CN104258389B - A kind of vaccine combination and its preparation method and application - Google Patents
A kind of vaccine combination and its preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of vaccine combination, wherein, the vaccine combination includes the Newcastle Disease Virus Antigen, avian infectious bronchitis virus antigen and chicken Mycoplasma synoviae antigen and carrier of immune amount.Present invention also offers vaccine combination ground preparation method and application.Triple vaccine immune effect of the present invention is better than new branch dyad inactivated vaccine and inactivates immune effect associated with single seedling vaccine with chicken Mycoplasma synoviae.Invention further provides a kind of vaccine combination, antigen also includes Mycoplasma Gallisepticum Antigen Recognized By Antibody during the vaccine combination removes above-mentioned three, and the vaccine combination is highly resistant to the attack of four kinds of cause of diseases.
Description
Technical field
The present invention relates to a kind of vaccine combination, with and its preparation method and application, belong to bioengineering field.
Background technology
Newcastle disease (New Castle disease) is the high degree in contact infectiousness as caused by paramyxovirus, lethal
Property disease, also known as philippine fowl disease or pseudo- checken pest, be in often acute sepsis shape.Be mainly characterized by expiratory dyspnea, just it is dilute, nerve it is disorderly
Disorderly, mucous membrane and serous coat bleeding, the death rate are high, endanger serious to poultry husbandry.Effective medicine is there is no, can only rely on and strictly disappear
Poison, isolation and with inactivated vaccine and live seedling vaccine inoculation prevention.
Infectious bronchitis is a kind of acute high degree in contact of the chicken as caused by infectious bronchitis virus
Respiratory infectious disease.The chicken of various ages in days is all susceptible, its face examine be characterized in expiratory dyspnea, send rale, cough, mouth breathing,
Sneeze, this sick incidence of disease is high, and the death rate of chick is up to more than 25%.After laying hen infection, egg production reduction, the product of egg
Matter declines.It is slow-growing after broiler chicken infection, and easily secondary respiratory tract bacterial disease.This disease is widely current in all over the world, is
The important epidemic disease of poultry husbandry.
Chicken Mycoplasma synoviae disease is to cause young age chicken by chicken Mycoplasma synoviae (Mycoplasma Synoviae, MS)
With a kind of infectious disease of turkey, also known as avian infectious synovitis (avian onfectious synovitis), it is characterized in that pass
Save enlargement, bursa synovialis and tendon inflammation and the enlargement of organa parenchymatosum.Due to this sick slower development, the course of disease is long, once in chicken group
Infection, eradicates highly difficult, therefore is spread for a long time in chicken group, causes low efficiency of feed utilization, growth retardation, mortality to increase
High, egg production decline etc..Chicken group easily occurs mixed infection, aggravated one's illness, the death rate increases as existed during the cause of disease, causes tight
The economic loss of weight.
Chicken Mycoplasma synoviae disease increases year by year in recent years, diseased chicken depauperation, and resistance declines, so as to trigger chicken new
City epidemic disease and infective bronchitis disease.
The content of the invention
To solve the deficiencies in the prior art, the invention provides a kind of new newcastle disease, infectious bronchitis of chicken, chicken
Mycoplasma synoviae vaccine combination, three kinds of epidemic diseases to current trend reach preferable protecting effect.
It is a primary object of the present invention to provide a kind of vaccine combination, wherein, the vaccine combination includes immune amount
Newcastle Disease Virus Antigen, avian infectious bronchitis virus antigen and chicken Mycoplasma synoviae antigen and carrier.
Preferably, described Newcastle Disease Virus Antigen is the totivirus antigen, the totivirus antigen of attenuation, inactivation of work
Totivirus antigen or subunit antigen；Described avian infectious bronchitis virus antigen is totivirus antigen living, attenuation
Totivirus antigen, the totivirus antigen of inactivation or subunit antigen；Described chicken Mycoplasma synoviae antigen is former for full branch living
Body antigen, the full mycoplasma antigen of attenuation, the full mycoplasma antigen of inactivation or subunit antigen.
Preferably, described Newcastle Disease Virus Antigen is Sota plants of totivirus antigens of La of inactivation, described chicken infection
Property bronchitis virus antigen be the M41 strain totivirus antigens of inactivation, described chicken Mycoplasma synoviae antigen is the HN1 that inactivates
The full mycoplasma antigen of strain.
Sota plants of newcastle disease virus La, M41 plants of avian infectious bronchitis virus are purchased from Chinese veterinary medicament supervision
Institute.
HN1 plants of chicken Mycoplasma synoviae (Mycoplasma synoviae strain HN1) preserving number：CCTCC NO：
V201425, is preserved in China typical culture collection center (CCTCC)；Preservation address：Wuhan, China Wuhan University, preservation
Date：On 08 19th, 2014.
The composition of composition or the amount of component of the present invention is preferably therapeutically effective amount.The therapeutically effective amount refers to
Their immunological role is played without causing excessive side effect institute necessary amounts in the host that composition is applied.Composition used and
The accurate amount of composition to be administered is by according to factor such as the type of disease treated, the type of animal to be treated and year
Age, the mode of administration, and other compositions in composition and change.
Preferably, described Newcastle Disease Virus Antigen content is >=108.0EID50/ 0.1ml, described avian infectious branch
Bronchitis virus antigenic content is >=106.0EID50/ 0.1ml, described chicken Mycoplasma synoviae antigenic content is >=109CCU/
mL。
Preferably, described Newcastle Disease Virus Antigen content is 108.0EID50/ 0.1ml~109.0EID50/ 0.1ml, institute
The avian infectious bronchitis virus antigenic content stated is 106.0EID50/ 0.1ml~107.0EID50/ 0.1ml, described chicken is slided
Liquid capsule mycoplasma antigen content is 109CCU/mL~1010CCU/mL。
Preferably, the carrier includes adjuvant, and the adjuvant includes oily adjuvant, water-soluble adjuvant, Alum adjuvant, cell because
Sub- adjuvant；It is further preferred that the adjuvant includes white-oil adjuvant..
Preferably, the vaccine combination further comprises adjuvant.
Term " adjuvant ", which refers to, to be added in the composition of the present invention with the material for the immunogenicity for increasing composition.It is known
Adjuvant includes, but are not limited to：Oily adjuvant, water-soluble adjuvant, Alum adjuvant, Cytokine adjuvant.
Term used herein " oily adjuvant " is also known as " oil adjuvant " or " oil emulsion adjuvant ", is by including vegetable oil, animal
One or more of compositions in oil, mineral oil, for delaying RT of the immunogene in body, are allowed to continue slowly to release
Put, strengthen phagocytosis and the sterilizing ability of macrophage.
Term used herein " water-soluble adjuvant " is also known as " water-based adjuvant " or " water adjuvant ", is a kind of polymeric water-soluble
Dispersion, effect and security for improving water-soluble vaccines can be by high molecular weight polypropylene acids synthetic polymer groups
Into.
Term used herein " Alum adjuvant ", also known as " aluminium glue adjuvant " or " aluminium adjuvant ", including aluminum hydroxide adjuvant and
Aluminium phosphate adjuvant, its major function is sustained release, but has the activation to immunocyte simultaneously.By antigen and aluminium hydroxide or
Aluminum phosphate hybrid injection, can make antigen be stored in injection site, with antigen sustained release and nonspecific immunity stimulation.
Term used herein " Cytokine adjuvant ", including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-10, IL-12, IL-15, IL-18, INF- γ, GM-CSF, TNF-α, TNF-β, TCA-3 etc., also known as " cell factor " or " thin
Born of the same parents' element ", is that the secretion of live body host cell by diffusion, cell contact or blood circulation reaches other cells of host, in body
A class non-immunoglobulin, local native protein or the glycoprotein played a role in liquid with extremely low concentration is also a class by body
The immunocyte of activation and some nonimmune cells are produced, secretion, can adjust cell growth, differentiation, with hematopoiesis, inflammatory reaction,
The general designation of the closely related high activity multi-functional small molecules albumen such as immune response and wound healing, can stimulate or suppress
Immunologic function, promotes cell development differentiation, regulation cell physiological function and cell-tocell transmission, immune in immune response
Very important regulating and controlling effect is played in system.
Amount suitable for the adjuvant of the composition of the present invention is preferably effective dose." effective dose " refers to adjuvant same
Played in host during antigen combined administration of the invention for their immunological role must or it is enough excessive without causing
Side effect institute necessary amounts.The accurate amount of adjuvant to be administered is by the class according to factor such as composition used and the disease for the treatment of
Type, the type of animal to be treated and age, the mode of administration, and other compositions in composition and change.
Preferably, described vaccine combination further comprises Mycoplasma Gallisepticum Antigen Recognized By Antibody, and the Mycoplasma Gallisepticum Antigen Recognized By Antibody contains
Measure as >=109CCU/mL。
It is highly preferred that described vaccine combination further comprises the full mycoplasma antigens of chicken virus mycoplasma CR of inactivation, institute
It is 10 to state Mycoplasma Gallisepticum Antigen Recognized By Antibody content9CCU/mL~1010CCU/mL。
It is further preferred that the ewcastle disease totivirus antigenic content is 109.0EID50/ 0.1ml, the avian infectious branch
Tracheitis totivirus antigenic content is 107.0EID50/ 0.1ml, the full mycoplasma antigen content of chicken Mycoplasma synoviae is
109.0CCU/mL, the full mycoplasma antigen content of chicken virus mycoplasma is 109.0CCU/mL。
Another object of the present invention is to provide a kind of preparation method of vaccine combination, wherein, methods described includes：
(1) newcastle disease virus, avian infectious bronchitis virus, chicken Mycoplasma synoviae are bred in culture respectively；(2) inactivation is walked respectively
Suddenly (1) described culture is bred newcastle disease virus, avian infectious bronchitis virus and chicken Mycoplasma synoviae；And (3)
By the newcastle disease virus totivirus antigen, avian infectious bronchitis virus totivirus antigen and chicken bursa synovialis branch of the inactivation
The full mycoplasma antigen mixing of substance, adds adjuvant.
Present patent application is by cultivation in the chick embryo culture newcastle disease virus and avian infectious bronchitis virus, with fermentation
Tank is prepared after chicken Mycoplasma synoviae (Pu Laike oneself separation), formalin-inactivated, and oily adjuvant triple inactivated vaccine is made, can be simultaneously
Prevent the generation of these three diseases.
It is still another object of the present invention to provide a kind of method for preparing the vaccine combination, wherein, methods described bag
Include：(1) culture propagation newcastle disease virus, avian infectious bronchitis virus, chicken Mycoplasma synoviae and the malicious branch of chicken are former respectively
Body；(2) newcastle disease virus, avian infectious bronchitis virus, the chicken bursa synovialis that inactivation step (1) culture is bred respectively
Mycoplasma and chicken virus mycoplasma；And (3) are by newcastle disease virus totivirus antigen, the infectious bronchitis of chicken of the inactivation
Viral totivirus antigen, the full mycoplasma antigen of chicken Mycoplasma synoviae and the full mycoplasma antigen mixing of chicken virus mycoplasma, add assistant
Agent.
Preferably, the adjuvant is white-oil adjuvant.
A further object of the present invention is that providing the vaccine combination is preparing prevention and/or treatment Newcastle disease
Application in the medicine of the relevant disease of poison, avian infectious bronchitis virus and the infection of chicken Mycoplasma synoviae.
The a further object of the present invention is that providing the vaccine combination is preparing prevention and/or treatment Newcastle disease
In poison, avian infectious bronchitis virus, the medicine of the relevant disease of chicken Mycoplasma synoviae and mycoplasma gallisepticum infection
Using.
The non-exhaustive list of the adaptable newcastle disease relevant disease of the present invention includes, and such as respiratory tract, alimentary canal glue
Film bleeding, egg production decline etc..
The non-exhaustive list of the adaptable infectious bronchitis of chicken relevant disease of the present invention includes, and such as breathes tired
Hardly possible, rale, cough, mouth breathing, sneeze, egg production decline etc..
The non-exhaustive list of the adaptable chicken Mycoplasma synoviae relevant disease of the present invention includes, such as arthrocele,
Bursa synovialis and tendon inflammation, the enlargement of organa parenchymatosum, growth retardation, egg production decline etc..
Term " prevention " refers to by itself and newcastle disease virus, infectious bronchitis virus and Mycoplasma synoviae phase
The infection of pass or the symptom of disease are blocked or postponed；Term " treatment " refer to by with newcastle disease virus, infectious bronchitis
The process that the symptom of scorching virus and the related infection of Mycoplasma synoviae or disease is alleviated or is completely eliminated.
Embodiment
The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature will be with description more
To be clear.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.Those skilled in the art
It should be understood that can be carried out without departing from the spirit and scope of the invention to the details and form of technical solution of the present invention
Modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.
The embodiment of the present invention is using LaSota plants of newcastle disease seed culture of viruses, infectious bronchitis of chicken strain as M41 plants, chicken synovia
Illustrate the present invention exemplified by HN1 plants of capsule mycoplasma, CR plants of chicken virus mycoplasma.
The preparation of the newcastle disease of embodiment 1, infectious bronchitis virus liquid and Mycoplasma synoviae bacterium solution
1. the selection of strain
LaSota plant of ewcastle disease seed culture of viruses of this product is manufactured, infective bronchitis strain is M41 plants, is purchased from Chinese veterinary drug
Supervision institute.Mycoplasma synoviae is that laboratory voluntarily separates identification, is HN1 plants.
2. it is prepared by virus liquid
2.1 Sota plants of newcastle disease virus La seedlings are prepared with virus liquid
2.1.1 inoculation
Production seed culture of viruses is taken, 10 are diluted to sterile saline-3, the susceptible chicken embryo of 10 ages in days is inoculated with through allantoic cavity, per embryo
0.1ml, is inoculated with rear enclosed pin hole, puts 36~37 DEG C and continues to be incubated, it is not necessary to egg-turning.
2.1.2 it is incubated and observation
After egg inoculation, often sunshine egg 1 time, chicken embryo dead before 48 hours is discarded, hereafter, every 8~12 hours photograph eggs 1
It is secondary, dead chicken embryo is taken out at any time, until 120 hours, it is no matter whether dead, all take out, air chamber is upright upwards, is placed in 2~8 DEG C
Cooling 12~24 hours.
2.1.3 harvest
By after cooling chicken embryo take out, first with the tincture of iodine, after with alcohol disinfecting after, by embryo harvest chicken embryo liquid.Per several chickens
Embryo is divided into one group, draws blastochyle and is put in same sterilization container, and every group of sampling determines HA-HI test, and HA-HI test is (micro less than 1: 128
Method) person should discard, and put less than -15 DEG C preservations, it should be no more than 30.
2.1.4 examine
2.1.4.1 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, the virus liquid asepsis growth of sampling.
2.2 M41 plants of avian infectious bronchitis virus seedlings are prepared with virus liquid
2.2.1 inoculation
Production seed culture of viruses is taken, 10 are diluted to sterile saline-3, the susceptible chicken embryo of 10 ages in days is inoculated with through allantoic cavity, per embryo
0.1ml.Rear enclosed pin hole is inoculated with, 36~37 DEG C is put and continues to be incubated, it is not necessary to egg-turning.
2.2.2 it is incubated and observation
Egg is shone after egg inoculation, before 24 hours 1 time, discard dead germ.Hereafter, every 8~12 hours photograph eggs 1 time, take out at any time
Dead chicken embryo, until 48 hours, it is no matter whether dead, all take out, air chamber is upright upwards, be placed in 2~8 DEG C of coolings 12~24 small
When.
2.2.3 harvest
By after cooling chicken embryo take out, first with the tincture of iodine, after with alcohol disinfecting after, by embryo harvest chicken embryo liquid.Per several chickens
Embryo is divided into one group, draws blastochyle and is put in same sterilization container, puts less than -15 DEG C preservations, should be no more than 30.
2.2.4 examine
2.2.4.1 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, the virus liquid asepsis growth of sampling.
3. the preparation of mycoplasma bacterium solution
3.1 culture medium prescription
Basic media components：PPLO meat soups 24.5g, lactoalbumin hydrolysate 7g, yeast extract powder 6g, niacinamide 0.3g, matter
Measure the phenol red solution 2ml deionized waters 800ml that concentration is 0.4%.
Finished product culture medium preparation method：By above-mentioned 121 DEG C of culture medium autoclaving 15 minutes, filtration sterilization is added after cooling
125ml Swine serums and 800,000 units of Penicillin, 0.01MNaOH adjust pH to 7.6~7.8, aseptic subpackaged, 2~8 DEG C of preservations are standby
With.
The propagation of 3.2 bacterium solutions
Chicken Mycoplasma synoviae is pressed 1：10 ratio is inoculated in chicken Mycoplasma synoviae finished product culture medium, 36~37
DEG C culture 24~48h harvest be used as primary seed solution；Primary seed solution is in kind pressed 1：10 ratio inoculates passage 1
Secondary harvest is used as secondary seed solution；Purely after the assay was approved, then in the same way culture is expanded.Determine chicken Mycoplasma synoviae
Bacterial concentration reaches 1011CCU/mL。
3.3 inactivation
The virus liquid of harvest is inactivated 20 hours for 37 DEG C through 0.1% formaldehyde, by chicken Mycoplasma synoviae bacterium solution, by bacteria liquid
Product total amount is slowly added to final concentration of 0.2% formalin (V/V), puts 37 DEG C of inactivations, therebetween every stirring in 4 hours once,
Taken out after 24h.
3.3.1 inactivation is examined
Take bacterium solution 10ml after inactivation to be inoculated in 37 DEG C of 4~7d of culture in 100ml fluid nutrient mediums, then transplanted by same ratio
37 DEG C of 4~7d of culture in above-mentioned culture medium 100ml；In addition, taking bacterium solution 0.5ml after inactivation to be inoculated in solid medium, 37 DEG C
Cultivate 7~10d.The equal asepsis growth of result of above two method culture.
3.3.2 steriling test
The bacterium solution inactivated is taken, is pressed《Republic of China Veterinary Pharmacopoeia》Annex page is carried out, as a result asepsis growth.
The concentration of 4 virus liquids
The sterilizing of 4.1 enrichment facilities
Pre-filtering post, container, pipeline etc. are sterilized 40 minutes for 121 DEG C using high steam, emptying film after the cleaning of milipore filter bag
Bag in moisture, with 2% formalin circulate 5~10 minutes, close circulating pump, make formalin kept in film bag 24 hours with
On, formalin is bled off before use, it is 7.0~7.2 to be rinsed repeatedly with sterile water for injection to pH value.
4.2 concentration
Prepared newcastle disease, avian infectious bronchitis virus in difference Example 1, then by virus liquid in nothing
Filtered under the conditions of bacterium with two grades of pre-filtering posts of sterilizing, remove the residue and other impurities of virus liquid.Above-mentioned ultrafiltration concentration is used again
Device, virus liquid is continuously circulated, moisture constantly oozes out, and completes viral concentration process.After concentration terminates, with distilled water by ultrafiltration
The residue of film bag is developed, and is then rinsed 30 minutes with 0.4mol/L NaOH solutions iterative cycles, is stopped circulation, is made
NaOH solution is kept for more than 10 hours in film bag, then is cleaned NaOH with distilled water, uses formalin soaking disinfection.
4.3 concentration restrovirus assays
By the newcastle disease virus after concentration, avian infectious bronchitis virus, EID is determined respectively50, it is new per 0.1ml chickens
City epidemic disease poison, avian infectious bronchitis virus content are respectively 1010、108。
The preparation of the vaccine of embodiment 2
It is prepared by 1 oil phase
94 parts of injection white oil is taken, after Jia Siben -806 parts of mixing, plus 2 parts of aluminum stearate, it is to transparent with stirring with adding
Only, it is standby after autoclaving, as oil phase.
It is prepared by 2 aqueous phases
Antigen is mixed in sterile chamber in proportion, the Tween-80 after adding 4% sterilizing and cooling down is stirring while adding,
Make untill Tween-80 is completely dissolved, as aqueous phase.
3 emulsifications
Take 3 parts of oil phase to put in oil phase tank, start motor stirring, 1 part of aqueous phase is then added slowly, emulsion tank is transferred to after adding
In, then with 2800r/min emulsifications 40 minutes.Emulsification terminate before add 1% thimerosal, make its final concentration of 0.01%.
The specific formula of vaccine such as following table.
The vaccine composition of table 1 and content
4 product inspections
4.1 character
After testing, character is as follows for vaccine 1, vaccine 2, vaccine 3 and vaccine 4：
Outward appearance：The uniform emulsion of milky.
Formulation：Water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and drips in cold water surface, in addition to the 1st drips, in oil droplet
Shape indiffusion.
Stability：Draw vaccine 10ml to add in centrifuge tube, centrifuged 15 minutes with 3000r/min, the aqueous phase that ttom of pipe is separated out
No more than 0.5ml.
Viscosity：By existing《Chinese veterinary pharmacopoeia》Annex is measured, and meets regulation.
4.2 loading quantity inspections are by existing《Chinese veterinary pharmacopoeia》Annex is checked, meets regulation.
4.3 steriling tests are by existing《Chinese veterinary pharmacopoeia》Annex is tested, asepsis growth.
4.4 safety verifications are subcutaneously injected vaccine 0.6ml, 14 are observed day by day with 14 age in days SPF chickens 10, each muscle or neck
Day.Occur without any locally and systemically adverse reaction as caused by vaccine.
4.5 efficacy test
4.5.1 vaccine 1, the efficacy test of vaccine 2
Newcastle disease part is divided to two groups with 30 age in days SPF chickens 25.Vaccine is subcutaneously injected in every group 10 each muscle respectively
1st, the μ l of vaccine 220, another 5 are only used as control.21 days after immune, every chicken is taken a blood sample, and separates serum, carries out HI antibody titer surveys
It is fixed.Immune chicken, control chicken HI antibody titers such as table 2.It can be seen that, the geometrical mean of immune chicken HI antibody titers is not less than
4log2, 2log all should be not higher than by compareing chicken HI antibody titers2, meet effect inspection standard.
The newcastle disease HI potency of table 2
| Group | Individual values (log2) | GMT(log2) |
| The immune group of vaccine 1 | 6、7、7、6、7、6、8、7、6、6 | 6.6 |
| The immune group of vaccine 2 | 7、7、8、8、6、6、7、7、6、7 | 6.9 |
| Control group | 0、0、0、0、0 | 0 |
Infectious bronchitis of chicken part is divided into two groups, every group 10, each collunarium is inoculated with chicken with 21 age in days SPF chickens 20
Infective bronchitis live vaccine (H120 plants) 1 plumage part, 21 days after inoculation, takes a blood sample respectively, separates serum, strengthens for two groups exempting from respectively
Epidemic disease is inoculated with (intramuscular injection) inactivated vaccine 1 and vaccine 20.3ml.28 days after immune, take a blood sample respectively, separate serum, will serum twice
HI experiments are done respectively, the results are shown in Table 3,1 group two of vaccine exempts from potency：One exempts from potency for 5.3:It is to exempt from that 1, i.e., two, which exempt from HI potency,
5.3 times, 2 group two of vaccine exempts from potency：One exempts from potency for 6.5:It is 6.5 times exempted from that 1, i.e., two, which exempt from HI potency, meets immune group
Chicken two exempts from the more first effect inspection mark for exempting from high 3 times of Serum HI antibody potency geometrical mean of Serum HI antibody potency geometrical mean
It is accurate.
The infectious bronchitis of chicken HI potency of table 3
Chicken Mycoplasma synoviae part is divided into two groups, every group 10, neck is subcutaneously noted respectively with 21 age in days SPF chickens 25
Vaccine 1, vaccine 20.3ml are penetrated, another 5 are not inoculated with, and are used as control.30 days after inoculation, each palmula connects simultaneously for immune chicken and control chicken
Breeder Mycoplasma synoviae culture 0.1ml, is cultivated 14, cut open inspection gambrel and palmula, 2 kinds of cotton swabs of aseptic collection, mixing
Afterwards, according to by existing《Chinese veterinary pharmacopoeia》The detection method of annex mycoplasma carries out mycoplasma separation, as a result two groups of immune group chickens
Mycoplasma synoviae separation rate is 1/10, and control group is 5/5.Meeting effect inspection standard, i.e. chicken Mycoplasma synoviae immune group should
Not higher than 2/10 separation rate, control group is not less than 4/5 separation rate.
4.5.2 the efficacy test of vaccine 3
Newcastle disease part 30 age in days SPF chickens 15,10 each muscle or the hypodermic injection μ l of vaccine 20, another 5 are only used as
Control.21 days after immune, every chicken is taken a blood sample, and separates serum, carries out HI antibody titer measure.Immune chicken, control chicken HI antibody
Potency such as table 4.It can be seen that, the geometrical mean of immune chicken HI antibody titers is not less than 4log2, control chicken HI antibody titers all should
Not higher than 2log2, meet effect inspection standard.
The newcastle disease HI potency of table 4
| Group | Individual values (log2) | GMT(log2) |
| Immune group | 6、6、6、5、7、6、7、7、6、8 | 6.4 |
| Control group | 0、0、0、0、0 | 0 |
Infectious bronchitis of chicken part is inoculated with infectious bronchitis of chicken work epidemic disease with 21 age in days SPF chickens 10, each collunarium
Seedling (H120 plants) 1 plumage part, 21 days after inoculation, takes a blood sample respectively, separates serum, immune group booster immunization inoculation (intramuscular injection) inactivation
Vaccine 0.3ml.28 days after immune, take a blood sample respectively, separate serum, HI experiments will be done respectively by serum twice, 5 are the results are shown in Table, two exempt from
Potency：One exempts from potency for 5.7:It is 5.7 times exempted from that 1, i.e., two, which exempt from HI potency, meets immune group chicken two and exempts from Serum HI antibody potency
The more first effect inspection standard for exempting from high 3 times of Serum HI antibody potency geometrical mean of geometrical mean.
The infectious bronchitis of chicken HI potency of table 5
| Group | Individual values (log2) | GMT(log2) |
| One exempts from | 4、5、4、5、4、6、4、5、5、4 | 4.6 |
| Two exempt from | 8、6、7、6、8、8、7、6、7、8 | 7.1 |
4.5.3 the efficacy test of vaccine 4
With 21 age in days SPF chickens 15, wherein vaccine 0.3ml is subcutaneously injected in 10 necks, another 5 are not inoculated with, and are used as control.
30 days after inoculation, chicken and control chicken each footpad inoculation chicken Mycoplasma synoviae culture 0.1ml simultaneously is immunized, cultivates 14, cuts open
Examine gambrel and palmula, 2 kinds of cotton swabs of aseptic collection, after mixing, according to by existing《Chinese veterinary pharmacopoeia》The inspection of annex mycoplasma
Survey method carries out mycoplasma separation, and as a result immune group chicken Mycoplasma synoviae separation rate is 2/10, and control group is 5/5.Meet effect
Inspection standard, i.e. chicken Mycoplasma synoviae immune group should be not higher than 2/10 separation rate, and control group is not less than 4/5 separation rate.
The newcastle disease of embodiment 3, infective bronchitis, Mycoplasma synoviae triple inactivated vaccine and newcastle disease, biography
The comparison of side reaction is immunized in metachromia bronchitis bigeminy killed vaccine+Mycoplasma synoviae inactivated vaccine
The packet of 1SPF chickens is with being immunized
Take 14 age in days SPF chickens 20, wherein the vaccine 1 that 10 subcutaneous inoculation embodiments 2 are prepared, 0.3ml/ only, in addition 10
Vaccine 3 (0.3ml/ is only) and vaccine 4 that only subcutaneous inoculation embodiment 2 is prepared simultaneously (0.3ml/ is only).Same age in days 10 is set in addition
SPF chickens are not immunized and compareed.
2 side reactions are compared
After immune 14 days, all chickens are weighed, as a result body weight result such as table 6 shows, triple vaccine immune group and control group phase
Than, after injection 14 days when, body weight shows weightening nothing of the triple vaccine to SPF chickens without significant difference (ANOVA, P ＞ 0.05)
Significantly affect.And newly branch dyad inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group is compared with control group, body weight is substantially inclined
It is low, and significant difference (ANOVA, P ＜ 0.05).It can be seen that, because triple vaccine is than new branch dyad inactivated vaccine+Mycoplasma synoviae inactivation
Vaccine injection dosage is few, stress be small to be immunized chicken, and immune chicken body weight is barely affected, thus it is speculated that go out the feed intake to chicken is immunized
Influence is smaller, so smaller on the production performance influence that chicken is immunized.Production performance influence after triple vaccine is immune on chicken is immunized will
Less than new branch dyad inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group.
Body weight after the age in days of SPF chicken immunes 14 is immunized in table 6
Then cut open and kill immune group chicken, observe vaccine injection site vaccine absorbing state, vaccine absorbing state criterion is 4
Divide system, specific criterion such as table 7.Immune group chicken is after immune 14 days, vaccine injection site vaccine absorbing state such as table 8, three
Join seedling immune group vaccine absorbing state better and poor than new branch dyad inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group effect
Different notable (ANOVA, P ＜ 0.05).It can be seen that, because triple vaccine is than new branch dyad inactivated vaccine+Mycoplasma synoviae inactivated vaccine note
Penetrate dosage few, vaccine absorbs very fast, after being immunized 14, injection site vaccine less residue, the influence to immune chicken is low.
The vaccine of table 7 absorbs criterion
| Vaccine absorbing state | Result of determination (is divided) |
| Fully absorb, injection site noresidue | 0 |
| Basic absorption is complete, have a small amount of residual, without obvious bulk thing | 1 |
| Absorption is incomplete, have a small amount of block vaccine residual | 2 |
| Vaccine has a large amount of block vaccine residuals substantially without absorption | 3 |
Table 8 is immunized chicken vaccine assimilation effect and compared
The newcastle disease of embodiment 4, infective bronchitis, Mycoplasma synoviae triple inactivated vaccine and newcastle disease, biography
The comparison of the immune egg production influence on laying hen of metachromia bronchitis bigeminy killed vaccine+Mycoplasma synoviae inactivated vaccine
1 packet is with being immunized
Vaccine 1 prepared by Example 2, and vaccine 3 and vaccine 4, with 150 ages in days female SPF chickens 30, are randomly divided into
3 groups：10/group.Wherein 10 subcutaneous inoculation vaccines 1,0.3ml/ only, in addition 10 simultaneously (the 0.3ml/ of subcutaneous inoculation vaccine 3
) and vaccine 4 (0.3ml/ is only).Set in addition same 10 SPF chickens of age in days be immunized compare.
2 attack poison
After immune 30 days, chicken and control chicken each footpad inoculation chicken Mycoplasma synoviae culture 0.1ml simultaneously is immunized
(108CCU/ml), carry out chicken Mycoplasma synoviae and attack poison.
2 egg productions compare
The chicken attacked after poison is further cultured for 14, calculates every chicken in next all egg productions such as table 9.As a result show, three go out
Live vaccine immune group chicken egg productivity is compared with control group, no significant difference (ANOVA, P ＞ 0.05)；But, triple inactivated vaccine
Immune group chicken egg productivity is apparently higher than new branch bivalent inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group, and significant difference
(ANOVA, P ＜ 0.05).It can be seen that, it is former that triple vaccine is better than new branch dyad inactivated vaccine+bursa synovialis branch to the immune effect of laying hen
Body inactivated vaccine immune group.
Table 9 is immunized 1 week interior egg production of chicken and compared
The newcastle disease of embodiment 5, infective bronchitis, Mycoplasma synoviae triple inactivated vaccine and newcastle disease, biography
Metachromia bronchitis bigeminy killed vaccine+Mycoplasma synoviae inactivated vaccine, which is immunized after chicken, attacks poison protection effect to Mycoplasma synoviae
The comparison of fruit
1 packet is with being immunized
Vaccine 1 prepared by Example 2, and vaccine 3 and vaccine 4, with 30 age in days SPF chickens 25, wherein 10 muscle
Immune vaccine 1,0.3ml/ only, 10 intramuscular immunisation vaccine 3 (0.3ml/ only) and vaccines 4 simultaneously in addition (0.3ml/ is only).Another 5
It is not inoculated with, is used as control.
2 attack poison
After immune 30 days, chicken and control chicken each footpad inoculation chicken Mycoplasma synoviae culture 0.1ml simultaneously is immunized
(108CCU/ml), after cultivating 14, observation gambrel and palmula swelling situation, and cut open inspection, if gambrel or palmula have yellow
Glue peptone sample mucus, you can judge this chicken fall ill, do not protected by vaccine.Difference aseptic collection cut open inspection gambrel and palmula
Cotton swab, after mixing, according to by existing《Chinese veterinary pharmacopoeia》The detection method of annex mycoplasma carries out mycoplasma separation, as a result such as
Table 10.Triple vaccine immune group can completely (10/10) with new branch bivalent inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group
The immune chicken of protection, but the virus isolated rate (5/10) of new branch bivalent inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group
But apparently higher than triple vaccine immune group (1/10).It can be seen that, triple vaccine immune group and new branch bivalent inactivated vaccine+Mycoplasma synoviae
Although quite, the virus purification of triple vaccine immune group is but significantly lower than inactivated vaccine immune group clinicing symptom observation protective rate
New branch bivalent inactivated vaccine+Mycoplasma synoviae inactivated vaccine immune group, therefore the immune effect of triple vaccine goes out better than new Zhi Erlian
Live vaccine+Mycoplasma synoviae inactivated vaccine immune effect.
The Mycoplasma synoviae of table 10 attacks poison protection result
From the above experiments, it was found that the step of triple vaccine simplifies vaccine inoculation, it is to avoid the trouble of multiple inoculation
The side effect easily produced, compared with traditional immunization mode, triple vaccine is immunized after chicken, and vaccine absorbs very fast, and residual is few, chicken
Body weight institute is impacted smaller；For laying hen, immune chicken can effectively resist the infection of chicken Mycoplasma synoviae, and egg production is not by shadow
Ring；In addition, the triple vaccine is for attack malicious protective rate and the traditional vaccine of chicken Mycoplasma synoviae, to attack malicious protective rate suitable, but chicken
Mycoplasma synoviae separation rate is but significantly lower than traditional vaccine, and immune chicken cause of disease carrying rate is low, can effectively control chicken bursa synovialis
The propagation of mycoplasma.
The newcastle disease of embodiment 6, infective bronchitis, chicken Mycoplasma synoviae, chicken virus mycoplasma tetrad inactivated vaccine
Preparation
The preparation of 1 chicken virus mycoplasma bacterium solution
1.1 strain
Make vaccine CR strains, effect inspection and attack malicious R plants, be purchased from China Veterinary Drugs Supervisory Inst..
1.2 culture medium prescription
According to《Republic of China Veterinary Pharmacopoeia》The compound method of mycoplasma fluid nutrient medium is matched somebody with somebody in annex page page 24
System, wherein per 1000ml culture mediums, the amount of Swine serum increases to 150ml, and PPLO gravy powders increase to 25g, aseptic subpackaged, 2~8
DEG C save backup.
The propagation of 1.3 bacterium solutions
Chicken virus mycoplasma is pressed 1：10 ratio is inoculated in chicken virus mycoplasma finished product culture medium, and 16 are cultivated at 36~37 DEG C
~24h harvests are used as primary seed solution；Primary seed solution is in kind pressed 1：10 ratio inoculates passage 1 time, 36
~37 DEG C of culture 24~48h harvests are used as secondary seed solution；Purely after the assay was approved, then in the same way culture is expanded.Survey
Determine chicken virus mycoplasma bacterial concentration and reach 1011CCU/mL。
1.4 inactivation
The virus liquid of harvest is inactivated 20 hours through 37 DEG C of 0.1% formaldehyde, it is total by bacterium solution volume by chicken virus mycoplasma bacterium solution
Amount is slowly added to final concentration of 0.2% formalin (V/V), puts 37 DEG C of inactivations, therebetween every stirring in 4 hours once, after 24h
Take out.
1.4.1 inactivation is examined
Take bacterium solution 10ml after inactivation to be inoculated in 37 DEG C of 4~7d of culture in 100ml fluid nutrient mediums, then transplanted by same ratio
37 DEG C of 4~7d of culture in above-mentioned culture medium 100ml；In addition, taking bacterium solution 0.5ml after inactivation to be inoculated in solid medium, 37 DEG C
Cultivate 7~10d.The equal asepsis growth of result of above two method culture.
1.4.2 steriling test
The bacterium solution inactivated is taken, is pressed《Republic of China Veterinary Pharmacopoeia》Annex page is carried out, as a result asepsis growth.
2 newcastle disease viruses, infectious bronchitis virus, the preparation of chicken Mycoplasma synoviae bacterium solution
It is former using the newcastle disease virus, infectious bronchitis virus liquid prepared in embodiment 1, and chicken bursa synovialis branch
Body bacterium solution prepares newcastle disease, infective bronchitis, chicken Mycoplasma synoviae, chicken virus mycoplasma tetrad inactivated vaccine.
The preparation of 3 vaccines
It is prepared by 3.1 oil phases
94 parts of injection white oil is taken, after Jia Siben -806 parts of mixing, plus 2 parts of aluminum stearate, it is to transparent with stirring with adding
Only, it is standby after autoclaving, as oil phase.
It is prepared by 3.2 aqueous phases
Antigen is mixed in sterile chamber in proportion, the Tween-80 after adding 4% sterilizing and cooling down is stirring while adding,
Make untill Tween-80 is completely dissolved, as aqueous phase.
3.3 emulsification
Take 3 parts of oil phase to put in oil phase tank, start motor stirring, 1 part of aqueous phase is then added slowly, emulsion tank is transferred to after adding
In, then with 2800r/min emulsifications 40 minutes.Emulsification terminate before add 1% thimerosal, make its final concentration of 0.01%.
The antigenic content ewcastle disease EID of prepared vaccine50/ 0.1ml is 109.0, infective bronchitis EID50/ 0.1ml is 107.0, it is sliding
Liquid capsule mycoplasma is 109.0CCU/mL, chicken virus mycoplasma is 109.0CCU/mL。
4 product inspections
4.1 character
Outward appearance：The uniform emulsion of milky.
Formulation：Water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and drips in cold water surface, in addition to the 1st drips, in oil droplet
Shape indiffusion.
Stability：Draw vaccine 10ml to add in centrifuge tube, centrifuged 15 minutes with 3000r/min, the aqueous phase that ttom of pipe is separated out
No more than 0.5ml.
Viscosity：By existing《Chinese veterinary pharmacopoeia》Annex is measured, and meets regulation.
4.2 loading quantity inspections are by existing《Chinese veterinary pharmacopoeia》Annex is checked, meets regulation.
4.3 steriling tests are by existing《Chinese veterinary pharmacopoeia》Annex is tested, asepsis growth.
4.4 safety verifications are subcutaneously injected vaccine 0.6ml, 14 are observed day by day with 14 age in days SPF chickens 10, each muscle or neck
Day.Occur without any locally and systemically adverse reaction as caused by vaccine.
4.5 efficacy test
Newcastle disease part is divided to two groups with 30 age in days SPF chickens 15.10 each hypodermic injection μ l of vaccine 20, another 5 are only used as
Control.21 days after immune, every chicken is taken a blood sample, and separates serum, carries out HI antibody titer measure.Immune chicken, control chicken HI antibody
Potency such as table 11.It can be seen that, the geometrical mean of immune chicken HI antibody titers is not less than 4log2, compare chicken HI antibody titers equal
2log should be not higher than2, meet effect inspection standard.
The newcastle disease HI potency of table 11
| Group | Individual values (log2) | GMT(log2) |
| Immune group | 7、7、7、5、7、6、7、6、7、6 | 6.5 |
| Control group | 0、0、0、0、0 | 0 |
Infectious bronchitis of chicken part infectious bronchitis of chicken part is inoculated with 21 age in days SPF chickens 10, each collunarium
Infectious bronchitis of chicken live vaccine (H120 plants) 1 plumage part, 21 days after inoculation, takes a blood sample respectively, separates serum, and immune group is strengthened exempting from
Epidemic disease inoculation (intramuscular injection) inactivated vaccine 0.3ml.28 days after immune, take a blood sample respectively, separate serum, HI will be respectively by serum twice
Experiment, the results are shown in Table 12, and two exempt from potency for 6.5log2, one exempts from potency for 4.3log2, i.e., two to exempt from HI potency be 4.6 times exempted from,
Meet immune group chicken two to exempt from Serum HI antibody potency geometrical mean more first to exempt from Serum HI antibody potency geometrical mean high 3 times
Effect inspection standard.
The infectious bronchitis of chicken HI potency of table 12
| Group | Individual values (log2) | GMT(log2) |
| One exempts from | 4、4、6、4、3、5、4、4、5、4 | 4.3 |
| Two exempt from | 8、6、6、7、6、7、7、7、6、5 | 6.5 |
Chicken Mycoplasma synoviae part is with 21 age in days SPF chickens 15, wherein vaccine 0.3ml, another 5 is subcutaneously injected in 10 necks
Only it is not inoculated with, is used as control.30 days after inoculation, chicken and control chicken each footpad inoculation chicken Mycoplasma synoviae culture simultaneously is immunized
0.1ml, is cultivated 14, cut open inspection gambrel and palmula, 2 kinds of cotton swabs of aseptic collection, after mixing, according to by existing《Chinese veterinary drug
Allusion quotation》The detection method of annex mycoplasma carries out mycoplasma separation, and as a result immune group chicken Mycoplasma synoviae separation rate is 1/10, right
It is 5/5 according to group.2/10 separation rate should be not higher than by meeting effect inspection standard, i.e. chicken Mycoplasma synoviae immune group, and control group is not low
In 4/5 separation rate.
Chicken virus mycoplasma part is referred to《Republic of China Veterinary Pharmacopoeia》Carry out, take 40 age in days SPF chickens 16,8 necks
Dorsal sc injection 0.3ml vaccines, another 8 be immunized compare.After immune 30 days, spray attacks chicken poison branch is former in isolator
R plants of culture 600ml (10 of body9.0CCU/1.0ml), the spraying duration is no less than 5 minutes, observes 14, cut open inspection, observes gas
Capsule lesion, and carry out lesion score.Score standard is shown in Table 13.Air bag lesion score the results are shown in Table 14, and control group chicken air bag is whole
There are more than 2 points lesions, protective rate is 83.3%, more than 60%, meet the effect inspection standard no less than 60%.
The air bag lesion scoring criteria of table 13
Protective rate calculation formula：(control chicken airsac disease change fraction/only-test chicken airsac disease change fraction/only)/control chicken gas
Capsule lesion fraction/only × 100%
The air bag lesion score of table 14
| Group | Individual values | Average value |
| Immune group | 0、1、0、1、0、1、1、0 | 0.5 |
| Control group | 3、3、2、3、3、4、3、4 | 3 |
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God is with principle, and any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (8)
1. a kind of vaccine combination, wherein, the vaccine combination includes the Newcastle Disease Virus Antigen, avian infectious of immune amount
Bronchitis virus antigen and chicken Mycoplasma synoviae antigen and carrier；Wherein, the Newcastle Disease Virus Antigen is to go out
Sota plants of antigens of newcastle disease virus La living, the avian infectious bronchitis virus antigen is the avian infectious branch of inactivation
M41 plants of antigens of bronchitis virus, the chicken Mycoplasma synoviae antigen is HN1 plants of antigens of chicken Mycoplasma synoviae of inactivation, is protected
Tibetan number is CCTCC NO.V201425；And described Newcastle Disease Virus Antigen content is 108.0EID50/ 0.1ml, it is described
Avian infectious bronchitis virus antigenic content is 106.0EID50/ 0.1ml, described chicken Mycoplasma synoviae antigenic content is
109CCU/mL。
2. vaccine combination according to claim 1, wherein, the carrier includes adjuvant, and the adjuvant includes oily adjuvant,
Water-soluble adjuvant, Alum adjuvant, Cytokine adjuvant.
3. vaccine combination according to claim 2, wherein, the adjuvant includes white-oil adjuvant.
4. a kind of vaccine combination, wherein, the vaccine combination includes the Newcastle Disease Virus Antigen, avian infectious of immune amount
Bronchitis virus antigen chicken Mycoplasma synoviae antigen and Mycoplasma Gallisepticum Antigen Recognized By Antibody and carrier；Wherein, the chicken new city
Epidemic disease viral antigen is Sota plants of antigens of newcastle disease virus La of inactivation, and the avian infectious bronchitis virus antigen is to go out
M41 plants of antigens of avian infectious bronchitis virus living, the chicken Mycoplasma synoviae antigen is former for the chicken bursa synovialis branch of inactivation
HN1 plants of antigens of body, preserving number is CCTCC NO.V201425, and the Mycoplasma Gallisepticum Antigen Recognized By Antibody is CR plants of the chicken virus mycoplasma of inactivation
Antigen；The newcastle disease totivirus antigenic content is 109.0EID50/ 0.1ml, the infectious bronchitis of chicken totivirus resists
Former content is 107.0EID50/ 0.1ml, the full mycoplasma antigen content of chicken Mycoplasma synoviae is 109.0CCU/mL, the chicken
The full mycoplasma antigen content of virus mycoplasma is 109.0CCU/mL。
5. a kind of method for preparing vaccine combination described in claim 1, wherein, methods described includes：
(1) Sota plants of newcastle disease virus La, M41 plants of avian infectious bronchitis virus, chicken bursa synovialis branch are bred in culture respectively
HN1 plants of substance, preserving number is CCTCC NO.V201425；
(2) newcastle disease virus, avian infectious bronchitis virus and the chicken synovia that inactivation step (1) culture is bred respectively
Capsule mycoplasma；And
(3) by newcastle disease virus totivirus antigen, avian infectious bronchitis virus totivirus antigen and the chicken of the inactivation
The full mycoplasma antigen mixing of Mycoplasma synoviae, adds adjuvant；Wherein, described Newcastle Disease Virus Antigen content is
108.0EID50/ 0.1ml, described avian infectious bronchitis virus antigenic content is 106.0EID50/ 0.1ml, described chicken is slided
Liquid capsule mycoplasma antigen content is 109CCU/mL。
6. a kind of method for preparing vaccine combination described in claim 4, wherein, methods described includes：
(1) Sota plants of newcastle disease virus La, M41 plants of avian infectious bronchitis virus, chicken bursa synovialis branch are bred in culture respectively
HN1 plants of substance, preserving number is CCTCC NO.V201425 and chicken virus mycoplasma；
(2) newcastle disease virus, avian infectious bronchitis virus, the chicken synovia that inactivation step (1) culture is bred respectively
Capsule mycoplasma and chicken virus mycoplasma；And
(3) the newcastle disease virus totivirus antigen, avian infectious bronchitis virus totivirus antigen, chicken of the inactivation are slided
The full mycoplasma antigen of liquid capsule mycoplasma and the full mycoplasma antigen mixing of chicken virus mycoplasma, add adjuvant, wherein, described chicken new city
Epidemic disease poison antigenic content is 109.0EID50/ 0.1ml, described avian infectious bronchitis virus antigenic content is 107.0EID50/
0.1ml, described chicken Mycoplasma synoviae antigenic content is 109CCU/mL, the full mycoplasma antigen content of chicken virus mycoplasma
For 109.0CCU/mL。
7. vaccine combination is preparing prevention and/or treatment newcastle disease virus, avian infectious branch gas according to claim 1
Application in the medicine of the scorching virus of pipe and the relevant disease of chicken Mycoplasma synoviae infection.
8. vaccine combination is preparing prevention and/or treatment newcastle disease virus, avian infectious branch gas according to claim 4
Application in the medicine of the relevant disease of the scorching virus of pipe, chicken Mycoplasma synoviae and mycoplasma gallisepticum infection.
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| CN105816868B (en) * | 2016-03-21 | 2020-01-03 | 青岛易邦生物工程有限公司 | Inactivated vaccine for mycoplasma synoviae |
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