阅读说明：本技术 一种羊肺炎支原体、a型羊多杀性巴氏杆菌和d型羊多杀性巴氏杆菌三联灭活疫苗 (Mycoplasma ovipneumoniae, type-A pasteurella multocida and type-D pasteurella multocida triple inactivated vaccine ) 是由 张月梅 赵世华 戴伶俐 王娜 宋越 张帆 周蕾 达来宝力格 刘威 杨斌 柴春霞 于 2020-05-23 设计创作，主要内容包括：本发明提供了一种羊肺炎支原体、A型羊多杀性巴氏杆菌和D型羊多杀性巴氏杆菌三联灭活疫苗,属于疫苗技术领域；所述三联灭活疫苗包括绵羊肺炎支原体MO_NM01的灭活菌液、A型羊多杀性巴氏杆菌PM-NM1的灭活菌液、D型羊多杀性巴氏杆菌P<-NM2的灭活菌液和免疫佐剂。本发明提供的羊肺炎支原体、A型羊多杀性巴氏杆菌和D型羊多杀性巴氏杆菌三联灭活疫苗与市售的绵羊肺炎支原体灭活疫苗相比,相应病原的免疫保护力基本相当,但可以达到一针多防、降低成本、减少羊只应激的目的。(The invention provides a mycoplasma hyopneumoniae, A type pasteurella multocida and D type pasteurella multocida triple inactivated vaccine, belonging to the technical field of vaccines; the triple inactivated vaccine comprises an inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, an inactivated bacterial liquid of A type pasteurella multocida PM-NM1, an inactivated bacterial liquid of D type pasteurella multocida P < -NM2 and an immunologic adjuvant. Compared with the commercial mycoplasma ovipneumoniae inactivated vaccine, the triple inactivated vaccine for mycoplasma ovipneumoniae, the type-A pasteurella multocida and the type-D pasteurella multocida provided by the invention has basically equivalent immune protection of corresponding pathogens, but can achieve the purposes of preventing more diseases with one needle, reducing cost and reducing stress of sheep.)

1. A triple inactivated vaccine of mycoplasma ovipneumoniae, A-type pasteurella multocida and D-type pasteurella multocida comprises an inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, an inactivated bacterial liquid of A-type pasteurella multocida PM-NM1, an inactivated bacterial liquid of D-type pasteurella multocida P < -NM2 and an immunologic adjuvant;

the collection number of the mycoplasma ovipneumoniae MO _ NM01 is CGMCC No. 19698;

the preservation number of the A-type sheep pasteurella multocida PM-NM1 is CGMCC No. 19798;

the preservation number of the D type pasteurella multocida P < -NM2 is CGMCC No. 19799;

the volume ratio of the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of the type A pasteurella multocida PM-NM1 and the inactivated bacterial liquid of the type D pasteurella multocida P < -NM2 is (0.8-1.2): (0.8-1.2): (0.8 to 1.2);

the thallus concentration of the mycoplasma ovipneumoniae MO _ NM01 in the triple inactivated vaccine is (1-9) × 10⁹CCU/mL；

The thallus concentration of the A-type sheep pasteurella multocida PM-NM1 in the triple inactivated vaccine is (1-9) × 10⁹CFU/mL；

D-type sheep pasteurella multocida P in triple inactivated vaccine<The bacterial concentration of-NM 2 is (1-9) × 10⁹CFU/mL。

2. The vaccine of claim 1, wherein the immune adjuvant comprises a freund adjuvant or an ISA-201-VG immune oil adjuvant.

3. The vaccine of claim 2, wherein the ratio of the total volume of the inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of Pasteurella multocida A PM-NM1 and the inactivated bacterial liquid of Pasteurella multocida D P < -NM2 to the volume of the immunoadjuvant is 1: (1-2).

4. The preparation method of the triple inactivated vaccine as described in any one of claims 1 to 3, comprising the following steps:

1) respectively culturing mycoplasma ovipneumoniae MO _ NM01, A-type pasteurella multocida PM-NM1 and D-type pasteurella multocida P < -NM2 to respectively prepare mycoplasma ovipneumoniae culture solution, A-type pasteurella multocida liquid and D-type pasteurella multocida liquid;

2) respectively inactivating the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01, the A type pasteurella multocida PM-NM1 and the D type pasteurella multocida P < -NM2, and then mixing to obtain an inactivated microbial preparation;

3) and mixing the inactivated microbial preparation and the immunologic adjuvant to obtain the triple inactivated vaccine.

5. The preparation method according to claim 4, wherein the method for inactivating the mycoplasma ovipneumoniae MO _ NM01 in the step 2) comprises the following steps: mixing the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01 with 0.2-0.5% by volume of formaldehyde aqueous solution, inactivating for 48 hours at 37 ℃, and shaking for 1-2 times during the inactivation period.

6. The preparation method of claim 4, wherein the method for inactivating the A type Pasteurella multocida PM-NM1 and the D type Pasteurella multocida P < -NM2 in step 2) comprises: respectively mixing the inactivated bacterial liquid of the A-type sheep pasteurella multocida PM-NM1 and the D-type sheep pasteurella multocida P < -NM2 with 0.5-1% by volume of formaldehyde aqueous solution, inactivating for 48h at 37 ℃, and shaking for 1-2 times during inactivation.

Technical Field

The invention relates to the technical field of vaccines, in particular to a mycoplasma ovipneumoniae, a type-A pasteurella multocida and a type-D pasteurella multocida triple inactivated vaccine.

Background

Mycoplasma ovipneumoniae, a type-A sheep pasteurella multocida and a type-D sheep pasteurella multocida are the most important conditional pathogenic pathogens harming sheep farming today, are often used as sheep infectious pleuropneumonia and hemorrhagic septicemia complicating or secondary infection pathogens, and seriously harm the healthy and sustainable development of the sheep farming in China and even in the world.

Disclosure of Invention

The invention aims to provide a mycoplasma ovipneumoniae, a type-A pasteurella multocida and a type-D pasteurella multocida triple inactivated vaccine, which has an immune effect on mycoplasma ovipneumoniae, type-A pasteurella multocida and type-D pasteurella multocida, so that the infectious pleuropneumonia of sheep can be more reliably prevented.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a mycoplasma ovipneumoniae, A type pasteurella multocida and D type pasteurella multocida triple inactivated vaccine, which comprises an inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, an inactivated bacterial liquid of A type pasteurella multocida PM-NM1, an inactivated bacterial liquid of D type pasteurella multocida P < -NM2 and an immunologic adjuvant;

the collection number of the mycoplasma ovipneumoniae MO _ NM01 is CGMCC No. 19698;

the preservation number of the A-type sheep pasteurella multocida PM-NM1 is CGMCC No. 19798;

the preservation number of the D type pasteurella multocida P < -NM2 is CGMCC No. 19799;

the volume ratio of the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of the type A pasteurella multocida PM-NM1 and the inactivated bacterial liquid of the type D pasteurella multocida P < -NM2 is (0.8-1.2): (0.8-1.2): (0.8 to 1.2);

the thallus concentration of the mycoplasma ovipneumoniae MO _ NM01 in the triple inactivated vaccine is (1-9) × 10⁹CCU/mL；

The thallus concentration of the A-type sheep pasteurella multocida PM-NM1 in the triple inactivated vaccine is (1-9) × 10⁹CFU/mL；

D-type sheep pasteurella multocida P in triple inactivated vaccine<The bacterial concentration of-NM 2 is (1-9) × 10⁹CFU/mL。

Preferably, the immunological adjuvant comprises Freund's adjuvant or ISA-201-VG immunological oil adjuvant.

Preferably, the ratio of the total volume of the inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of type A sheep pasteurella multocida PM-NM1 and the inactivated bacterial liquid of type D sheep pasteurella multocida P < -NM2 to the volume of the immune adjuvant is 1: (1-2).

The invention provides a preparation method of the triple inactivated vaccine in the scheme, which comprises the following steps:

1) respectively culturing mycoplasma ovipneumoniae MO _ NM01, A-type pasteurella multocida PM-NM1 and D-type pasteurella multocida P < -NM2 to respectively prepare mycoplasma ovipneumoniae culture solution, A-type pasteurella multocida liquid and D-type pasteurella multocida liquid;

2) respectively inactivating the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01, the A type pasteurella multocida PM-NM1 and the D type pasteurella multocida P < -NM2, and mixing to obtain an inactivated microbial preparation;

3) and mixing the inactivated microbial preparation and the immunologic adjuvant to obtain the triple inactivated vaccine.

Preferably, the method for inactivating the mycoplasma ovipneumoniae MO _ NM01 in step 2) comprises the following steps: mixing the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01 with 0.2-0.5% by volume of formaldehyde aqueous solution, inactivating for 48 hours at 37 ℃, and shaking for 1-2 times during the inactivation period.

Preferably, the method for inactivating the inactivated bacteria liquid of the type A Pasteurella multocida PM-NM1 and the inactivated bacteria liquid of the type D Pasteurella multocida P < -NM2 in the step 2) comprises the following steps: respectively mixing the inactivated bacterial liquid of the A-type sheep pasteurella multocida PM-NM1 and the D-type sheep pasteurella multocida P < -NM2 with 0.5-1% by volume of formaldehyde aqueous solution, inactivating for 48h at 37 ℃, and shaking for 1-2 times during inactivation.

The invention has the beneficial effects that: the invention provides a mycoplasma ovipneumoniae, A type pasteurella multocida and D type pasteurella multocida triple inactivated vaccine, which comprises an inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, an inactivated bacterial liquid of A type pasteurella multocida PM-NM1, an inactivated bacterial liquid of D type pasteurella multocida P < -NM2 and an immunologic adjuvant. Compared with the commercial mycoplasma ovipneumoniae inactivated vaccine, the triple inactivated vaccine for mycoplasma ovipneumoniae, the type-A pasteurella multocida and the type-D pasteurella multocida provided by the invention has basically equivalent immune protection of corresponding pathogens, but can achieve the purposes of preventing more diseases with one needle, reducing cost and reducing stress of sheep.

Biological preservation information

The mycoplasma ovipneumoniae MO _ NM01, the type A sheep pasteurella multocida PM-NM1 and the type D sheep pasteurella multocida P < -NM2 are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation time is 5 and 11 days in 2020, and the preservation address is the institute of microbiology of China academy of sciences No. 3 of the West Lu No.1 Hopkins of the sunward Yang district, Beijing city; the collection number of the mycoplasma ovipneumoniae MO _ NM01 is CGMCC No. 19698; the preservation number of the A-type sheep pasteurella multocida PM-NM1 is CGMCC No. 19798; the preservation number of the D type pasteurella multocida P < -NM2 is CGMCC No. 19799.

Detailed Description

The invention provides a mycoplasma ovipneumoniae, A type pasteurella multocida and D type pasteurella multocida triple inactivated vaccine, which comprises an inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, an inactivated bacterial liquid of A type pasteurella multocida PM-NM1, an inactivated bacterial liquid of D type pasteurella multocida P < -NM2 and an immunologic adjuvant;

the collection number of the mycoplasma ovipneumoniae MO _ NM01 is CGMCC No. 19698;

the preservation number of the A-type sheep pasteurella multocida PM-NM1 is CGMCC No. 19798;

the preservation number of the D type pasteurella multocida P < -NM2 is CGMCC No. 19799;

the volume ratio of the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of the type A pasteurella multocida PM-NM1 and the inactivated bacterial liquid of the type D pasteurella multocida P < -NM2 is (0.8-1.2): (0.8-1.2): (0.8 to 1.2), preferably 1: 1: 1;

the third mentionedThe bacterial concentration of the mycoplasma ovipneumoniae MO _ NM01 in the combined inactivated vaccine is (1-9) × 10⁹CCU/mL；

The thallus concentration of the A-type sheep pasteurella multocida PM-NM1 in the triple inactivated vaccine is (1-9) × 10⁹CFU/mL；

D-type sheep pasteurella multocida P in triple inactivated vaccine<The bacterial concentration of-NM 2 is (1-9) × 10⁹CFU/mL。

In the invention, the mycoplasma ovipneumoniae MO _ NM01 and the A-type and D-type pasteurella multocida are strains which are separated from mycoplasma ovipneumoniae pathogens collected from sheep farms and pastures in western, middle, northeast and other places of inner Mongolia in 2016-2018 years, and finally have better immunogenicity and virulence through morphological observation, culture characteristics, biochemical characteristics, serological tests, virulence and immunogenicity tests.

In the invention, the mycoplasma ovipneumoniae MO _ NM01 is characterized by the following morphological characteristics: the colony is raised under a 40 times magnifying glass and is mulberry-shaped without umbilicus; the mycoplasma solid plate can be seen with a round microcolony with the size of a needle tip by naked eyes, and the colony of the whole plate is fine sand.

In the invention, the A type sheep pasteurella multocida PM-NM1 and the D type sheep pasteurella multocida P < -NM2A are round and transparent on a solid plate culture medium and have light blue metal luster microcolonies.

In the present invention, the immunological adjuvant comprises Freund's adjuvant or ISA-201-VG immunological oil adjuvant (purchased from SEPPIC brand, France).

In the invention, the ratio of the total volume of the inactivated bacterial liquid of mycoplasma ovipneumoniae MO _ NM01, the inactivated bacterial liquid of type A pasteurella multocida PM-NM1 and the inactivated bacterial liquid of type D pasteurella multocida P < -NM2 to the volume of the immune adjuvant is preferably 1: (1-2).

The invention provides a preparation method of the triple inactivated vaccine in the scheme, which comprises the following steps:

1) respectively culturing mycoplasma ovipneumoniae MO _ NM01, A-type pasteurella multocida PM-NM1 and D-type pasteurella multocida P < -NM2 to respectively prepare mycoplasma ovipneumoniae culture solution, A-type pasteurella multocida liquid and D-type pasteurella multocida liquid;

2) respectively inactivating the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid, the A type pasteurella multocida PM-NM1 bacterial liquid and the D type pasteurella multocida P < -NM2 bacterial liquid, and mixing to obtain an inactivated microbial preparation;

3) and mixing the inactivated microbial preparation and the immunologic adjuvant to obtain the triple inactivated vaccine.

The method comprises the steps of firstly, respectively culturing the mycoplasma ovipneumoniae MO _ NM01, the type-A pasteurella multocida PM-NM1 and the type-D pasteurella multocida P < -NM2 to respectively prepare a mycoplasma ovipneumoniae culture solution, a type-A pasteurella multocida liquid and a type-D pasteurella multocida liquid.

In the invention, the preparation methods of the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid, the type A pasteurella multocida PM-NM1 bacterial liquid and the type D pasteurella multocida P < -NM2 bacterial liquid independently and preferably comprise first-stage seed propagation, second-stage seed propagation and fermentation culture.

In the present invention, the preparation method of the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid preferably includes the following steps:

s1, inoculating mycoplasma ovipneumoniae MO _ NM01 to a mycoplasma solid culture medium, culturing for 72-96 h at 37 ℃, selecting a single colony, inoculating to an improved KM2 liquid culture medium, and culturing for 45-50 h at 37 ℃ to obtain a mycoplasma ovipneumoniae MO _ NM01 first-level seed;

s2, inoculating the mycoplasma ovipneumoniae MO _ NM01 primary seeds prepared in the step S1 into an improved KM2 liquid culture medium according to the volume ratio of 1%, culturing for 45-50 h at 37 ℃, carrying out pure inspection according to the appendix of the existing Chinese veterinary pharmacopoeia, and taking the qualified mycoplasma ovipneumoniae MO _ NM01 secondary seeds;

s3, inoculating the secondary seeds of the mycoplasma ovipneumoniae MO _ NM01 obtained in the step S2 into an improved KM2 liquid culture medium according to the volume ratio of 1%, and culturing at 37 ℃ for 45-50 h to obtain a mycoplasma ovipneumoniae MO _ NM01 bacterial liquid.

The method comprises the steps of firstly inoculating mycoplasma ovipneumoniae MO _ NM01 into a mycoplasma solid culture medium, culturing for 72-96 h, preferably 80h, at 37 ℃, selecting a single colony, inoculating into an improved KM2 liquid culture medium, culturing for 45-50 h, preferably 48h, at 37 ℃, and obtaining a mycoplasma ovipneumoniae MO _ NM01 primary seed; the mode of inoculation is preferably streaking.

In the present invention, the mycoplasma solid culture medium preferably comprises the following components in 1L: 21g of Pflo broth, 5g of glucose, 5g of yeast powder, 10mL of thallium acetate with the mass percentage of 1% and the balance of water; the preparation method of the mycoplasma solid culture medium is not particularly limited, and the mycoplasma solid culture medium is obtained by uniformly mixing the raw materials and sterilizing at 116 ℃ for 20 min.

In the present invention, the modified KM2 liquid medium preferably comprises the following components in concentration: 1640 cell culture solution 500mL/L, hydrolyzed milk protein Hanks solution 300mL/L with mass percent of 1.7%, yeast liquid 20mL/L with mass percent of 25%, horse serum 200mL/L, penicillin 2mL of 20 ten thousand international units, thallium acetate 14mL/L with mass percent of 1%, phenol red 5mL/L with mass percent of 0.4%, glucose 4g/L and sodium pyruvate 2 g/L; the pH value is preferably 7.6-7.8. The preparation method of the improved KM2 liquid culture medium is not particularly limited, the raw materials are uniformly mixed, the pH is adjusted to 7.6-7.8, and the mixture is filtered by a 0.22 mu m filter to obtain the improved KM2 liquid culture medium; the reagent for adjusting the pH is preferably a 1mol/L aqueous solution of NaOH.

After the first-grade seed of the mycoplasma ovipneumoniae MO _ NM01 is obtained, the first-grade seed of the mycoplasma ovipneumoniae MO _ NM01 is inoculated into an improved KM2 liquid culture medium according to the volume ratio of 1%, the first-grade seed is cultured for 45-50 h, preferably 48h, the pure inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the qualified first-grade seed is used as the second-grade seed of the mycoplasma ovipneumoniae MO _ NM 01.

After the secondary seed of the mycoplasma ovipneumoniae MO _ NM01 is obtained, the obtained secondary seed of the mycoplasma ovipneumoniae MO _ NM01 is inoculated into an improved KM2 liquid culture medium according to the volume ratio of 1%, and the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid is obtained after the culture is cultured for 45-50 h, preferably 48h, at the temperature of 37 ℃.

In the invention, the preparation methods of the A type pasteurella multocida PM-NM1 bacterial liquid and the D type pasteurella multocida P < -NM2 bacterial liquid are preferably prepared by adopting the following methods:

p1, respectively streaking and inoculating A type sheep pasteurella multocida PM-NM1 and D type sheep pasteurella multocida P < -NM2 into a tryptone soybean agar culture medium containing 5% by volume of horse serum, culturing for 16-20 h at 37 ℃, selecting a single colony to be inoculated into tryptone soybean broth containing 5% by volume of horse serum, and culturing for 16-20 h at 37 ℃ to respectively obtain an A type sheep pasteurella multocida PM-NM1 primary seed and a D type sheep pasteurella multocida P < -NM2 primary seed;

p2, respectively inoculating the A-type sheep pasteurella multocida PM-NM1 primary seeds and the D-type sheep pasteurella multocida P < -NM2 primary seeds into tryptone soybean broth containing 5% by volume of horse serum according to the volume ratio of 1%, culturing for 16-20 h, preferably 18h at 37 ℃, carrying out pure inspection according to the supplement of the current Chinese veterinary pharmacopoeia, and taking the qualified seeds as A-type sheep pasteurella multocida PM-NM1 secondary seeds and D-type sheep pasteurella multocida P < -NM2 secondary seeds;

and P3, respectively inoculating the A-type sheep pasteurella multocida PM-NM1 secondary seeds and the D-type sheep pasteurella multocida P < -NM2 secondary seeds into tryptone soybean broth containing 5% by volume of horse serum according to the volume ratio of 1%, and culturing at 37 ℃ for 16-20 h, preferably 18h to respectively obtain the A-type sheep pasteurella multocida PM-NM1 bacterial liquid and the D-type sheep pasteurella multocida P < -NM2 bacterial liquid.

Firstly, streaking A type sheep pasteurella multocida PM-NM1 and D type sheep pasteurella multocida P < -NM2 respectively in a tryptone soybean agar culture medium containing 5% by volume of horse serum, culturing for 16-20 h, preferably 18h, at 37 ℃, selecting a single colony, inoculating in tryptone soybean broth containing 5% by volume of horse serum, culturing for 16-20 h, preferably 18h, at 37 ℃, and obtaining A type sheep pasteurella multocida PM-NM1 primary seeds and D type sheep pasteurella multocida P < -NM2 primary seeds respectively.

In the present invention, the tryptone soy agar medium (TSA) preferably comprises the following components: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 1000ml of purified water, wherein the pH value at 25 ℃ is preferably 7.1-7.5, and more preferably 7.3; the tryptone soy agar medium is preferably prepared by the following method: mixing tryptone and soybean peptone, sodium chloride and purified water, mixing, adjusting the pH value to be 7.1-7.5 at 25 ℃, adding agar, heating to melt, and sterilizing to obtain a tryptone and soybean agar culture medium; mixing the tryptone soy agar culture medium with 5% by volume of horse serum to obtain the tryptone soy agar culture medium containing 5% by volume of horse serum; the temperature of the mixing is preferably 60 ℃.

In the present invention, the Tryptone Soy Broth (TSB) preferably comprises the following components: tryptone 17g, soybean peptone 3g, D-glucose 2.5g, sodium chloride 5.0g, dipotassium hydrogen phosphate 2.5g and purified water; the proportion of the total mass of the tryptone, the soybean peptone, the D-glucose, the sodium chloride and the dipotassium hydrogen phosphate to the volume of the purified water is 30 g: 1000 ml; the pH value of the tryptone soybean broth is preferably 7.1-7.5, and is preferably 7.3; the preparation method of the tryptone soybean broth is not particularly limited, and the tryptone soybean broth containing 5% by volume of horse serum is obtained by mixing the raw materials, adjusting the pH value to 7.1-7.5, sterilizing at 121 ℃ for 15min, cooling to 60 ℃ and mixing with 5% by volume of horse serum.

After obtaining first-grade seeds of A type sheep pasteurella multocida PM-NM1 and first-grade seeds of D type sheep pasteurella multocida P < -NM2, the first-grade seeds of the A type sheep pasteurella multocida PM-NM1 and the first-grade seeds of the D type sheep pasteurella multocida P < -NM2 are respectively inoculated into tryptone soybean broth containing 5% by volume of horse serum according to the volume ratio of 1%, cultured for 16-20 h, preferably 18h, purely checked according to the appendix of the existing Chinese pharmacopoeia, and qualified as second-grade seeds of the A type sheep pasteurella multocida PM-NM1 and second-grade seeds of the D type sheep pasteurella multocida P < -NM 2.

After obtaining the A type sheep pasteurella multocida PM-NM1 secondary seeds and the D type sheep pasteurella multocida P < -NM2 secondary seeds, the invention respectively inserts the A type sheep pasteurella multocida PM-NM1 secondary seeds and the D type sheep pasteurella multocida P < -NM2 secondary seeds into tryptone soybean broth containing 5 percent of horse serum by volume ratio of 1 percent, and cultures for 16-20 h, preferably 18h at 37 ℃ to respectively obtain the A type sheep pasteurella multocida PM-NM1 bacterial liquid and the D type sheep pasteurella multocida P < -NM2 bacterial liquid.

After a mycoplasma ovipneumoniae culture solution, an A-type pasteurella multocida liquid and a D-type pasteurella multocida liquid are obtained, the mycoplasma ovipneumoniae MO _ NM01 liquid, the A-type pasteurella multocida PM-NM1 liquid and the D-type pasteurella multocida P < -NM2 liquid are inactivated and mixed to obtain an inactivated microbial preparation. In the present invention, the method for inactivating the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid is preferably: mixing the inactivated bacterial liquid of the mycoplasma ovipneumoniae MO _ NM01 with 0.2-0.5% by volume of formaldehyde aqueous solution, inactivating for 48 hours at 37 ℃, and shaking for 1-2 times during the inactivation period.

In the invention, the method for inactivating the A type sheep pasteurella multocida PM-NM1 bacterial liquid and the D type sheep pasteurella multocida P < -NM2 bacterial liquid comprises the following steps: respectively mixing the inactivated bacterial liquid of the A-type sheep pasteurella multocida PM-NM1 and the D-type sheep pasteurella multocida P < -NM2 with 0.5-1% by volume of formaldehyde aqueous solution, inactivating for 48h at 37 ℃, and shaking for 1-2 times during inactivation.

After inactivation, the method preferably further comprises sampling, and carrying out inactivation inspection according to the appendix of the current Chinese veterinary pharmacopoeia, so that no bacteria grow to be qualified. If not, the inactivation is continued.

The invention provides the mycoplasma ovipneumoniae MO _ NM01 bacterial liquid, the type-A sheep pasteurella multocida PM-NM1 bacterial liquid and the type-D sheep pasteurella multocida P<After the inactivation of the-NM 2 bacterial liquid, preferably, the method further comprises the step of concentrating each inactivated liquid respectively to obtain the inactivated mycoplasma ovipneumoniae MO _ NM01 bacterial liquidThe concentration of the mycoplasma ovipneumoniae MO _ NM01 is (1-9) × 10⁹CCU/mL, and the concentration of the A-type sheep pasteurella multocida PM-NM1 in the inactivated A-type sheep pasteurella multocida PM-NM1 bacterial liquid is (1-9) × 10⁹CFU/mL inactivated D-type sheep Pasteurella multocida P<D type Pasteurella multocida P in NM2<-NM2 concentration of (1-9) × 10⁹CFU/mL。

In the present invention, the concentration is preferably performed by resuspension after centrifugation.

After obtaining the inactivated microbial preparation, the inactivated microbial preparation and the immunologic adjuvant are mixed according to the mixing volume ratio of 1: 1, obtaining a triple inactivated vaccine; the mixing mode is not particularly limited, and the uniform mixing is taken as the standard.

In the specific implementation process of the invention, the equal volume and the concentration of 2 × 10 are added⁹CFU/mL bacterial liquid of type A sheep pasteurella multocida PM-NM1 and type D sheep pasteurella multocida P<-NM2 bacterial liquid, centrifugation to obtain precipitate, and mixing with the same volume and concentration of 2 × 10⁹CCU/mL mycoplasma ovipneumoniae MO _ NM01 bacterial liquid is used for re-suspending the precipitates of the two kinds of pasteurella, and the concentrations of the bacterial liquids of the two kinds of pasteurella in the obtained re-suspended liquid are respectively 2 × 10⁹CFU/ml, then adding an immunologic adjuvant with the same volume as that of the heavy suspension liquid to obtain the triple inactivated vaccine, wherein the thallus concentration of the mycoplasma ovipneumoniae MO _ NM01 in the triple inactivated vaccine is 1 × 10⁹CCU/mL, the thallus concentration of A type sheep pasteurella multocida PM-NM1 is 1 × 10⁹CFU/mL, D type Pasteurella multocida P<The cell density of-NM 2 was 1 × 10⁹CFU/mL。

The step of mixing in the scheme is not particularly limited, and the uniform mixing is taken as the standard.

The use method of the triple inactivated vaccine preferably comprises the following steps: intramuscular injecting the triple inactivated vaccine into weaned lambs, injecting 2-3 mL of the triple inactivated vaccine into each weaned lambs, and performing secondary immunization by using the same method and the same dose after 2-3 weeks; the week age of the weaned lamb is preferably 4-6 weeks old; the breed of weaned lambs preferably comprises sheep and goats.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The formulation of the culture medium employed in the examples of the present invention is as follows:

1. improved KM2 liquid culture medium

The formula is as follows: (g/L)

The components are adjusted to pH 7.6-7.8 by 1mol/L NaOH aqueous solution, filtered by a 0.22 mu m filter and then placed in a refrigerator at 4 ℃ for standby.

2. Mycoplasma solid culture medium

The formula is as follows: (g/L)

Mixing the above materials, heating to dissolve, packaging, sterilizing at 116 deg.C for 20min, and storing in 4 deg.C refrigerator. Heating 100mL of solid culture medium to dissolve, and pouring about 20mL into a sterile plate when the temperature is reduced to 55-60 ℃. After capping, the mixture was left to solidify at room temperature.

3. Tryptone soy agar Medium (TSA) + 5% horse serum

The formula is as follows: (g/L)

Removing agar from the above components, mixing, slightly heating to dissolve, adjusting pH to pH 7.3 + -0.2 at 25 deg.C after sterilization, adding agar, heating to melt, packaging, sterilizing, cooling to about 60 deg.C, adding horse serum with final concentration of 5%, and pouring into sterile plate with about 20 ml. After capping, the mixture was left to solidify at room temperature.

Plate culture and preservation: the prepared culture medium plate is preferably stored at 2-8 ℃, and is generally preferably performed for one week or according to the standard provided by the manufacturer.

4. Tryptone Soy Broth (TSB) + 5% horse serum

The formula is as follows: (g/L)

30g of medium powder were dissolved in 1000ml of deionized water. The pH was adjusted to 7.3. The TSB culture medium is sterilized by autoclaving at 121 deg.C for 15min, cooling, and adding horse serum with final concentration of 5%.

The strains adopted in the embodiment of the invention are as follows:

the mycoplasma ovipneumoniae MO _ NM01, the type A sheep pasteurella multocida PM-NM1 and the type D sheep pasteurella multocida P < -NM2 are preserved in the China general microbiological culture Collection center; the collection number of the mycoplasma ovipneumoniae MO _ NM01 is CGMCC No. 19698; the preservation number of the A type sheep pasteurella multocida PM-NM1 is CGMCC No. 19798; the preservation number of the D type pasteurella multocida P < -NM2 is CGMCC No. 19799.