CN113018425B - Mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological products for livestock, and particularly relates to preparation and application of a mycoplasma hyopneumoniae and haemophilus parasuis serum 4 type, 5 type, 12 type and 13 type bivalent pentavalent inactivated vaccine. The inactivated vaccine is prepared by inactivated mycoplasma hyopneumoniae HNMhy1 strain antigen liquid and inactivated haemophilus parasuis 4-type HNHPS1 strain, 5-type HN1570 strain, 12-type HN486 strain and 13-type HN1601 strain antigen liquid, and then adding an immunological adjuvant for livestock and poultry. The vaccine not only can well prevent infection of mycoplasma hyopneumoniae, haemophilus parasuis serum types 4, 5, 12 and 13, but also has good cross protection ability to other serotypes and haemophilus parasuis which are not typed.

Description

Mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine and application thereof

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to preparation and application of a mycoplasma hyopneumoniae and haemophilus parasuis serum 4 type, 5 type, 12 type and 13 type bivalent pentavalent inactivated vaccine.

Background

Mycoplasma hyopneumoniae @Mycoplasma hyopneumoniaeMhp is the major pathogen of mycoplasma hyopneumoniae (Mycoplasmal pneumonia of Swine, MPS). Mycoplasma pneumonia of pigs is a chronic and contact infectious disease, has the characteristics of high morbidity and low mortality, is mainly characterized by symptoms such as anorexia, fever, cough, asthma, dyspnea and the like, and the sick pigs grow slowly, have reduced feed conversion rate, often induce infection of other pathogens, particularly immunosuppressive pathogens such as porcine reproductive and respiratory syndrome virus (Porcinereproductive and respiratory syndrome, PRRSV) and porcine circovirus type 2 (Porcine cirovirus, PCV-2), cause immunosuppression induction, often cause death due to simultaneous secondary bacterial infection, and are one of the most important swine diseases in the world. The disease is widely present in all parts of the world, is continuously infected and is difficult to cure, and meanwhile, the pig industry is seriously harmed due to high treatment cost of infected pigs and production performance reduction.

Haemophilus parasuisHaemophilus parasuisHPS) is a pathogenic bacterium of haemophilus parasuis disease in pigs, mainly causing serositis, arthritis and meningitis in pigs. The disease widely exists worldwide, can infect pigs of various ages, mainly occurs after weaning and in the nursing stage, mainly infects pigs of 2 to 4 months of age, has the morbidity of 10 to 15 percent, and has the mortality rate of 80 percent when serious, and the disease becomes one of the main causes of death of weaned and nursing piglets in recent years, is one of the main pathogens of clinical mixed infection, causes great economic loss for pig industry and seriously affects the healthy development of pig industry.

Haemophilus parasuis has more clinical serotypes, 15 serotypes exist in standard typing, more than 20% of non-typing strains exist clinically, the serotypes of epidemic strains are different, and the immunological cross-protection capability among serotypes is lacked. Vaccination is the best way to control haemophilus parasuis and the corresponding monovalent or multivalent vaccine should be selected according to the prevailing serotype. The dominant serotypes of domestic haemophilus parasuis epidemic are mainly serotypes 4, 5, 1 and 13, and the commercial vaccines in China are mainly type 4 and 5 bivalent vaccines.

Mycoplasma hyopneumoniae is clinically mixed with haemophilus parasuis or secondarily infected, the symptoms of the Mycoplasma hyopneumoniae and haemophilus parasuis are similar, the drug treatment effect is poor, and the haemophilus parasuis type 4, type 5, type 12 and type 13 are serotypes with the highest clinical separation proportion, so that a new vaccine prepared by a strain with good safety and immunogenicity is needed to prevent and treat Mycoplasma pneumonia and haemophilus parasuis.

Disclosure of Invention

At present, mycoplasma hyopneumoniae and haemophilus parasuis are mixed to infect, immune cross protection is absent among serotypes of haemophilus parasuis, and the haemophilus parasuis inactivated vaccine only has the problem of resisting and protecting strains of the same serotype.

The invention solves the technical problems by adopting the scheme that: the bivalent and pentavalent inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis comprises inactivated mycoplasma hyopneumoniae, four haemophilus parasuis serotype antigens and an immunological adjuvant for livestock, wherein the mycoplasma hyopneumoniae antigens are inactivated mycoplasma hyopneumoniae HNMhy1 strain, and the four haemophilus parasuis serotype antigens are inactivated haemophilus parasuis 4 type HNHPS1 strain, 5 type HN1570 strain, 12 type HN486 strain and 13 type HN1601 strain antigens respectively.

The mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine and mycoplasma hyopneumoniaeMycoplasma hyopneumoniae) The HNMhy1 strain is preserved in China general microbiological culture Collection center (CGMCC) in the 5 th month of 2017 and 26 th day, and the preservation number is CGMCC NO:13858; haemophilus parasuisHaemophilus parasuis) The 4 HNHPS1 strain is preserved in China general microbiological culture Collection center (CGMCC) at the date of 2016, 11 and 22, and the preservation number is: 13335; haemophilus parasuisHaemophilus parasuis) The strain 5 HN1570 is preserved in China general microbiological culture Collection center (CGMCC) on the 11 th month 09 of 2018, and the preservation number is CGMCC NO:16802; haemophilus parasuisHaemophilus parasuis) The 12 HN486 strain is preserved in China general microbiological culture Collection center (CGMCC) in the 11 th month 09 of 2018, and the preservation number is CGMCC NO: 16505; haemophilus parasuisHaemophilus parasuis) 13 HN1601 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO:16806, deposit address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The ratio of mycoplasma hyopneumoniae to haemophilus parasuis bivalent pentavalent inactivated vaccine to mycoplasma hyopneumoniae, haemophilus parasuis type 4, type 5, type 12 and type 13 serotypes antigens is 1:1:1:1:1.

The content of mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine, mycoplasma hyopneumoniae, haemophilus parasuis serum type 4, type 5, type 12 and type 13 antigens is 1×10 ⁹ CFU/mL。

The mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine is characterized in that the immune adjuvant is a water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry.

The invention also provides a preparation method of the bivalent pentavalent inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis, which comprises the following steps:

(1) Proliferation: mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis serum 4 type HNHPS1 strain, type 5 HN1570 strain, type 12 HN486 strain and type 13 HN1601 strain are respectively propagated and cultured to obtain bacterial solutions of Mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain, and viable count is respectively carried out;

(2) And (3) inactivation: adding formaldehyde solution into the bacterial solutions of mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain obtained in the step a according to 0.2% of the volume of the bacterial solution, and inactivating for 48 hours at 37 ℃;

(3) Concentrating: concentrating and inactivating qualified mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain bacterial liquid by ultrafiltration device, and adjusting concentration final concentration to 1.0X10 by sterile physiological saline according to viable bacteria count before inactivation ¹⁰ CFU/mL；

(4) Preparation of vaccine: the multiplied, inactivated and concentrated mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain antigen liquid are uniformly mixed according to the ratio of 1:1:1:1:1:1, 30% of final volume of water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry is added, and the mixed solution is uniformly mixed to prepare the bivalent pentavalent inactivated vaccine of mycoplasma hyopneumoniae and haemophilus parasuis serum type 4, type 5, type 12 and type 13.

The preparation method of the bivalent pentavalent inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis comprises the following steps of:

(1) First-stage seed propagation: inoculating Mycoplasma hyopneumoniae HNMhy1 strain into PPLO solid culture medium, placing at 37deg.C and 5% CO ₂ Culturing in incubator, collecting culture after 3d, inoculating 4 HNHPS1 strain, 5 HN1570 strain and 12 HN486 strain of haemophilus parasuis, 13 HN1601 strain freeze-dried strain on TSA plate, culturing at 37deg.C for 18-24 hr, picking up the strainSubculturing the required typical colonies on TSA plates respectively, culturing at 37 ℃ for 24 hours, and taking the qualified colonies as first-stage seeds;

(2) Secondary seed propagation: inoculating Mycoplasma hyopneumoniae and four haemophilus parasuis primary seeds into PPLO and TSB liquid culture medium respectively, and inoculating 5% CO at 37deg.C ₂ Shaking table shaking and culturing for 3d and 18-24 hours respectively, and taking the seeds as secondary seeds after pure inspection is qualified;

(3) Inoculating secondary seeds of mycoplasma hyopneumoniae and four haemophilus parasuis strains into PPLO and TSB liquid culture medium at 1%, respectively at 37deg.C and 5% CO ₂ Shaking table shaking culture is carried out for 3d and 18-24 hours respectively, and bacterial liquid is obtained.

The invention relates to application of a mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine in preparation of a medicament for preventing and treating mycoplasma hyopneumoniae and haemophilus parasuis related diseases.

The invention has the beneficial effects that: the mycoplasma hyopneumoniae strain HNMhy1 is separated from a nursery pig with typical dyspnea and actual change of lung, has stronger pathogenicity to the nursery pig, can cause the nursery pig to generate typical wheezing symptoms, and has good immunogenicity.

According to the invention, dominant epidemic strains of haemophilus parasuis serum 4 HNHPS1 strain, 5 HN1570 strain, 12 HN486 strain and 13 HN1601 strain are selected from clinical isolates, wherein the strains of HNHPS1 strain, HN1570 strain, HN486 strain and HN1601 strain have strong pathogenicity to the nursery pig, and cause morbidity and mortality of the nursery pig; the four haemophilus parasuis are found to have good immunogenicity through experiments.

The inactivated vaccine prepared by using the mycoplasma hyopneumoniae strain HNMhy1 and haemophilus parasuis serum 4 HNHPS1 strain, HN1570 strain 5, HN486 strain 12 and HN601 strain 13 as strain antigens can well prevent clinical mycoplasma hyopneumoniae, haemophilus parasuis serum 4, 5, 12 and 13 infections, and has better cross protection capability on other serotypes 1, 2, 3, 6, 7, 8, 9, 10, 11, 15 and non-typed haemophilus parasuis, and meanwhile, the vaccine has the advantages of simple preparation process, good safety, good immune effect and long immune period.

Detailed Description

The invention is further illustrated below with reference to examples.

Example 1: isolation and identification of mycoplasma hyopneumoniae

1.1 Disease material collection

The disease material of mycoplasma hyopneumoniae is derived from the lung of a solid changed pig of a 2-month-old nursery pig which has severe pneumonia dyspnea in a large pig farm in Puyang city of Henan, 4 months of 2017.

1.2 Preparation of the culture Medium

PPLO liquid medium: PPLO broth powder 10.5 g, glucose 2.5 g, yeast powder 2.5 g, dissolved in 440 mL ultrapure water, sterilized at 115℃for 15min, and MEM medium 5 mL, horse serum 50mL, penicillin 8 ten thousand units, sterile 10% arginine 10 mL and 1% (w/v) phenol red 500. Mu.L were added. After 15min of sterilization at 115℃the culture was kept at 4℃for further use.

PPLO solid medium: PPLO liquid medium of 1.5% (w/v) agar powder was added. After 15min of sterilization at 115℃the culture was kept at 4℃for further use.

1.3 Isolated culture of mycoplasma hyopneumoniae

Taking a proper amount of pathological lung tissue, putting the pathological lung tissue into a grinder for shearing, adding 2mL of sterilized PBS buffer solution, grinding, and taking supernatant into an EP tube. Then centrifuging at 5000 r/m for 5min, collecting supernatant with needle tube, filtering with 0.22 μm filter membrane at needle tube part, adding into PPLO liquid culture medium, and concentrating at 37deg.C under 5% CO ₂ Culturing in an incubator. After nearly one week of culture, 1mL of transfer PPLO liquid culture medium is used for culturing again, the liquid color is changed from red to yellow after passage 3-5 generations, 100 mu L of coating PPLO solid culture medium surface is used, and the culture medium is placed at 37 ℃ and 5% CO ₂ Culturing in an incubator. After 5-7. 7 d, colorless and transparent needle-shaped colonies are observed by naked eyes, and the forms of the colonies on the solid medium are observed under a low power microscope, and are typical mycoplasma fried egg-like. Giemsa staining, observing thallus morphology under oil lens, forming into multiple forms, and forming into coccoid, curved filiform and spiral shape,designated as strain HNMhy1.

1.4 Identification of strains

1.4.1 Primer design

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

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

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

the expected amplified fragment size is 1502bp.

1.4.2 PCR identification

Inoculating strain HNMhy1 into PPLO liquid culture medium, and placing at 37deg.C and 5% CO ₂ After 3d culture in the incubator, the medium turns from red to yellow, the bacterial cells are collected, DNA of the strain HNMhy1 is extracted according to the operation steps of the DNA extraction kit, the concentration is 100 mug/mL by spectrophotometry, and PCR identification is carried out by using MB1 and MB2 amplification. Meanwhile, a mycoplasma hyopneumoniae vaccine strain 168 is set as a positive control.

The PCR amplification reaction system is 25 [ mu ] L:10 times of buffer solution 2.5 mu L,2.5mM dNTPs 0.5 mu L,10 mu M/L universal primers MB1, MB2 each 1 mu L, 5U/mu L rTaq 1 mu L,100 mu g/mL DNA template 1 mu L, and ddH is added ₂ O to 25 [ mu ] L.

Reaction conditions: pre-denaturation at 95 ℃ for 5min, and circulation: 95℃for 30 s,57℃for 30 s,72℃for 1 min, 35 cycles total, and finally 72℃for 10 min extension, and 4℃for preservation of the PCR product. And respectively carrying out electrophoresis on amplified products, adding 10 mu L of sample into each hole, carrying out agarose gel electrophoresis at 1%, observing the result under ultraviolet light, amplifying fragments conforming to the expected size from MB1 and MB2, and sequencing to obtain the strain with the homology of 78% to the strain 168 of the mycoplasma hyopneumoniae subculture attenuated vaccine strain (Genbank No. CP002274) and 100% to the strain ES-2 of Hubei isolate strain (Genbank No. CP038641). Proved that the separated strain HNMhy1 is mycoplasma hyopneumoniae @Mycoplasma hyopneumoniae）。

1.4.3 Mycoplasma hyopneumoniae virulence test

The test pigs were 8 healthy nursery pigs of 15 days of age, and were all negative for mycoplasma hyopneumoniae ELISA antibodies. Test animals are classified intoTwo groups, control group 4, test group 4, and two groups of test animals were randomly grouped. Inoculating HNMhy1 into PPLO liquid culture medium at 37deg.C with 5% CO ₂ Culturing in incubator at 3d, measuring thallus concentration, continuously diluting with 10 times of coated solid plate, counting viable bacteria under low power microscope, calculating thallus concentration of stock solution of culture, and adjusting to 10 ⁸ CFU/mL, animals in the test group were inoculated with 3 mL Mycoplasma hyopneumoniae broth by laryngeal airway, and animals in the control group were inoculated with 3 mL sterilized PPLO broth by laryngeal airway. The test period is 15 d, the clinical manifestations of the test animals are observed every day, the body temperature is measured, and if the test animals die, the test animals are immediately dissected and killed, and the respiratory tract pathological changes are observed to collect the disease materials. At the end of the 15 d test, all test animals were dissected.

Animals in the test group show symptoms such as oral nasal flow, dyspnea, cough and the like after being inoculated with mycoplasma 24h, have normal body temperature, are attenuated, open mouth and extend tongue, and have sitting type abdominal respiration, and die 1 after 7 d and die 1 after 10 d. Animals in the control group were normothermic and had no apparent respiratory symptoms during the trial. After the test is finished, animals in the test group are killed, and animals in the 4 control group are killed at the same time. Collecting the disease material and separating mycoplasma. The experimental group is examined by sectioning, and the tip leaf, the heart leaf and the partition leaf of the lung are subjected to actual transformation of bilateral symmetry meat-like transformation, and the limit of the experimental group and the surrounding tissue is obvious. No obvious pathological change occurs in the control group of the split examination, no mycoplasma is separated from 4 disease materials of the control group, mycoplasma hyopneumoniae is separated from 4 disease materials of the test group, the colony morphology is represented as a fried egg-like, and the PCR identification result is consistent with HNMhy1.

Example 2: isolation and identification of haemophilus parasuis

2.1 Source of disease material

In the period from 1 month 2016 to 12 months 2019, lung, heart blood, lymph node, spleen, brain, joint fluid and other diseases of pigs with clinical symptoms of suspected haemophilus parasuis disease in the provinces of Henan, shanxi, shandong, hubei, anhui, jiangsu, hebei and the like are collected together, and the ill pigs mostly show fever, dyspnea, joint swelling, lameness, skin and mucous membrane cyanosis, standing difficulty and even paralysis, stiff pigs or death.

2.2 Preparation of the culture Medium

TSB broth: TSB broth powder (Tryptic Soy broth, tryptone soy broth, BD company) 30g was dissolved in 1000mL ultrapure water and sterilized at 115 ℃ for 15min, and fetal bovine serum 50mL (Hychone company), sterile 1% nad (Nicotinamideadenine dinucleotide, nicotinamide adenine dinucleotide, coenzyme i, roche company) 1mL was added.

TSA solid medium: 40g of TSA Agar powder (Tryptic Soy Agar, BD company) was dissolved in 1000mL of ultra pure water, sterilized at 115℃for 15min, and 50mL of fetal bovine serum and 1mL of sterile 1% NAD were added; preserving at 4 ℃ for standby.

2.3 Isolated culture of haemophilus parasuis

Collecting the lung, heart blood, lymph node, spleen, brain, joint fluid and other diseases of the sick or dead pig under the aseptic condition, inoculating the disease materials on a TSA solid culture medium containing NAD, culturing 36 h in a constant temperature incubator at 37 ℃, selecting suspected colonies for subculture purification, culturing 24-48 h, observing the colony morphology of haemophilus parasuis, and growing uniform needle-tip-shaped, colorless, transparent, smooth and moist colonies with the diameter of 1-2 mm; purification and inoculation are carried out on a TSA solid culture medium without NAD, and growth cannot be carried out on the TSA solid culture medium without NAD; the purified individual colonies were picked for gram staining microscopy and the observed bacteria were characterized as gram negative bacteria, having a variety of different morphologies, ranging from individual cocci to elongate filamentous bacteria.

2.4 Identification of haemophilus parasuis and identification of serotypes

2.4.1 Primer design

The clinical isolated suspected strain is subjected to haemophilus parasuis identification and typing identification by Polymerase Chain Reaction (PCR) technology. The primer sequences and the amplified fragment lengths of the haemophilus parasuis types 1-15 are shown in Table 1.

2.4.2 PCR identification

The PCR reaction system was 25. Mu.L, which included 10X Tap PCR master mix. Mu.L, 1.0. Mu.L each of the upstream and downstream primers, 5.0. Mu.L of sterile water, and 5. Mu.L of template. The reaction conditions are as follows: 94. after denaturation at 5min, 30 cycles of 94℃for 30 s,54℃for 30 s,72℃for 40 s were performed, and finally extension at 72℃for 10 min, and the PCR product was stored at 4 ℃. 10 mu L of PCR products are taken, electrophoresis is carried out for 45 min under the voltage of 1% agarose gel 100V, and then a gel imaging system is used for analyzing and observing results, and positive results are sequenced and compared.

The 60 haemophilus parasuis strains are co-separated by bacterial separation, purification and identification of the collected suspected samples, and the homology of the separated strains and the 16SrRNA sequence of the standard HPS is over 99 percent through gene comparison.

And (3) carrying out PCR molecular serotyping identification on 60 haemophilus parasuis Henan isolates.

Identification result: serotype 5, serotype 13, serotype 10, serotype 4, serotype 9, serotype 12, serotype 8, serotype 2, serotype 6, serotype 7, serotype 5, serotype 1, serotype 4, serotype 14 have 2 strains and non-classifiable strains have 4 strains.

The results show that the type 5, 13, 4, 12, 2, 7, 1 and 14 are clinically the main prevalent serotypes, with the type 5, 13, 4 and 12 being the most prevalent.

4 strains of haemophilus parasuis isolated from typical disease cases were selected, wherein one strain 4 of haemophilus parasuis was designated as HNHPS1 strain, one strain 5 was designated as HN1570 strain, one strain 12 was designated as HN486 strain, one strain 13 was designated as HN1601 strain, and deposited at the chinese microbiological bacterial culture collection center, address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Wherein the 4-type HNHPS1 strain is preserved in the year of 2016, 11 and 22, and the preservation number is CGMCC NO:13335; the strain 5 HN1570 is preserved in the year 2018, 11 and 09, and the preservation number is CGMCC NO:16802;12 HN486 is preserved in 2018, 11 and 09 days, and the preservation number is CGMCC NO: 16505; the 13 type HN1601 strain is preserved in 2018, 11 and 09 days, and the preservation number is CGMCC NO:16806.

2.5 Toxicity test of haemophilus parasuis

And selecting 25 healthy nursery pigs of 9-10 weeks of weaning age as test animals. Will testAnimals were randomly assigned to 5 groups, the control group, and the 8 test groups, each group having 5 heads. Inoculating haemophilus parasuis HNHPS1, HN1570, HN486 and HN1601 respectively into TSB liquid culture medium, shake culturing at 37deg.C for 24h, measuring thallus concentration, continuously diluting 10 times, coating with solid plate, counting viable bacteria, calculating thallus concentration of stock solution, and adjusting to 10 ⁹ CFU/mL; animals in the 4 groups of test groups were inoculated intraperitoneally with 3 mLHNHPS1, HN1570, HN486, HN1601 Haemophilus parasuis liquid cultures, respectively, and animals in the control group were inoculated intraperitoneally with 3 mL sterilized TSB liquid culture medium. The number of morbidity and mortality was observed and recorded by continuous observation for 2 weeks after injection.

Observations find that: the 4 test groups showed symptoms such as wheezing, dyspnea, etc., elevated body temperature, cyanosis of ears, abdomen and buttocks, and circling after 24 hours inoculation with haemophilus parasuis. Death began to occur after 2 days. Animals in the control group were normothermic and had no apparent respiratory symptoms during the trial. The onset and mortality are shown in Table 2.

And after the test is finished, the animals of the test group are killed, 5 animals of the control group are killed at the same time, and the disease materials are collected for bacterial separation. The lung of the test group is dissected and examined to have a great deal of cellulose-like exudation, adhesion with pleura, pericardial effusion, choriocardious and cerebral hemorrhage; the 3 groups of thoracic cavities have small amount of cellulose exudation, pulmonary hemorrhage and other pneumonia symptoms, and pericardial effusion; no obvious pathological change occurs in the control group of the split examination, no haemophilus parasuis is separated from 5 disease materials of the control group, and haemophilus parasuis is separated from 20 disease materials of the test group, and the PCR identification result is consistent with the strains HNHPS1, HN1570, HN486 and HN 1601.

Based on the results of the toxicity test, it was confirmed that the haemophilus parasuis strains HN1570, HN486 and HN1601 were the most virulent strains and that HNHPS1 was the less moderately virulent strains.

Example 3: vaccine preparation, safety and efficacy testing

3.1 Preparation of vaccine

3.1.1 Bacterial strain and strain propagation

First seed propagation

Inoculating Mycoplasma hyopneumoniae HNMhy1 strain into PPLO solid culture medium, placing at 37deg.C and 5% CO ₂ Culturing in an incubator, and collecting the culture after 3 d.

The haemophilus parasuis 4 type HNHPS1 strain, 5 type HN1570 strain, 12 type HN486 strain and 13 type HN1601 strain freeze-dried strains are respectively inoculated on TSA flat plates containing new born calf serum (final concentration 5%) and NAD (final concentration 0.01%), cultured for 18-24 hours at 37 ℃, typical colonies meeting the requirements are selected, TSA flat plates containing new born calf serum (final concentration 5%) and NAD (final concentration 0.01%) are respectively inoculated in a passage way, cultured for 24 hours at 37 ℃, and the seeds are used as primary seeds after the seeds are checked to be qualified.

Secondary seed propagation

Inoculating Mycoplasma hyopneumoniae HNMhy1 primary seeds into PPLO culture medium respectively, placing at 37deg.C, 5% CO ₂ Shaking the culture medium to culture for 3d and 18-24 hr, and taking the culture medium as secondary mycoplasma hyopneumoniae seed after pure inspection;

first seed of Haemophilus parasuis 4-type HNHPS1 strain, 5-type HN1570 strain, 12-type HN486 strain and 13-type HN1601 strain are inoculated into TSB liquid culture medium containing new born calf serum (final concentration 5%) and NAD (final concentration 0.01%) at 37deg.C, 5% CO ₂ Shaking the culture medium to culture for 3d and 18-24 hr, and obtaining the second-stage haemophilus parasuis seed after pure inspection.

3.1.2 Culture of antigenic solution

Mycoplasma hyopneumoniae is inoculated into PPLO culture medium at 1% respectively at 37 ℃ and 5% CO ₂ Shaking the culture table to culture for 3d and 18-24 hr separately and obtain bacterial liquid as mycoplasma hyopneumoniae antigen bacterial liquid;

two-stage seeds of three haemophilus parasuis strains were inoculated at 1% respectively into TSB liquid culture medium containing neonatal bovine serum (final concentration 5%) and NAD (final concentration 0.01%), at 37℃respectively, 5% CO ₂ Shaking table shaking and respectively culturing for 3d and 18-24 h, and then obtaining bacterial liquid serving as haemophilus parasuis antigen bacterial liquid.

3.1.3 Viable count

According to the method of the annex of the current Chinese animal pharmacopoeia,live bacteria counts were performed on mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen respectively using PPLO solid medium suitable for mycoplasma hyopneumoniae growth and TSA medium containing 5% neonatal bovine serum and 0.01% NAD suitable for haemophilus parasuis growth. Shake flask culture is used, viable bacteria count concentration is achieved, and the viable bacteria content of five strains are all 1.0X10 ⁹ CFU/mL or more.

3.1.4 Bacterial liquid inactivation

To the culture solutions of mycoplasma hyopneumoniae and four haemophilus parasuis strains, 0.2% formaldehyde solution was added in total volume, and the mixture was inactivated at 37℃for 48 hours.

3.1.5 Inactivation test

0.2mL of an inactivated bacterial liquid of mycoplasma hyopneumoniae is inoculated in a PPLO culture medium, 0.2mL of an inactivated bacterial liquid of four haemophilus parasuis strains is respectively inoculated in a TSA flat plate (containing 5% of new born calf serum and 0.01% of NAD), and the culture is carried out for 48 hours at 37 ℃, so that no bacteria grow to be qualified.

3.1.6 Antigen concentration

And (5) respectively concentrating the five antigen bacterial liquids which are qualified in the inactivation test through an ultrafiltration system device. Based on the results of the viable bacteria count before inactivation, the concentrations of the antigens of the five bacteria were adjusted to 1.0X10 s by using sterile physiological saline ¹⁰ CFU/mL, and mix the five antigen solutions uniformly in a ratio of 1:1:1:1.

3.1.7 Adjuvant preparation

Split charging beasts and birds with water-in-oil-in-water adjuvant Summit-S550 into bottles, sterilizing at 121deg.C for 30 min, and storing at room temperature for use.

3.1.8 Vaccine preparation

Adding 30% of water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry according to the concentrated and uniformly mixed antigen liquid, stirring and fully uniformly mixing, and subpackaging to obtain the mycoplasma hyopneumoniae and haemophilus parasuis disease bivalent inactivated vaccine of the water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry. The final vaccine contains total number of live bacteria of five strains 1.0X10 before inactivation ¹⁰ CFU/mL。

3.2 Safety test of vaccine

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

Vaccine group: neck intramuscular injection of 4 mL of the vaccine;

control group: neck intramuscular injection of 4 mL sterilized normal saline. Animal body temperature was measured and clinical manifestations were observed for a total of 2 weeks.

The average body temperature rise of the nursery pigs in the vaccine group and the control group is not more than 1 ℃ as can be seen from the table 3, so that the inactivated vaccine of the invention is safe.

3.3 Efficacy test of vaccine

170 healthy piglets of 15 days old are selected, and a vaccine immunization group 17 and a control group 17 are established, wherein each group is provided with 5 piglets. Each vaccine group was injected with 2ml of the vaccine, and 3 weeks later, the vaccine group was given an equivalent dose of the vaccine without vaccination. The vaccine immunization group of 16 groups and the control group of 16 groups are respectively used for 10 days after the secondary immunization ⁹ Mycoplasma hyopneumoniae HNMhy1 of CFU, haemophilus parasuis 4-type HNHPS1 strain, HN1570 strain, HN486 strain 12, HN1601 strain 13, standard strain 1, standard strain 2, standard strain 3, standard strain 6, standard strain 7, standard strain 8, standard strain 9, standard strain 10, standard strain 11, standard strain 14, standard strain 15, strain liquid which is not subjected to typing is subjected to intraperitoneal toxicity attack. The clinical manifestations of each group of piglets were observed, the body temperature was measured daily, and the total observation was 2 weeks.

Calculating morbidity and mortality of experimental piglets in the whole observation period, measuring body temperature, and carrying out bacterial isolated culture on organs such as the lungs of dead pigs.

Observations find that: the vaccine group is obviously different from the control group, the next day after the virus attack, the piglets of the control group begin to have clinical symptoms, rise in body temperature, dyspnea and turn round, and begin to die after 2 days, and pigs of different strain virus attack groups have different degrees of morbidity and death. The control group is examined for lesions with different degrees, the pleural cavity and the abdominal cavity have effusions, and have cellulosic exudates, pericardial effusions, and pericardial cavity have cellulosic exudates and pericardial adhesion; the lung is swollen, the two sides of the lung become stagnant or bleeding, there is some solid change, and there is surface cellulosic exudates. Meningeal hyperemia and cerebrospinal fluid increase. In the 17 vaccine groups, the morbidity and mortality conditions are obviously different from those of the control group, and the immune toxicity attack results of different serotype strains are shown in Table 4 respectively.

As can be seen from the results of the immune challenge test in Table 4, the vaccine has 100% protection rate for 2ml of immune dose of the vaccine for two times of immune mycoplasma pneumonia and infection of haemophilus parasuis type 4, type 5, type 12 and type 13. The inactivated vaccine has excellent cross protection capability on the infection of serum type 1, type 2, type 3, type 6, type 7, type 8, type 9, type 11, type 14, type 15 standard strains and haemophilus parasuis which cannot be typed, and has better protection effect on the currently known haemophilus parasuis infection of different serum types.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Sequence listing

<110> institute of livestock and veterinary at academy of agricultural sciences in Henan province

<120> Mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine and application thereof

<141> 2021-02-07

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Claims (6)

1. The bivalent pentavalent inactivated vaccine for mycoplasma hyopneumoniae and haemophilus parasuis is characterized in that: the antigen is prepared from inactivated mycoplasma hyopneumoniae and four haemophilus parasuis serotype antigens and an immunological adjuvant for livestock, wherein the mycoplasma hyopneumoniae antigen is an inactivated mycoplasma hyopneumoniae HNMhy1 strain, and the four haemophilus parasuis serotype antigens are respectively an inactivated haemophilus parasuis 4-type HNHPS1 strain, a 5-type HN1570 strain, a 12-type HN486 strain and a 13-type HN1601 strain antigen; wherein the mycoplasma hyopneumoniae HNMhy1 strain is preserved in China general microbiological culture Collection center (CGMCC) in 5-month 26 of 2017, and the preservation number is CGMCC NO:13858; haemophilus parasuis 4 type HNHPS1 strain is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 22 days of 2016: 13335; haemophilus parasuis 5 type HN1570 strain is preserved in China general microbiological culture Collection center (CGMCC) in 11-09 of 2018, and the preservation number is CGMCC NO:16802; haemophilus parasuis 12 type HN486 is preserved in China general microbiological culture Collection center (CGMCC) in 11-09 of 2018, and the preservation number is CGMCC NO: 16505; haemophilus parasuis 13 type HN1601 strain is preserved in China general microbiological culture Collection center (CGMCC) at 11-09 of 2018, and the preservation number is CGMCC NO:16806, deposit address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

2. The mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine according to claim 1, wherein: mycoplasma hyopneumoniae, haemophilus parasuis type 4, type 5, type 12, type 13 serotype antigens in a ratio of 1:1:1:1:1.

3. The mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine according to claim 1, wherein: mycoplasma hyopneumoniae and haemophilus parasuis serum type 4, type 5, type 12 and type 13 antigens are all 1×10 in content ⁹ CFU/mL。

4. The mycoplasma hyopneumoniae and haemophilus parasuis bivalent pentavalent inactivated vaccine according to claim 1, wherein: the immune adjuvant is water-in-oil-in-water adjuvant Summit-S550 for livestock and fowl.

5. A method for preparing a bivalent pentavalent inactivated vaccine against mycoplasma hyopneumoniae and haemophilus parasuis according to any one of claims 1-4, which is characterized in that: the method comprises the following steps:

(1) Proliferation: mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis serum 4 type HNHPS1 strain, type 5 HN1570 strain, type 12 HN486 strain and type 13 HN1601 strain are respectively propagated and cultured to obtain bacterial solutions of Mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain, and viable count is respectively carried out;

(2) And (3) inactivation: adding formaldehyde solution into the bacterial solutions of mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain obtained in the step a according to 0.2% of the volume of the bacterial solution, and inactivating for 48 hours at 37 ℃;

(3) Concentrating: concentrating and inactivating qualified mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain bacterial liquid by ultrafiltration device, and adjusting concentration final concentration to 1.0X10 by sterile physiological saline according to viable bacteria count before inactivation ¹⁰ CFU/mL；

(4) Preparation of vaccine: the multiplied, inactivated and concentrated mycoplasma hyopneumoniae HNMhy1 strain, haemophilus parasuis HN1553 strain, HN1570 strain, HN486 strain and HN1601 strain antigen liquid are uniformly mixed according to the ratio of 1:1:1:1:1:1, 30% of final volume of water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry is added, and the mixed solution is uniformly mixed to prepare the bivalent pentavalent inactivated vaccine of mycoplasma hyopneumoniae and haemophilus parasuis serum type 4, type 5, type 12 and type 13.

6. The method for preparing the bivalent pentavalent inactivated vaccine against mycoplasma hyopneumoniae and haemophilus parasuis according to claim 5, which is characterized in that: the proliferation operation steps of the step (1) are as follows:

(1) First-stage seed propagation: inoculating Mycoplasma hyopneumoniae HNMhy1 strain into PPLO solid culture medium, placing at 37deg.C and 5% CO ₂ Culturing in an incubator, collecting cultures after 3d, inoculating 4-type HNHPS1 strain, 5-type HN1570 strain, 12-type HN486 strain and 13-type HN1601 strain freeze-dried strains of haemophilus parasuis respectively on a TSA plate, culturing for 18-24 hours at 37 ℃, picking up typical colonies meeting the requirements, respectively subculturing on the TSA plate, culturing for 24 hours at 37 ℃, and taking the cultured colonies as first-stage seeds after the culture is qualified;

(2) Secondary seed propagation: inoculating Mycoplasma hyopneumoniae and four haemophilus parasuis primary seeds into PPLO and TSB liquid culture medium respectively, and inoculating 5% CO at 37deg.C ₂ Shaking table shaking and culturing for 3d and 18-24 hours respectively, and taking the seeds as secondary seeds after pure inspection is qualified;

(3) Inoculating secondary seeds of mycoplasma hyopneumoniae and four haemophilus parasuis strains into PPLO and TSB liquid culture medium at 1%, respectively at 37deg.C and 5% CO ₂ Shaking table shaking culture is carried out for 3d and 18-24 hours respectively, and bacterial liquid is obtained.