CN117511816A - Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof - Google Patents
Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN117511816A CN117511816A CN202311609402.7A CN202311609402A CN117511816A CN 117511816 A CN117511816 A CN 117511816A CN 202311609402 A CN202311609402 A CN 202311609402A CN 117511816 A CN117511816 A CN 117511816A
- Authority
- CN
- China
- Prior art keywords
- mycoplasma
- culture
- strain
- live vaccine
- mycoplasma synoviae
- 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.)
- Pending
Links
- 241000202942 Mycoplasma synoviae Species 0.000 title claims abstract description 88
- 229960005486 vaccine Drugs 0.000 title claims abstract description 74
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 241000287828 Gallus gallus Species 0.000 claims abstract description 92
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 43
- 241000204031 Mycoplasma Species 0.000 claims abstract description 38
- 241001260012 Bursa Species 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 28
- 230000004151 fermentation Effects 0.000 claims abstract description 28
- 239000000427 antigen Substances 0.000 claims abstract description 27
- 108091007433 antigens Proteins 0.000 claims abstract description 27
- 102000036639 antigens Human genes 0.000 claims abstract description 27
- 239000006052 feed supplement Substances 0.000 claims abstract description 22
- 238000004108 freeze drying Methods 0.000 claims abstract description 16
- 239000003223 protective agent Substances 0.000 claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims description 45
- 241000204003 Mycoplasmatales Species 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 15
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 14
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 13
- 230000002829 reductive effect Effects 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000004445 quantitative analysis Methods 0.000 claims description 10
- 238000007865 diluting Methods 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 7
- 235000005152 nicotinamide Nutrition 0.000 claims description 7
- 229960003966 nicotinamide Drugs 0.000 claims description 7
- 239000011570 nicotinamide Substances 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 6
- 239000004475 Arginine Substances 0.000 claims description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 5
- 235000009697 arginine Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000000703 high-speed centrifugation Methods 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 4
- 229930064664 L-arginine Natural products 0.000 claims description 4
- 235000014852 L-arginine Nutrition 0.000 claims description 4
- 229920002125 Sokalan® Polymers 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 229960001631 carbomer Drugs 0.000 claims description 4
- 108010079058 casein hydrolysate Proteins 0.000 claims description 4
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 4
- 229940054269 sodium pyruvate Drugs 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 108010010803 Gelatin Proteins 0.000 claims description 3
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 238000003753 real-time PCR Methods 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 14
- 230000005847 immunogenicity Effects 0.000 abstract description 10
- 238000004321 preservation Methods 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 239000000047 product Substances 0.000 abstract description 3
- 235000013330 chicken meat Nutrition 0.000 description 86
- 239000001963 growth medium Substances 0.000 description 27
- 239000000243 solution Substances 0.000 description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 20
- 201000010099 disease Diseases 0.000 description 19
- 229940125575 vaccine candidate Drugs 0.000 description 18
- 231100000419 toxicity Toxicity 0.000 description 17
- 230000001988 toxicity Effects 0.000 description 17
- 241000204022 Mycoplasma gallisepticum Species 0.000 description 15
- 238000009630 liquid culture Methods 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 239000000463 material Substances 0.000 description 9
- 235000010633 broth Nutrition 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 230000008961 swelling Effects 0.000 description 7
- 210000004341 tarsal joint Anatomy 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 210000003437 trachea Anatomy 0.000 description 6
- 230000001018 virulence Effects 0.000 description 6
- 230000002238 attenuated effect Effects 0.000 description 5
- 210000003837 chick embryo Anatomy 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 238000010367 cloning Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 210000000416 exudates and transudate Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- 241000589945 Magnetospirillum magnetotacticum Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000004712 air sac Anatomy 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000003097 mucus Anatomy 0.000 description 3
- 238000001921 nucleic acid quantification Methods 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 210000001226 toe joint Anatomy 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- 206010028470 Mycoplasma infections Diseases 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003889 eye drop Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000001562 sternum Anatomy 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000003313 weakening effect Effects 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 206010006811 Bursitis Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 208000004760 Tenosynovitis Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000078 claw Anatomy 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000000832 effect on mycoplasma Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000005222 synovial tissue Anatomy 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 230000005570 vertical transmission Effects 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/35—Mycoplasma
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a chicken bursa mycoplasma live vaccine strain, a live vaccine and a preparation method thereof, belonging to the technical field of biological products for animals. The live vaccine strain of mycoplasma synoviae is named as RPMSA1301, the preservation number is CCTCC M20232235, and the preparation method of the live vaccine comprises the steps of carrying out subculture and fermentation culture on the live vaccine strain of mycoplasma synoviae RPMSA1301 after resuscitating to obtain fermentation broth; adding a feed supplement 1 in the process of subculture, and adding a feed supplement 2 in the process of fermentation culture; quantitatively detecting the antigen content of the obtained fermentation broth; and adding a heat-resistant freeze-drying protective agent into the obtained zymophyte liquid, and freeze-drying to prepare the chicken bursa mycoplasma live vaccine. The chicken bursa mycoplasma RPMSA1301 strain has good immunogenicity, safe use and stable genetic performance.
Description
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to a chicken bursa mycoplasma live vaccine strain, a live vaccine and a preparation method thereof.
Background
Mycoplasma synoviae (Mycoplasma synoviae, MS) causes infectious diseases characterized mainly by joint exudative synovitis and tenosynovitis. The weak chick rate is increased, the laying rate of the laying hen, the weight of the broiler chicken and the feed conversion rate are reduced, and when the medicine is used for treatment, the medicine resistance is easy to generate, and the effect is also unsatisfactory. The mycoplasma synoviae and mycoplasma gallisepticum are often mixed and infected, and are often mixed and infected with other bacteria or viruses, so that the utilization rate and the laying rate of the chicken feed are low, and the mycoplasma pathogen becomes the bottleneck for restricting the rapid development of the chicken industry due to the expensive drug treatment cost. The method mainly adopts drug treatment and immunization for preventing and treating mycoplasma disease, clinical practice proves that MS is extremely easy to generate drug resistance, and mycoplasma infection of chicken flocks cannot be cleared through antibiotic treatment; vaccination is thus a more effective measure in controlling the disease.
The common clinical mycoplasma gallisepticum vaccines are mainly divided into an inactivated vaccine and a attenuated vaccine, wherein the inactivated vaccine can reduce the vertical transmission of mycoplasma gallisepticum and relieve infection symptoms, but can not thoroughly remove wild toxins in chickens and prevent the invasion of the wild toxins. The attenuated vaccine can make organism produce local mucosa immunity, and its application is extensive. A variety of live Mycoplasma gallisepticum vaccines, such as strain F-36, strain TS-11, strain 6/85, have been marketed, whereas live Mycoplasma synoviae vaccines have only been marketed as MS-H strain imported from Australia.
In recent years, the infection condition of the chicken bursa of mycoplasma in China presents universality and is far more harmful than mycoplasma gallisepticum. Research on chicken bursa mycoplasma vaccines in China is concentrated on development of inactivated vaccines. Therefore, the chicken bursa mycoplasma attenuated live vaccine strain with good safety and strong immunogenicity is obtained, an MS animal infection model is successfully established, and the chicken bursa mycoplasma live vaccine is developed, so that the chicken bursa mycoplasma attenuated live vaccine strain is necessary for effectively preventing and controlling mycoplasma diseases.
Disclosure of Invention
The invention aims to provide a chicken bursa mycoplasma live vaccine strain, a live vaccine and a preparation method thereof, which are used for solving the problems of the prior art, and the chicken bursa mycoplasma RPMSA1301 strain is a epidemic strain which is automatically separated and screened by an inventor, is obtained through artificial domestication and weakening, has good immunogenicity, is safe to use and has stable genetic performance.
In order to achieve the above object, the present invention provides the following solutions:
the chicken mycoplasma synoviae live vaccine strain RPMSA1301 has a preservation number of CCTCC M20232235.
The invention also provides application of the chicken bursa mycoplasma live vaccine strain RPMSA1301 in preparation of chicken bursa mycoplasma vaccine.
The invention also provides a chicken mycoplasma synoviae live vaccine, and the effective components comprise the chicken mycoplasma synoviae live vaccine strain RPMSA1301 of claim 1.
A preparation method of chicken bursa mycoplasma live vaccine comprises the following steps:
s1, activating a mycoplasma synoviae live vaccine strain RPMSA1301 of claim 1, and then performing subculture and fermentation culture to obtain a fermentation broth;
adding a feed supplement 1 in the process of subculture, and adding a feed supplement 2 in the process of fermentation culture;
the feed supplement 1 comprises, by mass and volume percentage, 1-5% of gelatin, 1-3% of nicotinamide, 1-2% of arginine, 5-10% of glucose and 0.1-0.5% of sodium pyruvate;
the components of the feed supplement 2 comprise, by mass and volume percentage, 2-4% of sodium hydroxide, 1-3% of PEG 600010-20%, 1-3% of nicotinamide and 10-20% of DMEM;
s2, quantitatively detecting the antigen content of the fermentation broth obtained in the step S1, wherein the detection method is a nucleic acid quantitative method or a protein quantitative method, and diluting the fermentation broth according to a detection result;
s3, adding a heat-resistant freeze-drying protective agent into the bacterial liquid diluted in the step S2, and freeze-drying to prepare the chicken bursa mycoplasma live vaccine;
the heat-resistant freeze-drying protective agent comprises 2-4% of carbomer by mass and volume percentage, 1-4% of polyvinylpyrrolidone by mass and volume percentage, 2-8% of trehalose by mass and volume percentage, 4-12% of casein hydrolysate by mass and volume percentage, 0.1-0.2% of L-arginine by mass and volume percentage and 4-8% of ethylene glycol by volume concentration.
Further, in step S1, the feed 1 is added in an amount of 0.1 to 0.5% of the total culture volume when the pH is reduced to 6.9 during the subculture.
Further, in the step S1, when the pH is reduced to 6.7 in the fermentation culture process, adding the feed supplement 2, continuously culturing for 10-20 hours, adding the feed supplement 2 for the second time, culturing for 10-20 hours, and adding the feed supplement 2 for the third time; the addition amount of the feed supplement 2 in the fermentation culture process is 0.1-0.5% of the total culture volume.
Further, in step S1, the conditions for the subculture are constant temperature culture at 37 ℃; the fermentation culture condition is 0.04MPa, the rotating speed is 100r/min, and the constant temperature culture is carried out at 37 ℃.
Further, in step S2, a Mycoplasma synoviae bacterial liquid antigen is detected by a nucleic acid quantification methodContent, preferably fluorescence quantitative PCR method; concentrating antigen by 10-50 times by high-speed centrifugation or membrane filtration, quantitatively detecting, wherein the upstream primer, the downstream primer and the probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; diluting the concentrated bacterial liquid according to the detection result to ensure that the antigen content of the mycoplasma synoviae is (0.5-1.5) multiplied by 10 7.0 Copy number/plume.
Further, in the step S2, the antigen content of mycoplasma synoviae bacterial liquid is detected by a protein quantitative method, the antigen is concentrated by 10-50 times by a high-speed centrifugation or membrane filtration method, and then quantitative detection is carried out; and diluting the concentrated bacterial liquid according to the detection result to ensure that the antigen content of the mycoplasma synoviae is 200-500 mug/serving.
Further, in step S3, the volume ratio of the bacterial liquid to the heat-resistant freeze-drying protective agent is 1:1.
the invention discloses the following technical effects:
the chicken bursa mycoplasma RPMSA1301 strain is a epidemic strain which is automatically separated and screened by the inventor, is obtained through artificial domestication and weakening, has good immunogenicity, is safe to use and has stable genetic performance.
In the process of culturing chicken bursa mycoplasma, the invention improves the titer of MS fermentation culture to 10 by timely adding two nutrient solutions (the feed 1 and the feed 2) 10.0 The efficient culture process is beneficial to improving the immunity effect of the chicken bursa mycoplasma synoviae vaccine.
The quantitative detection method of the antigen content of the mycoplasma synoviae bacterial liquid is a nucleic acid quantitative method or a protein quantitative method, firstly, the antigen is concentrated 10 to 50 times by a high-speed centrifugation or membrane filtration method, then the quantitative detection is carried out, and the concentrated bacterial liquid is diluted according to the detection result; the method has the advantages that the antigen is accurately quantified before the vaccine preparation, the defect that the bacterial live vaccine can only quantify the antigen by a method of tracking and checking the CCU is overcome, and the stability among vaccine batches is improved.
According to the invention, the chicken bursa mycoplasma and the preferred protective agent are freeze-dried in an optimal ratio to form individual snails, so that good storage is provided for the mycoplasma, the vaccine has good stability, long-term storage and transportation at the temperature of 2-8 ℃ are realized, on the other hand, the protective agent has the effects of synergistic immunity and external adverse environment resistance, the vaccine can be used for nasal drip, eye drop, drinking water or spray immunity, even mixed material immunity can be performed, and the use is more convenient.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The conventional mycoplasma liquid culture medium adopted in the embodiment of the invention is Frey's solid or liquid culture medium, and the inventor prepares the mycoplasma liquid culture medium according to the formula of annex 32-33 pages in the pharmacopoeia of the animal of the people's republic of China (three parts of 2020 edition).
Example 1 isolation and identification of Mycoplasma synoviae
1. Disease material
The disease material is a suspected chicken bursa mycoplasma infection disease chicken trachea and a swelling tarsal joint cavity swab or a chicken embryo and a weak embryo with incomplete pecking shell.
2. Pathogen isolation culture and colony morphology observation
And (3) carrying out separation culture on the collected sample in Frey's liquid culture medium, placing the culture medium in a 37 ℃ incubator for culture, inoculating the culture medium into a new Frey's liquid culture medium for passage for 3-5 times when the culture medium turns orange, and separating the culture after the culture medium grows stably.
Isolated cultures whose colonies were Rayleigh stained and either spherical or elliptical under an oil microscope. Colonies appeared as tiny, smooth and dense microcolonies on Frey's solid medium, were semi-concave in the medium, and the colony morphology was observed under a low magnification mirror as characteristic "omelette".
3. Authentication
The isolated culture was able to break down glucose and maltose but not hydrolyze arginine and urea by passing through a 0.45 μm filter. The target fragment of 461bp can be obtained through 16S rRNA gene amplification, and the target fragment is identified as the mycoplasma synoviae by sequencing.
The plate agglutination test result shows that the isolate can be agglutinated with chicken mycoplasma synoviae standard positive serum, but not with chicken mycoplasma synoviae standard positive serum, indicating that the isolate is chicken mycoplasma synoviae.
4. Pure culture
The method comprises the steps of selecting a colony of a omelette sample in a Frey solid culture medium, placing the colony in a Frey liquid culture medium, placing the colony in a 37 ℃ incubator for culture, inoculating the colony into a new Frey liquid culture medium for passage when the culture solution turns orange, continuously carrying out passage for 5 times according to the 10% inoculation proportion to obtain a 6 th generation mycoplasma synoviae bacterial liquid, and determining the concentration of the mycoplasma synoviae viable bacteria by a CCU method.
5. Regression experiments
The purified and cultured mycoplasma synoviae bacterial liquid is respectively injected into SPF chick embryo and SPF chick of 7 days old, and the same amount of Frey's liquid culture medium is used as a control.
The result shows that the purified and cultured mycoplasma synoviae bacterial liquid can cause death of SPF chick embryos of 7 days old, the dead chick embryos are shown as weak or swollen, and the bacterial strain can be re-separated from the egg yolk liquid of the dead chick embryos; the chick embryos of the control group developed normally and were not isolated from mycoplasma synoviae.
The purified and cultured mycoplasma synoviae bacterial liquid can also cause SPF chicken to have characteristic pathological changes of mycoplasma synoviae, the sick chicken presents with swelling of the paw pad or tarsal joints, the synovial tissue has yellowish viscous exudates, and the joint liquid at the swelled joint can be separated into the mycoplasma synoviae again; the SPF chicken in the control group has normal appetite, obvious swelling at joints is not seen, and chicken bursa mycoplasma is not separated from joint fluid.
Example 2 determination of virulence of Mycoplasma synoviae isolate
The different MS strains isolated in example 1 were cultured to F6 generation, and the viable bacteria concentration was measured, which revealed that the viable bacteria concentration of each MS isolate was 10 7.0 ～10 10.0 In the CCU/mL range.
Taking bacterial liquid of each MS isolate, injecting SPF chicken of 49 days old through a claw pad with injection dosage of 0.2mL×10 7.0 CCU/only, 10 per group; a blank of 10 was additionally placed, and Frey's liquid medium, 0.2 mL/mouse, was injected via the paw pad. After the toxicity is removed, the chickens are routinely fed and observed, and the incidence of the experimental chickens is recorded. The results of the toxicity measurements for each MS isolate are shown in Table 1.
TABLE 1 toxicity test results of MS isolates
The MS isolate virulence assay results showed: SPF chickens in the group injected with MS culture medium (blank control group) did not develop diseases, and the groups injected with MS isolates developed diseases to different degrees, mainly manifested by swelling of the paw pad (toe joint), purulent mucus or cheese-like exudates were visible at the swollen part of the dissected chickens, and the disease peak was concentrated on days 7-13.
The culture titer of the living bacteria of MS-12# strain is 10 9.0 CCU/ml,49 day old SPF chicken foot pad injection 0.2ml x 10 7.0 CCU/individual, which resulted in 10/10 of the cases (containing 2 deaths) in 9 days, was significantly more severe than other MS isolates, was more virulent, and was a good virulent strain for challenge. The MS-12# strain is named RPMSA1303 strain, is preserved in China Center for Type Culture Collection (CCTCC) M20232237 at the 11 th month of 2023 and has a preservation address of university of Wuhan and Wuhan in China.
Viable cell culture titer of MS-1# strain and MS-10# strain 10 9.0 Over CCU/mL, SPF chicken foot pad of 49 days old is injected with 0.2mL×10 7.0 The CCU only causes 3/10 of the disease in 11-13 days, the disease degree is obviously lighter than other MS isolates, the toxicity is weaker, and the CCU is a good vaccine candidate strain.
Example 3 establishment of model for challenge of Mycoplasma synoviae
Culturing MS-12# strain (RPMSA 1303 strain) to F6 generation, and measuring the concentration of viable bacteria to 10 8.0 CCU/mL。
Taking F6 generation fresh culture bacterial liquid, and carrying out different ways to detoxify 49-day-old SPF chickens, wherein 10 chickens are selected in each group; setting 10 blank control groups; after the toxicity is removed, the chickens are routinely fed and observed, and the incidence of the experimental chickens is recorded. The experimental results are shown in Table 2.
TABLE 2 test results of MS-12# challenge model for 49 day old SPF chickens
Note that: the disease sites include left paw pad, toe joint, left tarsal joint, right paw pad, toe joint, right tarsal joint, sternum, left wing and right wing joint.
The experimental result shows that: the chicken has different disease time and disease degree in different ways and different dosage of toxicity attack experiments, and symptoms mainly appear as bursitis and arthrocele (one or more places), and purulent mucus or cheese-like exudates are detected by sectioning.
Wherein, the disease can be caused by injecting the group through the paw pad for 9-14 days (8-10)/10, but the disease symptoms are limited to the paw pad and do not completely accord with clinical symptoms; the intramuscular injection and intravenous injection of the toxicity attacking groups start to attack the toxicity after 7 days, the toxicity attacking peaks after 10 to 14 days, the attack symptoms are swelling of the paw pad, the tarsal joint or the wing joint, and the sternal cyst, and the attack degree is obviously positively related to the toxicity attacking amount. The attack of other approaches to the virus group is only (1-2)/10, and the model standard of the attack is not reached.
The toxin attacking model is determined as follows: 10 SPF chickens with age of 49 days, and the strain MS-12# (RPMSA 1303 strain) is challenged by intramuscular injection or intravenous injection with the concentration of 0.2mL×10 7.0～8.0 CCU/alone, at least 7 onset.
Example 4 live vaccine candidate strain of Mycoplasma synoviae is attenuated and safety verification
1. Cloning and attenuation of MS attenuated isolates
The MS vaccine candidate strains (MS-1 # strain and MS-10# strain) are inoculated with Frey's liquid culture medium for rejuvenation, and the bacterial liquid is diluted to 10 2.0 After CCU/mL, the culture medium is coated and inoculated to Frey's solid culture medium, the culture is carried out for 60 to 72 hours at 37 ℃, and single bacterial colony which is characteristic to mycoplasma is picked under a low-power mirror and inoculated to Frey's liquid culture medium for proliferation culture; repeating the steps for 3 times; the cultured bacterial solutions were subjected to virulence and immunogenicity verification by serial passage of 3 cloned MS-1# strain and MS-10# strain in Frey's liquid medium.
2. MS vaccine candidate strain virulence verification
The 105 th generation cultures of MS-1# strain and MS-10# strain were cultured in a ratio of 0.3mL×10 9.0 CCU/chicken, 10 vaccinated with 7 day old SPF chickens each; setting culture groups of 10 th generation and 50 th generation of vaccine candidate strains inoculated by the same dose and the same way respectively as controls; observing clinical symptoms, aseptically collecting lung, trachea and joint fluid of experimental chicken, performing MS separation, and identifying the separated product by PCRConfirm as MS; the animals were returned to SPF chicken experiments 4 times in the same manner as above.
The regression experimental results of the vaccine candidate strain MS-1# strain animal are shown in Table 3.
TABLE 3 regression (virulence verification) experiment results of MS-1# Strain in this animal
The results show that: after the 10 th generation, 50 th generation and 105 th generation culture groups of the vaccine candidate strain MS-1# strain are respectively inoculated, the typical morbidity symptoms (incidence rate is 2-4/10) of chicken bursa mycoplasmosis of different degrees appear in each group of experimental chickens, and MS can be separated from lung, trachea and joint fluid of the experimental chickens. Regression experiments show that the toxicity of the MS vaccine candidate strain MS-1# strain is not obviously reduced after cloning and continuous passage, and the strain is not suitable for being used as a live vaccine strain.
The regression test results of the vaccine candidate MS-10# strain animal are shown in Table 4.
Table 4 MS-10# Strain animal regression (virulence verification) experiment results
The results show that: the vaccine candidate strain MS-10# strain 10 generation 10 culture group has five times of regression incidence rates of animals of 4/10, 3/10, 4/10, 3/10 and 2/10 respectively, and can be separated into MS from the lung, the trachea and the air sac of the experimental chicken; the vaccine candidate strain MS-10# strain 50 th generation culture group has five times of animal regression incidence rates of 2/10, 1/10 and 2/10 respectively, and can be separated into MS from the lung, the trachea and the air sac of the experimental chicken; after the MS vaccine candidate strain MS-10# strain 105 th generation culture is inoculated, five times of animal regression experiments are carried out, no obvious disease symptoms appear in all groups of experimental chickens, and MS can be separated from the lung, the trachea and the air sac of the experimental chickens.
The animal regression experiment shows that the toxicity of the MS vaccine candidate strain MS-10# strain is obviously reduced after cloning and continuous passage, and the MS vaccine candidate strain MS-10# strain has good safety.
3. MS vaccine candidate strain immunogenicity verification
MS vaccine candidate strain MS-10# strain 105 th generation culture was diluted according to 0.3mL×10 8.0 CCU/animals were vaccinated with 10 7 day old SPF chickens by eye and nose drop, while a non-immunized group was set as a control. The MS-12# virulent isolate was used to detoxify experimental and control chickens 28 days after immunization, and the disease condition was determined by a split examination 14 days after detoxication.
The result of the immunogenicity verification experiment shows that the virus-attack control chicken is 9/10 ill, and the immunized chicken is only 2/10 ill.
The immunogenicity experiment shows that the 105 th generation culture of MS vaccine candidate strain MS-10# strain has good immunogenicity.
4. MS vaccine Strain determination
After cloning and continuous passage, the strain MS-10# of the MS vaccine candidate strain has obviously reduced toxicity of the 105 th-generation culture, good safety and immunogenicity, is a good MS live vaccine strain, and the artificially domesticated attenuated strain is named as RPMSA1301 strain.
The RPMSA1301 strain is preserved in China Center for Type Culture Collection (CCTCC) M20232235 at the 11 th and 15 th year 2023 and has a preservation address of university of Wuhan and Wuhan in China.
EXAMPLE 5 preparation of live vaccine against Mycoplasma synoviae
1. Preparing the culture medium fluid infusion and heat-resistant freeze-drying protective agent for mycoplasma
The mycoplasma fowl culture medium fluid infusion comprises a feed supplement 1 and a feed supplement 2.
Preparing a mycoplasma culture medium feed 1: respectively weighing a proper amount of nicotinamide, arginine, glucose and sodium pyruvate, dissolving in water, and filtering and sterilizing by a 0.22 mu m membrane to obtain a solution 1, wherein the nicotinamide accounts for 2-6%, the arginine accounts for 2-4%, the glucose accounts for 10-20%, and the sodium pyruvate accounts for 0.2-1.0% in terms of mass and volume percent (W: V); sterilizing gelatin solution with mass and volume percentage (W: V) of 2-10% by high pressure to obtain solution 2; and uniformly mixing the solution 1 and the solution 2 in equal proportion to obtain the feed supplement 1.
Preparing a mycoplasma culture medium feed 2: respectively weighing a proper amount of PEG6000 and sodium hydroxide, dissolving in water, and sterilizing at high pressure to obtain a solution 3, wherein the PEG6000 accounts for 20-40% and the sodium hydroxide accounts for 4-8% in terms of mass volume percent (W: V); respectively weighing a proper amount of nicotinamide and DMEM, dissolving the nicotinamide and DMEM in water to enable the nicotinamide to be 2-6%, filtering and sterilizing the nicotinamide and the DMEM to be 20-40% by using a 0.22 mu m membrane to obtain a solution 4; and uniformly mixing the solution 3 and the solution 4 in equal proportion to obtain the feed supplement 2.
Preparing a heat-resistant freeze-drying protective agent: respectively weighing proper amounts of carbomer and polyvinylpyrrolidone, dissolving in water, and sterilizing at 116 ℃ for 30min to obtain a solution 5, wherein the carbomer is 4-8% and the polyvinylpyrrolidone is 2-8% in terms of mass and volume percentage (W: V); respectively weighing or measuring a proper amount of trehalose, casein hydrolysate, L-arginine and ethylene glycol, dissolving in water to enable the trehalose to be 4-16%, the casein hydrolysate to be 8-24%, and the L-arginine to be 0.2-0.4%; the volume concentration (V: V) of the glycol is 4-8%, and the glycol is filtered and sterilized by a 0.22 mu m film to obtain a solution 6; and uniformly mixing the solution 5 and the solution 6 in equal proportion to obtain the heat-resistant freeze-drying protective agent.
Feed 1, feed 2 and heat-resistant freeze-drying protective agent of the mycoplasma fowl culture medium as shown in tables 5 to 7 were prepared according to the above methods, respectively.
TABLE 5 poultry mycoplasma culture medium feed 1
TABLE 6 poultry mycoplasma culture medium feed supplement 2
Table 7 heat resistant lyoprotectant
2. Mycoplasma synoviae bacterial liquid for cultivating chickens
The mycoplasma synoviae is cultivated, a Frey liquid medium of the mycoplasma avium is used, and nutrient solution feed 1 or feed 2 is required to be supplemented in the cultivation process. When the pH is reduced to 6.9 during subculture, adding the feed supplement 1 according to 0.1-0.5% of the total culture volume (V: V); when the pH is reduced to 6.7 during fermentation culture, adding the feed 2 according to 0.5-1.0% of the total culture volume (V: V), and feeding for 3 times.
Recovering chicken bursa of mycoplasma RPMSA1301 strain according to the volume ratio of 1:10 are inoculated in Frey's liquid culture medium, the pH value is regulated to 7.7-7.8, subculture is carried out at the constant temperature of 37 ℃, when the pH value of the culture solution is reduced to 6.9 (the culture time is 8-12 h), the feed supplement 1 is added according to 0.1-0.5% of the total culture volume (V: V), the pH value is regulated to 7.7-7.8, and the culture is continued; the bacterial liquid is harvested when the pH of the culture is reduced to 6.7 and is used as the primary seed liquid. The first seed liquid is prepared by the same method according to the following steps: 10, inoculating again for passage 1 time, and collecting bacterial liquid as secondary seed liquid.
According to the volume ratio of 1:10, inoculating the secondary seed liquid into Frey's liquid culture medium, fermenting and culturing in a mechanically stirring fermentation tank, regulating the pH value to 7.7-7.8, culturing at 37 ℃ and constant temperature, wherein the tank pressure is 0.04MPa, and the rotating speed is 100r/min; when the pH value is reduced to 6.7 (the culture time is 10-15 h), adding the feed 2 for the first time, wherein the addition amount is 0.5-1.0% of the total culture volume, adjusting the pH value to 7.7-7.8, and continuing to culture; and adding the supplementary material for 2 times repeatedly (the second fluid infusion time is 10-20 h after the first material infusion 2 is added, and the third fluid infusion time is 10-20 h after the second material infusion), culturing for 8-15 h, and harvesting the fermentation broth when the pH value is reduced to 6.4.
Fermentation broths as shown in table 8 were obtained according to the above methods, respectively.
TABLE 8 bacterial liquids obtained by different culture methods
Note that: the feed 1 and feed 2 were added in amounts of percentage of the total culture volume.
3. Preparation of chicken bursa mycoplasma live vaccine
The antigen content of the different fermentation broths obtained in the table 8 is quantitatively detected, and the detection method is a nucleic acid quantitative method or a protein quantitative method. The experiment adopts a nucleic acid quantification method, chicken bursa mycoplasma MS fermentation broth is concentrated by adopting a membrane filtration method, and after impurity removal by a 10 mu m filter membrane, the chicken bursa mycoplasma MS fermentation broth is concentrated by 50 times by using a 0.1 mu m hollow fiber column, sampling is carried out, the antigen content is detected by using the nucleic acid quantification method, and the upstream primer and the downstream primer and the probe are respectively as follows:
an upstream primer F5'-CTGCTAAAACAGAAGCTAAAACTGCTAT-3', SEQ ID NO.1;
the downstream primer R is 5'-GCAGATTCTGTTGTAGTTGCTTCAA-3', SEQ ID NO.2;
probe P5'-CTGCAGCAGAATTATCAGA-3', SEQ ID No.3.
The results show that: the content of the bacterial liquid-1, the bacterial liquid-2 and the bacterial liquid-3 antigens are respectively 3.0X10 8.0 Copy number/mL, 3.3X10 8.0 Copy number/mL, 3.1X10 8.0 Copy number/mL. Diluting the concentrated bacterial liquid according to the detection result to ensure that the MS antigen content of the mycoplasma synoviae is 1.0x10 7.0 Copy number/plume.
Different heat-resistant lyoprotectants (the volume ratio of the bacterial liquid to the lyoprotectant after dilution is 1:1) in Table 7 are added, and the mixture is lyophilized to prepare the chicken bursa mycoplasma live vaccine as shown in Table 9.
TABLE 9 live vaccine against Mycoplasma synoviae prepared from different bacterial solutions and different heat-resistant lyoprotectants
| Vaccine numbering | Bacterial liquid | Heat-resistant freeze-drying protective agent |
| Vaccine 1 | Bacterial liquid-1 | Protective agent-2 |
| Vaccine 2 | Bacterial liquid-2 | Protective agent-3 |
| Vaccine 3 | Bacterial liquid-3 | Protective agent-1 |
4. Evaluation of vaccine immunization Effect
SPF chickens of 7 days old were randomly immunized with the chicken bursa mycoplasma live vaccine of the invention in Table 9 by three modes of eye spot, drinking water and spraying, 20 animals per group, and a non-immunized group was set as an challenge control. And 6 weeks after vaccine immunization, respectively taking immunized chickens and control chickens, and carrying out mycoplasma synoviae virus attack by adopting an intravenous injection mode.
The toxin counteracting method comprises the following steps: the immunized chicken and the control chicken were placed at 3m 3 In a spacer (temperature of 22 ℃ C., relative humidity of 60%) of Mycoplasma synoviae RPMSA1303 strain, 0.2mL×10, was intravenously injected 7 CCU/plume, 14 days of observation, section examination, judging the disease condition, and calculating the relative protection rate. The toxicity attack control group should have at least 7 chicken onset, and the relative protection rate of the immune group should be not lower than 70%.
The test results are shown in Table 10: the toxicity attack control group comprises 10 chickens, the toxicity attack protection rate of the live vaccine of the mycoplasma synoviae is 90 percent of that of the eye-drop immune group, 80% of drinking water immunity group and 80% of aerosol immunity group, and the chicken bursa mycoplasma live vaccine has good protection effect on epidemic virulent strains.
Table 10 results of efficacy test of live vaccine against Mycoplasma synoviae
Note that: swelling of any point of sternum, left tarsal joint, right tarsal joint, left foot pad, right foot pad, left pterygoid lamina joint and right pterygoid lamina joint is judged as pathogenesis.
And (3) notes: a method for calculating the disease standard and relative protection rate of chicken bursa mycoplasma.
The relative protection rate calculation formula:
relative protection = (number of non-onset of immune group challenge-number of non-onset of immune group challenge)/total number of immune group challenge x 100%. The criteria for determining the onset of mycoplasma synoviae are shown in Table 11.
TABLE 11 criterion for determining the onset of mycoplasma synoviae in chickens
| Determination of | Decision criterion |
| No pathological changes | Normal or slightly enlarged appearance, a small amount of clear mucus was seen by dissection without turbidity and yellow cheese-like material; |
| lesions | Obvious swelling in appearance, visible turbid liquid or yellow cheese-like exudates; death occurred after the experimental chicken had been detoxified. |
Example 6 preparation of Mycoplasma gallisepticum, mycoplasma synoviae bivalent live vaccine and evaluation of immune Effect
1. Mycoplasma bacterial liquid culture
Bacterial solutions of mycoplasma gallisepticum MG (CR strain, purchased from Chinese veterinary drug administration) and mycoplasma gallisepticum MS (RPMSA 1301 strain) were cultivated separately. The cultivation method was the same as in example 5.
2. Preparation of mycoplasma gallisepticum and mycoplasma synoviae bivalent live vaccine
The antigen content of Mycoplasma Gallisepticum (MG) bacterial liquid and Mycoplasma Synoviae (MS) bacterial liquid is quantitatively detected, the experiment adopts a protein quantification method, bacterial liquid is concentrated by adopting a membrane filtration method, impurities are filtered by a 10 mu m membrane, the bacterial liquid is concentrated by 20 times by using a 0.1 mu m hollow fiber column, sampling is carried out, the antigen content is detected by using the protein quantification method, and the result shows that the antigen content of mycoplasma gallisepticum and mycoplasma synoviae is 1500 mu g/mL and 1200 mu g/mL respectively.
And diluting the concentrated bacterial liquid according to the detection result, so that the antigen content of mycoplasma gallisepticum and mycoplasma synoviae is 200 mug/plume. Adding a heat-resistant freeze-drying protective agent according to the volume ratio (V: V) of 1:1, and performing vacuum freeze-drying to prepare the mycoplasma gallisepticum and mycoplasma synoviae bivalent live vaccine.
3. Evaluation of vaccine Effect
Taking an SPF chicken of 7 days old, immunizing a mycoplasma gallisepticum and a bursa of chicken mycoplasma bigeminal live vaccine in an eye-spot mode, setting a non-immune group as a virus-attack control, taking the immunized chicken and the control chicken, and carrying out a strong virus-attack test by intramuscular injection virus-attack at the same time 6 weeks after vaccine immunization.
The mycoplasma gallisepticum and mycoplasma synoviae combined live vaccine has the mycoplasma gallisepticum toxicity attack protection rate of 83.3% and the mycoplasma synoviae toxicity attack protection rate of 90%. The bigeminal live vaccine has good protection effect on mycoplasma gallisepticum and mycoplasma synoviae.
The vaccine preparation method is suitable for preparing the chicken mycoplasma synoviae live vaccine, is not limited to preparing single vaccine of the chicken mycoplasma synoviae live vaccine, and is also suitable for preparing bivalent or multi-bivalent live vaccine with other pathogenic microorganisms.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The mycoplasma synoviae live vaccine strain RPMSA1301 is characterized in that the collection number of the mycoplasma synoviae live vaccine strain RPMSA1301 is CCTCC M20232235.
2. The use of the live mycoplasma synoviae vaccine strain RPMSA1301 according to claim 1 for the preparation of a mycoplasma synoviae vaccine.
3. A live mycoplasma synoviae vaccine, characterized in that the active ingredient comprises the live mycoplasma synoviae vaccine strain RPMSA1301 of claim 1.
4. The preparation method of the chicken bursa mycoplasma live vaccine is characterized by comprising the following steps of:
s1, resuscitating the mycoplasma synoviae live vaccine strain RPMSA1301 of claim 1, and then performing subculture and fermentation culture to obtain a fermentation broth;
adding a feed supplement 1 in the process of subculture, and adding a feed supplement 2 in the process of fermentation culture;
the feed supplement 1 comprises, by mass and volume percentage, 1-5% of gelatin, 1-3% of nicotinamide, 1-2% of arginine, 5-10% of glucose and 0.1-0.5% of sodium pyruvate;
the components of the feed supplement 2 comprise, by mass and volume percentage, 2-4% of sodium hydroxide, 1-3% of PEG 600010-20%, 1-3% of nicotinamide and 10-20% of DMEM;
s2, quantitatively detecting the antigen content of the fermentation broth obtained in the step S1, wherein the detection method is a nucleic acid quantitative method or a protein quantitative method, and diluting the fermentation broth according to a detection result;
s3, adding a heat-resistant freeze-drying protective agent into the bacterial liquid diluted in the step S2, and freeze-drying to prepare the chicken bursa mycoplasma live vaccine;
the heat-resistant freeze-drying protective agent comprises 2-4% of carbomer by mass and volume percentage, 1-4% of polyvinylpyrrolidone by mass and volume percentage, 2-8% of trehalose by mass and volume percentage, 4-12% of casein hydrolysate by mass and volume percentage, 0.1-0.2% of L-arginine by mass and volume percentage and 4-8% of ethylene glycol by volume concentration.
5. The method according to claim 4, wherein in step S1, the feed 1 is added in an amount of 0.1 to 0.5% of the total culture volume when the pH is lowered to 6.9 during the subculture.
6. The method according to claim 4, wherein in step S1, the feed 2 is added when the pH is reduced to 6.7 during the fermentation culture, and the feed 2 is added for the second time after the continuous culture for 10 to 20 hours, and the feed 2 is added for the third time after the continuous culture for 10 to 20 hours; the addition amount of the feed supplement 2 in the fermentation culture process is 0.1-0.5% of the total culture volume.
7. The method according to claim 4, wherein in step S1, the subculture conditions are constant temperature culture at 37 ℃; the fermentation culture condition is 0.04MPa, the rotating speed is 100r/min, and the constant temperature culture is carried out at 37 ℃.
8. The method according to claim 4, wherein in step S2, a fluorescent quantitative PCR method is used for detecting the antigen content of Mycoplasma synoviae bacteria liquid by a nucleic acid quantitative method; concentrating antigen by 10-50 times by high-speed centrifugation or membrane filtration, quantitatively detecting, wherein the upstream primer, the downstream primer and the probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; diluting the concentrated bacterial liquid according to the detection result to ensure that the antigen content of the mycoplasma synoviae is (0.5-1.5) multiplied by 10 7.0 Copy number/plume.
9. The method according to claim 4, wherein in step S2, the antigen content of the mycoplasma synoviae bacterial liquid is detected by a protein quantification method, and the antigen is concentrated by 10-50 times by a high-speed centrifugation or membrane filtration method and then quantitatively detected; and diluting the concentrated bacterial liquid according to the detection result to ensure that the antigen content of the mycoplasma synoviae is 200-500 mug/serving.
10. The method according to claim 4, wherein in step S3, the volume ratio of the bacterial liquid to the heat-resistant freeze-drying protective agent is 1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311609402.7A CN117511816A (en) | 2023-11-29 | 2023-11-29 | Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311609402.7A CN117511816A (en) | 2023-11-29 | 2023-11-29 | Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117511816A true CN117511816A (en) | 2024-02-06 |
Family
ID=89756540
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311609402.7A Pending CN117511816A (en) | 2023-11-29 | 2023-11-29 | Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117511816A (en) |
-
2023
- 2023-11-29 CN CN202311609402.7A patent/CN117511816A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105816868B (en) | Inactivated vaccine for mycoplasma synoviae | |
| CN112779193B (en) | Virulent strain of mycoplasma synoviae and application thereof | |
| CN1940063A (en) | Pseudo-rabies gE/gI-gene loss poison strain, killed vaccine containing it and use | |
| CN111000993B (en) | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof | |
| CN107513506B (en) | Mycoplasma hyopneumoniae, vaccine composition and application thereof | |
| CN104946600B (en) | A kind of H9 subtype avian influenza virus strain | |
| CN103497934B (en) | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof | |
| CN110680914B (en) | Triple inactivated vaccine and preparation method thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN117568226A (en) | Strong mycoplasma synoviae strain and application thereof | |
| CN109097340B (en) | Avian adenovirus, quadruple vaccine and preparation method thereof | |
| CN107384837B (en) | Chicken mycoplasma synoviae and application thereof | |
| CN111690554B (en) | Combined strain for preparing mycoplasma ovis pneumonia vaccine, mycoplasma ovis pneumonia trivalent inactivated vaccine and preparation method thereof | |
| CN109207436A (en) | One plant of 4 type aviadenovirus strain of I group and its application | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| CN109867713B (en) | Canine distemper genetic engineering subunit vaccine | |
| CN113957007B (en) | Inactivated vaccine for mycoplasma synoviae | |
| CN107446859B (en) | Mycoplasma gallisepticum and application thereof | |
| CN114395536B (en) | Avian adenovirus type 4, 8 and 11 trivalent vaccine and preparation method and application thereof | |
| CN117511816A (en) | Live vaccine strain of mycoplasma synoviae, live vaccine and preparation method thereof | |
| CN113278595B (en) | Duck adenovirus type 3 strain, duck adenovirus egg yolk antibody, and preparation methods and application thereof | |
| CN116042537A (en) | Avian reovirus strain QYH2020-QD, virus liquid, preparation method and application | |
| CN110713987B (en) | Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof | |
| CN112063596A (en) | Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof | |
| CN108624522B (en) | Acinetobacter paragallinarum strain and application 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 |