[0046] The PCR amplification reaction system was 25 μL: 5 μL of extracted DNA, 2 μL of upstream primer MhF: 5′-AGCCGCGGTAATACGGGGGGT-3′ (25 pmol / μL), 2 μL of downstream primer MhR: 5′-GAAGTAGATAGCTTAACCGA-3′ (25 pmol / μL), 5 μL of 5×PCR Buffer, 5 μL of 2.5 mmol / L dNTPs, 0.5 μL of Taq enzyme (5 U / μL), and 25 mM MgCl 2 3.5μL, DEPC water 2μL, mix thoroughly and centrifuge briefly.

[0047] Reaction conditions: pre-denaturation at 94°C for 5 min, then cycle at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for a total of 35 cycles, and finally extension at 72°C for 10 min. The PCR products were stored at 4°C.

[0048] The amplified product was electrophoresed, 5 μL was added to each well, and electrophoresed on 1% agarose gel, and the results were observed under ultraviolet light (see Figure 3 ). Figure 3 It can be seen that MhF and MhR amplified fragments of expected size, and the homology with the hemolytic Mannheimia strain HNYJWLT19 (Genbank No. GCF004342115.1) was 100% after sequencing. The sequencing results are as follows, proving that the isolated strain HNYJWLT19 is hemolytic Mannheimia.

[0049] TACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGGAACCCTGATGCAGCCATGCCGCGTGAATGAAGAAGGCCTTCGGGTTGTAAAGTTCTTTCGGTGACGAGGAAGGCGATTGTTTTAATAGAACAGTCGATTGACGTTAATCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGGTGCGAGCGTTAATCGGAATAACTGGGCGTAAAGGGCACGCAGGCGGTTGTTTAAGTGAGGTGTGAAAGCCCCGGGCTTAACCTGGGAATTGCATTTCAGACTGGACAACTAGAGTACTTTAGGGAGGGGTAGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGAAGGCGAAGGCAGCCCCTTGGGAATGTACTGACGCTCATGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGCTGTCGATTTGGGGATTGGGGTTTAACTCTGGTGCCCGTAGCTAACGTGATAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCATGGAATCTTGTAGAGATATGAGAGTGCCTTCGGGAACCATGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTAGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAGAGGGCGGCGAGCCAGCGATGGGGAGCGAATCTCAGAAAGTACGTCTAAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAAATCA GAATGTTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGTACCAGAAGTAGATAGCTTAACCGAAAGGGGGGGCGTTTACCACGGTATGAT(SEQ ID NO.3).

[0050] (3) Serotype identification of strain HNYJWLT19

[0051] In order to further clarify the serotype of the strain, the corresponding serotype primers were synthesized for the serotype, and the primer sequences are as follows:

[0052] Table 1 Primers for serotyping of Mannheimia hemolytica

[0053]

[0054] The strain HNYJWLT19 was inoculated into liquid culture medium and cultured in a 37°C incubator for 24 hours. The bacteria were collected and the DNA of the strain HNYJWLT19 was extracted according to the operation steps of the DNA extraction kit. The concentration was determined by spectrophotometer to be 100 μg / mL. MhF1F / R, MhF2F / R and MhF3F / R were used for PCR identification. At the same time, the hemolytic Mannheimia standard strain was set as a positive control.

[0055] The PCR amplification reaction system was 25 μL: 5 μL of extracted DNA, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of 5×PCR Buffer, 5 μL of 2.5 mmol / L dNTPs, 0.5 μL of Taq enzyme (5 U / μL), 25 mM MgCl 2 3.5μL, DEPC water 2μL, mix thoroughly and centrifuge briefly.

[0056] Reaction conditions: pre-denaturation at 94°C for 5 min, then cycle at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for a total of 35 cycles, and finally extension at 72°C for 10 min. The PCR products were stored at 4°C.

[0057] The amplified product was electrophoresed, 5 μL was added to each well, and electrophoresed on 1% agarose gel, and the results were observed under ultraviolet light (see Figure 4 ). Figure 4It can be seen that MhF3 and MhR3 amplified fragments of expected size, proving that the isolated strain HNYJWLT19 is Mannheimia hemolytica type 3.

[0058] (4) Identification of virulence factors

[0059] Virulence factors The virulence factors of strain HNYJWLT19 were analyzed by whole genome sequencing, and the database used was VFDB. The VFDB core virulence factor database was compared to obtain virulence factor gene annotations. A total of 258 virulence factors were obtained and classified. Figure 5 and Table 2.

[0060] Table 2 Virulence factors associated with pathogenicity in the genome of HNYJWLT19 strain

[0061]

[0062]

[0063] from Figure 5 As can be seen from Table 2, nutritional and metabolic factors account for a relatively high proportion, followed by immune regulation, adhesion, exotoxin, regulation, biofilm, stress survival, effector delivery system, exoenzyme, invasion, motility, antimicrobial activity / competitive advantage and protein post-translational modification virulence factors.

[0064] (5) Toxicity test

[0065] Sixteen mice from the same batch were randomly divided into two groups, with 8 mice in each group. The strain HNYJWLT19 prepared in advance was cultured and the content was measured and adjusted to 1×10 9 CFU / mL, the experimental group was injected with 1mL of bacterial solution by subcutaneous injection in the neck, and the virus was challenged for 3 consecutive days, once a day; the control group was injected with 1mL of sterile saline at the same location and frequency.

[0066] After the challenge is completed, the test group and the control group are placed separately to prevent aerosol transmission and cross infection from affecting the accuracy of the test. The first day of the challenge test is recorded as day 0, and the test period is 14 days. The diet and mental state of the animals are observed every morning, noon and evening. After 14 days of the test, the surviving mice in all groups were killed by injecting air into the ear vein, and the lungs were removed and the lung lesions were observed. After the mice were killed, the lungs were dissected to isolate the pathogens.

[0067] The results are recorded as follows: On the 0th day of infection, all mice had good mental state and appetite without any abnormalities; on the 1st day, there was no change in the experimental group and the control group; on the 2nd day, the mice in the experimental group coughed, had a loss of appetite, and had nasal discharge, while there was no change in the control group; on the 7th day, the experimental group had wheezing and milky white mucus outside the nasal cavity, and had a poor appetite, until the 14th day, there was no change in the control group; the lungs of the experimental group showed obvious fleshy changes; the lungs of the control group were pink, with a smooth surface, and no pathological changes.

[0068] No hemolytic Mannheimia was isolated from the 8 lung samples of the control group, while hemolytic Mannheimia was isolated from the 8 lung samples of the test group. The colony morphology was "spherical", and the PCR identification results of the test group were all positive. Therefore, it was shown that the strain HNYJWLT19 had a strong virulence.

[0069] Example 2: Vaccine preparation

[0070] 1. Vaccine preparation

[0071] (1) Primary seed propagation

[0072] The hemolytic Mannheimia strain HNYJWLT19 was inoculated into a tryptone soy broth (containing 10% fetal bovine serum) medium and cultured in a 37° C. incubator for 1 to 2 days as a primary seed.

[0073] (2) Secondary seed propagation

[0074] After the first-level seeds are inspected and found to be free of pollution, they are inoculated into a tryptone soy broth (containing 10% fetal bovine serum) medium and cultured at 37°C for 2 to 3 days for second-level seed propagation. After passing the inspection, they are used as second-level seeds.

[0075] (3) Preparation of bacterial solution

[0076] 2% to 4% of the seeds were inoculated into tryptone soy broth (containing 10% fetal bovine serum) medium and cultured at 37°C for 3 to 4 days. Samples were taken daily to detect the number of hemolytic Mannheimia. When the growth titer of hemolytic Mannheimia reached 1 to 5 × 10 10 CFU / mL, terminate the culture, perform a purity test and store for later use.

[0077] (4) Concentration and inactivation

[0078] After passing the test, concentrate to 50% of the original bacterial solution; add formaldehyde to the concentrated bacterial solution to a final concentration of 0.2%, mix thoroughly, inactivate at 37°C for 12 hours, and shake twice during the process.

[0079] (5) Seedling matching

[0080] After the sterility test and inactivation test are passed, add sterilized propolis ethanol extract to make each milliliter of bacterial solution contain 10 mg of propolis. Shake while adding to make it emulsified, and then package it to make the hemolytic Mannheimia inactivated vaccine.

[0081] Example 3: Safety and efficacy testing of vaccines

[0082] 1. Vaccine safety testing

[0083] Eight rabbits weighing 1.5-2.0 kg were injected subcutaneously or intramuscularly with 1 × 10 10 1mL of vaccine with CFU / mL was administered and observed for 10 days, and all of them were alive.

[0084] Eight healthy susceptible long-haired goats of about 12 months old were injected with 4 mL of the vaccine subcutaneously in the neck and observed for 30 days. No adverse reactions such as loss of appetite, mental depression, temperature reaction, and local inflammation after injection were observed. This indicates that the inactivated vaccine of the present invention is safe.

[0085] 2. Vaccine efficacy test

[0086] Twelve healthy susceptible long-haired goats of about 12 months old were divided into an immunization group and a control group, and 2 mL of vaccine was injected subcutaneously in the neck of each group. 30 days after vaccination, 10 mL (5×10 8 CFU / mL) and observed for 30 days.

[0087] There were significant differences between the immunized group and the control group after the challenge. The goats in the immunized group showed no obvious symptoms and all survived. On the second day of the challenge, the goats in the control group began to show clinical symptoms, with fever, dyspnea, and cough. Death began on the third day, with nasal discharge from the mouth and nose before death. All died on the 9th day. The goats in the control group were autopsied and had pleural effusion, peritoneal effusion, pericardial effusion, thoracoabdominal adhesions, fibrinous villous heart, and lung enlargement. All the goats in the control group became ill and died, while all the immunized goats survived and were protected, indicating that the inactivated vaccine of the present invention has a good protective effect.

[0088] In order to further verify the efficacy of the vaccine, 72 healthy susceptible long-haired goats of about 12 months old were used to set up 6 vaccine immunization groups and 6 control groups, with 6 goats in each group. Each vaccine group was injected with 2 mL of vaccine, and each control group was injected with the same dose of sterile saline. 30 days after vaccination, 10 mL of HNYJWLT19 strain culture was inoculated into the trachea of ​​each immunization group and control group. The immunization dose and challenge dose of each group are shown in Table 3 below. The clinical manifestations of goats in each group were observed for a total of 30 days.

[0089] Table 3 Efficacy experiments of different immunization doses and different challenge doses

[0090]

[0091] As can be seen from Table 3, under different immunization doses and different challenge doses, all the animals in the control group died, and the immune protection rate of the immunization group reached 100%. Even under the conditions of high challenge dose and low immunization dose, the protection rate was still good, indicating that the vaccine prepared by the strain of the present invention has an excellent immune protection effect.

[0092] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Mannheimia haemolytica, characterized in that: The hemolytic Mannheimia is hemolytic Mannheimia HNYJWLT19, its preservation number is CGMCC No: 31259, the preservation date is July 10, 2024, the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The hemolytic Mannheimia according to claim 1, characterized in that: The hemolytic Mannheimia HNYJWLT19 is type 3 hemolytic Mannheimia.

3. Use of the hemolytic Mannheimia according to claim 1 in the preparation of an inactivated vaccine for preventing hemolytic Mannheimia infection.

4. The use according to claim 3, characterized in that: The inactivated vaccine is prepared from inactivated hemolytic Mannheimia HNYJWLT19 strain and an adjuvant.

5. The use according to claim 3, characterized in that: The adjuvant is propolis adjuvant.

6. The use according to claim 3, characterized in that: The concentration of the inactivated strain HNYJWLT19 in the inactivated vaccine is 1-3×10 10 CFU / mL.

7. The use according to claim 3, characterized in that: The preparation method of the inactivated vaccine comprises the following steps: (1) Primary seed propagation: The hemolytic Mannheimia strain HNYJWLT19 was inoculated into a tryptone soy broth medium containing 10% fetal bovine serum and cultured in a 37°C incubator for 1 to 2 days as primary seeds; (2) Secondary seed propagation: After the primary seeds have passed the pollution inspection, they are inoculated into a tryptone soy broth medium containing 10% fetal bovine serum and cultured at 37°C for 2 to 3 days for secondary seed propagation. After passing the inspection, they are used as secondary seeds. (3) Preparation of bacterial solution: 2% to 4% of the seed volume was inoculated into tryptone soy broth medium containing 10% fetal bovine serum and cultured at 37°C for 3 to 4 days. Samples were taken daily to detect the number of hemolytic Mannheimia. When the growth titer of hemolytic Mannheimia reached 1 to 5 × 10 10 CFU / mL, terminate the culture, perform purity test and store for later use; (4) Concentration and inactivation: After passing the test, concentrate the bacterial solution to 50% of the original volume, add formaldehyde to the concentrated bacterial solution to a final concentration of 0.2%, mix thoroughly, and inactivate at 37°C for 12 h; (5) Vaccine preparation: After the sterility test and inactivation test are passed, add sterilized propolis ethanol extract to make each milliliter of bacterial solution contain 10 mg of propolis. Shake while adding to make it emulsified. Then divide the mixture into portions to make the hemolytic Mannheimia inactivated vaccine.

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