CN114015658B - Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma - Google Patents

Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma Download PDF

Info

Publication number
CN114015658B
CN114015658B CN202111186799.4A CN202111186799A CN114015658B CN 114015658 B CN114015658 B CN 114015658B CN 202111186799 A CN202111186799 A CN 202111186799A CN 114015658 B CN114015658 B CN 114015658B
Authority
CN
China
Prior art keywords
mycoplasma
virus
avian influenza
strain
influenza
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111186799.4A
Other languages
Chinese (zh)
Other versions
CN114015658A (en
Inventor
陈吉龙
颜世红
李训良
王松
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fujian Agriculture and Forestry University
Original Assignee
Fujian Agriculture and Forestry University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fujian Agriculture and Forestry University filed Critical Fujian Agriculture and Forestry University
Priority to CN202111186799.4A priority Critical patent/CN114015658B/en
Publication of CN114015658A publication Critical patent/CN114015658A/en
Application granted granted Critical
Publication of CN114015658B publication Critical patent/CN114015658B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Communicable Diseases (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pulmonology (AREA)

Abstract

The invention discloses a bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma, which comprises an antigen and a vaccine adjuvant, wherein the antigen is inactivated influenza A virus FZ strain and bursa mycoplasma MS-FJ01; FZ strain and MS-FJ01 strain belong to typical representative strains of Fujian province; the bivalent inactivated vaccine of the scheme can induce chickens to generate higher levels of H9N2 subtype avian influenza antibodies and mycoplasma synoviae antibodies, the antibody duration is long, the toxin expelling of the immune chicken H9N2 subtype avian influenza viruses can be effectively reduced after immunization, the incidence of mycoplasma synoviae of chickens can be obviously reduced, and the epidemic of the H9N2 subtype avian influenza and the mycoplasma synoviae of chickens can be simultaneously and effectively prevented. At present, no H9N2 subtype avian influenza and chicken bursa mycoplasma combined inactivated vaccine is marketed in China, and the H9N2 subtype avian influenza and chicken bursa mycoplasma combined inactivated vaccine of the scheme can effectively prevent and control two avian diseases and can also make up for the current market vacancy of poultry.

Description

Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to a bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma.
Background
The H9N2 subtype avian influenza virus (Avian Influenza Virus, AIV) is the main subtype of the current avian influenza epidemic in China, and the infectious chicken flock can cause egg laying reduction or reduce chicken flock resistance, and causes secondary and mixed infection phenomena, so that the H9N2 avian influenza causes great economic loss to the poultry industry, and becomes one of the main epidemic diseases which plague and endanger the large-scale poultry farm in China. In addition, the H9N2AIV host is wide, can be used as an internal gene donor of other novel influenza viruses, accelerates the sustainable variation of the influenza viruses, promotes the generation and the epidemic of the novel influenza viruses, and has a great potential threat in public health.
The mycoplasma synoviae (Mycoplasma synoviae, MS) is an acute or chronic infectious disease mainly causing respiratory tract symptoms and infectious synovitis symptoms of chickens and turkeys, pathogens of the mycoplasma synoviae can directly invade chicken cartilage tissues, so that the chicken joint is swelled and swelled, synoviae and tendon sheaths are inflamed, lameness and paralysis are caused, the infection of the mycoplasma synoviae also can reduce laying rate of laying hens and influence egg quality, and the mycoplasma synoviae can be vertically transmitted, so that great difficulty is brought to pathogen prevention and control. At present, H9N2AIV and MS widely exist in chicken farms worldwide, and cross infection of the two pathogens is easy to occur, especially in the fowls province area with high humidity and high heat, the mixed infection of mycoplasma and influenza is frequently generated clinically, and serious economic loss is caused for poultry farming industry.
One of the main measures for preventing and controlling avian influenza is to use vaccine immunization, but the traditional inactivated vaccine cannot meet the current immunization and epidemic prevention requirements in many areas of China because the classical H9N2 subtype commercial vaccine has poor immune effect on new strains and variant strains, so that the development of a vaccine with higher matching property with the main epidemic strains in Fujian province is urgently needed. For diseases and economic losses caused by mycoplasma synoviae, three different measures are currently mainly used for preventing and controlling: chicken farm purification, antibiotic treatment and vaccination. However, due to the different scale and management level of the Chinese poultry farm, the purification cost is high, the breeding hens without MS infection are difficult to establish and maintain, and the antibiotic treatment method not only easily causes the bacterial strain to generate drug resistance, but also has the risk that the drug residue affects the food safety. Vaccination is thus also the best choice for controlling MS.
At present, no H9N2 subtype avian influenza and chicken bursa mycoplasma bigeminal inactivated vaccine is marketed in China. Therefore, the research and development of the efficient bivalent inactivated vaccine meets the market demands of the current poultry industry, and has important significance for preventing and controlling H9N2 subtype avian influenza and chicken bursa mycoplasma in China.
Disclosure of Invention
In view of the above, the invention aims to provide a dual inactivated vaccine of H9N2 subtype avian influenza and chicken bursa mycoplasma, which is reliable and effective in implementation, convenient to prepare and can be used for immunoprophylaxis of chicken bursa mycoplasma and H9N 2.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a bivalent inactivated vaccine against H9N2 subtype avian influenza and mycoplasma synoviae, comprising an antigen and a vaccine adjuvant.
As a possible embodiment, further, the antigen is an inactivated antigen comprising an inactivated influenza a virus FZ strain and an inactivated mycoplasma synoviae MS-FJ01; wherein, the influenza A virus FZ strain is preserved in China center for type culture Collection, the preservation address is China WU, the preservation number is CCTCC NO: V202171, and the preservation date is 2021, 9 and 26 days; the bursa of mycoplasma MS-FJ01 is preserved in China center for type culture Collection, the preservation address is Wuhan, the preservation number is CCTCC NO: M2021210, and the preservation date is 2021, 3, 8 days.
As a preferred implementation choice, it is preferred that the influenza A virus FZ strain and the mycoplasma synoviae MS-FJ01 are inactivated with formaldehyde.
Based on the vaccine composition scheme, the invention also provides a preparation method of the bivalent inactivated vaccine for the H9N2 subtype avian influenza and the chicken bursa mycoplasma, which comprises the following steps:
1) Diluting influenza A virus FZ strain with sterilized PBS 1000 times, inoculating into 9-11 day-old SPF chick embryo allantoic cavity, incubating at 37deg.C, collecting chick embryo virus allantoic liquid after 48 hr, performing hemagglutination test with 1% chick erythrocyte, aseptically collecting virus allantoic liquid with HA titer not less than 256, and storing in-80deg.C (such as low temperature refrigerator); then taking a chicken mycoplasma synoviae culture medium with the pH value of 7.6-7.8, adding the mycoplasma synoviae MS-FJ01 in a ratio of 10 to 1, then placing the chicken mycoplasma synoviae culture medium in an environment with the culture temperature of 37 ℃ and the shaking speed of 180r/min, culturing until the bacterial liquid turns orange, and then placing the chicken mycoplasma synoviae culture medium at 4 ℃ for preservation;
2) Antigen inactivation: adding formaldehyde solution with the final concentration of 0.1% into the allantoic fluid of the chick embryo virus prepared in the step 1), and shaking the chick embryo virus with the formaldehyde solution to fully mix the chick embryo virus; then placing the mixture in a shaking table at a constant temperature of 37 ℃ for shaking and inactivating for 12 hours at a speed of 200-220 r/min, taking out the mixture, and storing the mixture in an environment (such as a refrigerator) at a temperature of 4 ℃ for later use; the bacterial liquid containing the mycoplasma synoviae MS-FJ01 is placed in an ultra-high-count centrifuge for centrifugal treatment, and the bacterial liquid is concentrated until the content of viable bacteria per milliliter is not less than 5 multiplied by 10 13 Adding formaldehyde with the final concentration of 0.2% into the concentrated bacterial liquid immediately after concentration, and carrying out oscillation inactivation for 24 hours under the conditions that the culture temperature is 37 ℃ and the oscillation speed is 180 r/min;
3) 1, the method comprises the following steps: 1:5.62, respectively adding the inactivated influenza A virus FZ strain virus liquid, the bursa of mycoplasma MS-FJ01 bacterial liquid and the vaccine adjuvant in the step 2), and gradually emulsifying from the A grade to the D grade by using an HR-500 dispersing emulsifying machine, so that the emulsion is emulsified for 30 minutes at 15000r/min under the low temperature condition.
As a preferred implementation choice, in the step 1), preferably, the preparation method of the mycoplasma synoviae culture medium comprises the following steps: 21g of brain-heart leaching liquid broth, 5.6g of yeast leaching powder, 2mL of 1wt% phenol red solution, 2mL of 50mg/mL of L-cysteine solution, 8mL of 50mg/mL of arginine solution and 0.4mL of 250mg/mL of coenzyme I solution are taken and mixed, the volume is fixed to 900mL, 100mL of inactivated fetal calf serum is added, 80 ten thousand units/L of penicillin is added, the pH is adjusted to 7.6-7.8, and finally a 0.22 mu m filter is used for filtering to a sterile container to complete the preparation.
Furthermore, the invention also provides application of the influenza A virus FZ strain and the mycoplasma synoviae MS-FJ01 in preparing positive serum reagents for diagnosis of the avian influenza virus.
Furthermore, the invention also provides a poultry bivalent inactivated vaccine, which comprises the bivalent inactivated vaccine for the H9N2 subtype avian influenza and the chicken bursa mycoplasma.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that:
1. the vaccine of the scheme of the invention can induce chickens to generate higher-level antibodies, has long duration, obviously reduces the morbidity of H9N2 avian influenza and chicken bursa mycoplasma after immunization, and can effectively prevent the epidemic of chicken bursa mycoplasma.
2. The dual inactivated vaccine of the H9N2 subtype avian influenza and chicken mycoplasma synoviae is not marketed in China, and the dual inactivated vaccine prepared by the influenza A virus FZ strain and the mycoplasma synoviae MS-FJ01 can effectively prevent and control two infectious diseases and can also fill in market vacancies of the poultry industry.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the analysis of the evolutionary tree of the HA gene of avian influenza subtype H9N2 according to the scheme of the present invention;
FIG. 2 is a graph showing the change in profile of SPF-infected chickens infected with avian influenza subtype H9N2 according to the present invention.
FIG. 3 is a graph showing clinical symptoms of the present invention after challenge of Mycoplasma synoviae to SPF chickens;
FIG. 4 is a diagram showing the analysis of the phylogenetic tree of the Mycoplasma synoviae 16s rRNA gene according to the scheme of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. It is specifically noted that the following examples are only for illustrating the present invention, but do not limit the scope of the present invention. Likewise, the following examples are only some, but not all, of the examples of the present invention, and all other examples, which a person of ordinary skill in the art would obtain without making any inventive effort, are within the scope of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used in the following examples are commercially available.
Example 1, identification of representative strains of H9N2 subtype avian influenza
Isolation and identification of H9N2 subtype avian influenza strain
1. Sample collection: collecting throat and cloaca swabs of chickens, ducks, muscovy ducks, geese and the like in fowls and poultry clinics, and viscera such as lungs, air pipes and the like of the examination cases. The collected samples were stored in PBS with penicillin and streptomycin, transported to the laboratory at low temperature, and immediately subjected to virus isolation, identification and culture.
2. Separation and identification of H9N2 subtype avian influenza virus: the collected sample is vortex vibrated, centrifuged at 12000rpm/min for 1min, the supernatant is inoculated with 9-day-old SPF chick embryo, and the inoculated chick embryo is placed in a 37 ℃ incubator for culture. Collecting allantoic fluid of non-lethal chick embryo after 48H, preparing 1% chick erythrocyte suspension, performing hemagglutination test, obtaining allantoic fluid with hemagglutination value, and then using H9 single factor serum of laboratory as hemagglutination inhibition test to identify HA subtype. After serum identification, the H9 subtype avian influenza virus is determined to be separated, allantoic fluid is inoculated to SPF chick embryo of 10 days old for 5 times, and stable hemagglutination positive virus allantoic fluid is obtained and frozen in a refrigerator at-80 ℃.
Sequencing analysis of (II) H9N2 subtype avian influenza strains
1. HA genome sequencing: and (3) extracting viral RNA from the separated and purified H9 subtype avian influenza virus, and carrying out reverse transcription by using a 12bp primer conserved by the influenza virus to obtain cDNA. The HA fragment of avian influenza was then amplified with fragment specific primers and sequenced with H9N2 subtype specific sequencing primers after purification.
2. Genetic evolution analysis: sequencing results are spliced by using Seqman software in DNAStar software package, and after the sequences are spliced into complete sequences, the sequences are analyzed and compared by using Mega Align. And drawing an AIV genetic evolution tree by using Mega 7, and carrying out genotyping on viruses according to the genetic differences. As shown in FIG. 1, the genetic relationship between the vaccine candidate strain FZ strain and the H9N2AIV epidemic strain of Fujian province in recent years is relatively close.
Pathogenicity analysis of (III) H9N2 subtype avian influenza strains
1. Expanding and propagating strains: the allantoic fluid of the preserved H9N2 strain is melted at room temperature, diluted and inoculated into the allantoic cavity of 10-day-old SPF chick embryo according to 0.1 mL/embryo, and the chick embryo is placed in a constant temperature incubator at 37 ℃ for incubation after inoculation. Observing every 12 hours, discarding the dead chick embryo within 24 hours, and collecting chick embryo allantoic fluid with the hemagglutination titer of more than or equal to 256 in 48 hours.
2. Strain EID 50 Determination of H9N 2-containing Prostrain FZ was subjected to ten-fold specific dilution with PBS from 10 -5 To 10 -9 The method comprises the steps of carrying out a first treatment on the surface of the Inoculating 5 SPF chick embryos of 10 days old at each dilution, wherein each embryo is 0.1mL; taking chick embryos every 12 hours, discarding the chick embryos dead within 24 hours; preserving the dead chick embryo at 4 ℃ after 24 hours; collecting allantoic fluid for 72h for hemagglutination test, wherein the hemagglutination is greater than 1:8 were judged to be positive, and the half-number of infection of the virus liquid chick embryo was calculated to be 10 according to the Reed-Muench method 8.5 EID 50 /0.1mL。
3. Animal infection assay: H9N2 subtype avian influenza FZ virus liquid was administered by intravenous route in an amount of 0.2mL (containing 10 6.0 EID 50 ) Clinical observations showed that the blank group was well-conditioned and no visible clinical symptoms were observed during the observation period. Only some chickens in the FZ test group showed slight respiratory symptoms such as head flick, sneeze, etc., and the mortality was 0. The results of the section change are shown in figure 2, mucus exists in the trachea and the larynx of the chicken in the FZ test group, the larynx has bleeding points, and other tissues and organs have no obvious change. Each group of throat swabs are randomly collected 5d after virus attack, and the detection is carried out by a chick embryo inoculation methodThe result shows that the toxin expelling rate of the FZ test group 5d reaches 100 percent.
The test results show that the separated H9N2 subtype avian influenza virus strain belongs to a weak strain and accords with the characteristics of the H9N2 subtype avian influenza virus; it was named: the influenza A virus FZ strain is preserved in China center for type culture collection, the preservation address is China Wuhan, the preservation number is CCTCC NO: V202171, and the preservation date is 2021, 9 and 26 days.
Example 2 isolation and identification of Mycoplasma synoviae
Isolation of Mycoplasma synoviae
Collecting joint liquid and contents in a swelling tarsal joint cavity of a sick chicken suspected of infecting chicken bursa of mycoplasma in Fuzhou chicken farm, inoculating in an improved Frey liquid culture medium under aseptic operation condition, placing in a constant-temperature shaking culture medium at 37 ℃ for 48 hours, filtering the bacterial liquid by a 0.22 mu m filter when the bacterial liquid turns from red to yellow, re-inoculating in the improved Frey liquid culture medium according to the volume ratio of the filtrate to the improved Frey liquid culture medium of 1:10, and culturing again, and collecting the 2 nd-generation bacterial liquid for identifying pathogenic bacteria.
(II) identification of Mycoplasma synoviae
1. And (3) identifying and observing the bacterial forms: the separated 2 nd generation bacterial liquid is smeared, and is subjected to Giemsa staining and microscopic examination, and the observed bacterial form is characterized by a sphere or a club. Inoculating the filtered strain on modified Frey's solid culture medium, and placing in CO with volume fraction of 5% 2 In the incubator, the culture was carried out at 37℃for 7 days, and the microcolonies were observed under a microscope to be fine, smooth and dense, which were expressed as "omelette-like", and were semi-dented in the medium.
2. Identification of thallus L type: inoculating the separated 2 nd generation bacterial liquid into an improved Frey's liquid culture medium without penicillin and thallium acetate, continuously subculturing for 10 times, inoculating the culture bacterial liquid into an improved Frey's solid culture medium, culturing at a constant temperature of 37 ℃, observing colony morphology, and performing smear microscopy. The results showed that the isolated bacteria were bacterial L-type negative.
3. Identification of artificial infection: taking 0.5mL of the separated 2 nd generation bacterial liquid, injecting and inoculating SPF chicks through nose drops, eyes and foot pads, and after 40d, infecting the chicks to have obvious clinical symptoms such as lameness, paralysis, arthrocele, cheese-like sediment at keels and the like (see figure 3).
4. Treeing analysis of 16s rRNA genes: amplifying the 16s rRNA gene of the mycoplasma synoviae strain, sequencing the amplified gene sequence, and comparing with other known several mycoplasma synoviae strains and analyzing the evolutionary tree. As shown in FIG. 4, the Mycoplasma synoviae strain has high homology with Mycoplasma synoviae strain of Asian.
The identification results show that the separated mycoplasma synoviae strains all accord with the mycoplasma synoviae characteristics; it was designated as Mycoplasma synoviae MS-FJ01; the Latin name is Mycoplasmanoviae, the bursa of sliding mycoplasma MS-FJ01 is preserved in China center for type culture Collection, the preservation address is China Wuhan, the preservation number is CCTCC NO: M2021210, and the preservation date is 2021, 3 months and 8 days.
Wherein, the improved Frey's liquid culture medium and the improved Frey's solid culture medium are both conventional products sold in the market and will not be described in detail.
Example 3, H9N2 subtype avian influenza and chicken bursa mycoplasma bigeminal inactivated vaccine
Seed liquid preparation
The purified allantoic fluid of H9N2 strain FZ strain virus (obtained in the step (I) of the example 1) is diluted and inoculated into allantoic cavity of SPF chick embryo of 10 days old according to 0.1 mL/embryo, and the inoculated chick embryo is placed in a constant temperature incubator at 37 ℃ for incubation. Observing every 12h, discarding the dead chick embryo within 24h, collecting chick embryo allantoic fluid with the hemagglutination titer of more than or equal to 256 for 48h, collecting 5 to 15 generations as seed batches, and storing at-80 ℃.
Adding mycoplasma synoviae MS-FJ01 into a mycoplasma synoviae culture medium according to the proportion of 1 to 10, culturing at 37 ℃ at the shaking speed of 180r/min, collecting bacterial liquid after culturing until the bacterial liquid changes from red to orange to logarithmic phase, recording actual culturing time, collecting bacterial liquid as mycoplasma synoviae seed liquid, and storing at 4 ℃.
The preparation method of the chicken bursa mycoplasma culture medium comprises the following steps: 21g of brain-heart leaching liquid broth, 5.6g of yeast leaching powder, 2mL of 1wt% phenol red solution, 2mL of 50mg/mL of L-cysteine solution, 8mL of 50mg/mL of arginine solution and 0.4mL of 250mg/mL of coenzyme I solution are taken and mixed, the volume is fixed to 900mL, 100mL of inactivated fetal calf serum is added, 80 ten thousand units/L of penicillin is added, pH 7.6-7.8 is regulated, and finally a 0.22 mu m filter is used for filtering to a sterile container to complete the preparation.
Amplification culture of the second toxic Strain
The allantoic fluid of the preserved H9N2 strain is melted at room temperature, inoculated into allantoic cavity of SPF chick embryo of 9-11 days old according to 0.1 mL/embryo, and placed in a constant temperature incubator at 37 ℃ for incubation after inoculation. Observing every 12h, discarding the dead chick embryo within 24h, collecting chick embryo allantoic fluid for 48-72h, and displaying that the collected allantoic fluid has a hemagglutination titer of 256 or more and a virus antigen content of 10 or more 8.5 EID 50 /0.1mL。
Adding mycoplasma synoviae seed liquid into a mycoplasma synoviae culture medium according to the proportion of 1 to 10, culturing at 37 ℃ at the shaking speed of 180r/min, culturing until the bacterial liquid changes from red to orange to obtain bacterial liquid, sampling, and performing viable bacteria determination: the cultured MS-FJ01 strain bacterial liquid is serially diluted to 10 by 10 times by using a chicken bursa mycoplasma culture medium 24 The viable bacteria count tubes were each placed at 37℃and cultured with shaking at 180r/min, and observed for 7 days (final tube with medium color changing from scarlet to orange as the highest concentration of viable bacteria). The result shows that the living bacterial load of MS-FJ01 strain is not less than 10 12 CCU/mL。
(III) concentration of mycoplasma bacteria liquid
Centrifuging the collected Mycoplasma synoviae bacterial liquid at 15000g and 4deg.C for 30min with ultra-high speed refrigerated centrifuge, and concentrating the Mycoplasma synoviae bacterial liquid until the viable bacteria content per ml is not less than 5×10 13 CCU, immediately inactivated after concentration.
(IV) bacterial liquid and strain inactivation
Adding formaldehyde solution with the final concentration of 0.1% into the allantoic fluid of the sterile H9N2 subtype AIV FZ strain virus prepared in the step (II), and shaking with the addition to ensure that the formaldehyde solution is fully mixed. Placing the mixture in a shaking table at the constant temperature of 37 ℃ for shaking and inactivating for 12 hours at 200-220 r/min, taking out the mixture, and storing the mixture in a refrigerator at the temperature of 4 ℃ for later use.
Adding formaldehyde with the final concentration of 0.2% into the concentrated mycoplasma gallisepticum bacterial solution prepared in the step (three), fully mixing, and placing the mixture in an incubator with the culture temperature of 37 ℃ and the shaking speed of 180r/min for shaking and inactivating for 24 hours.
(fifth) inactivation test
Inoculating an inactivated virus liquid into the allantoic cavity of the SPF chick embryo of the age of 10 days according to the standard of 3 pieces per serving and 0.1mL per piece, sealing and marking, placing the chick embryo in a 37 ℃ incubator for culturing, continuously observing for 5d for 2 times/d, and harvesting the allantoic liquid of the chick embryo after 5d, and carrying out HA measurement, wherein the result shows that the virus liquid can be fully inactivated by shaking and inactivating for 12 hours by using 0.1% formaldehyde in a shaking table at the constant temperature of 37 ℃ for 200-220 r/min.
Inoculating the inactivated bacterial liquid into a mycoplasma synoviae culture medium according to the proportion of 1 to 10, culturing for about 14 days, and observing the growth condition of the bacterial body. The result shows that the bacteria can be fully inactivated by shaking in an incubator with the shaking speed of 180r/min at 37 ℃ and 0.2% formaldehyde.
(six) vaccine preparation
In a sterile container, 1:2.81 of the total amount of the inactivated H9N2 subtype AIV FZ strain virus solution (the virus antigen content of the vaccine is 10) 8.0 EID 50 /mL) and vaccine adjuvants, the vaccine 1 preparation was completed using HR-500 dispersing emulsifying machine gradually starting from a-stage to E-stage for 30 minutes.
In a sterile container, 1:2.81 respectively adding inactivated mycoplasma synoviae bacterial liquid and vaccine adjuvant (the bacterial antigen content of vaccine is not less than 1×10) 12 CCU/mL), emulsification was gradually started from a gear a to a gear E using HR-500 dispersion emulsifying machine for 30 minutes to complete vaccine 2 preparation.
In a sterile container, 1:1:5.62 adding inactivated H9N2 subtype AIV FZ strain virus solution (virus antigen content of vaccine 10) 8.0 EID 50 /mL), mycoplasma synoviae (bacterial antigen content of vaccine is not less than 1×10) 12 CCU/mL) and vaccine adjuvants using HR-500 dispersing emulsifying machineEmulsification was gradually started from a gear to E gear for 30 minutes to complete vaccine 3 preparation.
(seventh) inspection of finished products
1. Appearance: 5mL of each vaccine is taken and placed in a clean glass tube, and the color of the vaccine is observed to be unchanged, free of impurities and the like.
2. Dosage form: taking a cleaning straw, sucking a small amount of vaccine, dripping the vaccine onto the surface of cold water, and observing that the vaccine is not diffused.
3. Stability: 10mL of each batch of vaccine is sucked and added into a centrifuge tube, and the mixture is centrifuged for 15 minutes at 3000r/min, so that the vaccine is observed to be not layered.
4. And (3) sterile inspection: according to the annex of the current Chinese animal pharmacopoeia, no bacteria grow.
5. Pure test: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and all the tests accord with the regulations.
6. Measurement of residual formaldehyde: the measurement is carried out according to the annex of the current Chinese animal pharmacopoeia, and the result shows that the method accords with the rule of general rules of biological products for animals.
7. And (3) safety inspection: 10 SPF chickens of 14 days old are taken, the vaccine of the invention is injected subcutaneously into the neck, each dose is 0.5mL, the spirit, feeding and drinking water of all the test chickens are observed continuously for 14 days, no obvious abnormality is seen, and no local or systemic adverse reaction is caused by injecting the vaccine.
8. Serum antibody titer assay: 40 SPF chickens of 14 days of age were randomly divided into 4 groups of 10 chickens. Group 1 cervical subcutaneous vaccine 1, group 2 cervical subcutaneous vaccine 2, group 3 cervical subcutaneous vaccine 3, and group 4 non-immunized as controls. The first group, the third group and the control chicken are subjected to blood sampling at intervals of one week after immunization, serum is separated, and after total collection of 6 weeks of serum, the H9N2 subtype avian influenza HI antibody and chicken bursa mycoplasma ELISA antibody titer S/P value are respectively measured. The results of the H9N2 subtype avian influenza HI antibody show that the average HI titer of chicken serum in a control group is less than 2, the average HI titer of 14 days after immunization is more than or equal to 256, the average HI titer of 21 days after immunization is more than or equal to 512, the antibody level reaches a peak value after immunization for 21 days, the average HI titer is more than or equal to 512, and the chicken serum tends to be stable after immunization, and the high-level serum antibody is kept. ELISA antibody results of chicken bursa of mycoplasma show that the average S/P value is less than 0.1 in the chicken serum of the control group; the average S/P value of the immunized chicken serum after immunization is more than or equal to 1 in 14 days, the average S/P value of the immunized chicken serum after immunization is more than or equal to 1.5 in 21 days, the antibody level reaches a peak value in 28 days after immunization, the average S/P value of the immunized chicken serum is more than or equal to 2.5, and the immunized chicken serum is stable and accords with the ELISA antibody detection kit standard.
9. Efficacy test: the immunization of 40 SPF chickens at 14 days of age was controlled by injecting vaccine 1 into the neck of group 1, vaccine 2 into the neck of group 2, vaccine 3 into the neck of group 3, and no immunization of group 4. After 14 days of immunization, group 1 was intravenously inoculated with 0.2mL of H9N2 subtype avian influenza virus (10 6.0 EID 50 Group 2 nasal drop point eye inoculation of chicken bursa mycoplasma synoviae culture 0.5mL (10) 12 CCU/mL), group 3 infected chicken bursa mycoplasma synoviae cultures 0.5mL (10) 12 CCU/mL) and 0.2mL (10) of H9N2 subtype avian influenza virus 6.0 EID 50 /mL). Collecting throat and cloaca swabs of each group of chickens after the toxin is attacked to carry out embryo grafting detection of H9N2 subtype avian influenza, and collecting throat swabs of each group of chickens to carry out PCR detection of chicken bursa of mycoplasma. The embryo grafting detection results of the H9N2 subtype avian influenza are shown in Table 1, and the PCR detection results of the chicken bursa mycoplasma are shown in Table 2.
TABLE 1 statistics of 3 and 5d detoxification detection results after immune challenge of H9N2 subtype avian influenza
Figure BDA0003299579960000111
TABLE 2 statistics of 3 and 5d detoxification detection results of Mycoplasma synoviae of immunized challenge chickens
Figure BDA0003299579960000112
The foregoing description is only a partial embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes using the descriptions and the drawings of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.

Claims (5)

1. An avian influenza H9N2 subtype virus strain characterized by: it is named influenza A virusAvian Influenza Virus) FZ strain is preserved in China center for type culture Collection, with preservation address of Chinese Wuhan, preservation number of CCTCC NO: V202171, and preservation date of 2021, 9 months and 26 days.
2. A bivalent inactivated vaccine against H9N2 subtype avian influenza and mycoplasma synoviae, characterized in that it comprises an antigen and a vaccine adjuvant; the antigen is an inactivated antigen, and comprises an inactivated influenza A virus FZ strain and an inactivated mycoplasma synoviae @ strainMycoplasma synoviae）MS-FJ01；
Wherein, the influenza A virus FZ strain is preserved in China center for type culture Collection, the preservation address is China WU, the preservation number is CCTCC NO: V202171, and the preservation date is 2021, 9 and 26 days; the bursa of mycoplasma MS-FJ01 is preserved in China center for type culture Collection, the preservation address is Wuhan, the preservation number is CCTCC NO: M2021210, and the preservation date is 2021, 3, 8 days.
3. The bivalent inactivated vaccine against H9N2 subtype avian influenza and mycoplasma synoviae of claim 2, wherein the influenza a virus FZ strain and mycoplasma synoviae MS-FJ01 are inactivated with formaldehyde.
4. The method for preparing the bivalent inactivated vaccine against H9N2 subtype avian influenza and chicken bursa mycoplasma as claimed in claim 3, which comprises the following steps:
1) Diluting influenza A virus FZ strain with sterilized PBS 1000 times, inoculating into 9-11 day-old SPF chick embryo allantoic cavity, incubating at 37 deg.C, collecting chick embryo virus allantoic liquid after 48 hr, performing hemagglutination test with 1% chick erythrocyte, aseptically collecting virus allantoic liquid with HA titer not less than 256, and storing at-80deg.C; then taking a chicken mycoplasma synoviae culture medium with the pH value of 7.6-7.8, adding the mycoplasma synoviae MS-FJ01 in a ratio of 10 to 1, then placing the chicken mycoplasma synoviae culture medium in an environment with the culture temperature of 37 ℃ and the shaking speed of 180r/min, culturing until the bacterial liquid turns orange, and then placing the chicken mycoplasma synoviae culture medium at 4 ℃ for preservation;
2) Antigen inactivation: adding formaldehyde solution with the final concentration of 0.1% into the allantoic fluid of the chick embryo virus prepared in the step 1), and shaking the chick embryo virus with the formaldehyde solution to fully mix the chick embryo virus; then placing the mixture in a constant temperature shaking table at 37 ℃ for shaking and inactivating for 12 hours at 200-220 r/min, taking out the mixture, and storing the mixture in an environment at 4 ℃ for later use; the bacterial liquid containing the mycoplasma synoviae MS-FJ01 is placed in an ultra-high-count centrifuge for centrifugal treatment, and the bacterial liquid is concentrated until the content of viable bacteria per milliliter is not less than 5 multiplied by 10 13 Adding formaldehyde with the final concentration of 0.2% into the concentrated bacterial liquid immediately after concentration, and carrying out oscillation inactivation for 24 hours under the conditions that the culture temperature is 37 ℃ and the oscillation speed is 180 r/min;
3) 1, the method comprises the following steps: 1:5.62, respectively adding the inactivated influenza A virus FZ strain virus liquid, the bursa of mycoplasma MS-FJ01 bacterial liquid and the vaccine adjuvant in the step 2), and gradually emulsifying from the A grade to the D grade by using an HR-500 dispersing emulsifying machine, so that the emulsion is emulsified for 30 minutes at 15000r/min under the low temperature condition.
5. The method for preparing the bivalent inactivated vaccine against the H9N2 subtype avian influenza and the mycoplasma synoviae according to claim 4, wherein in the step 1), the preparation method of the mycoplasma synoviae culture medium is as follows: the preparation is completed by taking brain-heart leaching liquor broth 21g, yeast leaching powder 5.6g, 1wt% phenol red solution 2mL, 50mg/mL L-cysteine solution 2mL, 50mg/mL arginine solution 8mL and 250mg/mL coenzyme I solution 0.4mL, mixing, then fixing the volume to 900mL, adding inactivated fetal calf serum 100mL, adding 80 ten thousand units/L penicillin, adjusting the pH to 7.6-7.8, and finally filtering to a sterile container by using a 0.22 μm filter.
CN202111186799.4A 2021-10-12 2021-10-12 Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma Active CN114015658B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111186799.4A CN114015658B (en) 2021-10-12 2021-10-12 Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111186799.4A CN114015658B (en) 2021-10-12 2021-10-12 Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma

Publications (2)

Publication Number Publication Date
CN114015658A CN114015658A (en) 2022-02-08
CN114015658B true CN114015658B (en) 2023-06-09

Family

ID=80055664

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111186799.4A Active CN114015658B (en) 2021-10-12 2021-10-12 Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma

Country Status (1)

Country Link
CN (1) CN114015658B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008115785A2 (en) * 2007-03-16 2008-09-25 Wyeth Multivalent avian influenza vaccines and methods
CN102805864A (en) * 2012-09-03 2012-12-05 江苏省农业科学院 Newcastle disease and H9N2 subtype avian influenza bivalent inactivated vaccine and preparation method thereof
CN103937753A (en) * 2014-04-14 2014-07-23 吉林正业生物制品股份有限公司 H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof
WO2017129048A1 (en) * 2016-01-29 2017-08-03 程龙 Vaccine used for preventing and treating influenza, avian influenza and upper respiratory tract infection
CN110575539A (en) * 2018-06-11 2019-12-17 洛阳惠中生物技术有限公司 Avian influenza virus-like particle vaccine, and preparation method and application thereof
CN112574958A (en) * 2019-09-28 2021-03-30 普莱柯生物工程股份有限公司 H9 subtype avian influenza virus isolate and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008115785A2 (en) * 2007-03-16 2008-09-25 Wyeth Multivalent avian influenza vaccines and methods
CN102805864A (en) * 2012-09-03 2012-12-05 江苏省农业科学院 Newcastle disease and H9N2 subtype avian influenza bivalent inactivated vaccine and preparation method thereof
CN103937753A (en) * 2014-04-14 2014-07-23 吉林正业生物制品股份有限公司 H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof
WO2017129048A1 (en) * 2016-01-29 2017-08-03 程龙 Vaccine used for preventing and treating influenza, avian influenza and upper respiratory tract infection
CN110575539A (en) * 2018-06-11 2019-12-17 洛阳惠中生物技术有限公司 Avian influenza virus-like particle vaccine, and preparation method and application thereof
CN112574958A (en) * 2019-09-28 2021-03-30 普莱柯生物工程股份有限公司 H9 subtype avian influenza virus isolate and application thereof

Also Published As

Publication number Publication date
CN114015658A (en) 2022-02-08

Similar Documents

Publication Publication Date Title
CN105816868B (en) Inactivated vaccine for mycoplasma synoviae
CN112779193B (en) Virulent strain of mycoplasma synoviae and application thereof
CN110251671B (en) Preparation method of goose astrovirus egg yolk antigen-antibody complex
CN111905100B (en) Goose astrovirus bivalent inactivated vaccine and yolk antibody and preparation method thereof
CN104946600B (en) A kind of H9 subtype avian influenza virus strain
CN111000993A (en) Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof
CN105247043A (en) Scale drop disease (SDD) causative virus and derivatives thereof
CN105733987A (en) Mycoplasma synoviae
CN109097340B (en) Avian adenovirus, quadruple vaccine and preparation method thereof
CN108465107B (en) Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine
CN105770881A (en) Mycoplasma gallisepticum (MG) and mycoplasma synoviae (MS) combined inactivate vaccine
CN113957007B (en) Inactivated vaccine for mycoplasma synoviae
CN109055320B (en) Infectious bronchitis virus isolate and application thereof in vaccine preparation
CN109207436A (en) One plant of 4 type aviadenovirus strain of I group and its application
CN108939063B (en) Muscovy duck triple inactivated vaccine
CN114015658B (en) Bivalent inactivated vaccine for H9N2 subtype avian influenza and chicken bursa mycoplasma
CN106065030A (en) Yolk antibody of chicken inclusion body hepatitis and preparation method thereof
CN113278595B (en) Duck adenovirus type 3 strain, duck adenovirus egg yolk antibody, and preparation methods and application thereof
CN112063596A (en) Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof
CN110079509A (en) One kind I group of strain of 11d type aviadenovirus, inactivated vaccine and preparation method thereof
CN111084150A (en) Method for establishing animal model infected by avian infectious bronchitis virus
CN111150840B (en) Tetrad vaccine for poultry and preparation method and application thereof
CN109022372A (en) A kind of cultural method of Porcine epidemic diarrhea virus
CN109679926B (en) Avian influenza virus H9 subtype JXD strain and application thereof
CN116875564B (en) H9 subtype avian influenza virus strain and culture method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant