Quintuptuple inactivated vaccine for sheep multiple lung rot disease and preparation method of quintuptuple inactivated vaccine

Abstract

The invention provides a quintuplet inactivated vaccine for sheep multiple lung rot disease and a preparation method thereof, and belongs to the technical field of multi-combined vaccines. According to the invention, five pathogens are combined in one vaccine, so that animals can generate effective antibodies aiming at multiple pathogens while one vaccine is used for immunization, the effect of preventing multiple diseases by one injection is achieved, and the prevention and control effect of the lung rot disease is improved. Meanwhile, the quintuplet inactivated vaccine can avoid frequent capture of activities of the sheep due to injection of various monovalent vaccines, reduces animal stress caused by driving and fixation, reduces the working intensity of immunization personnel, has very important clinical application value and wide market prospect, and can generate huge social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of multi-unit vaccines, and in particular relates to a five-unit inactivated vaccine for multiple "lung rot" in sheep and a preparation method thereof. Background Art

[0002] In recent years, the sheep industry in Inner Mongolia Autonomous Region has developed rapidly, and the sheep breeding industry has become one of the advantageous industries driving the development of animal husbandry. However, in the process of transforming from traditional grazing to intensive, large-scale indoor or semi-indoor feeding, the problems of disease hazards and prevention and control have become increasingly prominent. Due to the severe cold in winter, spring and late autumn in Inner Mongolia Autonomous Region, the changeable climate, the large temperature difference between day and night, especially the frequent occurrence of extreme weather in the transition season, cold and heat stressors, the incidence of respiratory infectious diseases in sheep has greatly increased, becoming an important factor that seriously restricts large-scale breeding production and efficiency improvement. According to surveys and statistics, 75% of the cases of diseases with observable clinical symptoms are related to pneumonia, and 45% to 55% of the cases are directly related to pneumonia at the mortality level. In recent years, according to literature reports, the serum positive rate of sheep pneumonia Mycoplasma in 10 major sheep-raising provinces in northern China has reached 36% to 62%.

[0003] Vaccination with vaccines is the core technology for preventing the occurrence and spread of sheep pneumonia. Although there are monovalent or bivalent inactivated vaccines against goat and sheep pneumonia Mycoplasma in China, the pathogen components are single, the immune protection effect is not ideal, and the prevention effect is low. Summary of the invention

[0004] The purpose of the present invention is to provide a pentavalent inactivated vaccine for multiple ovine lung rot and a preparation method thereof, which can achieve one vaccine immunization to enable animals to produce effective antibodies against multiple pathogens, achieve the effect of "one shot for multiple protections", and improve the prevention and control effect.

[0005] The present invention provides a five-in-one inactivated vaccine for multiple ovine "lung rot", wherein the inactivated pathogens include Mycoplasma ovipneumoniae, Mycoplasma mycoides subspecies caprine, Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subspecies zooepidemicus.

[0006] Preferably, the Mycoplasma ovipneumoniae includes Mycoplasma ovipneumoniae MO_BM13D004 strain, with a deposit number of CGMCC No.45420;

[0007] The mycoplasma mycoides subspecies caprae includes the mycoplasma mycoides subspecies caprae subspecies Mm_NM02 strain, with a deposit number of CGMCC No.45419;

[0008] The hemolytic Mannheimia A2 serotype includes the hemolytic Mannheimia A2 serotype MS-NM2 strain, with a deposit number of CGMCC No.19797;

[0009] The D-type Pasteurella multocida of sheep includes Pasteurella multocida PmD100 strain, and its deposit number is CGMCC No.26636;

[0010] The Streptococcus equi subspecies zooepidemicus includes the Streptococcus equi subspecies zooepidemicus SeZ100 strain, and its preservation number is CGMCC No.26635.

[0011] Preferably, the inactivated pathogens include inactivated pathogenic bacteria liquid, and the culture medium of the inactivated pathogenic bacteria liquid includes a modified KM2 liquid culture medium and a TSB liquid culture medium containing 5% horse serum.

[0012] Preferably, the inactivated pathogenic bacteria liquid includes: an inactivated bacterial liquid obtained by inactivating Mycoplasma ovipneumoniae after culturing in a modified KM2 liquid culture medium, an inactivated bacterial liquid obtained by inactivating Mycoplasma mycoides subspecies in a modified KM2 liquid culture medium, an inactivated bacterial liquid obtained by inactivating Mannheimia hemolytica serotype A2 after culturing in a TSB liquid culture medium containing 5% horse serum, an inactivated bacterial liquid obtained by inactivating Pasteurella multocida type D after culturing in a TSB liquid culture medium containing 5% horse serum, and an inactivated bacterial liquid obtained by inactivating Streptococcus equi subspecies zooepidemicus after culturing in a TSB liquid culture medium containing 5% horse serum.

[0013] Preferably, each L of the improved KM2 medium comprises the following components: 500 mL of 1640 cell culture medium, 300 mL of 1.7% by weight Hanks solution of hydrolyzed milk protein, 20 mL of 25% by weight yeast solution, 200 mL of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL of 1% by weight thallium acetate, 5 mL of 0.4% by weight phenol red, 4 g of glucose and 2 g of sodium pyruvate, and the pH value is 7.6-7.8;

[0014] Each liter of the TSB liquid culture medium containing 5% horse serum includes the following components: 17.0 g of trypticase peptone, 3.0 g of soy peptone, 2.5 g of D-glucose, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate and 200 mL of horse serum.

[0015] Preferably, the amount of each pathogen in the pentavalent inactivated vaccine is (1-9)×10 8 CFU / mL or (1~9)×10 8 CCU / mL.

[0016] Preferably, the pentavalent inactivated vaccine further comprises an adjuvant;

[0017] The types of adjuvants include water-in-oil-in-water adjuvants, water-in-oil adjuvants, microemulsion adjuvants, aluminum salt adjuvants or Freund's adjuvants.

[0018] Preferably, the volume ratio of the inactivated bacterial liquid containing the inactivated pathogen to the adjuvant is 1:(1-2).

[0019] The present invention also provides a method for preparing the above-mentioned pentavalent inactivated vaccine, comprising the following steps: culturing Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies caprae in a modified KM2 liquid culture medium and then inactivating them to obtain an inactivated bacterial solution;

[0020] Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subspecies zooepidemicus are cultured in TSB liquid medium containing 5% horse serum and then inactivated to obtain inactivated bacterial liquid;

[0021] Mix all the inactivated bacterial solutions in a volume ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2) to obtain an inactivated bacterial solution containing all inactivated pathogens;

[0022] The inactivated bacterial solution containing all inactivated pathogens is mixed with an immune adjuvant and then emulsified to obtain the pentavalent inactivated vaccine.

[0023] Preferably, the culturing comprises inoculating the secondary seed liquid of Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies capricolum into a modified KM2 medium respectively, and culturing at 37° C. for 48 to 72 hours;

[0024] The secondary seed liquids of Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subsp. zooepidemicus were inoculated into TSB medium containing 5% horse serum respectively, and cultured at 37°C for 16 to 24 hours.

[0025] Beneficial effects: The present invention innovatively combines five pathogens, namely, Mycoplasma ovipneumoniae, Mycoplasma mycoides subspecies caprine, Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi zooepidemicus subspecies, into one vaccine, which can achieve one vaccine immunization while enabling animals to produce effective antibodies against multiple pathogens, achieving the effect of "one shot for multiple preventions" and improving the prevention and control effect of "lung rot". In the embodiment of the present invention, serological studies are also conducted on the five-in-one inactivated vaccine, which confirms that the five-in-one inactivated vaccine can cause the animal body to produce an effective immune response and generate a higher antibody titer. The attack protection test confirms that the vaccine can effectively protect the body from infection by the corresponding pathogens. At the same time, the five-in-one inactivated vaccine can avoid the frequent capture of sheep due to the need to inject multiple monovalent vaccines, reduce the animal stress caused by driving and restraining, and reduce the work intensity of immunization personnel. It has very important clinical application value and broad market prospects, and will produce huge social and economic benefits.

[0026] Biological deposit information

[0027] Pasteurella multocida PmD100 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on February 21, 2023. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26636.

[0028] Mycoplasma ovipneumoniae MO-BM13D004 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on February 21, 2023. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45420.

[0029] Streptococcus equisubsp.zooepidemicus SeZ100 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on February 21, 2023. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 26635.

[0030] Mannheimia haemolytica MS-NM2 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on May 11, 2020. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.19797.

[0031] The Mycoplasma mycoides subsp.capri Mm_NM02 strain was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on February 21, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNo.45419. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the result of serum antibody test for Mycoplasma ovipneumoniae;

[0033] Figure 2 This is the colony morphology of Mycoplasma mycoides subspecies capricolum;

[0034] Figure 3This is the result of the serum antibody test of Mycoplasma mycoides subspecies caprine;

[0035] Figure 4 This is the result of Gram staining of Mannheimia hemolytica;

[0036] Figure 5 This is a graph showing the results of serum antibody testing against Mannheimia hemolytica;

[0037] Figure 6 This is a picture of the colony morphology of Pasteurella multocida;

[0038] Figure 7 This is the Blast result diagram after sequencing of Mycoplasma ovipneumoniae;

[0039] Figure 8 This is the Blast result diagram after sequencing of Mycoplasma mycoides subspecies caprine;

[0040] Fig. 9 This is the Blast result diagram after sequencing of Mannheimia hemolytica;

[0041] Fig.10 This is the Blast result diagram after sequencing of type D sheep Pasteurella multocida;

[0042] Fig.11 This is a graph showing the results of the serum antibody test for type D Pasteurella multocida;

[0043] Fig.12 This is the Blast result diagram after sequencing of Streptococcus equi subsp. zooepidemicus;

[0044] Fig.13 This is the result of Gram staining of Streptococcus equi subsp. zooepidemicus;

[0045] Fig.14 The results of the streptococcal complement-dependent cytotoxicity test in different volumes of serum are shown in Figure A: negative serum; B: 10 μL positive serum; C: 20 μL positive serum; D: 40 μL positive serum;

[0046] Fig.15 For Fig.14 After the colonies were counted on the plates, significance analysis was performed and a bar graph was drawn. DETAILED DESCRIPTION

[0047] The present invention provides a five-in-one inactivated vaccine for multiple ovine "lung rot", wherein the inactivated pathogens include Mycoplasma ovipneumoniae, Mycoplasma mycoides subspecies caprine, Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subspecies zooepidemicus.

[0048] The Mycoplasma ovipneumoniae of the present invention preferably includes Mycoplasma ovipneumoniae MO_BM13D004 strain, with a deposit number of CGMCC No.45420; the Mycoplasma mycoides subspecies caprae preferably includes Mycoplasma mycoides subspecies caprae Mm_NM02 strain, with a deposit number of CGMCC No.45419; the hemolytic Mannheimia A2 serotype preferably includes the hemolytic Mannheimia A2 serotype MS-NM2 strain, with a deposit number of CGMCC No.19797; the D-type Pasteurella multocida of sheep preferably includes Pasteurella multocida PmD100 strain, with a deposit number of CGMCC No.26636; the Streptococcus equi zooepidemicus subspecies preferably includes the Streptococcus equi zooepidemicus subspecies SeZ100 strain, with a deposit number of CGMCC No.26635. The Mycoplasma ovipneumoniae, Mycoplasma mycoides subspecies caprine, Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi zooepidemicus subspecies described in the present invention are all collected by the inventor from sheep farms and pastures in western, central and northeastern Inner Mongolia between 2018 and 2023, and diseased materials such as lungs, tracheal nasopharyngeal swabs and the like were isolated from multiple pathogen strains. The isolated strains were subjected to morphological observation, culture characteristics, biochemical characteristics, serological tests, virulence and immunogenicity tests, and finally strains with good immunogenicity and virulence were screened.

[0049] The present invention preferably uses the inactivated bacterial liquid of the above five pathogens as the inactivated pathogen, and the culture liquid of the inactivated pathogen liquid includes a modified KM2 liquid culture medium and a TSB liquid culture medium containing 5% horse serum. The composition of the culture medium described in the present invention preferably includes: each L of the modified KM2 culture medium includes the following components: 500 mL of 1640 cell culture liquid, 300 mL of Hanks liquid with a mass percentage of 1.7% hydrolyzed milk protein, 20 mL of yeast liquid with a mass percentage of 25%, 200 mL of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL of 1% thallium acetate, 5 mL of 0.4% phenol red, 4 g of glucose and 2 g of sodium pyruvate, and the pH value is 7.6-7.8;

[0050] Each liter of the TSB liquid culture medium containing 5% horse serum includes the following components: 17.0 g of trypticase peptone, 3.0 g of soy peptone, 2.5 g of D-glucose, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate and 200 mL of horse serum.

[0051] In the present invention, the inactivated bacterial liquid obtained by inactivating Mycoplasma ovipneumoniae after culturing in a modified KM2 liquid medium, the inactivated bacterial liquid obtained by inactivating Mycoplasma mycoides subspecies after culturing in a modified KM2 liquid medium, the inactivated bacterial liquid obtained by inactivating Mannheimia hemolytica serotype A2 after culturing in a TSB liquid medium containing 5% horse serum, the inactivated bacterial liquid obtained by inactivating Pasteurella multocida type D after culturing in a TSB liquid medium containing 5% horse serum, and the inactivated bacterial liquid obtained by inactivating Streptococcus equi subspecies zooepidemicus after culturing in a TSB liquid medium containing 5% horse serum, the above five inactivated bacterial liquids are mixed in a volume ratio of 0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2) as inactivated pathogens, and the amount of each pathogen in the pentavalent inactivated vaccine is (1-9)×10 8 CFU / mL or (1~9)×10 8 CCU / mL, the concentration of Mycoplasma ovipneumoniae MO-BM13D004 in the pentavalent inactivated vaccine is (1~9)×10 8 CCU / mL; the concentration of Mycoplasma mycoides subspecies Mm-NM02 in the pentavalent inactivated vaccine is (1~9)×10 8 CCU / mL; the concentration of hemolytic Mannheimia serotype A2 in the pentavalent inactivated vaccine is (1~9)×10 8 CFU / mL; the bacterial concentration of Pasteurella multocida type D in the pentavalent inactivated vaccine is (1~9)×10 8 CFU / mL; the bacterial concentration of Streptococcus equi subspecies zooepidemicus SeZ100 in the pentavalent inactivated vaccine is (1~9)×10 8 CFU / mL.

[0052] The pentavalent inactivated vaccine of the present invention preferably further comprises an adjuvant, and the type of the adjuvant preferably comprises a water-in-oil-in-water adjuvant, a water-in-oil adjuvant, a microemulsion adjuvant, an aluminum salt adjuvant or a Freund's adjuvant. In the pentavalent inactivated vaccine of the present invention, the volume ratio of the inactivated bacterial solution containing the inactivated pathogen to the adjuvant is preferably 1:(1-2).

[0053] The present invention also provides a method for preparing the above-mentioned pentavalent inactivated vaccine, comprising the following steps: culturing Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies caprae in a modified KM2 liquid culture medium and then inactivating them to obtain an inactivated bacterial solution;

[0054] Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subspecies zooepidemicus are cultured in TSB liquid medium containing 5% horse serum and then inactivated to obtain inactivated bacterial liquid;

[0055] Mix all the inactivated bacterial solutions in a volume ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2) to obtain an inactivated bacterial solution containing all inactivated pathogens;

[0056] The inactivated bacterial solution containing all inactivated pathogens is mixed with an immune adjuvant and then emulsified to obtain the pentavalent inactivated vaccine.

[0057] The culture of the present invention preferably includes inoculating the secondary seed liquid of Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies capricolum into a modified KM2 medium, and culturing at 37°C for 48 to 72 hours; inoculating the secondary seed liquid of Mannheimia hemolytica A2 serotype, Pasteurella multocida type D and Streptococcus equi zooepidemicus subspecies into a TSB medium containing 5% horse serum, and culturing at 37°C for 16 to 24 hours. The present invention does not specifically limit the preparation method of the secondary seed liquid of each bacterium, and it can be obtained by conventional activation and expansion culture methods in the art.

[0058] To further illustrate the present invention, the pentavalent inactivated vaccine for multiple lung rot in sheep provided by the present invention and its preparation method are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0059] The culture medium ratio and preparation method involved in the embodiments of the present invention are as follows:

[0060] Each L of tryptone soy broth (TSB) comprises the following components: 17 g / L of tryptone casein, 3 g / L of soy peptone, 2.5 g / L of D-glucose, 5.0 g / L of sodium chloride and 2.5 g / L of dipotassium hydrogen phosphate; the raw materials are mixed and dissolved in 1000 mL of purified water, the pH value is adjusted to 7.1-7.5, sterilized at 121° C. for 15 min, cooled to 60° C. and mixed with 5% horse serum by volume to obtain a tryptone soy broth containing 5% horse serum by volume;

[0061] Each liter of the improved KM2 liquid culture medium includes the following components: 500 mL of 1640 cell culture medium, 300 mL of Hanks solution with a mass percentage of 1.7% hydrolyzed milk protein, 20 mL of yeast solution with a mass percentage of 25%, 200 mL of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL of 1% thallium acetate, 5 mL of 0.4% phenol red, 4 g of glucose and 2 g of sodium pyruvate; the pH value is 7.6-7.8. After the raw materials are evenly mixed, the pH is adjusted to 7.6-7.8 with NaOH, and filtered through a 0.22 μm filter to obtain the improved KM2 liquid culture medium.

[0062] Example 1 Mycoplasma ovipneumoniae MO_BM13D004 strain

[0063] Lung lesions were collected from a farm in Hohhot and sent to the laboratory at low temperature. Under sterile conditions, the surface of the sample was disinfected by wiping with alcohol, and then the lesions at the junction of the lung lesions were separated using sterile instruments. After adding 1 mL of normal saline, the tissue was homogenized. After the homogenate was filtered through a 0.22 μm filter, it was inoculated into a modified KM2 medium supplemented with double antibodies and thallium acetate, and cultured in an incubator at 37°C for 72 hours. When the culture medium turned yellow, the whole genome DNA of the pathogen was extracted, and 16S rDNA universal primers were used for PCR amplification and then sent for sequencing. The sequencing results are shown in SEQ ID No. 1, and the pathogen was confirmed to be Mycoplasma ovipneumoniae ( Figure 7 ), named as strain MO_BM13D004.

[0064] 1) Colony morphology: Under a microscope at 40 times magnification, the colonies have obvious protrusions and very small transparent colonies can be seen. The colonies appear as round protrusions, without a navel, and are mulberry-shaped.

[0065] 2) Biochemical characteristics: The isolated strain could ferment glucose, reduce methylene blue, and liquefy serum; it could not hydrolyze urea and arginine; and the colony adsorption to erythrocytes was negative (Table 1).

[0066] Table 1 Biochemical identification results

[0067] Test items MO-BM13D004 Hydrolyzed Arginine Negative Hydrolyzed urea Negative Meilan reduction Positive Fermentation of glucose Positive Horse serum liquefaction Positive Adsorption of red blood cells Negative

[0068] 3) Toxicity

[0069] Use 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 Four healthy one-month-old lambs were injected intratracheally with CCU / mL bacterial solution and observed for 21 consecutive days. 9 and 1×10 8 All the patients in the CCU / mL challenge group developed symptoms of cough, runny nose, and fever. The autopsy showed adhesions in the lungs and obvious pneumonia symptoms. 7 CCU / mL challenge group half of the disease, 1×10 6 There was no disease in the CCU / mL challenge group, and the lungs were normal after autopsy. The pathogen Mycoplasma ovipneumoniae could be isolated from the lungs and trachea of ​​the diseased sheep.

[0070] 4) Immunogenicity

[0071] The inactivated bacterial solution was obtained by inactivating the Mycoplasma ovipneumoniae strain after culturing, and the inactivated bacterial solution was mixed with the adjuvant in a volume ratio of 1:1 to prepare the inactivated vaccine. The inactivated vaccine was used to immunize rabbits twice with an interval of 14 days, and 1 mL was used per rabbit for each immunization. The immunogenicity test showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 21 to 35 days, and the peak period could last for 5 to 7 weeks, and then slowly declined ( Figure 1 ).

[0072] Example 2 Mycoplasma mycoides subsp. caprae

[0073] Lung lesions were collected from a farm in Chifeng City and sent to the laboratory at low temperature. Under sterile conditions, the surface of the sample was disinfected by wiping with alcohol, and then the lesions at the junction of the lung lesions were separated using sterile instruments. After adding 1 mL of normal saline, the tissue was homogenized. After filtering the homogenate through a 0.22 μm filter, it was inoculated into a modified KM2 medium supplemented with double antibodies and thallium acetate, and cultured in an incubator at 37°C for 72 hours. When the culture medium turned yellow, the whole genome DNA of the pathogen was extracted, and 16S rDNA universal primers were used for PCR amplification and then sent for sequencing. The sequencing sequence is shown in SEQ ID No. 2. Blast confirmed that the pathogen was Mycoplasma mycoides subspecies capricolum ( Figure 8 ), named as Mm-NM02 strain.

[0074] 1) Colony morphology: Under a microscope at 40 times magnification, the colonies have obvious protrusions, and very small transparent colonies can be seen. The colonies appear as round protrusions, shaped like fried eggs ( Figure 2 ).

[0075] 2) Biochemical characteristics: The isolated strain can ferment glucose, digest casein, reduce methylene blue, and liquefy serum; it cannot hydrolyze urea and arginine; and the colony adsorption to erythrocytes is negative (Table 2).

[0076] Table 2 Biochemical identification results

[0077] Test items Mm-NM02 Hydrolyzed Arginine Negative Hydrolyzed urea Negative Meilan reduction Positive Fermentation of glucose Positive Horse serum liquefaction Positive Adsorption of red blood cells Negative Digesting Casein Positive

[0078] 3) Toxicity

[0079] Use 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 Four healthy one-month-old lambs were injected intratracheally with CCU / mL bacterial solution and observed for 21 consecutive days. 9 , 1×10 8 and 1×10 7 All the patients in the CCU / mL challenge group developed symptoms of cough, runny nose, and fever. The chest wall and lungs were adhered to each other, with fibrinous exudate and obvious pneumonia symptoms. 6Half of the CCU / mL challenged sheep became ill, and the chest wall and lungs were found to be adhered to each other during autopsy. The pathogen Mycoplasma mycoides subspecies was isolated from the lungs and trachea of ​​the infected sheep.

[0080] 4) Immunogenicity

[0081] The inactivated bacterial solution was obtained by culturing and inactivating the Mycoplasma capricolum subspecies strain, and the inactivated bacterial solution was mixed with the adjuvant in a volume ratio of 1:1 to prepare the inactivated vaccine. The inactivated vaccine was used to immunize rabbits twice with an interval of 14 days, 1 mL per rabbit each time, and immunogenicity was tested. The results showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 28 to 35 days, and the peak period could last for 6 to 8 weeks, and then slowly declined ( Figure 3 ).

[0082] Example 3 Hemolytic Mannheimia

[0083] Lung lesions were collected from a farm in Hohhot and sent to the laboratory at low temperature. Under sterile conditions, the surface of the sample was disinfected by wiping with alcohol, and then the surface of the tissue at the junction of the lung lesions was cut open with sterile instruments. The lesions were clamped with sterile tweezers and smeared on TSA solid culture medium, and cultured in an incubator at 37°C for 24 hours. Suspected colonies were picked and purified and cultured three times, then examined by Gram staining, and the whole genome DNA of the pathogen was extracted. PCR amplification was performed with 16S rDNA universal primers and then sent for sequencing. The sequencing sequence is shown in SEQ ID No. 3. Blast confirmed that the pathogen was hemolytic Mannheimia, named MS-NM2 strain ( Fig. 9 ).

[0084] 1) Gram staining characteristics of bacteria: coccobacilli or short rods, with blunt ends, Gram staining negative ( Figure 4 ).

[0085] 2) Biochemical characteristics: The isolate fermented glucose, lactose, maltose, L-arabinose and other sugars, fermented mannitol, but not mannose. It was positive for oxidase and catalase, but negative for urease (Table 3).

[0086] Table 3 Biochemical identification results

[0087] Test items MS-NM2 Fermentation of glucose Positive Fermented lactose Positive Fermented maltose Positive Fermentation of L-arabinose Positive Fermented mannitol Positive Fermented mannose Negative Oxidase and catalase reactions Negative Urease activity Negative

[0088] 3) Toxicity

[0089] Use 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 Four healthy lambs of one month old were injected into the trachea with the bacterial solution and observed for 21 consecutive days. 9 and 1×10 8All the patients in the CFU / mL challenge group died, with obvious clinical symptoms of cough, runny nose, and fever. The autopsy showed adhesions in the lungs and obvious large-area consolidation in the lungs. 7 CFU / mL and 1×10 6 All the patients in the challenge group developed disease, including 1×10 7 Three mice died in the CFU / mL group, and 1×10 6 One sheep died in the CFU / mL group. The pathogen of Mannheimia hemolytica could be isolated from the lungs and trachea of ​​the diseased sheep.

[0090] 4) Immunogenicity

[0091] The inactivated bacterial solution was obtained by culturing and inactivating the hemolytic Mannheimia strain. The inactivated bacterial solution was mixed with the adjuvant in a volume ratio of 1:1 to prepare the inactivated vaccine. The inactivated vaccine was used to immunize rabbits twice with an interval of 14 days, with 1 mL per rabbit each time. The immunogenicity test showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 28 to 49 days, and the peak period could last for 8 to 10 weeks ( Figure 5 ).

[0092] Example 4 Type D Pasteurella multocida

[0093] Lung lesions were collected from a farm in Hohhot and sent to the laboratory at low temperature. Under sterile conditions, the surface of the sample was disinfected by wiping with alcohol, and then the surface of the tissue at the junction of the lung lesions was cut open with sterile instruments. The lesions were clamped with sterile tweezers and smeared on TSA solid culture medium, and cultured in an incubator at 37°C for 24 hours. Suspected colonies were picked and purified and cultured three times, then examined by Gram staining, and the whole genome DNA of the pathogen was extracted. PCR amplification was performed with 16S rDNA universal primers and then sent for sequencing. The sequencing sequence is shown in SEQ ID No. 4. The pathogen was confirmed by Blast to be type D Pasteurella multocida of sheep, named PmD100 strain ( Fig.10 ).

[0094] 1) Colony morphology: round, slightly raised, neatly edged, smooth surface, blue and translucent, with a slight metallic luster ( Figure 6 ).

[0095] 2) Biochemical characteristics: The isolated strain fermented glucose, mannitol, and D-mannose, but did not ferment mannose, lactose, maltose, and L-arabinose. It was positive for oxidase and catalase, but negative for urease. (Table 4)

[0096] Table 4 Biochemical identification results

[0097] Test items PmD100 Fermentation of glucose Positive Fermented lactose Negative Fermented maltose Negative Fermentation of L-arabinose Negative Fermented mannitol Positive Fermented D-Mannose Positive Oxidase and catalase reactions Positive Urease activity Negative

[0098] 3) Toxicity

[0099] Use 1×10 9, 1×10 8 , 1×10 7 , 1×10 6 Four healthy lambs of one month old were injected into the trachea with the bacterial solution and observed for 21 consecutive days. 9 All the patients in the CFU / mL challenge group died, with obvious clinical symptoms of cough, runny nose, and fever. The autopsy showed adhesions in the lungs and obvious large-area consolidation in the lungs. 8 , 1×10 7 and 1×10 6 All the patients in the challenge group developed disease, including 1×10 8 and 1×10 7 CFU / mL infection group 2 deaths, 1×10 6 One sheep died in the CFU / mL challenge group. Pasteurella multocida pathogens were isolated from the lungs and trachea of ​​the diseased sheep.

[0100] 4) Immunogenicity

[0101] The inactivated bacterial solution was obtained by culturing and inactivating the Pasteurella multocida strain. The inactivated bacterial solution was mixed with the adjuvant in a volume ratio of 1:1 to prepare the inactivated vaccine. The inactivated vaccine was used to immunize rabbits twice with an interval of 14 days, 1 mL per rabbit each time, and immunogenicity was tested. The results showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 21 to 42 days, and the peak period could last for 9 to 11 weeks ( Fig.11 ).

[0102] Example 5 Streptococcus equi subsp. zooepidemicus

[0103] Lung lesions were collected from a farm in Bayannur City and sent to the laboratory at low temperature. Under sterile conditions, the surface of the sample was disinfected by wiping with alcohol, and then the surface of the tissue at the junction of the lung lesions was cut open with sterile instruments. The lesions were clamped with sterile tweezers and smeared on TSA solid culture medium, and cultured in an incubator at 37°C for 24 hours. Suspected colonies were picked and purified and cultured three times, then examined by Gram staining, and the whole genome DNA of the pathogen was extracted. PCR amplification was performed with 16S rDNA universal primers and then sent for sequencing. The sequencing sequence is shown in SEQ ID No. 5. The pathogen was identified as Streptococcus equi subspecies zooepidemicus by Blast, named SeZ100 strain ( Fig.12 ).

[0104] 1) Gram staining characteristics of bacteria: The bacteria are spherical, arranged in pairs or chains, and Gram staining is positive ( Fig.13 ).

[0105] 2) Biochemical characteristics: The isolate fermented glucose, sucrose, lactose and other sugars, fermented arginine, but did not ferment trehalose, raffinose and mannitol. (Table 5)

[0106] Table 5 Biochemical identification results

[0107] Test items SeZ100 Fermented trehalose Negative Fermented lactose Positive Hydrolyzed Arginine Positive Fermented mannitol Negative Fermented Raffinose Negative Fermentation of glucose Positive Fermentation of sucrose Positive

[0108] 3) Toxicity

[0109] Use 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 Four healthy lambs of one month old were injected into the trachea with the bacterial solution and observed for 21 consecutive days. 9 , 1×10 8 and 1×10 7 All the patients in the CFU / mL challenge group died, with obvious clinical symptoms of cough, runny nose, and fever. The autopsy showed lung adhesions, a large amount of serous fibrinous exudate in the chest cavity, and obvious large-area consolidation in the lungs. 6 All the CFU / mL challenged sheep became ill, and 2 died. Streptococcus equi subsp. zooepidemicus pathogens were isolated from the lungs and trachea of ​​the infected sheep.

[0110] 4) Immunogenicity

[0111] The inactivated bacterial liquid was obtained by inactivating the Streptococcus equi subsp. zooepidemicus strain after culturing, and the inactivated bacterial liquid was mixed with the adjuvant in a volume ratio of 1:1 to prepare the inactivated vaccine. The inactivated vaccine was used to immunize rabbits twice with an interval of 14 days, 1 mL per rabbit each time, and immunogenicity was tested. The results of the complement-dependent cytotoxicity experiment showed that the high immune serum could effectively inhibit the growth of Streptococcus equi subsp. zooepidemicus strains in the complement environment ( Fig.14 , Fig.15 ).

[0112] Example 6 Production and preparation of multi-link inactivated vaccine

[0113] 1. Preparation of strains for the production of Mycoplasma ovipneumoniae MO_BM13D004 and Mycoplasma mycoides subspecies capricolum Mm_NM02

[0114] 1. Preparation of production strains

[0115] 1) Primary seed propagation

[0116] Mycoplasma ovipneumoniae strain MO_BM13D004 and Mycoplasma mycoides subspecies caprine strain Mm_NM02 were inoculated in improved KM2 medium at 10% subculture, cultured at 37°C for 72h, and used as primary seeds after passing the inspection.

[0117] 2) Secondary seed propagation

[0118] The primary seeds of each mycoplasma strain were inoculated into the improved KM2 medium at 10%, and cultured in a shaking incubator at 37°C for 72 hours. After passing the purity test, they were used as secondary seeds.

[0119] 2 Antigen culture

[0120] 2.1 Preparation of bacterial culture

[0121] The secondary seeds of the two mycoplasma strains were inoculated into the modified KM2 medium at 10%, respectively, and cultured at 37°C with shaking for 72 hours, and then the bacterial liquid was harvested.

[0122] The mycoplasma solution prepared in 2.1 is tested for purity and viable bacteria count according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine. If the result is pure, it is qualified.

[0123] 2.2 Antigen inactivation

[0124] Live bacteria count: Use the conventional CCU counting method to count, the bacterial solution concentration can reach 5×10 8 and 1×10 9 CCU / mL.

[0125] Purity test: Take samples from the two cultured bacterial liquids mentioned above, culture them using PPLO solid medium containing 20% ​​horse serum, pyruvic acid, and glucose, and conduct purity test with reference to the appendix of the "Pharmacopoeia of the People's Republic of China", which should be pure.

[0126] 2.3 Inactivation of bacterial liquid

[0127] 0.2% β-propiolactone in total volume was added to the culture fluid of the two mycoplasma strains respectively, and the culture fluid was inactivated at 4°C for 48 hours, mixed once every 6 hours, and then incubated at 37°C and 150 rpm for 2 hours to degrade β-propiolactone.

[0128] Inactivation test

[0129] Take 0.2 mL of the inactivated bacterial liquid of the two strains respectively and inoculate it into the improved KM2 liquid culture medium, culture it at 37℃, 150rpm for 7 days, and at the same time inoculate it into PPLO solid culture medium and culture it continuously for 7 days. According to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", it is qualified if there is no mycoplasma growth.

[0130] 2. Preparation of strains for the production of Mannheimia hemolytica serotype A2 MS-NM2, Pasteurella multocida type D PmD100, and Streptococcus equi subspecies zooepidemicus SeZ100

[0131] 1. Preparation of production strains

[0132] 1) Primary seed propagation

[0133] The hemolytic Mannheimia A2 serotype strain MS-NM2, the type D Pasteurella multocida strain PmD100, and the Streptococcus equi subsp. zooepidemicus strain SeZ100 were inoculated at 1% in TSB+5% horse serum medium, cultured at 37°C for 18 hours, and used as primary seeds after passing the inspection.

[0134] 2) Secondary seed propagation

[0135] The above-mentioned first-level seeds of Mannheimia hemolytica, Pasteurella multocida and Streptococcus were inoculated into TSB+5% horse serum culture medium at 1% respectively, cultured in a shaking incubator at 37°C for 18 hours, and used as second-level seeds after passing the purity test.

[0136] 2 Antigen culture

[0137] 2.1 Preparation of bacterial culture

[0138] 1% of the qualified secondary seeds of Mannheimia hemolytica, Pasteurella multocida and Streptococcus were inoculated into TSB+5% horse serum medium, and cultured at 37°C on a shaking incubator for 18 hours, and then the bacterial liquid was harvested.

[0139] The bacterial solution prepared in 2.1 is tested for purity and viable bacteria count according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine. If the result is pure, it is qualified.

[0140] 2.2 Antigen inactivation

[0141] Live bacteria count: Use the conventional CFU counting method to count, the bacterial solution concentration can reach 8×10 8 , 5×10 8 and 3×10 8 CFU / mL.

[0142] Purity test: Take samples from the three cultured bacterial liquids mentioned above, culture them using TSA solid medium containing 5% horse serum, and conduct purity test with reference to the appendix of the "Pharmacopoeia of the People's Republic of China". It should be pure.

[0143] 2.3 Inactivation of bacterial liquid

[0144] To the culture fluids of Mannheimia hemolytica, Pasteurella multocida and Streptococcus strains, add 0.5% formaldehyde aqueous solution by volume at 1% of the total bacterial fluid, mix well and place at 37°C for 48 hours, shaking once during the period.

[0145] Inactivation test

[0146] Take 0.2 mL of the inactivated bacterial solution of the above strains and inoculate it into 5 mL TSB + 5% horse serum medium, incubate it at 37°C, 150rpm for 3 days, and at the same time inoculate TSA solid medium and culture it continuously for 3 days. According to the appendix of the current "Chinese Veterinary Pharmacopoeia", it is qualified if there is no bacterial growth.

[0147] 3. Preparation of Pentavalent Inactivated Vaccine

[0148] The five antigen bacterial liquids that passed the inactivation test were evenly mixed in a volume ratio of 1:1:1:1:1, and then emulsified with water-in-oil-in-water mineral oil adjuvant in a mass ratio of 1:1.2. The emulsification conditions were: 31°C, 300 rpm, and gentle stirring for 30 min. Microscopic examination showed that the vaccine droplets were small and uniform, and the emulsified vaccine diffused evenly after being dropped into water.

[0149] 3.1 Sterility test

[0150] Take the above vaccines and conduct sterility test according to the appendix of the Veterinary Pharmacopoeia of the People's Republic of China. Sterile growth should be observed.

[0151] 3.2 Safety Inspection

[0152] Take the above vaccine and inject it subcutaneously into the neck of 3 SPF Balb / c mice, 0.2mL each. All of them are alive and well within 7 days. They are qualified. Take 3 rabbits weighing 1.5-2kg and inject them subcutaneously at multiple points on the back, 2mL each. Observe for 10 days and all should be alive. Take the above vaccine and inject it subcutaneously into the neck of 3 sheep, 10mL each. All of them are alive and well within 7 days. They are qualified.

[0153] Example 7 Vaccine efficacy study of pentavalent inactivated vaccine

[0154] 1 Experimental design

[0155] 1.1 Vaccine Immunization

[0156] The pentavalent inactivated vaccine prepared in Example 6 was used, and 5 healthy weaned goat lambs aged 4 to 6 weeks were injected subcutaneously in the neck of each head with 3 mL, and the second vaccination was performed 3 weeks after the first vaccination, with the same vaccination dose. At the same time, 5 healthy weaned goat lambs were immunized with PBS in each group as a control.

[0157] 1.2 Attack

[0158] Three weeks after the second immunization, the pathogen was challenged by tracheal injection with a virulent strain (Mycoplasma ovipneumoniae MO-BM13D004 (1×10 8 CCU / mL); Mycoplasma mycoides subspecies capricolum Mm-NM02 (1×10 7 CCU / mL); Mannheimia hemolytica serotype A2 MS-NM2 (1×10 8CFU / mL); Pasteurella multocida type D PmD100 (1×10 9 CFU / mL); Streptococcus equi subsp. zooepidemicus SeZ100 (1×10 7 CFU / mL)) 5mL / head by tracheal injection; the attack groups are as shown in Table 6.

[0159] Table 6 Attack group

[0160]

[0161] 1.3 Effectiveness test results

[0162] After the challenge of Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies, the clinical symptoms and the number of deaths in the immunized group and the control group were recorded for 2 months; after the challenge of hemolytic Mannheimia A2 type, D type sheep multocida Pasteurella and equine Streptococcus zooepidemicus subspecies, the clinical symptoms and the number of deaths in the immunized group and the control group were recorded for 1 month, and the clinical symptoms were coughing and fever in the sheep. The results are shown in Table 7: in the mycoplasma challenge group, the protection rate of the immunized group after the experimental animals were dissected was 80%, and the control group was not protected. The sheep lungs all had lesions, and the incidence rate was 100%; in the hemolytic Mannheimia A2 type and equine Streptococcus zooepidemicus subspecies, one experimental animal in the immunized group died, the protection rate of the immunized group was 80%, and all experimental animals in the control group died, with a mortality rate of 100%; in the D type sheep multocida Pasteurella challenge group, all experimental animals in the immunized group survived, the protection rate of the immunized group was 100%, and all experimental animals in the control group died, with a mortality rate of 100%. The pentavalent inactivated vaccine of the present invention can achieve the purpose of providing multiple protections with one injection, reducing costs, and reducing sheep stress.

[0163] Table 7 Efficacy test results

[0164]

[0165]

[0166] 1.4 Comparison with commercial vaccines

[0167] Comparison of pentavalent inactivated vaccine and commercial vaccine in immunization and virus attack:

[0168] Vaccine A: Example 6 Preparation of Pentavalent Inactivated Vaccine

[0169] Vaccine B: Commercial inactivated vaccine

[0170] The pentavalent inactivated vaccine prepared in Example 6 was used, and 4 healthy weaned goat lambs aged 4 to 6 weeks were injected subcutaneously in the neck of each head with 3 mL, and the second immunization was performed 3 weeks after the first immunization, with the same immunization dose. At the same time, 4 healthy weaned goat lambs in each group were immunized with commercial inactivated vaccines and PBS as controls.

[0171] Three weeks after the second immunization, the pathogen was challenged by tracheal injection with a virulent strain (Mycoplasma ovipneumoniae MO-BM13D004 (1×10 8 CCU / mL); Mycoplasma mycoides subspecies capricolum Mm-NM02 (1×10 7 CCU / mL); Mannheimia hemolytica serotype A2 MS-NM2 (1×10 8 CFU / mL); Pasteurella multocida type D PmD100 (1×10 9 CFU / mL); Streptococcus equi subsp. zooepidemicus SeZ100 (1×10 7 CFU / mL)) 5mL / head by tracheal injection; the attack groups are as shown in Table 8.

[0172] Observation after virus attack: There was a significant difference in immune protection between the vaccine group and the non-immunized group and the commercial vaccine group. After virus attack, all the non-immunized group and the commercial vaccine group became ill or died, and the protection rate of the multi-vaccine group was higher than that of the non-immunized blank control group and the commercial vaccine group. Within one month after immunization with the commercial vaccine group, the titer of Mycoplasma ovipneumoniae antibodies in the experimental animals was extremely low, and the antibody titer could not reach the immune protection level during virus attack; at the same time, the commercial vaccine did not contain Mycoplasma mycoides, Pasteurella, Mannheimia and Streptococcus antigens, and the experimental animals could not produce corresponding antibodies, and could not provide corresponding protection during virus attack, so all of them became ill or died.

[0173] Table 8 Comparison of immunization with pentavalent inactivated vaccine and commercial vaccine

[0174]

[0175]

[0176] Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A five-in-one inactivated vaccine against multiple lung rot in sheep, characterized in that: The inactivated pathogens include Mycoplasma ovipneumoniae, Mycoplasma mycoides subsp. capricolum, Mannheimia haemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subsp. zooepidemicus.

2. The pentavalent inactivated vaccine according to claim 1, characterized in that: The Mycoplasma ovipneumoniae includes the Mycoplasma ovipneumoniae MO_BM13D004 strain, with a deposit number of CGMCC No.45420; The mycoplasma mycoides subspecies caprae includes the mycoplasma mycoides subspecies caprae subspecies Mm_NM02 strain, with a deposit number of CGMCC No.45419; The hemolytic Mannheimia A2 serotype includes the hemolytic Mannheimia A2 serotype MS-NM2 strain, with a deposit number of CGMCC No.19797; The D-type Pasteurella multocida of sheep includes Pasteurella multocida PmD100 strain, and its deposit number is CGMCC No.26636; The Streptococcus equi subspecies zooepidemicus includes the Streptococcus equi subspecies zooepidemicus SeZ100 strain, and its preservation number is CGMCC No.26635.

3. The pentavalent inactivated vaccine according to claim 2, characterized in that: The inactivated pathogens include inactivated pathogenic bacteria liquid, and the culture medium of the inactivated pathogenic bacteria liquid includes improved KM2 liquid culture medium and TSB liquid culture medium containing 5% horse serum.

4. The pentavalent inactivated vaccine according to claim 3, characterized in that: The inactivated pathogenic bacteria liquid comprises: an inactivated bacteria liquid obtained by inactivating Mycoplasma ovipneumoniae after being cultured in a modified KM2 liquid culture medium, an inactivated bacteria liquid obtained by inactivating Mycoplasma mycoides subspecies after being cultured in a modified KM2 liquid culture medium, an inactivated bacteria liquid obtained by inactivating Mannheimia hemolytica serotype A2 after being cultured in a TSB liquid culture medium containing 5% horse serum, an inactivated bacteria liquid obtained by inactivating Pasteurella multocida type D after being cultured in a TSB liquid culture medium containing 5% horse serum, and an inactivated bacteria liquid obtained by inactivating Streptococcus equi subspecies zooepidemicus after being cultured in a TSB liquid culture medium containing 5% horse serum.

5. The pentavalent inactivated vaccine according to claim 3 or 4, characterized in that: Each liter of the modified KM2 medium includes the following components: 500 mL of 1640 cell culture medium, 300 mL of 1.7% by weight Hanks solution of hydrolyzed milk protein, 20 mL of 25% by weight yeast solution, 200 mL of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL of 1% by weight thallium acetate, 5 mL of 0.4% by weight phenol red, 4 g of glucose and 2 g of sodium pyruvate, and the pH value is 7.6-7.8; Each liter of the TSB liquid culture medium containing 5% horse serum includes the following components: 17.0 g of trypticase peptone, 3.0 g of soy peptone, 2.5 g of D-glucose, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate and 200 mL of horse serum.

6. The pentavalent inactivated vaccine according to claim 1, characterized in that: The amount of each pathogen in the five-in-one inactivated vaccine is (1-9)×10 8 CFU / mL or (1~9)×10 8 CCU / mL.

7. The pentavalent inactivated vaccine according to claim 1, characterized in that: The pentavalent inactivated vaccine also includes an adjuvant; The types of adjuvants include water-in-oil-in-water adjuvants, water-in-oil adjuvants, microemulsion adjuvants, aluminum salt adjuvants or Freund's adjuvants.

8. The pentavalent inactivated vaccine according to claim 7, characterized in that: The volume ratio of the inactivated bacteria liquid containing the inactivated pathogen to the adjuvant is 1:(1-2).

9. The method for preparing the pentavalent inactivated vaccine according to any one of claims 1 to 8, characterized in that: The following steps are involved: Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies capricolum were cultured in improved KM2 liquid medium and then inactivated to obtain inactivated bacterial liquid; Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subspecies zooepidemicus are cultured in TSB liquid medium containing 5% horse serum and then inactivated to obtain inactivated bacterial liquid; Mix all the inactivated bacterial solutions in a volume ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2) to obtain an inactivated bacterial solution containing all inactivated pathogens; The inactivated bacterial solution containing all inactivated pathogens is mixed with an immune adjuvant and then emulsified to obtain the pentavalent inactivated vaccine.

10. The preparation method according to claim 9, characterized in that: The culture comprises inoculating the secondary seed liquid of Mycoplasma ovipneumoniae and Mycoplasma mycoides subspecies capricolum into the improved KM2 culture medium respectively, and culturing at 37° C. for 48 to 72 hours; The secondary seed liquids of Mannheimia hemolytica serotype A2, Pasteurella multocida type D and Streptococcus equi subsp. zooepidemicus were inoculated into TSB medium containing 5% horse serum respectively, and cultured at 37°C for 16 to 24 hours.

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