CN110124022B

CN110124022B - Mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine and application thereof

Info

Publication number: CN110124022B
Application number: CN201910318204.2A
Authority: CN
Inventors: 徐引弟; 王治方; 张青娴; 朱文豪; 许峰; 焦文强; 李海利; 郎利敏; 张立宪; 游一; 王克领
Original assignee: Institute of Animal Husbandry and Veterinary Medicine of Henan Academy of Agricultural Sciences
Current assignee: Institute of Animal Husbandry and Veterinary Medicine of Henan Academy of Agricultural Sciences
Priority date: 2019-04-19
Filing date: 2019-04-19
Publication date: 2023-01-31
Anticipated expiration: 2039-04-19
Also published as: CN110124022A

Abstract

The invention relates to the technical field of biological products for veterinary use, in particular to a mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine which comprises an inactivated mycoplasma hyopneumoniae strain HNMhy1 serotype antigen, an inactivated haemophilus parasuis strain HNHPS1 serotype antigen, an inactivated haemophilus parasuis strain HN1570 serotype antigen, an inactivated streptococcus suis strain HNSS1 serotype antigen, an inactivated actinobacillus pleuropneumoniae strain HNAPP1 serotype antigen and an immunologic adjuvant.

Description

Mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine and application thereof

The technical field is as follows:

the invention belongs to the technical field of biological products for veterinary use, and particularly relates to preparation and application of a mycoplasma hyopneumoniae and haemophilus parasuis serotype 4, serotype 5, streptococcus suis serotype 2 and actinobacillus pleuropneumoniae serotype 1 quadruple inactivated vaccine.

Background art:

mycoplasma hyopneumoniae (Mhp) is the main cause of Mycoplasma hyopneumoniae (MPS). The swine mycoplasmal pneumonia is a chronic and contact infectious disease, has the characteristics of high morbidity and low mortality, is mainly characterized by symptoms of anorexia, fever, cough, asthma, dyspnea and the like, the sick swine grow slowly, the feed conversion rate is reduced, infection of other pathogens is often induced, particularly immunosuppressive pathogens such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine circovirus type 2 (PCV-2) cause immunosuppression induction, death is often caused by simultaneous secondary bacterial infection, and the swine mycoplasmal pneumonia is one of the most important swine diseases in the world. The disease is widely existed in all parts of the world, the infection is persistent and difficult to cure, and meanwhile, the infected pigs have serious harm to the pig industry due to higher treatment cost and reduced production performance.

Haemophilus parasuis (Haemophilus parasuis) (II)Haemophilus parasuisHPS) is a gram-negative, polymorphous, small bacterium of the pasteuriaceae family, a pathogenic bacterium of haemophilus parasuis, mainly causing multiple serositis, arthritis and meningitis in pigs. The disease is widely existed in the world, and Glsser, german scholars, first reported in 1910, is also called as the leather Laplace disease (Glsser's disease). The haemophilus parasuis can infect pigs of various ages, mainly occurs after weaning and in the nursing stage, mainly infects pigs of 2 weeks to 4 months of age, the morbidity is generally 10-15%, and the death rate can reach 50% in serious cases.

The clinical serotypes of the haemophilus parasuis are more, the standard typing has 15 serotypes, more than 20 percent of non-typing strains exist clinically, the pathogeny is distributed worldwide, the serotypes of popular strains are different, and the immune cross protection capability among the serotypes is lacked. Vaccination is the best way to control haemophilus parasuis disease and the corresponding monovalent or multivalent vaccine should be selected according to the prevailing serotype.

Serotype

4 and 5 of haemophilus parasuis are the most popular serotypes reported in China.

Streptococcus suis (Streptococcus SuisSS) is a second kind of animal epidemic disease prescribed by the state, which is a kind of acute and febrile infectious diseases of zoonosis, is the main pathogen causing swine streptococcosis, and mainly causes swine meningitis, septicemia and other epidemic diseases. The streptococcus suis capsular polysaccharide antigen can be divided into 35 serotypes according to the antigen,i.e., types 1 to 34 and 1/2. The

types

1,2, 1/2, 7 and 9 are more pathogenic to pigs. Of these, streptococcus suis type 2 is the most prevalent and most pathogenic serotype, followed by types 9, 7, and 1. In recent years, streptococcosis suis caused by group 2 streptococcus suis has been on the rise. The disease is mainly acute septicemia, is acute in morbidity and fast in death, causes huge economic loss to the pig industry, and the streptococcus suis type 2 can cause infection and death of people. In addition, the disease is often mixed with porcine circovirus, porcine reproductive and respiratory syndrome, porcine eperythrozoonosis and atypical swine fever, so that once the disease occurs, the disease is difficult to control, thereby causing great economic and property loss to farmers and bringing great difficulty to the development of the pig industry in China. Therefore, the research on the disease is of great significance to the animal husbandry and public health.

Infectious actinobacillus pleuropneumoniae: (A)Actinobacillus PleuropneumoniaeAPP) is a pathogen causing Porcine infectious Pleuropneumonia (Porcine infectious Pleuropneumonia), is a highly contact infectious respiratory disease of pigs, is mainly manifested as fulminant epidemic, is characterized by cellulosic, hemorrhagic and necrotizing pneumonia, has an incidence rate of 8.5-100% and a mortality rate of 0.4-100%, and is one of the main causes of death of fattening pigs and breeding pigs at present. Therefore, the prevention and treatment of the infectious actinobacillus pleuropneumoniae have great significance for the healthy development of the pig industry.

The serotype of the actinobacillus pleuropneumoniae is as many as 15, and at present, a plurality of clinical isolates which can not be typed exist, effective cross immune protection is lacked among the serotypes, and the dominant serotypes popular in different countries, regions and pig farms are different, so that the separation, identification and typing of APP are particularly important in the prevention and treatment of the disease. The serotype prevailing in China is mainly 1,2,3,5,7, the APP exotoxin plays an important role in pathogenesis and immunity, the APP has 4 exotoxins in total, namely, the toxins ApxI, apxII, apxIII and ApxIV, and the toxins ApxI and ApxII play a key role in the APP toxicity aspect. Exotoxins secreted by different serotypes differ. The serotype 1 can secrete toxins ApxI, apxII and ApxIV at the same time, is a strain with the strongest toxicity and the best immunogenicity of all serotype strains, and is also a dominant serotype strain popular in China.

The mycoplasma hyopneumoniae is clinically often mixed with haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae to cause infection or secondary infection, the symptoms of the mycoplasma hyopneumoniae are similar to those of the mycoplasma hyopneumoniae, the streptococcus suis and the actinobacillus pleuropneumoniae are all similar, and the medicament treatment effect is not good, so that a vaccine prepared from a novel multivalent strain with safety and good immunogenicity is urgently needed to prevent and treat mycoplasma pneumonia, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae.

The invention content is as follows:

aiming at the problems that at present, mycoplasma hyopneumoniae is mixed with haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae to infect, and immune cross protection is lacked among serotypes, the invention provides a quadruple inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis of

types

4 and 5, streptococcus suis of type 2 and actinobacillus pleuropneumoniae of type 1.

The technical scheme adopted by the invention for solving the technical problems is as follows: a vaccine for the four-linkage inactivated vaccine of mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae comprises an inactivated mycoplasma hyopneumoniae strain HNMhy1 serotype antigen, an inactivated haemophilus parasuis strain HNHPS1 serotype antigen, an inactivated haemophilus parasuis strain HN1570 serotype antigen, an inactivated streptococcus suis strain HNSS1 serotype antigen and an inactivated actinobacillus pleuropneumoniae strain HNAPP1 serotype antigen.

In the inactivated vaccine for mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple, the strain HNMhy1 is mycoplasma hyopneumoniae, is stored in China general microbiological culture Collection center (CGMCC) within 2017, 5 and 26 days, and has the address of No. 3 of Beijing city North Chen West Lu No. 1 of the sunny region, and the storage number is CGMCC NO:13858; the strain HNHPS1 is serum 4 type Haemophilus parasuis (Haemophilus parasuis), is preserved in the China general microbiological culture Collection center in 2016, 11 and 22 days, and has the address of No. 3 Hospital No. 1, xilu, beijing, the Chaoyang area, and the preservation number of CGMCC NO:13335; the strain HN1570 is serum 5 type Haemophilus parasuis (Haemophilus parasuis), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 11 months and 09 days in 2018, is No. 3 of No. 1 Xilu Beijing, the address of the Beijing market, the Kingyang district, and has the preservation number of CGMCC NO:16802; the strain HNSS1 is Streptococcus suis type 2 (Streptococcus suis), is preserved in the China general microbiological culture Collection center at 2016, 11 and 22 days, and is No. 3 of Xilu No. 1 of Beijing Kogyo-Yang district, with the preservation number of CGMCC NO:13334, adding a solvent; the strain HNAPP1 is actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) type 1, is preserved in the common microorganism center of China general microbiological culture Collection center in 2016, 11 and 22 days, and has the address of No. 3 of Xilu No. 1 of Beijing Korean district, the preservation number is CGMCC NO:13333.

the inactivated mycoplasma hyopneumoniae strain HNMhy1 serotype antigen, the inactivated haemophilus parasuis strain HNHPS1 serotype antigen, the inactivated haemophilus parasuis strain HN1570 serotype antigen, the inactivated streptococcus suis strain HNSS1 serotype antigen and the inactivated actinobacillus pleuropneumoniae strain HNAPP1 serotype antigen are in a ratio of 1: 1.

The invention also provides a preparation method of the mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine, which comprises the following steps:

a. and (3) proliferation: respectively propagating and culturing mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis 4 type HNHPS1 strain, haemophilus parasuis 5 type HN1570 strain, streptococcus suis 2 type HNSS1 strain and actinobacillus pleuropneumoniae 1 type HNAPP1 strain to obtain mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, haemophilus parasuis HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain liquid, and respectively counting viable bacteria;

b. inactivation: adding formaldehyde solution into the bacterial solutions of the mycoplasma hyopneumoniae HNMhy1 strain, the haemophilus parasuis HNHPS1 strain, the haemophilus parasuis HN1570 strain, the streptococcus suis HNSS1 strain and the actinobacillus pleuropneumoniae HNAPP1 strain obtained in the step a according to 0.2 percent of the volume of the bacterial solutions, and inactivating the bacterial solutions at 37 ℃ for 24 hours;

c. concentration: respectively concentrating and inactivating bacterial solutions of qualified mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, haemophilus parasuis HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain by using an ultrafiltration device, and adjusting the concentration final concentration to be 1.0 x 10 by using sterile physiological saline according to the viable count before inactivation ¹⁰ CFU/mL；

d. Preparing a vaccine:

(1) Preparation of oil phase: adding 92 parts of white oil for injection into an oil phase tank according to the volume ratio, adding 1 part of aluminum stearate into the tank, stirring while adding until the mixture is completely transparent, adding span-80 parts, sterilizing at 116 ℃ for 40min under high pressure, cooling to room temperature, adding 0.5 part of each of vitamin A and vitamin E, and uniformly mixing to ensure that the concentration of the vitamin A and the vitamin E is 300IU/Ml to obtain an oil phase for later use;

(2) Preparation of the aqueous phase: mixing antigen solutions of mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain obtained after concentration uniformly according to the proportion of 1:1 to prepare antigen solutions, taking 95 parts of the antigen solutions according to the volume part ratio, adding sterilized Tween-80 parts, and fully stirring until the antigen solutions are completely dissolved to obtain a water phase for later use;

(3) Emulsification: according to the proportion of the water phase and the oil phase 1:2, the oil phase is firstly added into an emulsification tank, the mixture is stirred at the rotating speed of 3000-4000rpm, then the water is added into the emulsification tank, the mixture is continuously stirred, mixed and emulsified, and the separated packaging and bottling are carried out after the emulsification is finished.

In the above preparation method of the inactivated vaccine for four-linkage of mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae, the operation steps of propagation and culture in the step a are as follows:

(1) First-order seed propagation: inoculating Mycoplasma hyopneumoniae HNMhy1 strain to PPLO solid medium, standing at 37 deg.C, and 5% CO ₂ Culturing in incubator, collecting culture after 3d, haemophilus parasuis type 4 HNHPS1 strain, type 5 HN1570 strain, streptococcus suis type 2 HNSS1 strain andrespectively inoculating freeze-dried strains of actinobacillus pleuropneumoniae 1 type HNAPP1 strains on a TSA plate, culturing at 37 ℃ for 18-24 hours, selecting typical colonies meeting requirements, respectively subculturing on the TSA plate, culturing at 37 ℃ for 24 hours, and checking to be qualified to serve as first-stage seeds; the TSA plate is added with newborn bovine serum with a final concentration of 5% and NAD with a final concentration of 0.01%;

(2) And (3) secondary seed propagation: inoculating primary seeds of Mycoplasma hyopneumoniae and Haemophilus parasuis type 4 HNHPS1 strain, haemophilus parasuis type 5 HN1570 strain, streptococcus suis type 2 HNSS1 strain and Actinobacillus pleuropneumoniae type 1 HNAPP1 strain to PPLO and TSB liquid culture medium respectively, and culturing at 37 deg.C and 5% CO ₂ Shaking the table to culture for 3d and 18-24 h respectively, and taking the seeds as secondary seeds after pure inspection is qualified; the TSB liquid culture medium is added with newborn bovine serum with the final concentration of 5% and NAD with the final concentration of 0.01%;

(3) Inoculating 1% of secondary seeds of Mycoplasma hyopneumoniae and Haemophilus parasuis type 4 HNHPS1 strain, type 5 HN1570 strain, streptococcus suis type 2 HNSS1 strain and Actinobacillus pleuropneumoniae type 1 HNAPP1 strain to PPLO and TSB liquid culture medium respectively, and respectively culturing at 37 deg.C and 5% CO ₂ Shaking the shaking table to culture for 3d and 18-24 hr separately and harvesting bacteria liquid; the TSB liquid culture medium is added with newborn bovine serum with a final concentration of 5% and NAD with a final concentration of 0.01%.

The application of the mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine in preparing the medicine for preventing and treating diseases related to mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae.

The invention has the beneficial effects that:

1. the mycoplasma hyopneumoniae strain HNMhy1 is separated from the nursery pig with typical dyspnea and lung consolidation, has strong pathogenicity on the nursery pig, can cause the typical gasp symptom of the nursery pig, and has good immunogenicity.

2. The invention screens out dominant epidemic strains of haemophilus parasuis serum 4 type HNHPS1, 5 type HN1570, streptococcus suis 2 type HNSS1 and actinobacillus pleuropneumoniae 1 type HNAPP1 from clinical isolates, wherein the strains of HNHPS1, HN1570, HNSS1 and HNAPP1 have stronger pathogenicity to nursery piglets and cause the death of nursery pigs; the four strains are found to have good immunogenicity through experiments.

3. The inactivated vaccine prepared by using the mycoplasma hyopneumoniae strain HNMhy1 and the haemophilus parasuis serum 4 type HNHPS1 strain, the 5 type HN1570 strain, the streptococcus suis 2 type HNSS1 strain and the actinobacillus pleuropneumoniae 1 type HNAPP1 strain as strain antigens can well prevent the infection of clinical mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae types, and meanwhile, the vaccine is simple in preparation process, good in safety, good in immune effect and long in immune period, and can achieve the purposes of preventing multiple diseases, reducing cost and reducing stress by one injection.

Description of the drawings:

FIG. 1 is a 4 Xmicroscopic morphology of colonies of strain HNMhy 1;

FIG. 2 shows the pattern of strain HNMhy1 with Giemsa stain of 100 Xmicroscopic;

FIG. 3 shows the PCR identification result of the strain HNMhy1, wherein the Marker in the figure is DL 2000 bp, and 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp from top to bottom; lane 1 is the result of amplification of mycoplasma hyopneumoniae vaccine strain 168 with primers MB1 and MB 2;

lane

2,3 is the amplification result of the strain HNMhy1 by the primers MB1 and MB 2; lane 4 is a negative control;

FIG. 4 shows the colony morphology of the strain HNHPS 1;

FIG. 5 shows the gram stain pattern of the strain HNHPS1 under 100 Xmicroscope;

FIG. 6 shows the PCR identification and serotype PCR identification results of the strain HNHPS1, wherein the Marker in the figure is DL 2000 bp, and 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp from top to bottom; lane 1 shows the amplification results of the primers HPS3 and HPS4 on the type 4 Haemophilus parasuis standard strain; lane 2 shows the amplification results of the primers HPS3 and HPS4 on the strain HNHPS 1; lane 3 is the amplification result of the primers HPS1 and HPS2 on the type 4 Haemophilus parasuis standard strain; lane 4 shows the amplification result of the strain HNHPS1 by the primers HPS1 and HPS 2; 5 is negative control;

FIG. 7 shows the colony morphology of strain HN 1570;

FIG. 8 shows the gram-stained 100 Xmicroscopic morphology of strain HN 1570;

FIG. 9 shows the PCR identification and serotype PCR identification of strain HN1570, wherein the Marker in the figure is DL 2000 bp, and 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp,100 bp from top to bottom; lane 1 shows the result of amplifying HN1570 strain 16S rRNA gene with primers HPS1 and HPS 2; lane 2 shows the results of amplifying the wciP gene of HN1570 strain with the primers HPS5 and HPS 6; lane 3 is a negative control;

FIG. 10 shows the colony morphology of strain HNSS 1;

FIG. 11 shows the gram-stained 100 Xmicroscopic morphology of strain HNSS 1;

FIG. 12 shows the results of PCR identification and serotype PCR identification of strain HNSS1, wherein Marker in the figure is DL 2000 bp, and 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp,100 bp from top to bottom; lane 1 shows the result of gdh gene amplification by primers SS1 and SS 2; lane 2 shows the result of cps2J gene amplification by primers SS3 and SS 4; lane 3 shows the amplification of mrp gene by primers mrp1 and mrp 2; lane 4 shows the result of sly gene amplification using primers sly, sly 2; 5 is the result of amplifying epf gene by the primers epf, epf;

FIG. 13 is the colony morphology of strain HN APP 1;

FIG. 14 shows the gram-stained 100 Xmicroscopic morphology of the strain HN APP 1;

FIG. 15 shows the results of PCR identification and serotype PCR identification of strain HN APP1, wherein Marker in the figure is DL 2000 bp, and 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp,100 bp from top to bottom;

lane

1,4 is a negative control; lane 2 is the amplification result of the primers APP3 and APP4 on the type 1 infectious actinobacillus pleuropneumoniae standard strain, and lane 3 is the amplification result of the primers APP3 and APP4 on the strain HNAPP 1; lane 5 is the amplification result of the primers APP1 and APP2 on the type 1 infectious A pleuropneumoniae standard strain; lane 6 shows the result of amplification of the strain HNAPP1 by the primers APP1 and APP 2.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples in conjunction with the drawings.

Example 1 isolation and characterization of Mycoplasma hyopneumoniae

1.1 Collection of pathological material

The disease material comes from the lung of a growing pig of a 2-month-old nursery pig which has severe pneumonia and dyspnea in a large pig farm of Puyang city, henan province in 2017 for 4 months.

1.2 preparation of the culture Medium

PPLO liquid medium: PPLO bouillon powder 10.5 g, glucose 2.5 g, yeast powder 2.5 g, dissolved in 440 mL ultra pure water, sterilized at 115 deg.C for 15min, MEM medium 5mL, horse serum 50mL, penicillin 8 ten thousand units, sterile 10% arginine 10 mL, and 1% (w/v) phenol red 500. Mu.L were added. Sterilizing at 115 deg.C for 15min, and storing at 4 deg.C;

PPLO solid medium: PPLO liquid medium supplemented with 1.5% (w/v) agar powder. The culture medium is sterilized at 115 deg.C for 15min, and stored at 4 deg.C for use.

1.3 Isolation culture of Mycoplasma

Cutting appropriate amount of diseased lung tissue in grinder, adding 2mL sterilized PBS buffer solution, grinding, collecting supernatant in EP tube, centrifuging at low speed of 5000 r/m for 5min, collecting supernatant with needle tube, filtering with 0.22 μm filter membrane, adding PPLO liquid culture medium, adding 5% CO at 37 deg.C ₂ Culturing in an incubator; culturing for about one week, culturing in 1mL transfer PPLO liquid culture medium again, subculturing 3~5 generation, changing liquid color from red to yellow, coating 100 μ L onto PPLO solid culture medium surface, standing at 37 deg.C and 5% CO ₂ Culturing in an incubator; after 5 to 7 days, colorless transparent needle-tip-shaped colonies can be observed by naked eyes, and the morphology of the colonies on the solid culture medium is observed under a low power microscope, wherein the morphology is a typical mycoplasma' fried egg-shaped "(see figure 1); giemsa staining, observing the shape of thallus under oil microscope, and taking the shape of multiple forms, such as coccoid, curved filamentous, and spiral (see FIG. 2), and named as strain HNMhy1.

1.4 Identification of strains

1.4.1 design of primers

A pair of universal primers is designed according to the sequence of the 16S rRNA of the mycoplasma hyopneumoniae and is used for amplifying the mycoplasma hyopneumoniae, and the primer sequences are as follows:

MB1：5’- ACGCGTCGACAGAGTTTGATCCTGGCT-3’（SEQ ID NO.1）

MB2：5’- CGCGGATCCGCTACCTTGTTACGACTT-3’（SEQ ID NO.2）

the expected amplified fragment size was 1502bp.

1.4.2 PCR identification

Inoculating strain HNMhy1 into PPLO liquid culture medium, standing at 37 deg.C and 5% CO ₂ Culturing in an incubator for 3 days, changing the culture medium from red to yellow, collecting thallus, extracting DNA of the strain HNMhy1 according to the operation steps of the DNA extraction kit, measuring the concentration by a spectrophotometer to be 100 mug/mL, and performing PCR identification by using amplification of MB1 and MB 2; meanwhile, mycoplasma hyopneumoniae vaccine strain 168 is set as a positive control.

The PCR amplification reaction system is 25 muL: 2.5 muL of 10 Xbuffer solution, 0.5 muL of 2.5mM dNTPs, 1 muL of 10 muM/L universal primers MB1 and MB2, 1 muL of 5U/muL rTaq 1 muL and 1 muL of 100 mug/mL DNA template, and ddH is added ₂ O to 25 mu L.

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, entering circulation: 95 ℃ 30 s,57 ℃ 30 s,72 ℃ 1 min for 35 cycles, final 72 ℃ extension 10min, PCR product 4 ℃ storage.

Respectively carrying out electrophoresis on the amplification products, carrying out sample application of 10 mu L per well, carrying out electrophoresis on 1% agarose gel, and observing the result under ultraviolet light (see figure 3); FIG. 3 shows that the template PCR amplified fragment is about 1502bp, which proves that the amplified fragment is the target fragment, the MB1 and MB2 amplified fragments with expected sizes have homology of 78% with the mycoplasma hyopneumoniae subculture attenuated vaccine strain 168 (Genbank No. CP002274) through sequencing, and the sequencing result is as follows, which proves that the isolated strain HNMhy1 is mycoplasma hyopneumoniae (M.hyopneumoniae)Mycoplasma hyopneumoniae）。ACGCGTCGACAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAAGCATCTTCGGATGCTTAGTGGCGAACGGGTGAGTAACACGTAGATAACCTACCTTTAACTCGAGGATAACTCCGGGAAACTGGAGCTAATACTGGATAGGATGTGTGCATGAAAAAAACACATTTAAAGATTTATCGGTTTAAGAGGGGTCTGCGGCGCATTAGTTAGTTGGTGGGGTAAGAGCCTACCAAGACGATGATGCGTAGCCGGACTGAGAGGTCTACCGGCCACATTGGGACTGAGAACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATTTTCGGCAATGGGGGAAACCCTGACCGAGCAACGCCGCGTGAACGACGAAGTACTTCGGTATGTAAAGTTCTTTTATATGGGAAGAAAAATTAAAAATTGACGGTACCATATGAATAAGCCCCGGCTAACTATGTGCCAGCAGCCGCGGTAATACATAGGGGGCGAGCGTTATCCGGATTTACTGGGCGTAAAGGGTGCGTAGGTGGTTATAAAAGTTTGTGGTGTAAGTGCAGTGCTTAACGCTGTGAGGCTATGAAAACTATATAACTAGAGTGAGACAGAGGCAAGTGGAATTCCATGTGTAGCGGTAAAATGCGTAAATATATGGAGGAACACCAGTGGCGAAGGCGGCTTGCTGGGTCTATACTGACACTGATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGAACTAAGTGTTGGCCATAAGGTCAGTGCTGCAGTTAACGCATTAAGTTCTCCGCCTGAGTAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGACCCCGCACAAGCGGTGGATCATGTTGTTTAATTCGAAGATACACGAAAAACCTTACCAGGTCTTGACATACTCTGCAAAGGCTTAGAAATAAGTTCGGAGGCTAACAGATGTACAGGTGGTGCACGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGCTAGTTACCATCATTAAGTTGGGGACTCTAGCGAGACTGCCAGTGATAAATTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACAAACGTGATACAATGGCTGGAACAAAGAGAAGCGATAGGGTGACCTGGAGCGAAACTCACAAAAACAGTCTCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAAATCAGCATGTTGCGGTGAATACGTTCTCGGGGTTTGTACACACCGCCCGTCAAACCACGAAAGTGGGCAATACCCAACGCCGGTGGCCTAACCCGAAAGGGAGGGAGCCGTCTAAGGTAGGGTCCATGATTGGGGTTAAGTCGTAACAAGGTAGCGGATCCGCG（SEQ ID NO.3）。

Example 2: isolation and characterization of the Strain HNHPS1

2.1 Collection of pathological material

The disease is from 2-month-old nursery pigs with severe pneumonia, dyspnea and neurosis death in a large pig farm in Ruyang county, henan province in 2016 (11 months).

2.2 preparation of the culture Medium

TSB liquid medium: dissolving 30g of TSB broth powder (Tryptic Soy broth) in 1000mL ultrapure water, sterilizing at 115 ℃ for 15min, adding 50mL fetal bovine serum and sterile 1% NAD (Nicotinamide adenine dinucleotide, coenzyme I) 1 mL;

TSA solid medium: 40g of TSA Agar powder (Tryptic Soy Agar, tryptone Soy Agar) was dissolved in 1000mL of ultrapure water, sterilized at 115 ℃ for 15min, added with 50mL of fetal bovine serum and 1mL of sterile NAD (1 mL), and stored at 4 ℃ until use.

2.3 Isolated culture of bacteria

Aseptically collecting brain tissue of sick pigs, inoculating the brain tissue on a TSA solid culture medium containing NAD, culturing in a 37 ℃ constant temperature incubator to obtain 36 h, and growing colonies with consistent needle tip shape size, colorless transparency, smoothness, humidity and diameter size of 1-2 mm (see figure 4); purifying and inoculating on a TSA solid culture medium without NAD, and not growing on the TSA culture medium without NAD; gram-stained, gram-negative bacteria (see fig. 5), with a variety of morphologies, from single coccobacillus to elongated filamentous bacteria, designated the strain HNHPS1.

2.4 Identification of the strains

2.4.1 design of primers

A pair of universal primers is designed according to the sequence of the Haemophilus parasuis 16S rRNA gene and is used for the amplification of the Haemophilus parasuis, and the primer sequences are as follows:

HPS1：5’- GTGATGAGGAAGGGTGGTGT -3’（SEQ ID NO.4）

HPS2：5’- GGCTTCGTCACCCTCTGT -3’（SEQ ID NO.5）

the amplified fragment size was 822bp.

Meanwhile, a pair of specific primers of the haemophilus parasuis type 4 is designed according to the sequence of the haemophilus parasuis wciP and is used for the amplification of the haemophilus parasuis type 4, and the primer sequences are as follows:

HPS3：5’-GGTTAAGAGGTAGAGCTAAGAATAGAGG-3’（SEQ ID NO.6）

HPS4：5’-CTTTCCACAACAGCTCTAGAAACC-3’（SEQ ID NO.7）

the amplified fragment size was 349bp.

2.4.2 PCR identification

Extracting DNA of the bacterial strain HNHPS1 according to the operation steps of the DNA extraction kit, measuring the concentration by a spectrophotometer to be 100ug/mL, and carrying out PCR identification; meanwhile, a type 4 haemophilus parasuis standard strain is set as a positive control, and a type 2 streptococcus suis standard strain is set as a negative control.

The PCR amplification reaction system is 25 muL: 2.5 muL of 10 Xbuffer solution, 0.5 muL of 2.5mM dNTPs, 1 muL of 10 muM/L general primer HPS1 and 1 muL of HPS2 respectively, 5U/muL rTaq 1 muL and 100ugAdding ddH into 1 mu L/mL DNA template ₂ O is 25 muL;

the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, entering circulation: 95 ℃ 1 min,57 ℃ 1 min,72 ℃ 30 s for 35 cycles, final 72 ℃ extension 10min, PCR product preservation at 4 ℃.

The amplified products were subjected to electrophoresis, 10. Mu.L of sample was applied to each well, 1% agarose gel electrophoresis was performed, the results were observed under ultraviolet light (see FIG. 6), and 822bp fragments were amplified as a result, sequencing was 100% homologous to Haemophilus parasuis SH0165 strain (GenBank NO: CP 001321), ZJ0906 strain (GenBank NO: CP 005384), KL0318 strain (GenBank NO: CP 009237), and 99.6% homologous to SC1401 strain (GenBank NO: CP 015099), and sequencing results were as follows, demonstrating that the strain HNHPS1 is Haemophilus parasuis ((C. Sub.) (C.) (H.) (Hps))Haemophilus parasuis）。

GTGATGAGGAAGGGTGGTGTTTTAATAGAGCATTACATTGACGTTAGTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATGACTGGGCGTAAAGGGCACGCAGGCGGTGACTTAAGTGAGATGTGAAAGCCCCGAGCTTAACTTGGGAATTGCATTTCATACTGGGTTGCTAGAGTATTTTAGGGAGGGGTAGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGAAGGCGAAGGCAGCCCCTTGGGAAAATACTGACGCTCATGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGCTGTCGATTTGGGGATTGGGCTTTATGTTTGGTGCCCGTAGCTAACGTGATAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCTAAGAAGCTTTCAGAGATGAGAGTGTGCCTTCGGGAACTTAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGTGCATACAGAGGGTGACGAAGCC（SEQ ID NO.8）。

2.4.3 serotype identification

Referring to the method 2.4.2, a fragment of size 349bp was amplified by using specific primers HPS3 and HPS4 of Haemophilus parasuis type 4 (see FIG. 6), and the sequence was 100% homologous to the Haemophilus parasuis type 4 SW124 strain (GenBank NO: KC 795356), thus confirming that the strain HNHPS1 is Haemophilus parasuis type 4.

GGTTAAGAGGTAGAGCTAAGAATAGAGGATATATCaCTCcAaGTGaATTAGGATGTACGTTAAGTCATTTTTCCGTGTATCGTGATTTTTTAAATAGTGATAAGGAATGGTTATTAGTTCTAGAAGATGATGTAACTATAAATAGTAATTTATTTTTTCTATTGGAGAGTATTATTAATCAAAATTATAGTGACTATATTAATATTCTGGGGGGACAGGAGGGGTTGAAAAGACCTAGAGTGCTAGAGTTTCTTTTTCGAGAAAATATAAAAATTCTTAATTCCCCTTTTTATGGATTCTTTTTATATCGAACGTGTAGTTATTTGGTTTCTAGAGCTGTTGGTGGAAAG（SEQ ID NO.9）。

Example 3: isolation and characterization of Strain HN1570

3.1 preparation of the culture Medium

TSB liquid medium and TSA solid medium were prepared by referring to the method in example 2.

3.2 isolation and culture of HN1570 Strain

The applicant collects the heart blood of a nursery pig with typical multiple serositis in 2017 under the aseptic condition, inoculates the heart blood on a TSA solid culture medium containing NAD, cultures 36 h in a constant-temperature incubator at 37 ℃, picks suspected colonies for subculture and purification, observes the colony morphology of haemophilus parasuis after culturing for 24-48 hours, and grows colonies with consistent needle-tip shape size, colorless transparency, smoothness and humidity and diameter size of 1-2 mm (see figure 7); while purifying and inoculating on TSA solid culture medium without NAD, the TSA culture medium without NAD can not grow;

the single colonies were picked for gram-stauroscopy and the morphology of the colonies was observed to be characterized as gram-negative bacteria with a variety of different morphologies, ranging from single coccobacillus to elongated filamentous bacteria (see FIG. 8). The strain HN1570 is preliminarily judged to belong to the genus Haemophilus parasuis according to the measurement results of morphological and biochemical characteristics of the strain HN 1570.

3.3 identification of the Strain HN1570

3.3.1 Primer design

The design of the general primer of the haemophilus parasuis is the same as HNHPS1.

Designing a pair of specific primers of the haemophilus parasuis type 5 according to the sequence of the haemophilus parasuis wciP, and using the primers for the amplification of the haemophilus parasuis type 5, wherein the primer sequences are as follows:

HPS5：5’-CCACTGGATAGAGAGTGGCAGG-3’（SEQ ID NO.10）

HPS6：5’-CCATACATCTGAATTCCTAAGC-3’（SEQ ID NO.11）

the amplified fragment size was 450bp.

3.3.2 PCR identification

DNA of the strain HN1570 was extracted according to the procedure of the DNA extraction kit, and the strain HN1570 was PCR-amplified according to the PCR amplification method described in example 2.

The amplified product is subjected to electrophoresis, 10 muL of sample is added into each hole, the electrophoresis is carried out by 1% agarose gel, the result is observed under ultraviolet light (see figure 9), the result is shown in the figure, the strain to be detected in the embodiment, namely the strain HN1570, is the same as the standard strain of haemophilus parasuis, and the sequencing of the strain HN1570 is 99% homologous with the haemophilus parasuis CLSH0104 strain (GenBank NO: CP 024412), 29755 strain (GenBank NO: CP 021644) and KL0318 strain (GenBank NO: CP 009237); demonstration that Strain HN1570 is Haemophilus parasuis (II)Haemophilus parasuis）。

3.3.3 Serotype identification

Referring to the method 2.4.2, the strain HN1570 was type 5 Haemophilus parasuis as a result of amplification with specific primers HPS5 and HPS6 of Haemophilus parasuis type 5, and as a result of sequencing, a 450bp fragment was amplified (see FIG. 9), and the sequencing result was shown below, and the sequencing was 99% homologous to Haemophilus parasuis type 5 KL0318 strain (GenBank NO: CP 009237).

CCTCCATACATTCGAATTCCTAAGCCAAAAATATATTTTATTTCTAGGGAACCAGCCATGACTTAAAAAATCAACTGGTTTATCATGAAAGAAAAAGCGCTCTTTATTGACACCCCTCATAATAATCACATCATCATTTAAATATACAAAATTTTCAGATAGAGCATCTATATTACCCAAATTCATTTCAATTGAACTGGAATTAAAAATAGGAAGGTGTTCTTTCTCATAATAAAGCTGATTATGTGTAATTAAAAATATTTTTTCATTATTTATATCTAACCATTCAGGAAAGTGTCCCTCAGTAATTAAATATATTCTATTATACCAAGGGCAGTTTTTTTCTATAGATCTTAGAACATATCTTAGAGTTCCCATATCTCTATATCTTGATTCAGAATTTGAACTAGTTTCTTTTAAATTTATATTACTATAGAAACTTTTTTTTGCCTGCCACTCTCTTATCCAGTGGA（SEQ ID NO.12）。

Example 4: isolation and characterization of the Strain HNSS1

4.1 preparation of the culture Medium

4.2 isolation and culture of the Strain HNSS1

The diseased material comes from a 2-month-old nursery pig with severe pneumonia, dyspnea and neurological symptom death in a large pig farm in Dryowa county, henan province in 2016 years, the brain tissue of the diseased pig is aseptically collected and inoculated on a TSA solid culture medium containing NAD, the diseased pig is cultured in a constant-temperature incubator at 37 ℃ for 24 hours, and consistent needle tip-shaped size is grown, a single colony on a blood plate is 0.5-1 mm, grey white color is semi-transparent, round, smooth and neat edge is in alpha hemolysis (see figure 10); after purification and inoculation, gram staining is carried out, the bacteria are gram positive bacteria, circular or oval, and are arranged in a chain shape or double in a chain shape, the chain lengths are different (see figure 11), and the bacteria are named as a strain HNSS1.

4.3 Identification of strains

4.3.1 design of primers

A pair of universal primers is designed according to the sequence of the gdh gene of the streptococcus suis and is used for amplifying the streptococcus suis, and the primer sequences are as follows:

SS1：5’-GCAGCGTATTCTGTCAAACG-3’（SEQ ID NO.13）

SS2：5’-CCATGGACAGATAAAGATGG-3’（SEQ ID NO.14）

the amplified fragment size was 689bp.

Designing a pair of streptococcus suis type 2 specific primers according to the sequence of streptococcus suis cps2J gene, and using the primers for amplifying the streptococcus suis type 2, wherein the primer sequences are as follows:

SS3：5’-TGATAGTGATTTGTCGGGAGGG-3’（SEQ ID NO.15）

SS4：5’-GAGTATCTAAAGAATGCCTATTG-3’（SEQ ID NO.16）

the amplified fragment size was 557bp.

A pair of primers is designed according to the sequence of the swine streptococcal extracellular factor (epf) gene and is used for amplification of the swine streptococcal extracellular factor (epf) gene, and the sequences of the primers are as follows:

epf1：5’-GCTACGACGGCCTCAGAAATC-3’ （SEQ ID NO.17）

epf2：5’-TGGATCAACCACTGGTGTTAC-3’（SEQ ID NO.18）

the amplified fragment size was 626 bp.

A pair of primers is designed according to the sequence of streptococcus suis lysozyme releasing protein (mrp) gene and is used for amplifying the streptococcus suis lysozyme releasing protein (mrp) gene, and the sequences of the primers are as follows:

mrp1：5’-ATCAGAATCACCACTTTTGG-3’ （SEQ ID NO.19）

mrp2：5’-TCATACCCAGTAAATACACG-3’ （SEQ ID NO.20）

the amplified fragment size was 885 bp.

A pair of primers is designed according to the sequence of the swine streptolysin (sly) gene and is used for amplification of the swine streptolysin (sly) gene, and the primer sequences are as follows:

sly1：5’-ATGAGAAAAAGTTCGCACTTGATT-3’ （SEQ ID NO.21）

sly2：5’-TTGCCAGATTACTCTATCA-3’ （SEQ ID NO.22）

the amplified fragment size was 1502bp.

4.3.2 PCR identification

Extracting DNA of the bacterial strain HNSS1 according to the operation steps of the DNA extraction kit, measuring the concentration by a spectrophotometer to be 100ug/mL, and carrying out PCR identification.

The PCR amplification reaction system is 25 muL: 10 Xbuffer solution 2.5 muL, 2.5mM dNTPs 0.5 muL, 10 muM/L universal primer SS1, SS 21 muL, 5U/muL rTaq 1 muL and 100ug/mL DNA template 1 muL respectively, and ddH is added ₂ And O is 25 muL.

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, entering circulation: 95 ℃ for 1 min,57 ℃ for 1 min,72 ℃ for 30 s for 35 cycles, final 72 ℃ extension for 10min, and storage of PCR products at 4 ℃.

Respectively carrying out electrophoresis on the amplified products, adding 10 mu L of sample per well, carrying out 1% agarose gel electrophoresis, observing the result under ultraviolet light (see figure 12), amplifying a 689bp fragment, sequencing the product to be 100% homologous with the Streptococcus suis SS2-1 strain (GenBank NO: CP 018908), and verifying that the strain HNSS1 is the Streptococcus suis (see figure 12)Streptococcus suis）。

GCAGCGTATTCTGTCAAACGAGCGCGGCGTTTTTCTTTGATGTCCACCAAGAGGTCGAAGTCGATACCAGTTTCGTCAATGATGTAACCATTTGAGTCTGAAACAGAAATAACTTTTGCACCAAGTTCAGTCGCTTTTTGAACAGCATATTGGGCAACGTTACCAGAACCTGAGATAAGGACAGTTTGGTCTTTGAAGGATTTACCGTTTGCTGCCAACATGTTATCAGTGAAGTAAACCAAACCGTAACCAGTTGCTTCTGGGCGGATCAATGAACCACCGAAGCCAAGAGGTTTACCAGTCAAGACACCTGCATCAAACTGGCGGAGGCGTTTGTATTGACCGTACATGTAACCGATCTCACGACCACCGACACCGATGTCACCAGCAGGGACGTCAAGTGAAGGTCCGATGTGTTTTTGCAATTCAGTCATGAAGCTTTGGCAGAAGCGCATGATTTCAGCATCAGTTTTTCCTTTAGGATCAAAGTCTGAACCACCTTTACCACCGCCGATTGGAAGACCAGTCAAGACGTTTTTGAAGATTTGCTCAAAACCGAGGAACTTCAAGATGGATTGGTTTACAGTTGGGTGGAAGCGAAGACCGCCTTTATAAGGACCTACAGCTGAGTTGAACTGAACACGGTAGCCACGGTTGACTTGAACATTTCCATCTTTATCTGTCCATGG（SEQ ID NO.23）。

4.3.3 serotype identification

Referring to the method of 4.3.2, the strain HNSS1 is proved to be the type 2 streptococcus suis by using primers SS3 and SS4 specific to the type 2 streptococcus suis to amplify a 557bp size fragment (see figure 12) and sequencing the fragment with 100 percent homology with the type 2 streptococcus suis strain SS2-1 (GenBank NO: CP 018908).

TGATAGTGATTTGTCGGGAGGGTTACTTGCTACTTTTGATGGAAATTATCAAGAATCTGAGCTGCAAAAGTGTCAAATTGATTTGGAAGAGATAAAAGAGGTGCGAGACTTAGGAAATGAAAATTTTCCAAATCATTATATGAGCGGTATCTTTAATAGCCCTTGTTGCAAACTTTATAAGAATATATATATAAACAAAGGTTTTGACACTGAACAGTGGTTAGGAGAGGACTTATTATTTAATCTAAATTATTTAAAGAATATAAAAAAAGTCAGCTATGTAAACAGAAATCTTTATTTTGCTAGAAGAGGTATACAAAGTACTACAAATACGTTTAAAAAAGATGTTTTTATTCAATTAGAAAATTTAGAAGAAAAAACTTTTGATTTGTTTGTTAAAATATTTGGTGGACAATATGAATTTTCTGTTTTTAAAGAGACGCTACAGTGGCATATTATTTATTATAGCTTATTAATGTTCAAAAATGGAGATGAATCGCTTCCAAAGAAATTGCATATATTTAAGTATTTATACAATAGGCATTCTTTAGATACTC (SEQ ID NO.24）。

4.3.4 virulence factor identification

Referring to the method of 4.3.2, primers of Streptococcus suis extracellular factor (epf) gene, lysozyme releasing protein (mrp) gene and hemolysin (sly) gene are used for amplification, so that 626 bp, 885 bp and 1502bp size fragments (shown in figure 12) are respectively amplified, the sequencing is 100 percent homologous with Streptococcus suis SS2-1 strain (GenBank NO: CP 018908), and the genotype of the separated virulence factor of the Streptococcus suis serotype 2 is proved to be sly ⁺ mrp ⁺ epf ⁺ 。

Example 5 isolation and characterization of the Strain HNAPP1

5.1 Collection of pathological material

The disease is from a fattening pig of 5 months old which has severe pneumonia and dyspnea acute death in a large pig farm in Ruzhou city, henan province in 2016 and 11 months.

5.2 Preparation of the culture Medium

5.3 Isolation and culture of bacteria

Aseptically collecting heart blood of sick pigs, inoculating the heart blood on a TSA solid culture medium containing NAD, culturing 24h in a constant-temperature incubator at 37 ℃, growing consistent round convex, semitransparent, smooth and moist bacterial colonies with the diameter of 1-1.5 mm, observing the bacterial colonies with obvious gold red blue fluorescence under 45-degree refracted light (see figure 13), purifying and inoculating the bacterial colonies on the TSA solid culture medium containing no NAD, and not growing on the TSA culture medium containing no NAD; gram-negative bacteria, coccobacillus, microbacterium, partially filamentous thallus designated the strain HNAPP1 (see fig. 14).

5.4 Identification of strains

5.4.1 Design of primers

A pair of universal primers is designed according to the sequence of an Actinobacillus pleuropneumoniae ApxIV gene and is used for amplifying the Actinobacillus pleuropneumoniae, and the primer sequences are as follows:

APP1：5’-GCTCACCAACGTTTGCTCAT-3’（SEQ ID NO.25）

APP2：5’- GGGGACGTAACTCGGTGATT -3’ （SEQ ID NO.26）

the amplified fragment size was 377 bp.

Meanwhile, a pair of specific primers of the actinobacillus pleuropneumoniae type 1 are designed according to the sequence of the actinobacillus pleuropneumoniae capsule CPS1B gene, and are used for amplifying the actinobacillus pleuropneumoniae type 1, wherein the primer sequences are as follows:

APP3：5’-CTGGAGTAATTACGGCGACTATTCC-3’（SEQ ID NO.27）

APP4：5’- AGGAGAAGCTAGTAGTACTTGCATTTTC -3’（SEQ ID NO.28）

the amplified fragment size was 959 bp.

5.4.2 PCR identification

Extracting DNA of the strain HNAPP1 according to the operation steps of the DNA extraction kit, measuring the concentration by a spectrophotometer to be 100ug/mL, and carrying out PCR identification; meanwhile, a type 1 actinobacillus pleuropneumoniae standard strain is set as a positive control, and a type 4 haemophilus parasuis standard strain is set as a negative control.

The PCR amplification reaction system is 25 muL: 2.5 muL of 10 Xbuffer solution, 0.5 muL of 2.5mM dNTPs, 1 muL of 10 muM/L general primer APP1, 1 muL of APP2, 1 muL of 5U/muL rTaq 1 muL and 1 muL of 100ug/mL DNA template are added with ddH ₂ And O is 25 muL.

And (4) respectively carrying out electrophoresis on the amplification products, adding 10 mu L of sample into each hole, carrying out 1% agarose gel electrophoresis, and observing the result under ultraviolet light. As a result, a 377bp fragment (see FIG. 15) was amplified, and the sequencing was 100% homologous to the A.pleuropneumoniae 8392 ApxIVA gene (GenBank NO: AF 188867), and the sequencing result was as follows, demonstrating that the strain HNAPP1 is A.pleuropneumoniae ((A) (A. Pleuropneumoniae))Actinobacillus pleuropneumoniae）。

GGGGACGTAACTCGGTGATTGATGCCGGTGCGGGTAATGATACGGTTAATGGCGGTAATGGCGATGACACCCTCATCGGCGGCAAAGGTAATGATATTCTAAGAGGTGGCTACGGTGCGGACACCTATATCTTTAGCAAAGGACACGGACAGGATATCGTTTATGAAGATACCAATAATGATAACCGCGCAAGAGATATCGACACCTTAAAATTTACTGATATTAATTTATCCGAACTTTGGTTTAGCCGAGAAAATAACGATTTGATTATTAAATCATTATTAAGTGAGGATAAAGTCACGGTTCAAAATTGGTATTCACACCAAGATCATAAAATAGAAAATATTCGTTTATCGAATGAGCAAACGTTGGTGAGC（SEQ ID NO.29）

5.4.3 serotype identification

Referring to the method of 5.4.2, a 959 bp size fragment was amplified by amplification using specific primers APP3 and APP4 of Actinobacillus pleuropneumoniae type 1 (see FIG. 15), and sequencing was 100% homologous to Actinobacillus pleuropneumoniae type 1S 4074 strain (GenBank NO: AF 518558), thus confirming that the strain HNAPP1 is Actinobacillus pleuropneumoniae type 1.

CTGGAGTAATTACGGCGACTATTCCTGAATATAACTCAATAGTAGAAGAACTAAATAAATATAAAATTGGTAAAAAGGAAACATTTTTAACAGGATTTCCTCGCCATGATAAATTACTATCTGGAAATATAAAAGGAGCTAAGACAATTCTCATCGTACCTACATGGCGACATTATATTATGGGGACTCAAATTGGAAAAGGAGCCAATACACGCGAGCTAAATAAAGCCTTTATGACAACAAATTATGCTAAAGCTTGGTATAATTTATTACATAGTCAGGAATTAAAAAATTTAATTAAAAATTTAGGATATAAAGTTATTTTTGCACCACACCCTAATATTGAACCATATTTAAATGAGTTTAACATCCCCCAATATATTGATGTGTGGAAAAGTGCAATATCAAGAGAAAGTATGCAAAGTTTATTCCAACAATCAAATCTATTGATTACGGACTATTCATCTATTGCATTTGAAATGGCATTTCTAGGAAAACAAACAATCTATTACCAATTTGATAAAGAGGAATTTAGATCTGGAATTCATACATATCAACAAGGATACTTTGAATACGAGAAAGATGGATTTGGTCCTGTAGCTGAAACATTAGATGATTTATTTATTCACCTAGATAAATTCGTAAACGGTGAAAATGATTACATAAATATTTATCAATCTCGTATACAAAAAACATTTAAATATCGGGATACGAATAATT

GCCAACGTGTTTATGAAGCTATTATTAACTTAGATATGCCGGATAAAGATATTAATAAAAATATTATCTTAAATGCTTTAGAATCTGCTTACAAAGCTCAAGACTGGAATTTAGTTATCTCTCGTGCTGAAGCTTTATTAGCAGAAATACCTAATCATTCATTTGCTAAGAGTGTGTTATTGAAGCAGTGATAGCTTCAAACAATAAAGAGAAAATGCAAGTACTACTAGCTTCTCCT（SEQ ID NO.30）。

Example 6: toxicity test

6.1 Virulence test of Mycoplasma hyopneumoniae HNMhy1

The test pigs are 8 healthy nursery pigs with 15 days of age and are negative to mycoplasma hyopneumoniae ELISA antibody. The test animals are divided into two groups, namely 4 test animals in a control group and 4 test animals in a test group, and the two groups of test animals are randomly grouped; inoculating HNMhy1 into PPLO liquid culture medium, at 37 deg.C, with 5% CO ₂ Culturing 3d in incubator, measuring thallus concentration, continuously diluting and coating solid plate by 10 times, counting viable bacteria under low power lens, calculating thallus concentration of culture stock solution, and adjusting to 10 ⁸ CFU/mL, test group animals inoculated 3mL Mycoplasma hyopneumoniae liquid culture through laryngotracheae, control group animals inoculated 3mL sterilized PPLO liquid culture medium through laryngotracheae, test period was 15 d, clinical performance of test animals was observed every day, body temperature was measured, test animals were immediately killed if they died, respiratory lesions were observed to collect disease material, and at the end of test 15 d, all test animals were killed.

After being inoculated with mycoplasma 7 d, animals in the test group show symptoms such as oral-nasal foam, dyspnea, cough and the like, and have normal body temperature, reduced appetite, mouth opening and tongue stretching and dog sitting-type abdominal respiration; the control animals were normothermic and had no obvious respiratory symptoms during the test period.

After the test is finished, killing animals in the test group, killing 4 animals in the control group at the same time, collecting disease materials, and separating mycoplasma; the experimental group is analyzed, the tip leaf, heart leaf and septal leaf of the lung have the real change of bilateral symmetry 'flesh sample change', and the boundary with the surrounding tissues is obvious; no obvious pathological change occurs in the autopsy of the control group, mycoplasma is not separated from 4 pathological materials of the control group, mycoplasma hyopneumoniae is separated from 4 pathological materials of the test group, the colony morphology is expressed as a fried poached egg sample, and the PCR identification result is consistent with the HNMhy1.

Virulence test of haemophilus parasuis

Selecting 12 weaned healthy nursery pigs of 9 to 10 weeks as test animals. Dividing the test animals into 4 groups at random, 4 control groups, 4

test groups

1 and 4 test groups 2; respectively inoculating Haemophilus parasuis strain HNHPS1 strain and HN1570 strain to TSB liquid culture medium, shake culturing at 37 deg.C for 24h, measuring thallus concentration, continuously diluting by 10 times, coating solid plate, counting viable bacteria under low-power microscope, calculating stock solution thallus concentration, adjusting to 10 ⁸ CFU/mL; the animals of

experiment

1 and 2 groups were inoculated with 3 mLHNHPS1 and HN1570 Haemophilus parasuis liquid cultures through the trachea, respectively, and the animals of control group were inoculated with 3mL sterilized TSB liquid culture medium through the trachea. After injection, the observation was continued for 2 weeks, and the number of morbidities and deaths were observed and recorded.

The observation shows that: the groups of

trials

1 and 2 showed symptoms of asthma, dyspnea, etc. after 24h inoculation with haemophilus parasuis, elevated body temperature, cyanosis of ears, abdomen and buttocks, and appearance of rolling circles. Test 1 group died 1 after 2 days and 2 after 3 days; test 2 groups died 2 after 2 days and 2 after 3 days. The control animals were normothermic and had no obvious respiratory symptoms during the test period; and (3) killing animals in the test group after the test is finished, killing 4 animals in the control group at the same time, collecting disease materials, and carrying out bacteria separation.

Experiments

1 and 2 groups of autopsy lung has a large amount of fibrin-like exudation, adhesion to pleura, pericardial effusion, villous heart and cerebral hemorrhage; no obvious pathological change occurs in the autopsy of a control group, haemophilus parasuis is not separated from 4 pathological materials of the control group, haemophilus parasuis is separated from 4 pathological materials of the

test groups

1 and 2, and the PCR identification result is consistent with the strains HNHPS1 and HN 1570; and (4) integrating virulence test results to determine that the porcine haemophilus strains HNHPS1 and HN1570 are shown as strains with the strongest virulence.

Virulence test of Streptococcus suis HNSS1

The test animals were 8 weaned healthy nursery pigs of 4~7 weeks of age. The test animals were divided into two groups, 4 control groups and 4 test groups. Inoculating HNSS1 into TSB liquid culture medium, shake culturing at 37 deg.C for 18 h, measuring thallus concentration, continuously diluting by 10 times, coating on solid plate, counting viable bacteria under low power lens, calculating stock solution thallus concentration, and adjusting to 10 ⁸ CFU/mL, experimental animals were inoculated with 3mL liquid culture of Streptococcus suis by cervical intramuscular injection, and control animals were inoculated with 3mL sterilized TSB liquid culture medium by cervical intramuscular injection. After injection, the observation was continued for 2 weeks, and the number of morbidities and deaths were observed and recorded.

After inoculation of streptococcus suis 24h, animals in the test group showed obvious symptoms of high fever, joint swelling, opisthotonus, limb watery, dyspnea, nostril bleeding and the like, and all the animals died within 3 days. The control animals were normothermic and had no obvious respiratory symptoms during the test period. After the test, the animals in the test group were killed, and 4 animals in the control group were killed. Collecting disease material, and separating streptococcus suis. The experimental group was examined for pulmonary hemorrhage, hydrarthrosis, and cerebral hemorrhage. No obvious pathological change occurs in the control group in the caesarean section, no streptococcus suis is separated from 4 disease materials of the control group, streptococcus suis is separated from 4 disease materials of the test group, and the PCR identification result is consistent with the HNSS1.

Virulence test of actinobacillus pleuropneumoniae HNAPP1

The test animals were 8 weaned healthy nursery pigs of 2 months of age. The test animals were divided into two groups, 4 control groups and 4 test groups. Inoculating HNAPP1 into TSB liquid culture medium, culturing at 37 deg.C by shaking table for 18 h, measuring thallus concentration, continuously diluting and coating solid plate by 10 times, counting viable bacteria under low-power lens, calculating stock solution thallus concentration, and adjusting to 10 ⁸ CFU/mL, passage of experimental animalsThe nasal drops were infected with 3mL of actinobacillus pleuropneumoniae liquid culture, and the control animals were inoculated with 3mL of sterilized TSB liquid culture medium by intratracheal inoculation. After injection, the observation was continued for 2 weeks, and the number of morbidities and deaths were observed and recorded.

The animals of the test group showed symptoms of dyspnea after inoculation of actinobacillus pleuropneumoniae 12 h, increased body temperature, cyanosis of ears, abdomen and buttocks, finally staying down on bed, muscle twitching, death by asphyxia, and before death, oronasal outflow of blood with foam. Death was 1 at

day

2 and 3 at 2 days later. The animals in the control group were normal in appetite, spirit and body temperature during the test period. After the test, the animals in the test group were killed, and 4 animals in the control group were killed. Collecting disease material, and separating actinobacillus pleuropneumoniae. The experimental group was examined for cardiopulmonary cellulose exudation, pericardial adhesion, purplish red lung, juicy section, bleeding, and trachea and bronchus filled with foam and bloody mucus. No obvious pathological change occurs in the control group in the way of autopsy, no actinobacillus pleuropneumoniae is separated from 4 disease materials of the control group, actinobacillus pleuropneumoniae is separated from 4 disease materials of the test group, and the PCR identification result is consistent with HNAPP1.

Example 7: vaccine preparation, safety and efficacy testing

7.1 Preparation of vaccines

7.1.1 strains and strain propagation

First order seed reproduction

Inoculating Mycoplasma hyopneumoniae HNMhy1 strain to PPLO solid medium, placing at 37 deg.C, and 5% ₂ Culturing in an incubator for 3 days, collecting culture, respectively inoculating freeze-dried strains of haemophilus parasuis type 4 HNHPS1, type 5 HN1570, streptococcus suis type 2 HNSS1 and actinobacillus pleuropneumoniae type 1 HNAPP1 on a TSA plate containing newborn bovine serum (final concentration 5%) and NAD (final concentration 0.01%), culturing at 37 ℃ for 18-24h, selecting typical bacterial colonies meeting requirements, respectively subculturing a TSA plate containing newborn bovine serum (final concentration 5%) and NAD (final concentration 0.01%), culturing at 37 ℃ for 24h, and taking the obtained product as a primary seed after passing inspection.

Two stage seed reproduction

Inoculating the primary seeds of each plant to PPLO,5% CO in TSB broth containing newborn bovine serum (final concentration 5%) and NAD (final concentration 0.01%) (37 ℃ C.) ₂ Shaking and culturing for 3d and 18 to 24h by using a shaking table, and taking the obtained product as a secondary seed after passing a pure inspection.

7.1.2 culture of antigen bacteria

Inoculating 1% of secondary seeds of Mycoplasma hyopneumoniae, two Haemophilus parasuis, streptococcus suis, and Actinobacillus pleuropneumoniae strains into PPLO, TSB liquid culture medium containing newborn calf serum (final concentration 5%) and NAD (final concentration 0.01%), respectively, and performing 5% CO conversion at 37 deg.C ₂ And performing shaking culture on the shaking table for 3d and 18-24 h to obtain a bacterial liquid.

7.1.3 viable count

Viable cell counts were performed according to the method in the appendix of the current "Chinese veterinary pharmacopoeia" using a PPLO solid medium suitable for growth of the present cells, a TSA medium containing 5% newborn bovine serum and 0.01% NAD. Using shake flask culture, the viable count concentration, the viable bacteria content of five strains are all 1.0 × 10 ⁹ CFU/mL or more.

7.1.4 inactivation of bacterial liquid

Separately, a 0.2% formaldehyde solution was added to the culture broth of each of the mycoplasma hyopneumoniae, two haemophilus parasuis, streptococcus suis, and actinobacillus pleuropneumoniae strains in a total volume amount, and inactivated at 37 ℃ for 24 hours.

7.1.5 inactivation assay

Inoculating 0.2mL of inactivated bacterial solution of Mycoplasma hyopneumoniae, two haemophilus parasuis, streptococcus suis, and Actinobacillus pleuropneumoniae strains, respectively, into PPLO or TSA plates (containing 5% neonatal bovine serum and 0.01% NAD), placing at 37 deg.C, 5% CO ₂ Culturing for 3d and 48h, and determining that no bacteria grows to be qualified.

Concentration of 7.1.6 antigen

And (3) respectively concentrating the bacterial liquid of the five antigens which are qualified in the inactivation test through an ultrafiltration system device. According to the counting result of viable bacteria before inactivation, the concentration of five bacterial antigens is adjusted to 1.0 multiplied by 10 by using sterile normal saline ¹⁰ CFU/mL, and mixing the five antigen solutions in a ratio of 1.

7.1.7 vaccine preparation

(1) Preparing an oil phase: adding 92 parts of white oil for injection into an oil phase tank, adding 1 part of aluminum stearate into the tank, stirring while adding until the mixture is completely transparent, adding span-80 parts, sterilizing at 116 ℃ for 40min under high pressure, cooling to room temperature, adding 0.5 part of each of vitamin A and vitamin E, and uniformly mixing to obtain an oil phase for later use;

(2) Preparation of an aqueous phase: mixing antigen solutions of mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain obtained after concentration uniformly according to the proportion of 1:1 to prepare antigen solutions, taking 95 parts of the antigen solutions according to the volume part ratio, adding sterilized Tween-80 parts, and fully stirring until the antigen solutions are completely dissolved to obtain a water phase for later use;

Or adding 10% of aluminium hydroxide adjuvant into the concentrated and uniformly mixed antigen solution according to the volume ratio, stirring and fully and uniformly mixing, and subpackaging to obtain the aluminium hydroxide gel adjuvant-containing mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine.

7.2 Safety testing of vaccines

10 healthy piglets of 14 days old are selected and divided into 2 groups, and 5 piglets are selected.

Vaccine groups: injecting the vaccine 4 mL through neck muscle;

control group: 4 mL sterilized normal saline is injected intramuscularly in the neck. Animal body temperature was measured and clinical performance was observed for 2 weeks.

The observation shows that the respiratory, appetite and mental state of the nursery pigs of the vaccine group and the control group are normal in the whole observation period, and the table 1 shows that the average body temperature rise does not exceed 1 ℃, which indicates that the inactivated vaccine of the invention is safe.

7.3 immunization challenge test of vaccines

Selecting 125 healthy piglets of 15 days old, which are negative to the ELISA antibodies of mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae; the vaccine immunity challenge test is provided with 5 groups of a mycoplasma hyopneumoniae HNMhy1 strain challenge test group, a haemophilus parasuis HNHPS1 strain challenge test group, a haemophilus parasuis HN1570 strain challenge test group, a streptococcus suis HNSS1 strain and an actinobacillus pleuropneumoniae HNAPP1 strain challenge test group; for each large group of trials, 25 piglets were selected and divided into 5 groups, each with 5 piglets. The specific grouping is as follows: vaccine 1 group, vaccine 2 group, vaccine 3 group and vaccine 4 group, wherein each vaccine group is injected with 0.5ml, 1ml, 1.5ml and 2ml of the vaccine respectively; group 5 was a control group, and 2mL sterile saline was injected. The vaccine group and the control group were injected with the same dose of vaccine or sterilized normal saline at 3 weeks after the immunization. 10 is respectively used in each group of experiments 14 days after the secondary immunization ⁹ The bacterial liquid of the bacterial strains of CFU mycoplasma hyopneumoniae HNMhy1, haemophilus parasuis HNHPS1, HN1570, streptococcus suis HNSS1 and actinobacillus pleuropneumoniae HNAPP1 adopts the nose-dropping toxin-attacking; observing the clinical manifestations of the piglets of each group, measuring the body temperature every day, and observing for 2 weeks; calculating the morbidity and mortality of the experimental piglets in the whole observation period, measuring the body temperature, and performing bacteria isolation culture on organs such as the lung of the dead piglets.

As a result: the vaccine group is obviously different from the non-immune group, and clinical symptoms, running nose, dyspnea, anorexia and emaciation begin to appear on the 7 th day of the non-immune group after the virus challenge of the mycoplasma hyopneumoniae HNMhy 1; the autopsy finds that: the tip, heart and septal lobe parts of the lung have real changes of bilateral symmetry 'flesh-like changes', and have obvious boundary with surrounding tissues; in the vaccine group, 2 piglets in 5 piglets in the 0.5mL vaccine group showed clinical symptoms and case changes; 1 of 5 piglets in the 1mL vaccine group presented with clinical symptoms and case changes; 1.5 No disease was observed in the mL and 2mL vaccine groups of 5 piglets;

on the 2 nd day after the two haemophilus parasuis detoxication, piglets in the non-immunized group begin to have clinical symptoms at the 2 nd day, rise in body temperature, dyspnea and circling, begin to die after 2 days, and die for 5 after 2 weeks; performing autopsy on dead pigs in an uninmmunized group, wherein the dead pigs have effusion in the chest and abdominal cavities, cellulosic exudate, pericardial effusion, cellulosic exudate in the pericardial cavity, pericardial adhesion, lung enlargement, extravasated blood or hemorrhage of lung lobes on two sides, surface cellulosic exudate, meningeal congestion and cerebrospinal fluid increase; in the vaccine group, 1 of 5 piglets in the 0.5ml vaccine group has clinical symptoms and case changes; 5 piglets of 1ml, 1.5ml and 2ml vaccine groups did not develop disease;

the streptococcus suis vaccine group is obviously different from the non-immune group, clinical symptoms, body temperature rise, dyspnea and arcus vertebrae reverse tension begin to appear in the non-immune group on the second day after 2 days after virus attack, and 5 patients die in 1 week; and (4) carrying out autopsy on the dead pig lung, swelling, blood stasis or bleeding, joint effusion and meningeal bleeding. In the vaccine group, 1 of 5 piglets in the 0.5ml vaccine group has clinical symptoms and case changes; 5 piglets in 1ml, 1.5ml, 2ml vaccine groups did not develop disease;

the actinobacillus pleuropneumoniae vaccine group is obviously different from the non-immune group, the clinical symptoms, the body temperature rise and the breathing difficulty of the piglets of the non-immune group begin to appear the next day after the challenge, the piglets die after the 2 nd day, the mouth and nose bleeding before death, and the piglets die in 5 days in total within 3 days. Performing autopsy on dead pigs in an uninmmunized group, wherein the dead pigs have effusion in the chest and abdominal cavities, cellulosic exudates, pericardial adhesion, pulmonary swelling and bleeding, and bloody foams filled in the trachea; in the vaccine group, 2 piglets in 5 piglets in the 0.5ml vaccine group have clinical symptoms and case changes; 5 piglets of the 1ml, 1.5ml and 2ml vaccine groups had no disease.

The immune toxicity-combating results of mycoplasma hyopneumoniae HNMhy1, haemophilus parasuis HNHPS1, HN1570, streptococcus suis HNSS1 and actinobacillus pleuropneumoniae HNAPP1 are shown in tables 2,3, 4, 5 and 6, respectively.

The result of the immune challenge test shows that the vaccine with the immune dose of 1ml can resist the infection of mycoplasma hyopneumoniae, haemophilus parasuis serogroups 4 and 5, streptococcus suis 2 and actinobacillus pleuropneumoniae 1 by two times of immunization, and the inactivated vaccine has good protection effect.

The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of zootechnics of academy of agricultural sciences of Henan province

<120> mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine and application thereof

<141> 2019-04-19

<160> 30

<170> SIPOSequenceListing 1.0

<210> 28

<211> 27

<212> DNA

<213> Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae)

<400> 28

acgcgtcgac agagtttgat cctggct 27

<210> 29

<211> 27

<212> DNA

<213> Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae)

<400> 29

cgcggatccg ctaccttgtt acgactt 27

<210> 30

<211> 1483

<212> DNA

<213> Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae)

<400> 30

acgcgtcgac agagtttgat cctggctcag gatgaacgct ggcggcgtgc ctaatacatg 60

caagtcgaac gaagcatctt cggatgctta gtggcgaacg ggtgagtaac acgtagataa 120

cctaccttta actcgaggat aactccggga aactggagct aatactggat aggatgtgtg 180

catgaaaaaa acacatttaa agatttatcg gtttaagagg ggtctgcggc gcattagtta 240

gttggtgggg taagagccta ccaagacgat gatgcgtagc cggactgaga ggtctaccgg 300

ccacattggg actgagaacg gcccaaactc ctacgggagg cagcagtagg gaattttcgg 360

caatggggga aaccctgacc gagcaacgcc gcgtgaacga cgaagtactt cggtatgtaa 420

agttctttta tatgggaaga aaaattaaaa attgacggta ccatatgaat aagccccggc 480

taactatgtg ccagcagccg cggtaataca tagggggcga gcgttatccg gatttactgg 540

gcgtaaaggg tgcgtaggtg gttataaaag tttgtggtgt aagtgcagtg cttaacgctg 600

tgaggctatg aaaactatat aactagagtg agacagaggc aagtggaatt ccatgtgtag 660

cggtaaaatg cgtaaatata tggaggaaca ccagtggcga aggcggcttg ctgggtctat 720

actgacactg atgcacgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgagaacta agtgttggcc ataaggtcag tgctgcagtt aacgcattaa 840

gttctccgcc tgagtagtac gtacgcaagt atgaaactca aaggaattga cgggaccccg 900

cacaagcggt ggatcatgtt gtttaattcg aagatacacg aaaaacctta ccaggtcttg 960

acatactctg caaaggctta gaaataagtt cggaggctaa cagatgtaca ggtggtgcac 1020

ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080

attgctagtt accatcatta agttggggac tctagcgaga ctgccagtga taaattggag 1140

gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca aacgtgatac 1200

aatggctgga acaaagagaa gcgatagggt gacctggagc gaaactcaca aaaacagtct 1260

cagttcggat tggagtctgc aactcgactc catgaagtcg gaatcgctag taatcgcaaa 1320

tcagcatgtt gcggtgaata cgttctcggg gtttgtacac accgcccgtc aaaccacgaa 1380

agtgggcaat acccaacgcc ggtggcctaa cccgaaaggg agggagccgt ctaaggtagg 1440

gtccatgatt ggggttaagt cgtaacaagg tagcggatcc gcg 1483

<210> 4

<211> 20

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 4

gtgatgagga agggtggtgt 20

<210> 5

<211> 18

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 5

ggcttcgtca ccctctgt 18

<210> 6

<211> 28

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 6

ggttaagagg tagagctaag aatagagg 28

<210> 7

<211> 24

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 7

ctttccacaa cagctctaga aacc 24

<210> 8

<211> 822

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 8

gtgatgagga agggtggtgt tttaatagag cattacattg acgttagtca cagaagaagc 60

accggctaac tccgtgccag cagccgcggt aatacggagg gtgcgagcgt taatcggaat 120

gactgggcgt aaagggcacg caggcggtga cttaagtgag atgtgaaagc cccgagctta 180

acttgggaat tgcatttcat actgggttgc tagagtattt tagggagggg tagaattcca 240

cgtgtagcgg tgaaatgcgt agagatgtgg aggaataccg aaggcgaagg cagccccttg 300

ggaaaatact gacgctcatg tgcgaaagcg tggggagcaa acaggattag ataccctggt 360

agtccacgct gtaaacgctg tcgatttggg gattgggctt tatgtttggt gcccgtagct 420

aacgtgataa atcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga 480

cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta 540

cctactcttg acatcctaag aagctttcag agatgagagt gtgccttcgg gaacttagag 600

acaggtgctg catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac 660

gagcgcaacc cttatccttt gttgccagcg attcggtcgg gaactcaaag gagactgcca 720

gtgataaact ggaggaaggt ggggatgacg tcaagtcatc atggccctta cgagtagggc 780

tacacacgtg ctacaatggt gcatacagag ggtgacgaag cc 822

<210> 9

<211> 350

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 9

ggttaagagg tagagctaag aatagaggat atatcactcc aagtgaatta ggatgtacgt 60

taagtcattt ttccgtgtat cgtgattttt taaatagtga taaggaatgg ttattagttc 120

tagaagatga tgtaactata aatagtaatt tattttttct attggagagt attattaatc 180

aaaattatag tgactatatt aatattctgg ggggacagga ggggttgaaa agacctagag 240

tgctagagtt tctttttcga gaaaatataa aaattcttaa ttcccctttt tatggattct 300

ttttatatcg aacgtgtagt tatttggttt ctagagctgt tggtggaaag 350

<210> 10

<211> 22

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 10

ccactggata gagagtggca gg 22

<210> 11

<211> 22

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 11

ccatacatct gaattcctaa gc 22

<210> 12

<211> 473

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 12

cctccataca ttcgaattcc taagccaaaa atatatttta tttctaggga accagccatg 60

acttaaaaaa tcaactggtt tatcatgaaa gaaaaagcgc tctttattga cacccctcat 120

aataatcaca tcatcattta aatatacaaa attttcagat agagcatcta tattacccaa 180

attcatttca attgaactgg aattaaaaat aggaaggtgt tctttctcat aataaagctg 240

attatgtgta attaaaaata ttttttcatt atttatatct aaccattcag gaaagtgtcc 300

ctcagtaatt aaatatattc tattatacca agggcagttt ttttctatag atcttagaac 360

atatcttaga gttcccatat ctctatatct tgattcagaa tttgaactag tttcttttaa 420

atttatatta ctatagaaac ttttttttgc ctgccactct cttatccagt gga 473

<210> 14

<211> 20

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 14

gcagcgtatt ctgtcaaacg 20

<210> 14

<211> 20

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 14

ccatggacag ataaagatgg 20

<210> 15

<211> 22

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 15

tgatagtgat ttgtcgggag gg 22

<210> 16

<211> 23

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 16

gagtatctaa agaatgccta ttg 23

<210> 17

<211> 21

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 17

gctacgacgg cctcagaaat c 21

<210> 19

<211> 21

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 19

tggatcaacc actggtgtta c 21

<210> 19

<211> 20

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 19

atcagaatca ccacttttgg 20

<210> 20

<211> 20

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 20

tcatacccag taaatacacg 20

<210> 21

<211> 24

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 21

atgagaaaaa gttcgcactt gatt 24

<210> 22

<211> 19

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 22

ttgccagatt actctatca 19

<210> 23

<211> 689

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 23

gcagcgtatt ctgtcaaacg agcgcggcgt ttttctttga tgtccaccaa gaggtcgaag 60

tcgataccag tttcgtcaat gatgtaacca tttgagtctg aaacagaaat aacttttgca 120

ccaagttcag tcgctttttg aacagcatat tgggcaacgt taccagaacc tgagataagg 180

acagtttggt ctttgaagga tttaccgttt gctgccaaca tgttatcagt gaagtaaacc 240

aaaccgtaac cagttgcttc tgggcggatc aatgaaccac cgaagccaag aggtttacca 300

gtcaagacac ctgcatcaaa ctggcggagg cgtttgtatt gaccgtacat gtaaccgatc 360

tcacgaccac cgacaccgat gtcaccagca gggacgtcaa gtgaaggtcc gatgtgtttt 420

tgcaattcag tcatgaagct ttggcagaag cgcatgattt cagcatcagt ttttccttta 480

ggatcaaagt ctgaaccacc tttaccaccg ccgattggaa gaccagtcaa gacgtttttg 540

aagatttgct caaaaccgag gaacttcaag atggattggt ttacagttgg gtggaagcga 600

agaccgcctt tataaggacc tacagctgag ttgaactgaa cacggtagcc acggttgact 660

tgaacatttc catctttatc tgtccatgg 689

<210> 24

<211> 557

<212> DNA

<213> Streptococcus suis (Streptococcus suis)

<400> 24

tgatagtgat ttgtcgggag ggttacttgc tacttttgat ggaaattatc aagaatctga 60

gctgcaaaag tgtcaaattg atttggaaga gataaaagag gtgcgagact taggaaatga 120

aaattttcca aatcattata tgagcggtat ctttaatagc ccttgttgca aactttataa 180

gaatatatat ataaacaaag gttttgacac tgaacagtgg ttaggagagg acttattatt 240

taatctaaat tatttaaaga atataaaaaa agtcagctat gtaaacagaa atctttattt 300

tgctagaaga ggtatacaaa gtactacaaa tacgtttaaa aaagatgttt ttattcaatt 360

agaaaattta gaagaaaaaa cttttgattt gtttgttaaa atatttggtg gacaatatga 420

attttctgtt tttaaagaga cgctacagtg gcatattatt tattatagct tattaatgtt 480

caaaaatgga gatgaatcgc ttccaaagaa attgcatata tttaagtatt tatacaatag 540

gcattcttta gatactc 557

<210> 25

<211> 20

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 25

gctcaccaac gtttgctcat 20

<210> 26

<211> 20

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 26

ggggacgtaa ctcggtgatt 20

<210> 27

<211> 25

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 27

ctggagtaat tacggcgact attcc 25

<210> 28

<211> 28

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 28

aggagaagct agtagtactt gcattttc 28

<210> 29

<211> 377

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 29

ggggacgtaa ctcggtgatt gatgccggtg cgggtaatga tacggttaat ggcggtaatg 60

gcgatgacac cctcatcggc ggcaaaggta atgatattct aagaggtggc tacggtgcgg 120

acacctatat ctttagcaaa ggacacggac aggatatcgt ttatgaagat accaataatg 180

ataaccgcgc aagagatatc gacaccttaa aatttactga tattaattta tccgaacttt 240

ggtttagccg agaaaataac gatttgatta ttaaatcatt attaagtgag gataaagtca 300

cggttcaaaa ttggtattca caccaagatc ataaaataga aaatattcgt ttatcgaatg 360

agcaaacgtt ggtgagc 377

<210> 30

<211> 958

<212> DNA

<213> Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae)

<400> 30

ctggagtaat tacggcgact attcctgaat ataactcaat agtagaagaa ctaaataaat 60

ataaaattgg taaaaaggaa acatttttaa caggatttcc tcgccatgat aaattactat 120

ctggaaatat aaaaggagct aagacaattc tcatcgtacc tacatggcga cattatatta 180

tggggactca aattggaaaa ggagccaata cacgcgagct aaataaagcc tttatgacaa 240

caaattatgc taaagcttgg tataatttat tacatagtca ggaattaaaa aatttaatta 300

aaaatttagg atataaagtt atttttgcac cacaccctaa tattgaacca tatttaaatg 360

agtttaacat cccccaatat attgatgtgt ggaaaagtgc aatatcaaga gaaagtatgc 420

aaagtttatt ccaacaatca aatctattga ttacggacta ttcatctatt gcatttgaaa 480

tggcatttct aggaaaacaa acaatctatt accaatttga taaagaggaa tttagatctg 540

gaattcatac atatcaacaa ggatactttg aatacgagaa agatggattt ggtcctgtag 600

ctgaaacatt agatgattta tttattcacc tagataaatt cgtaaacggt gaaaatgatt 660

acataaatat ttatcaatct cgtatacaaa aaacatttaa atatcgggat acgaataatt 720

gccaacgtgt ttatgaagct attattaact tagatatgcc ggataaagat attaataaaa 780

atattatctt aaatgcttta gaatctgctt acaaagctca agactggaat ttagttatct 840

ctcgtgctga agctttatta gcagaaatac ctaatcattc atttgctaag agtgtgttat 900

tgaagcagtg atagcttcaa acaataaaga gaaaatgcaa gtactactag cttctcct 958

Claims

1. A mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine is characterized in that: comprises inactivated mycoplasma hyopneumoniae strain HNMhy1 serotype antigen, inactivated haemophilus parasuis strain HNHPS1 serotype antigen, inactivated haemophilus parasuis strain HN1570 serotype antigen, inactivated streptococcus suis strain HNSS1 serotype antigen and inactivated actinobacillus pleuropneumoniae strain HNAPP1 serotype antigen;

wherein: the strain HNMhy1 is mycoplasma hyopneumoniae and is preserved in China general microbiological culture Collection center in 2017, 5 and 26 months, wherein the preservation number is CGMCC NO:13858; the strain HNHPS1 is serum 4 type haemophilus parasuisHaemophilus parasuisAnd is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 11 and 22, with the preservation number of CGMCC NO:13335; the strain HN1570 is serum 5 type haemophilus parasuisHaemophilus parasuisAnd the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 09 days in 2018, wherein the preservation number is CGMCC NO:16802; the strain HNSS1 is streptococcus suis type 2Streptococcus suisAnd is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 11 and 22, with the preservation number of CGMCC NO:13334, adding a solvent; the strain HNAPP1 is actinobacillus pleuropneumoniae type 1Actinobacilus pleuropneumoniaeAnd is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 11 and 22, with the preservation number of CGMCC NO:13333.

2. the inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis, and actinobacillus pleuropneumoniae in quadruple form, according to claim 1, wherein: the ratio of the inactivated mycoplasma hyopneumoniae strain HNMhy1 serotype antigen, the inactivated haemophilus parasuis strain HNHPS1 serotype antigen, the inactivated haemophilus parasuis strain HN1570 serotype antigen, the inactivated streptococcus suis strain HNSS1 serotype antigen and the inactivated actinobacillus pleuropneumoniae strain HNAPP1 serotype antigen is 1: 1.

3. A method for preparing the inactivated vaccine of mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae in quadruple according to claim 1, which comprises the following steps:

and (3) proliferation: respectively propagating and culturing mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis 4 type HNHPS1 strain, haemophilus parasuis 5 type HN1570 strain, streptococcus suis 2 type HNSS1 strain and actinobacillus pleuropneumoniae 1 type HNAPP1 strain to obtain mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, haemophilus parasuis HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain liquid, and respectively counting viable bacteria;

inactivation: adding formaldehyde solution into the bacterial solutions of the mycoplasma hyopneumoniae HNMhy1 strain, the haemophilus parasuis HNHPS1 strain, the haemophilus parasuis HN1570 strain, the streptococcus suis HNSS1 strain and the actinobacillus pleuropneumoniae HNAPP1 strain obtained in the step a according to 0.2 percent of the volume of the bacterial solutions, and inactivating the bacterial solutions at 37 ℃ for 24 hours;

concentration: respectively concentrating and inactivating bacterial solutions of qualified mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HNHPS1 strain, haemophilus parasuis HN1570 strain, streptococcus suis HNSS1 strain and actinobacillus pleuropneumoniae HNAPP1 strain by using an ultrafiltration device, and adjusting the concentration final concentration to be 1.0 x 10 by using sterile physiological saline according to the viable count before inactivation ¹⁰ CFU/mL；

Preparing a vaccine:

(3) Emulsification: according to the proportion of the water phase and the oil phase 1:2, the oil phase is added into an emulsification tank firstly, the mixture is stirred at the rotating speed of 3000-4000rpm, then the water is added into the emulsification tank, the mixture is stirred, mixed and emulsified successively, and the emulsification is subpackaged and bottled after being completed.

4. The method for preparing the inactivated vaccine for four combinations of mycoplasma hyopneumoniae and haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae according to claim 3, wherein the propagation culture in the step a comprises the following steps:

(1) First-order seed propagation: inoculating Mycoplasma hyopneumoniae HNMhy1 strain to PPLO solid medium, standing at 37 deg.C, and 5% CO ₂ Culturing in an incubator for 3 days, collecting cultures, respectively inoculating freeze-dried strains of haemophilus parasuis type 4 HNHPS1, type 5 HN1570, streptococcus suis type 2 HNSS1 and actinobacillus pleuropneumoniae type 1 HNAPP1 on a TSA plate, culturing at 37 ℃ for 18-24 hours, selecting typical colonies meeting requirements, respectively subculturing on the TSA plate, culturing at 37 ℃ for 24 hours, and checking to be qualified to serve as first-level seeds; the TSA plate is added with newborn bovine serum with the final concentration of 5% and NAD with the final concentration of 0.01%;

(3) Mixing pig mycoplasma pneumoniaSecondary seeds of H.parasuis type 4 HNHPS1, H.parasuis type 5 HN1570, S.suis type 2 HNSS1 and A.pleuropneumoniae type 1 HNAPP1 strains were inoculated at 1% into PPLO and TSB liquid medium, respectively, at 37 deg.C and 5% CO ₂ Shaking the shaking table to culture for 3d and 18-24 hr separately and harvesting bacteria liquid; the TSB liquid medium is added with newborn bovine serum with a final concentration of 5% and NAD with a final concentration of 0.01%.