Triple inactivated vaccine for sheep multiple lung rot disease and preparation method thereof

Abstract

The invention discloses a triple inactivated vaccine for sheep multiple lung rot diseases and a preparation method, and belongs to the technical field of animal vaccines. The three pathogenic bacteria of the mycoplasma ovipneumoniae, the D-type sheep pasteurella multocida and the streptococcus equi subsp. Zooepidemicus are combined in one vaccine, so that the immunization of one vaccine can be realized, meanwhile, an effective antibody for immunizing various pathogens is generated by animals, the effect of preventing multiple diseases by one injection is achieved, and the prevention and control effect of the disease is improved. The triple inactivated vaccine disclosed by the invention can enable an animal body to generate effective immune response and generate relatively high antibody titer. The vaccine provided by the invention can reduce the working intensity of immunization personnel, has very important clinical application value and wide market prospect, and will produce huge social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of animal vaccines, and in particular relates to a triple inactivated vaccine against 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 stall feeding or semi-stalling, 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.

[0003] Although there are monovalent or bivalent inactivated vaccines for goat and sheep pneumonia in China, the antigen composition is single and the prevention effect is low. According to the feedback from sheep farms in major breeding areas across the country, the protection rate of these vaccines against sheep infectious pleuropneumonia (lung rot) is within 45% to 70%. Experimental research and diagnosis and treatment practice show that the antigen spectrum of existing commercial vaccines is targeted at sheep pneumonia, and the types of common infectious pathogens in the clinic are not completely symmetrical. The immune antibodies produced cannot fully protect against mixed pathogen infections. This is a bottleneck problem in the core prevention and control technology itself, which needs to be broken through. Summary of the invention

[0004] The purpose of the present invention is to provide a triple inactivated vaccine against multiple "lung rot" in sheep and a preparation method, which is suitable for multiple "lung rot" in sheep caused by local multi-pathogen mixed infection in cold and arid areas in the north. One vaccine can immunize animals and simultaneously produce effective antibodies against multiple pathogens, achieving the effect of "one shot for multiple protections".

[0005] The present invention provides an antigen composition for multiple "lung rot" in sheep, comprising inactivated strains, inactivated bacteria, inactivated bacterial liquid or inactivated fermentation products of Mycoplasma ovipneumoniae MO-BM13D004 strain, D-type Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain;

[0006] The deposit number of the Mycoplasma ovipneumoniae MO-BM13D004 strain is CGMCC No.45420, the deposit number of the D-type Pasteurella multocida PmD100 strain is CGMCC No.26636, and the deposit number of the Streptococcus equi subspecies zooepidemicus SeZ100 strain is CGMCC No.26635.

[0007] The present invention also provides the use of the antigen composition in preparing a vaccine for preventing and / or treating multiple "lung rot" in sheep.

[0008] The present invention also provides a vaccine against multiple ovine lung rot, comprising the above antigen composition and an adjuvant, wherein the types of the adjuvant include water-in-oil-in-water adjuvant, water-in-oil adjuvant, microemulsion adjuvant, aluminum salt adjuvant and Freund's adjuvant.

[0009] Preferably, the volume ratio of the total volume of the inactivated bacterial solution of the antigen composition to the volume of the adjuvant is 1:(1-2).

[0010] Preferably, the volume ratio of the inactivated bacterial solution of Mycoplasma ovipneumoniae MO-BM13D004 strain, type D Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain is (0.8-1.2):(0.8-1.2):(0.8-1.2).

[0011] The present invention also provides a method for preparing the triple inactivated vaccine, comprising the following steps: inoculating Mycoplasma ovipneumoniae MO_BM13D004 strain, Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain into a culture medium for culturing respectively, mixing inactivated bacterial liquids that have passed the inspection to obtain an inactivated bacterial liquid mixture;

[0012] The inactivated bacterial liquid mixture is mixed with an adjuvant and then emulsified to prepare the triple inactivated vaccine.

[0013] Preferably, the culturing comprises inoculating the Mycoplasma ovipneumoniae MO_BM13D004 strain into a modified KM2 medium and culturing at 37° C. for 48 to 72 hours;

[0014] Pasteurella multocida PmD100 strain and Streptococcus equi subsp. zooepidemicus SeZ100 strain were inoculated into TSB medium containing 5% horse serum, respectively, and cultured at 37°C for 48 to 72 hours.

[0015] Preferably, the concentration of Mycoplasma ovipneumoniae MO-BM13D004 strain in the triple inactivated vaccine is (1-9)×10 8 CCU / mL;

[0016] The bacterial concentration of the D-type Pasteurella multocida PmD100 strain in the triple inactivated vaccine is (1-9)×10 8 CFU / mL;

[0017] The bacterial concentration of Streptococcus equi subspecies zooepidemicus SeZ100 in the triple inactivated vaccine is (1-9)×10 8 CFU / mL.

[0018] Preferably, the improved KM2 culture medium includes components in the following concentrations: 500 mL / L of 1640 cell culture fluid, 300 mL / L of Hanks solution with a mass percentage of 1.7% hydrolyzed milk protein, 20 mL / L of yeast solution with a mass percentage of 25%, 200 mL / L of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL / L of 1% thallium acetate, 5 mL / L of 0.4% phenol red, 4 g / L of glucose and 2 g / L of sodium pyruvate, and the pH value is 7.6-7.8.

[0019] Preferably, the TSB medium containing 5% horse serum comprises components in the following concentrations: 17 g / L tryptic peptone, 3 g / L soy peptone, 2.5 g / L D-glucose, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate and 5% horse serum by volume, and a pH value of 7.1-7.5.

[0020] Beneficial effects: The present invention provides three pathogens, Mycoplasma ovipneumoniae, Pasteurella multocida type D and Streptococcus equi zooepidemicus, which are suitable for multiple "lung rot" in sheep caused by local multi-pathogen mixed infection in cold and arid areas in the north. The above three pathogens are combined in one vaccine, respectively, so that one vaccine can be used for immunization while animals can produce effective antibodies against multiple pathogens, which has the effect of "one shot for multiple protections" and improves the prevention and control effect of the disease. It has been confirmed by relevant serological test data that the triple inactivated vaccine of the present invention can make the animal body produce an effective immune response and generate a higher antibody titer. The attack protection test confirms that the triple inactivated vaccine can effectively protect the body from infection by the corresponding pathogens. At the same time, the triple inactivated vaccine of the present invention can avoid the activities of frequently catching 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.

[0021] Biological deposit information

[0022] 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.

[0023] 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.

[0024] The Streptococcus equisubsp. zooepidemicus SeZ100 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 CGMCC No. 26635. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of serum antibody test of Mycoplasma ovipneumoniae MO-BM13D004 strain;

[0026] Figure 2 This is the colony morphology of the D-type Pasteurella multocida strain PmD100;

[0027] Figure 3 This is a test chart of serum antibodies against the D-type sheep Pasteurella multocida strain PmD100;

[0028] Figure 4 This is a Gram staining result of Streptococcus equi subspecies zooepidemicus SeZ100 strain;

[0029] Figure 5 This is a graph showing the results of a streptococcal complement-dependent cytotoxicity test, in which A represents negative serum, B represents 10 μL positive serum, C represents 20 μL positive serum, and D represents 40 μL positive serum;

[0030] Figure 6 For Figure 5 After the colonies were counted on the plate, the significance analysis was performed and a bar graph was drawn;

[0031] Figure 7 This is a comparison chart of the sequencing results of Mycoplasma ovipneumoniae MO_BM13D004 strain;

[0032] Figure 8 This is a comparison chart of sequencing results of the D-type sheep Pasteurella multocida strain PmD100;

[0033] Fig. 9 This is a comparison chart of the sequencing results of Streptococcus equi subspecies zooepidemicus SeZ100 strain. DETAILED DESCRIPTION

[0034] The present invention provides an antigen composition for multiple "lung rot" in sheep, comprising inactivated strains, inactivated bacteria, inactivated bacterial liquid or inactivated fermentation products of Mycoplasma ovipneumoniae MO-BM13D004 strain, D-type Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain;

[0035] The deposit number of the Mycoplasma ovipneumoniae MO-BM13D004 strain is CGMCC No.45420, the deposit number of the D-type Pasteurella multocida PmD100 strain is CGMCC No.26636, and the deposit number of the Streptococcus equi subspecies zooepidemicus SeZ100 strain is CGMCC No.26635.

[0036] The above three strains of the present invention were isolated from the lungs, tracheal nasopharyngeal swabs and other diseased materials of sick sheep in sheep farms and pastures in western, central and northeastern Inner Mongolia, and multiple pathogen strains were isolated. The isolated strains were subjected to morphological observation, culture characteristics, biochemical characteristics, serological tests, virulence and immunogenicity tests, and finally the above three strains with good immunogenicity and virulence were screened.

[0037] The present invention also provides the use of the antigen composition in preparing a vaccine for preventing and / or treating multiple "lung rot" in sheep.

[0038] The present invention uses the Mycoplasma ovis pneumoniae MO-BM13D004 strain to prepare an inactivated vaccine, and specific antibodies can be detected in immunized rabbits 7 days later, and the antibodies reach a peak in 21-35 days, and the peak period can last for 5-7 weeks, and then slowly decline; the D-type Pasteurella multocida PmD100 strain is used to prepare an inactivated vaccine, and specific antibodies can be detected in immunized rabbits 7 days later, and the antibodies reach a peak in 21-42 days, and the peak period can last for 9-11 weeks; the Streptococcus equi zooepidemicus SeZ100 strain is used to prepare an inactivated vaccine, and immunogenicity detection is performed on immunized rabbits. The complement-dependent cytotoxicity experiment results show that the high-immune serum can effectively inhibit the growth of Streptococcus equi zooepidemicus strains in a complement environment. Therefore, the above three strains can be prepared into vaccines.

[0039] The present invention also provides a vaccine against multiple ovine lung rot, comprising the above antigen composition and an adjuvant, wherein the adjuvant includes a water-in-oil-in-water adjuvant.

[0040] The antigen composition used in the vaccine of the present invention is preferably the inactivated bacterial solution of the above three strains, and the volume ratio of the inactivated bacterial solution of Mycoplasma ovipneumoniae MO-BM13D004 strain, type D sheep Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain is preferably (0.8-1.2): (0.8-1.2): (0.8-1.2), more preferably 1:1:1. The total volume of the inactivated bacterial solution of the antigen composition of the present invention and the volume ratio of the adjuvant are preferably 1: (1-2), and the adjuvant is preferably ISA-201-VG immune oil adjuvant. The bacterial concentration of Mycoplasma ovipneumoniae MO-BM13D004 strain in the triple inactivated vaccine of the present invention is (1-9)×10 8 CCU / mL; the bacterial concentration of the D-type sheep Pasteurella multocida PmD100 strain in the triple inactivated vaccine is (1-9)×10 8 CFU / mL; the bacterial concentration of Streptococcus equi subspecies zooepidemicus SeZ100 in the triple inactivated vaccine is (1-9)×10 8 CFU / mL. The triple inactivated vaccine of the present invention has been confirmed by relevant serological test data to produce an effective immune response in the animal body and generate a higher antibody titer. The attack protection test confirmed that the vaccine can effectively protect the body from infection by the corresponding pathogen. At the same time, the triple 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.

[0041] The present invention also provides a method for preparing the triple inactivated vaccine, comprising the following steps: inoculating Mycoplasma ovipneumoniae MO_BM13D004 strain, Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain into a culture medium for culturing respectively, mixing inactivated bacterial liquids that have passed the inspection to obtain an inactivated bacterial liquid mixture;

[0042] The inactivated bacterial liquid mixture is mixed with an adjuvant and then emulsified to prepare the triple inactivated vaccine.

[0043] The culture of the present invention preferably includes inoculating the Mycoplasma ovipneumoniae MO_BM13D004 strain into a modified KM2 medium and culturing at 37°C for 48 to 72 hours; inoculating the Pasteurella multocida PmD100 strain and the Streptococcus equi subspecies zooepidemicus SeZ100 strain into a TSB medium containing 5% horse serum, respectively, and culturing at 37°C for 48 to 72 hours. The emulsification conditions are: 31°C, 300 rpm, gentle stirring for 30 minutes, and microscopic observation of the vaccine emulsion droplets is small and uniform, and the emulsified vaccine diffuses evenly after being dropped into water. The improved KM2 medium of the present invention preferably includes the following components in concentration: 500mL / L of 1640 cell culture medium, 300mL / L of Hanks solution with a mass percentage of 1.7% hydrolyzed milk protein, 20mL / L of yeast solution with a mass percentage of 25%, 200mL / L of horse serum, 2mL of 200,000 international units of penicillin, 14mL / L of 1% thallium acetate, 5mL / L of phenol red with a mass percentage of 0.4%, 4g / L of glucose and 2g / L of sodium pyruvate, and a pH value of 7.6 to 7.8. The present invention has no special restrictions on the preparation method of the improved KM2 liquid culture medium. In the embodiment, after mixing all the above raw materials evenly, the pH is adjusted to 7.6 to 7.8, and filtered through a 0.22μm filter to obtain the improved KM2 liquid culture medium; preferably, a 1mol / L NaOH aqueous solution is used to adjust the pH value.

[0044] The TSB medium containing 5% horse serum of the present invention preferably comprises the following components: 17 g / L of trypticase peptone, 3 g / L of soy peptone, 2.5 g / L of D-glucose, 5.0 g / L of sodium chloride, 2.5 g / L of dipotassium hydrogen phosphate and 5% horse serum by volume, and the pH value is 7.1-7.5, preferably 7.3. The present invention does not specifically limit the preparation method of the TSB medium containing 5% horse serum. In the embodiment, the remaining raw materials except horse serum are preferably mixed, the pH value is adjusted, sterilized at 121° C. for 15 min, cooled to 60° C. and mixed with 5% horse serum by volume to obtain trypticase soy broth (TBS) containing 5% horse serum by volume.

[0045] In order to further illustrate the present invention, a triple inactivated vaccine for multiple "lung rot" in sheep and a preparation method provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] 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 sequence is shown in SEQ ID No.1, and the pathogen was confirmed to be Mycoplasma ovipneumoniae ( Figure 7 ), named as strain MO_BM13D004.

[0048] 1. Mycoplasma ovipneumoniae strain MO_BM13D004

[0049] 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.

[0050] 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).

[0051] Table 1 Biochemical identification results

[0052] 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

[0053] 3) Toxicity

[0054] 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.

[0055] 4) Immunogenicity

[0056] The inactivated vaccine was prepared with Mycoplasma ovipneumoniae strains and the immunogenicity test was performed on rabbits. The results showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 21-35 days. The peak period lasted for 5-7 weeks and then slowly decreased. Figure 1 ).

[0057] Example 2

[0058] Lung disease samples were collected from a farm in Hohhot and sent to the laboratory at low temperature. Under sterile conditions, the sample surface 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 diseased samples 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 with 16S rDNA universal primers was performed and sent for sequencing. The sequencing sequence is shown in SEQ ID No. 2, and the pathogen was confirmed by Blast to be type D sheep Pasteurella multocida ( Figure 8 ), and named as strain PmD100.

[0059] Pasteurella multocida type D

[0060] 1) Colony morphology: round, slightly raised, neatly edged, smooth surface, blue and translucent, with a slight metallic luster ( Figure 2 ).

[0061] Figure 2 Pasteurella multocida colony morphology

[0062] 2) Biochemical characteristics: The isolate fermented glucose, mannitol, and D-mannose, but not mannose, lactose, maltose, and L-arabinose. It was positive for oxidase and catalase, but negative for urease (Table 2).

[0063] Table 2 Biochemical identification results

[0064] 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

[0065] 3) Toxicity

[0066] Use 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 Four healthy lambs of one month old were injected intratracheally with 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 6All 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.

[0067] 4) Immunogenicity

[0068] The inactivated vaccine was prepared with Pasteurella multocida strains and immunogenicity was tested by immunizing rabbits. The results showed that specific antibodies could be detected 7 days after immunization, and the antibodies reached a peak at 21-42 days, and the peak period could last for 9-11 weeks ( Figure 3 ).

[0069] Example 3

[0070] 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 with 16S rDNA universal primers was performed and sent for sequencing. The sequencing sequence is shown in SEQ ID No. 3. The pathogen was Streptococcus equi subsp. zooepidemicus ( Fig. 9 ), named as SeZ100 strain.

[0071] Streptococcus equi subsp. zooepidemicus SeZ100

[0072] 1) Gram staining characteristics of bacteria: The bacteria are spherical, arranged in pairs or chains, and Gram staining is positive ( Figure 4 ).

[0073] 2) Biochemical properties: The isolate fermented glucose, sucrose, lactose and other sugars, fermented arginine, and did not ferment trehalose, raffinose and mannitol (Table 3).

[0074] Table 3 Biochemical identification results

[0075] 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

[0076] 3) Toxicity

[0077] 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 , 1×108 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 patients in the challenge group developed disease, including 1×10 7 CFU / mL group. Streptococcus equi subspecies zooepidemicus pathogens can be isolated from the lungs and trachea of ​​diseased sheep.

[0078] 4) Immunogenicity

[0079] The inactivated vaccine was prepared with Streptococcus equi subsp. zooepidemicus strains, and the immunogenicity test was performed on rabbits. The results of complement-dependent cytotoxicity experiments showed that the high-immune serum could effectively inhibit the growth of Streptococcus equi subsp. zooepidemicus strains in the complement environment ( Figure 5 and Figure 6 ).

[0080] Example 4

[0081] Production and preparation of triple inactivated vaccine

[0082] 1. Preparation of Mycoplasma ovipneumoniae MO_BM13D004 production strain

[0083] 1. Preparation of production strains

[0084] 1) Primary seed propagation

[0085] The strain of Mycoplasma ovipneumoniae MO_BM13D004 was inoculated into the improved KM2 medium at 10% subculture, cultured at 37°C for 72h, and used as the primary seed after passing the inspection.

[0086] 2) Secondary seed propagation

[0087] The first-level mycoplasma seeds were inoculated into the modified 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 the second-level seeds.

[0088] 2 Antigen culture

[0089] 2.1 Preparation of bacterial culture

[0090] The secondary seeds of the mycoplasma strain were inoculated into the modified KM2 medium at 10%, and the culture was harvested after shaking and culturing at 37°C for 72 hours.

[0091] 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.

[0092] 2.2 Antigen inactivation

[0093] Live bacteria count: Use the conventional CCU counting method to count, the bacterial solution concentration can reach 5×10 8 CCU / mL

[0094] Purity test: Take a sample from the above cultured bacterial liquid and culture it with PPLO solid medium containing 20% ​​horse serum, pyruvic acid and glucose. Perform purity test with reference to the appendix of the "Pharmacopoeia of the People's Republic of China". It should be pure.

[0095] Bacterial liquid inactivation

[0096] 0.2% β-propiolactone in total volume was added to the culture medium of the Mycoplasma ovipneumoniae strain, and the culture medium was inactivated at 4°C for 48 hours, mixed every 6 hours, and then incubated at 37°C and 150 rpm for 2 hours to degrade β-propiolactone.

[0097] Inactivation test

[0098] Take 0.2 mL of the inactivated bacterial solution and inoculate it into the modified KM2 liquid culture medium, incubate it at 37°C, 150 rpm for 7 days, and at the same time inoculate it into the PPLO solid culture medium and continue to culture for 7 days. According to the appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", it is qualified if there is no mycoplasma growth.

[0099] 2. Preparation of Pasteurella multocida type D PmD100 and Streptococcus equi subsp. zooepidemicus SeZ100 production strains

[0100] 1. Preparation of production strains

[0101] 1) Primary seed propagation

[0102] The D-type Pasteurella multocida strain PmD100 and Streptococcus equi subsp. zooepidemicus strain SeZ100 were inoculated in TSB+5% horse serum medium at 1% respectively, cultured at 37°C for 18h, and used as primary seeds after passing the inspection.

[0103] 2) Secondary seed propagation

[0104] The above-mentioned Pasteurella multocida and Streptococcus primary seeds were inoculated at 1% in TSB + 5% horse serum culture medium, shaken and cultured at 37°C for 18 hours, and used as secondary seeds after passing the purity test.

[0105] 2 Antigen culture

[0106] 2.1 Preparation of bacterial culture

[0107] 1% of the qualified secondary seeds of Pasteurella multocida and Streptococcus strains 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.

[0108] 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.

[0109] 2.2 Antigen inactivation

[0110] Live bacteria count: Use the conventional CFU counting method to count, the bacterial solution concentration can reach 5×10 8 and 3×10 8 CFU / mL.

[0111] 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.

[0112] Bacterial liquid inactivation

[0113] To the culture fluid of 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.

[0114] Inactivation test

[0115] 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.

[0116] 2. Preparation of triple inactivated vaccine

[0117] The three antigen bacterial liquids that passed the inactivation test were evenly mixed in a volume ratio of 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 minutes. Microscopic examination showed that the vaccine droplets were small and uniform, and the emulsified vaccine diffused evenly after being dropped into water.

[0118] Sterility testing

[0119] 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.

[0120] Safety inspection

[0121] 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. 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.

[0122] 3. Immune protection test of triple inactivated vaccine

[0123] 1 Experimental design

[0124] 1.1 Vaccine Immunization

[0125] The triple inactivated vaccine prepared above was used to subcutaneously inject 3 mL of the vaccine into the neck of 5 healthy weaned goat lambs aged 4 to 6 weeks in each group. The second vaccination was performed 3 weeks after the first vaccination, and the immunization dose was the same. At the same time, 5 healthy weaned goat lambs in each group were immunized with PBS as a control.

[0126] 1.2 Attack

[0127] Three weeks after the second immunization, the pathogens were challenged by intratracheal injection. The virulent strains (Mycoplasma ovipneumoniae MO-BM13D004; Pasteurella multocida type D PmD100; Streptococcus equi subsp. zooepidemicus SeZ100) were injected into the trachea at 5 mL / head (100 MID). The challenge groups are shown in Table 4.

[0128] Table 4: Grouping of virus attack

[0129]

[0130] 1.2 Efficacy test results

[0131] After the challenge with Mycoplasma ovipneumoniae and Mycoplasma mycoides subsp. goat, the animals were observed for 2 months, and the clinical symptoms and the number of deaths in the immunized group and the control group were recorded; after the challenge with Mannheimia hemolytica type A2, type D Pasteurella multocida and Streptococcus equi subsp. zooepidemicus, the animals were observed for 1 month, and the clinical symptoms and the number of deaths in the immunized group and the control group were recorded. The clinical symptoms were coughing and fever in the sheep. The results are shown in Table 5.

[0132] Table 5 Efficacy test results

[0133]

[0134] The results showed that in the mycoplasma challenge group, the protection rate of the immunized group after the experimental animals were dissected was 80%, the control group had no protection, the sheep lungs all had lesions, and the incidence rate was 100%; in the Streptococcus equi zooepidemicus challenge group, 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 Pasteurella multocida 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 triple inactivated vaccine of the present invention can achieve the purpose of one shot for multiple protections, reduce costs, and reduce sheep stress.

[0135] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of 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. An antigen composition for multiple "lung rot" in sheep, characterized in that: Including inactivated strains, inactivated bacteria, inactivated bacterial liquid or inactivated fermentation products of Mycoplasma ovipneumoniae MO-BM13D004 strain, Pasteurella multocida type D strain PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain; The deposit number of the Mycoplasma ovipneumoniae MO-BM13D004 strain is CGMCC No.45420, the deposit number of the D-type Pasteurella multocida PmD100 strain is CGMCC No.26636, and the deposit number of the Streptococcus equi subspecies zooepidemicus SeZ100 strain is CGMCC No.26635.

2. Use of the antigen composition according to claim 1 in the preparation of a vaccine for preventing and / or treating multiple lung rot in sheep.

3. A vaccine against multiple ovine lung rot, characterized in that: The method comprises the antigen composition according to claim 1 and an adjuvant, wherein the types of the adjuvant include water-in-oil-in-water adjuvant, water-in-oil adjuvant, microemulsion adjuvant, aluminum salt adjuvant and Freund's adjuvant.

4. The triple inactivated vaccine according to claim 3, characterized in that: The volume ratio of the total volume of the inactivated bacterial solution of the antigen composition to the volume of the adjuvant is 1:(1-2).

5. The triple inactivated vaccine according to claim 4, characterized in that: The volume ratio of the inactivated bacterial solution of Mycoplasma ovipneumoniae MO-BM13D004 strain, type D Pasteurella multocida PmD100 strain and Streptococcus equi subspecies zooepidemicus SeZ100 strain is (0.8-1.2): (0.8-1.2): (0.8-1.2).

6. The method for preparing the triple inactivated vaccine according to any one of claims 3 to 5, characterized in that: The following steps are involved: Mycoplasma ovis pneumoniae MO_BM13D004 strain, Pasteurella multocida PmD100 strain and Streptococcus equi subsp. zooepidemicus SeZ100 strain were inoculated into culture medium for cultivation respectively, and qualified inactivated bacterial liquids were mixed to obtain inactivated bacterial liquid mixture; The inactivated bacterial liquid mixture is mixed with an adjuvant and then emulsified to prepare the triple inactivated vaccine.

7. The preparation method according to claim 6, characterized in that: The culture comprises inoculating the Mycoplasma ovipneumoniae MO_BM13D004 strain into a modified KM2 culture medium and culturing at 37° C. for 48 to 72 hours; Pasteurella multocida PmD100 strain and Streptococcus equi subsp. zooepidemicus SeZ100 strain were inoculated into TSB medium containing 5% horse serum, respectively, and cultured at 37°C for 48 to 72 hours.

8. The preparation method according to claim 6, characterized in that: The bacterial concentration of Mycoplasma ovipneumoniae MO-BM13D004 strain in the triple inactivated vaccine is (1-9)×10 8 CCU / mL; The bacterial concentration of the D-type Pasteurella multocida PmD100 strain in the triple inactivated vaccine is (1-9)×10 8 CFU / mL; The bacterial concentration of Streptococcus equi subspecies zooepidemicus SeZ100 in the triple inactivated vaccine is (1-9)×10 8 CFU / mL.

9. The preparation method according to claim 7, characterized in that: The improved KM2 culture medium includes components with the following concentrations: 500 mL / L of 1640 cell culture fluid, 300 mL / L of Hanks solution with a mass percentage of 1.7% hydrolyzed milk protein, 20 mL / L of yeast solution with a mass percentage of 25%, 200 mL / L of horse serum, 2 mL of 200,000 international units of penicillin, 14 mL / L of 1% thallium acetate, 5 mL / L of 0.4% phenol red, 4 g / L of glucose and 2 g / L of sodium pyruvate, and a pH value of 7.6-7.

8.

10. The preparation method according to claim 7, characterized in that: The TSB culture medium containing 5% horse serum comprises the following components: 17 g / L trypsin casein, 3 g / L soy peptone, 2.5 g / L D-glucose, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate and 5% horse serum by volume, and the pH value is 7.1-7.5.

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