CN104415330A - Vaccine composition and preparation method and application thereof - Google Patents
Vaccine composition and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a vaccine composition, comprising an immunizing dose of avian pasteurella multocida antigen, an immunizing dose of newcastle disease antigen and an immunizing dose of H9 subtype avian influenza antigen, and one or more adjuvants acceptable in veterinary medicine. The vaccine composition can be used for simplifying the immune procedure, thus the multi-prevention purpose by one injection is achieved; protective immunity is generated for avian pasteurella multocida, newcastle disease and H9 subtype avian influenza; the side effect of lumps generated on the injection part caused by multiple times of immunization and the side effect of reduction of the laying rate can also be reduced; and the mass mortality of chicken flocks caused when the three diseases are infected in a mixed manner can also be lowered.
Description
Technical field
The present invention relates to a kind of vaccine combination and preparation method thereof and application.
Background technology
Fowl pasteurellosis has another name called fowl cholera, is a kind of contact septic infectious disease of the birdss such as main infringement chicken, turkey, duck, the goose caused by pasteurella multocida (Pasteurellamultocida, Pm).The poultry of this disease to each age group all has susceptibility, mainly betides laying hen group, often shows as the symptom such as acute sepsis and acute dysentery, and simultaneously with respiratory tract infection, mortality rate is 20%-30% or higher normally; The performance of poultry of survival after infecting declines, and there is the risk of the malicious recessive infection of band.This disease all can occur throughout the year, is the highest with high temperature, high humidity, rainy season in autumn in summer two sickness rate, is one of main bacteria disease having a strong impact on aviculture development.On market, existing avian pasteurella multocida disease vaccine mainly contains inactivated vaccine and live vaccine two kinds, but the adjuvant that avian pasteurella multocida inactivated vaccine uses is often aluminium glue adjuvant, oily adjuvant, and injection site can be made after immune animal to produce the side effect such as lump; And the safety issue caused by attenuated live vaccines can not be ignored.
Newcastle is commonly called as fowl plague, that a kind of birds of being caused by Avian pneumo-encephalitis virus is acute, septic, high degree in contact sexually transmitted disease, endanger the most serious one in all fowl disease, main through respiratory tract and digestive tract infection, principal character is dyspnea, serious dysentery, the course of disease slightly long with nervous symptoms.The chicken of various age in days all can infect, the most serious with chickling, also can cause the egg drop reduction of disease fowl.At present for the prevention and control of newcastle, mainly based on newcastle disease inactivated vaccine, but it uses adjuvant to be often oily adjuvant, and injection site can be made after immune animal to produce the side effect such as lump.
Bird flu (AI) causes the one of birds and other birds to infect and/or disease syndrome by orthomyxoviridae family's influenza A.H9 subtype avian influenza metainfective drylot feeding fowl (comprising poultry) can show as inferior clinical symptom, light respiratory system infection to multiple popular form such as asymptomatic band poison.At present, the inoculation of vaccine is still the most effective means of birds flu-preventing Occurrence & epidemic.Existing avian influenza vaccine mainly inactivated vaccine on market, the adjuvant used is often oily adjuvant, and injection site can be made after immune animal to produce the side effect such as lump.
Eggs crack detection itself is present in healthy body, belongs to conditionality pathogenic bacterium.Newcastle disease vaccine is seeded in and controls the popular on a large scale of this disease to a great extent, but the immune programme for children adopted due to raiser is unreasonable or immunization method is improper, and newcastle infects and still happens occasionally.H9 subtype avian influenza breaks out and usually causes relatively mild clinical symptoms, lower M & M in birds, but when after organism infection H9 subtype avian influenza, resistance is often poor, now just can cause high incidence and the high mortality of birds with other epidemic disease co-infection.In recent years, in clinical diagnosis, the while that the chicken that dies of illness detecting, the phenomenon of mixed infection eggs crack detection, newcastle, H9 subtype avian influenza three kinds of epidemic diseases increases year by year, according to clinical statistics, the more single epidemic disease of three's co-infection infects and causes body mortality rate significantly to rise, and be often the death without the sudden large quantities of chicken group of any symptom, bring huge economic loss to poultry breeding industry.And not yet have a kind of vaccine that simultaneously can prevent three kinds of epidemic diseases at present.
Patent application CN1724068A discloses a kind of preparation method of intensified inactivated cholera fowl vaccine, and isolation identification eggs crack detection CVCC458 makes strain; Separation of bacterial is inoculated in improvement BPY culture medium as seed liquor, inoculates instar chicken embryo on the 11st through allantoic cavity, and hatching results Embryo Gallus domesticus, smashs to pieces, deactivation, add white-oil adjuvant, intensified inactivated cholera fowl vaccine, every 0.5m1 mycetome 10,000,000,000.But the fowl cholera strengthening vaccine adjuvant used prepared by this invention is common white-oil adjuvant, absorb not exclusively after white-oil adjuvant injection, side reaction is large.
Patent application CN1724068A discloses the preparation method of a kind of newcastle disease, H9 subtype avian influenza bivalent inactivated vaccine, 9-10 age in days susceptible Embryo Gallus domesticus is inoculated respectively after NDV La Sota strain being produced seed culture of viruses, the dilution of AIV HL strain production seed culture of viruses, hatch, results, deactivation; Get NDV La Sota strain virus liquid and the AIV HL strain virus liquid mixed in equal amounts of deactivation, add white-oil adjuvant emulsifying.But this vaccine protective antigen composition is few, is only the sick antigen of two-strain, can not provides and protect completely.In addition adopt white-oil adjuvant, vaccine side reaction is larger.
Summary of the invention
In order to solve the deficiencies in the prior art; the invention provides a kind of vaccine combination; this vaccine combination can simplify immune programme for children; reach the object that how anti-a pin is; namely protective immunity is produced to eggs crack detection, newcastle, H9 subtype avian influenza three kinds of diseases; can reduce again repeatedly immunity makes injection site produce the side effect such as lump, laying rate reduction, chicken group massive mortality caused when can also reduce by three kinds of disease mixed infection.
Main purpose of the present invention is to provide a kind of vaccine combination, and described vaccine combination comprises: the H9 subtype avian influenza antigen of the eggs crack detection antigen of immunity amount, the newcastle antigen of immunity amount, immunity amount; And one or more veterinarily acceptable adjuvants.
" eggs crack detection antigen " of the present invention refers to when described antigen-immunized animal, and especially, described animal is chicken, during administration, can induce, stimulates or strengthen the immunne response that immune animal opposing eggs crack detection infects.
" newcastle antigen " of the present invention refers to when described antigen-immunized animal, and especially, described animal is chicken, during administration, can induce, stimulates or strengthen the immunne response that immune animal opposing newcastle infects.
" H9 subtype avian influenza antigen " of the present invention refers to when described antigen-immunized animal, and especially, described animal is chicken, during administration, can induce, stimulates or strengthen the immunne response that immune animal opposing H9 subtype avian influenza infects.
Preferably, described eggs crack detection antigen is deactivated form, the form of work of improvement or the eggs crack detection antigen of attenuated forms thereof; Described newcastle antigen is deactivated form, the form of work of improvement or the newcastle antigen of attenuated forms thereof; Described H9 subtype avian influenza antigen is deactivated form, the form of work of improvement or the H9 subtype avian influenza antigen of attenuated forms thereof.
More preferably, described eggs crack detection antigen is the full bacterium antigen of eggs crack detection C48-2 strain of deactivation, described newcastle antigen is the Avian pneumo-encephalitis virus LaSota strain totivirus antigen of deactivation, and described H9 subtype avian influenza antigen is the H9 subtype avian influenza antigen HL strain totivirus antigen of deactivation.
Eggs crack detection antigen used in the present invention can also comprise any one antigen in following compositions, as: the eggs crack detection inactivated vaccine (1502 strain) of Binzhou, Shandong Wo Hua biological engineering company limited, the eggs crack detection inactivated vaccine (C48-2 strain) of Shandong Lvdu Bio Sicience & Technology Co., Ltd..
H9 subtype avian influenza antigen used in the present invention can also comprise any one antigen in following compositions, as: inactivated avian influenza vaccine (the H9 hypotype of Vacbio Co., Ltd., F strain), inactivated avian influenza vaccine (the H9 hypotype of Harbin Wei Ke biotechnology development company, SD696 strain), inactivated avian influenza vaccine (the H9 hypotype of high-tech branch company of Tianjin Ruipu Biotechnology Co., Ltd, Sy strain), inactivated avian influenza vaccine (the H9 hypotype of Beijing Xinde Weite Science Co., Ltd, SS strain), inactivated avian influenza vaccine (the H9 hypotype of Shandong Dong Bao health product company limited, LG1 strain).More preferably, described eggs crack detection antigen is the full bacterium antigen of eggs crack detection C48-2 strain of deactivation, described newcastle antigen is the Avian pneumo-encephalitis virus LaSota strain totivirus antigen of deactivation, and described H9 subtype avian influenza antigen is the H9 subtype avian influenza antigen HL strain totivirus antigen of deactivation.
Eggs crack detection C48-2 strain is in National Veterinary Microbiological Culture Collection administrative center (referred to as CVCC) preservation, and preservation date is on June 25th, 1964, and preserving number is CVCC44802, buys in China Veterinery Drug Inspection Office.
Newcastle LaSota strain is in National Veterinary Microbiological Culture Collection administrative center (referred to as CVCC) preservation, and preservation date is on March 12nd, 1973, and preserving number is CVCC AV1615, buys in China Veterinery Drug Inspection Office.
Document is shown in H9 subtype avian influenza antigen HL strain: Sun Jinzhong etc., bird flu virus (H9N2 hypotype, HL strain) and Some Domestic economize the research of bird flu virus (H9 hypotype) epidemic strain antigen dependency and Immunogenicity, animal and veterinary association nd Annual Meeting collection in 2007,2007,35-38.
Preferably, described adjuvant comprises one or more in aqueous adjuvants, propolis adjuvant; More preferably, described aqueous adjuvants is Gel adjuvant.
Term used herein " aqueous adjuvants ", also known as " water-based adjuvant " or " water-soluble adjuvant ", is a kind of polymeric water-soluble dispersion, for improving effect and the safety of water-soluble vaccines, in instillation water can rapidly and water miscible.
Preferably, described adjuvant is the 5%-25%V/V of vaccine combination, is more preferably 10%-15%V/V.
Preferably, described eggs crack detection antigenic content is>=5 × 10
9cFU/ml; Described newcastle antigenic content is>=6 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is>=6 × 10
8.5eID
50/ ml.
More preferably, described eggs crack detection antigenic content is 1 × 10
11cFU/ml; Described newcastle antigenic content is 7.5 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is 7.5 × 10
8.5eID
50/ ml.
Another object of the present invention is to provide a kind of method preparing described vaccine combination, described preparation method comprises: (1) cultivates propagation described eggs crack detection, described Avian pneumo-encephalitis virus, described H9 subtype avian influenza virus respectively, deactivation respectively; (2) be mixed in proportion described eggs crack detection full bacterium antigen, described newcastle totivirus antigen, described H9 subtype avian influenza totivirus antigen, and add adjuvant.
Preferably, described antigen ratio is described eggs crack detection antigenic content is>=5 × 10
9cFU/ml; Described newcastle antigenic content is>=6 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is>=6 × 10
8.5eID
50/ ml.
Another object of the present invention is to provide described vaccine combination to prevent and/or treat the application in the medicine of eggs crack detection, newcastle, the infection of H9 subtype avian influenza in preparation.
Based on this, the present invention has following outstanding advantage;
(1) vaccine combination of the present invention can simplify immune programme for children, the object that a pin can prevent and/or treat eggs crack detection, H9 subtype avian influenza, newcastle three kinds of diseases can be reached, just can prevent and/or treat compared with the situation of these three kinds of diseases with the existing single vaccine of a few pin of beating more, technical solutions according to the invention are practical more economically, and have certain social benefit;
(2) vaccine combination of the present invention can just reduce existing single vaccine repeatedly immunity make injection site produce the side effect such as lump;
(3) chicken group massive mortality caused when vaccine combination of the present invention can also reduce by three kinds of disease mixed infection.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
" immunity amount " used in the embodiment of the present invention refers to the immunizing dose provided for influenza vaccines, depends primarily on following factor: the vaccine whether accepting to resist same virus before the species of immunized animal, kind, age, weight size, health status and animal.
" single Seedling " described in the embodiment of the present invention refers to the eggs crack detection antigen only containing immunity amount, a kind of vaccine in newcastle antigen or H9 subtype avian influenza antigen, " Combined vaccine " refers to the eggs crack detection antigen and newcastle antigen Combined vaccine only measured containing immunity, the eggs crack detection antigen of immunity amount and H9 subtype avian influenza antigen Combined vaccine, a kind of vaccine in newcastle antigen and H9 subtype avian influenza antigen Combined vaccine, can be used for the immune efficacy evaluating vaccine combination in the present invention, the foundation of three kinds of disease mixed infection counteracting toxic substances models, different adjuvant is to vaccine combination immune efficacy.
In the embodiment of the present invention, for eggs crack detection C48-2 strain, newcastle antigen LaSota strain, H9 subtype avian influenza antigen HL strain, the immune efficacy of vaccine combination, the foundation of three kinds of disease mixed infection counteracting toxic substances models, different adjuvant are explained vaccine combination immune efficacy.
The eggs crack detection C48-2 strain used in embodiments of the present invention, no matter this embodiment does not under any circumstance all form limitation of the invention.
The newcastle antigen LaSota strain used in embodiments of the present invention, no matter this embodiment does not under any circumstance all form limitation of the invention.
The H9 subtype avian influenza antigen HL strain used in embodiments of the present invention, no matter this embodiment does not under any circumstance all form limitation of the invention.
In embodiments of the present invention for eggs crack detection, newcastle, H9 subtype avian influenza three kinds of viruses use counteracting toxic substances strain be respectively eggs crack detection C48-1 strain (preserving number be CVCC44801, be preserved in National Veterinary Microbiological Culture Collection administrative center), newcastle Beijing Strain (preserving number is CVCC AV1611, and depositary institution is National Veterinary Microbiological Culture Collection administrative center), H9 subtype avian influenza HL strain.
Experimental technique described in following embodiment, if without specified otherwise, is conventional method; Described biomaterial, if without specified otherwise, all can obtain from commercial channels.
The preparation of embodiment 1 eggs crack detection, newcastle, H9 subtype avian influenza antigen
1.1 produce the preparation with seed (seed culture of viruses)
1.1.1 the preparation of eggs crack detection production seed
The breeding of first order seed: eggs crack detection C48-2 strain is inoculated in containing in 0.1% cracking blood cell whole blood martin's bouillon, put 36-37 DEG C and cultivate 24h, then streak inoculation is on the improvement Martin agar plate containing 0.1% cracking blood cell whole blood and 4% healthy animal serum, cultivate 16-22h for 36-37 DEG C, select more than 5 colonies typicals, inoculation some, blood agar inclined-plane, puts 36-37 DEG C and cultivates 18-24h, as first order seed.2-8 DEG C of preservation, storage life is no more than 14 days.Culture medium goes down to posterity, was no more than for 5 generations.
Secondary seed is bred: get first order seed and be inoculated in containing in 0.1% cracking blood cell whole blood martin's bouillon, puts 36-37 DEG C and cultivates 24h, purely check by " Chinese veterinary pharmacopoeia " annex, qualified rear as secondary seed, 2-8 DEG C of preservation, is no more than 4 days.
1.1.2 prepared by newcastle production seed culture of viruses
Newcastle La Sota strain seed culture of viruses physiological saline solution is done 10
-3-10
-4dilution, inoculates 10 age in days SPF Embryo Gallus domesticus through allantoic cavity, every embryo 0.1ml.Select death between the rear 72-120h of inoculation and the obvious Embryo Gallus domesticus of lesion, gather in the crops Embryo Gallus domesticus liquid (allantoic fluid and amniotic fluid) respectively, be loaded in sterile chamber.To check aseptic qualified and to 1% chicken erythrocyte suspension agglutination titer >=1:512(micromethod) Embryo Gallus domesticus liquid mixing, quantitative separating, freezen protective.Indicate harvest date, seed culture of viruses algebraically etc.
1.1.3H9 prepared by subtype avian influenza production seed culture of viruses
H9 subtype avian influenza seed culture of viruses HL strain physiological saline solution is done 10
-3-10
-4dilution, inoculates 10 age in days SPF Embryo Gallus domesticus through allantoic cavity, every embryo 0.1ml.Put 37 DEG C to continue to hatch, choosing inoculates death between rear 48-96h and the obvious Embryo Gallus domesticus of lesion, gathers in the crops Embryo Gallus domesticus liquid (allantoic fluid and amniotic fluid) respectively, is loaded in sterile chamber.To check aseptic and to 1% chicken erythrocyte suspension agglutination titer >=1:512(micromethod) Embryo Gallus domesticus liquid mixing, quantitative separating in ampoule, freezen protective.Indicate harvest date, seed culture of viruses algebraically etc.
Prepared by 1.2 seedlings bacterium liquid (virus liquid)
1.2.1 eggs crack detection seedling bacterium solution preparation
Biological fermentation tank is adopted to carry out the preparation of eggs crack detection A type bacterial strain bacterium liquid.Qualified secondary seed solution is inoculated in ferment tank culture medium with the inoculum concentration of 1%-2%, 37 DEG C, 200rpm, by constantly increasing the mode fermentation culture 14-20h of ventilation and flow feeding, results bacterium liquid.Count plate is carried out in sampling, and purely checks by existing " Chinese veterinary pharmacopoeia " annex, should be pure.
1.2.2 the preparation of newcastle seedling virus liquid
Get production seed culture of viruses, do 10 with physiological saline solution
-3-10
-4dilution, inoculates 10 age in days susceptible Embryo Gallus domesticus through allantoic cavity, every embryo 0.1ml, puts 37 DEG C and continue to hatch, and after egg inoculation, per sunshine, egg 1 time, discarded Embryo Gallus domesticus dead before 48h.After this, every 4h shines egg 1 time, and dead embryo takes out at any time, until 120h, no matter death whether, is all taken out, after being placed in 2-8 DEG C of cooling 12-24h, and sterile working's results Embryo Gallus domesticus liquid.
1.2.3H9 the preparation of subtype avian influenza seedling virus liquid
Get production seed culture of viruses, do 10 with physiological saline solution
-3-10
-4dilution, inoculates 10 age in days susceptible Embryo Gallus domesticus through allantoic cavity, every embryo 0.1ml, puts 37 DEG C and continue to hatch, and after egg inoculation, per sunshine, egg 1 time, discarded Embryo Gallus domesticus dead before 48h.After this, every 4h shines egg 1 time, and dead embryo takes out at any time, until 96h, no matter death whether, is all taken out, after being placed in 2-8 DEG C of cooling 12-24h, and sterile working's results Embryo Gallus domesticus liquid.
Concentrating of 1.3 bacterium liquid (virus liquid)
The bacterium liquid be up to the standards pure property and the qualified virus liquid of steriling test adopt ultrafiltration and concentration technology to concentrate respectively, and after concentrated, each antigenic content is in table 1.Get concentrated rear sample sample and do the inspection of pure property and steriling test respectively, should be qualified.
Content after table 1 antigen is concentrated
| Antigen | Deactivation pro-antigen content |
| Eggs crack detection A type (C48-2 strain) | 1×10 11CFU/ml |
| Newcastle (La Sota strain) | 3×10 8.5EID 50/0.1ml |
| H9 subtype avian influenza (HL strain) | 3×10 8.5EID 50/0.1ml |
The deactivation of 1.4 bacterium liquid (virus liquid)
The eggs crack detection C48-2 strain bacterium liquid be purely up to the standards and the qualified newcastle La Sota strain of steriling test, H9 subtype avian influenza HL strain, add formalin (V/V) by the 0.2%-0.4% of bacterium liquid (virus liquid) total amount respectively, 37 DEG C of deactivation 16-24h, constantly shake therebetween.Then sampling carries out deactivation inspection by existing " Chinese veterinary pharmacopoeia " annex, and assay shows: without colony growth.
The preparation of the different vaccine combination of embodiment 2
The preparation of 1 antiseptic
Thimerosal aqueous solution 1%(W/V): 1g thimerosal is dissolved in 100ml purified water, 121 DEG C of autoclaving 30min are for subsequent use.
The preparation of 2 diluent
Aseptic PBS buffer solution: dissolve 8g sodium chloride, 0.25g potassium chloride, 3.63g sodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate in 900ml purified water, be then settled to 1L, 121 DEG C of autoclaving 30min are for subsequent use.
3 vaccine adjuvant process
Gel adjuvant sterilizing: being proceeded to by Gel adjuvant can in sterilization container, and 121 DEG C of autoclaving 30min are for subsequent use.
4 join Seedling
Mentioned component is mixed by a certain percentage, namely by sterile working, after eggs crack detection prepared by embodiment 1, H9 subtype avian influenza, newcastle concentrated antigen and adjuvant, antiseptic, mixing diluents, with mulser stirring at low speed 15min.Prepared different vaccine combinations are as follows:
The vaccine proportioning specific embodiments (V/V) that table 2 is different
The inspection of the different vaccine combination of embodiment 3
3.1 steriling tests and safety verification
The different vaccine combinations prepared according to embodiment 2 carry out steriling test and safety verification according to the vaccine of 2001 editions " People's Republic of China's veterinary biologics quality standard " methods to preparation.
Steriling test and the display of safety verification result: each vaccine combination all conforms with the regulations standard.
3.2 efficacy test
3.2.1 eggs crack detection efficacy test
Get 2 monthly age SPF chicken 48, wherein make blank for 3, remain 45 and be divided into 9 groups at random, often organize 5, neck dorsal sc injects two groups of vaccines respectively, 1mL/ only, exempt from rear 21d, each intramuscular injection lethal dose eggs crack detection C48-1 strain bacterium liquid carries out challenge test, observes 14d, record each group of morbidity, death condition, the results are shown in Table 3.
The eggs crack detection assay of table 3 vaccine potency inspection
As shown in Table 3: vaccine 1, vaccine 2, vaccine 3, vaccine 4, vaccine 5, vaccine 8, vaccine 9, vaccine 11 and vaccine 12 pairs of eggs crack detection all have good protective effect, and protective rate is 80%-100%.
3.2.2 newcastle efficacy test
Get 30 age in days SPF chicken 95, wherein 5 as blank; Remain 90 and be divided into 9 groups at random, often organize 10, neck dorsal sc injects each group of vaccine 20 μ l respectively, exempts from rear 21d and takes a blood sample, simultaneously every chicken muscle injection 10
5.0eLD
50newcastle Beijing Strain (passing for 1 generation with SPF embryo) 1.0ml, observes 14 days, records each immune group and matched group is survived and death condition, the results are shown in Table 4.
The newcastle assay of table 4 vaccine potency inspection
As shown in Table 4: vaccine 1, vaccine 2, vaccine 3, vaccine 4, vaccine 6, vaccine 8, vaccine 10, vaccine 11 and vaccine 12 pairs of newcastles all have protective effect, and protective rate is all up to 100%.
3.2.3H9 subtype avian influenza efficacy test
Get 30 age in days SPF chicken 95, wherein 5 remain 90 be divided into 9 groups at random as blanks, and often organize 10, neck dorsal sc injects each group of triple vaccine respectively, and 0.3mL/ only.After immunity 21d, blood sampling measures H9 subtype avian influenza respectively, and the seed culture of viruses that 21d 1:10 dilutes after exempting from carries out intravenous injection, every chicken 0.2ml.After counteracting toxic substances the 5th day, gather the larynx of every chicken, cloacal swab, aggregate sample is called after mixing, through allantoic cavity inoculation 10 ~ 11 age in days SPF Embryo Gallus domesticus 5 pieces, every embryo 0.2ml, hatches observation 5, and no matter dead germ, embryo of living all should measure Embryo Gallus domesticus liquid HA-HI test, 1 piece or the HA of Embryo Gallus domesticus liquid that is greater than 1 piece of Embryo Gallus domesticus is had to tire >=1:16(micromethod in 5 pieces of Embryo Gallus domesticus of each swab samples inoculation), the virus purification positive can be judged to.To the sample of virus purification feminine gender, judge again after answering blind passage 1 time.During successful immunization, immune group should have at least 9 chicken virus purification negative, and matched group should be all positive.Measurement result is in table 5.
The H9 subtype avian influenza assay of table 5 vaccine potency inspection
As shown in Table 5: the antibody titer that vaccine 1, vaccine 2, vaccine 3, vaccine 4, vaccine 7, vaccine 9, vaccine 10, vaccine 11 and vaccine 12 produce all>=7log
2, and matched group is negative, show that each group of vaccine all has protective effect to H9 subtype avian influenza, and protective rate is all up to 100%.
Generally speaking, prepared eggs crack detection, newcastle, H9 subtype avian influenza triple inactivated vaccine compositions all can produce good protective effect to corresponding cause of disease, and antigenic content is larger, and protective effect is stronger.
The single Seedling conbined usage of embodiment 4 three kinds and triple vaccine immunity contrast test
Choosing is in egg-laying peak and the laying hen (200 age in days) 180 of nonreactive three kinds of disease antibodies, wherein randomly draw 30 and be divided into 2 groups, 1st group of immunity 0.5ml vaccine 3(is prepared by embodiment 2), 2nd group of immune vaccine 5, vaccine 6 and vaccine 7(are all prepared by embodiment 2) each 0.5ml, before exempting from respectively at one, one exempts within latter 14 days and one, to exempt from blood sampling in latter 28 days, separation of serum, agar diffusion method (ID) is adopted to carry out the detection (according to People's Republic of China (PRC) agricultural industry criteria NY/T563-2002) of immune antibody to eggs crack detection, HI method is adopted to measure newcastle in serum, H9 subtype avian influenza antibody horizontal, testing result is in table 6.
The single Seedling conbined usage of three kinds, table 6 and triple vaccine immunity comparative test result
As shown in Table 6: the antibody horizontal that the antibody produced after triple vaccine immunity and three kinds of single Seedling conbined usage produce is suitable.
Meanwhile, extract 50 in contrast from residue 150 chickens, all the other 100 are divided into two groups of respectively immunity connection Seedlings and single Seedlings at random, and before record one is exempted from, one exempt within latter 7 days, one, to exempt from all laying rate that latter 14 days and exempt from latter 21 days, comparing result is in table 7.
The single Seedling conbined usage of three kinds, table 7 and all average egg production comparing results of triple vaccine
As shown in Table 7: make all average egg productions of laying hen apparently higher than after the immunity of three kinds of single Seedling conbined usage after triple vaccine immunity, show that the impact of triple vaccine on Layer Production Performance is less.
Generally speaking, the antibody horizontal produced after triple vaccine and the immunity of three kinds of single Seedling conbined usage is suitable, especially, and the side reaction after the use of triple vaccine reduces multiple injection, laying hen produced, thus laying rate is obviously raised, also reduce labor intensity and epidemic prevention cost.
Embodiment 5 avian pasteurella multocida, newcastle, H9 subtype avian influenza mixed infection research
Get 80 80 age in days SPF chickens to raise in isolator, randomly drawing 10 is left intact as a control group, remain 70 and be divided into 7 groups, respectively with avian pasteurella multocida C48-1 strain, newcastle Beijing Strain, H9 subtype avian influenza HL strain separately or combination carry out counteracting toxic substances, concrete grouping and counteracting toxic substances situation are in table 8.To 14d after counteracting toxic substances after counteracting toxic substances, observe morbidity and the death condition of statistics each group of chicken day by day, the results are shown in Table 8.
The grouping of table 8 counteracting toxic substances and incidence statistics
Note: "-" expression does not carry out this; MLD is minimal lethal dose
As shown in Table 8: (1) the 1st group is carried out counteracting toxic substances with avian pasteurella multocida, the strong poison of bird flu H9N2 respectively with the 3rd group, all can cause relatively mild clinical symptoms, and the phenomena of mortality do not occur after counteracting toxic substances; (2) the 2nd groups are carried out counteracting toxic substances with Virulent Newcastle Disease Virus, and the chicken infected in 14d after counteracting toxic substances is all dead, cut open inspection and have found the typical cytopathic of newcastle; (3) the 4th groups are carried out counteracting toxic substances with avian pasteurella multocida and Virulent Newcastle Disease Virus simultaneously, occur mortality after co-infection chicken, cut open the dead chicken of inspection and can occur the metainfective clinical symptoms of obvious avian pasteurella multocida; (5) the 5th groups with the 6th group respectively poison strong with bird flu H9N2 with the strong poison of avian pasteurella multocida and bird flu H9N2, Virulent Newcastle Disease Virus while counteracting toxic substances, the death time of chicken that makes after discovery co-infection significantly shifts to an earlier date; (6) and when three simultaneously infected chicken time (the 7th group), can massive mortality be there is in chicken group in 1 ~ 3d.
As can be seen here, as avian pasteurella multocida, newcastle, H9 subtype avian influenza co-infection chicken group, significantly can shift to an earlier date the death time of chicken group, increase chicken group case fatality rate; Especially, although avian pasteurella multocida is chicken infected separately, chicken group can not be caused dead, and when jointly existing with other virulence factor, can form synergism, causing chicken group mortality, deducibility avian pasteurella multocida lures chicken group main causes of death into.
The vaccine combination that embodiment 6 is different is studied for the immune effect of three kinds of pathogen mixed infections
Choose 60 age in days SPF chicken 100, be divided into 9 groups at random, often organize 10 (see table 9).Wherein the 18th group is not carried out Immunization, as Normal group; All the other groups by the vaccine that intramuscular injection path immunity is different, are specially all simultaneously: 9-11 group is immune vaccine 5, vaccine 6, vaccine 7 three kinds of single Seedlings (all preparing according to embodiment 2) respectively; 12-14 group is immune vaccine 8, vaccine 9, vaccine 10 3 kinds of bigeminy vaccines (all preparing according to embodiment 2) respectively; 15th group, the triple vaccine of immune vaccine 3; 16th group, carry out immunity with vaccine 5, vaccine 6, the single Seedling conbined usage of vaccine 7 three kinds; 17th group not immune, as counteracting toxic substances matched group.
21d after immunity, except Normal group, 9-17 group carries out counteracting toxic substances according to the Infection route of the 7th group in the table 8 of embodiment 5 and dosage respectively, and observe and add up 14d after the death condition of respectively organizing chicken every day to counteracting toxic substances, result of the test is in table 9.
The different vaccine combination of table 9 is for the immune effect result of study of three kinds of pathogen mixed infections
As shown in Table 9: in the counteracting toxic substances model that these three kinds of pathogen co-infection build, the use of single Seedling is substantially without immune effect, and each group case fatality rate is at 80%-100%; The use of Combined vaccine is better than single Seedling, but still can not provide and protect completely; And the conbined usage of trigeminy vaccine and single Seedling can play good counteracting toxic substances protective effect to the mixed infection of three kinds of pathogen.
It can thus be appreciated that: only have and the immunity with three kinds of pathogen corresponding antigens compositions is carried out to chicken group, effective protection could be provided to three kinds of pathogen mixed infections.
The different adjuvant immunity assimilation effect of embodiment 7 compares
Prepare vaccine 3, vaccine 4 respectively according to embodiment 2, and prepare vaccine A and vaccine B simultaneously.Wherein, vaccine A is identical with vaccine 3 antigenic content, and adjuvant is white oil content is 39%(V/V); Vaccine B is also identical with vaccine 3 antigenic content, and adjuvant is aluminium hydroxide gel, content 10%(V/V).
Choose SPF chicken 40 in 3 week age, be divided into 4 groups at random, respectively immune vaccine 3, vaccine 4, vaccine A and vaccine B, 2 weeks, interval, carries out booster immunization, observes untoward reaction after immunity, and cutd open inspection observation to injection site after 1 month.Untoward reaction after immunity and cut open inspection and the results are shown in Table 10.
Untoward reaction and cut open inspection result after the different Adjuvanted vaccines immunity of table 10
As shown in Table 10: in the present invention, the use of Gel adjuvant does not all affect assimilation effect and the safety of vaccine, and there is good absorbing, reduce the advantage such as untoward reaction.
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. a vaccine combination, described vaccine combination comprises: the H9 subtype avian influenza antigen of the eggs crack detection antigen of immunity amount, the newcastle antigen of immunity amount, immunity amount; And one or more veterinarily acceptable adjuvants.
2. vaccine combination according to claim 1, wherein, described eggs crack detection antigen is deactivated form, the form of work of improvement or the eggs crack detection antigen of attenuated forms thereof; Described newcastle antigen is deactivated form, the form of work of improvement or the newcastle antigen of attenuated forms thereof; Described H9 subtype avian influenza antigen is deactivated form, the form of work of improvement or the H9 subtype avian influenza antigen of attenuated forms thereof.
3. vaccine combination according to claim 2, wherein, described eggs crack detection antigen is the full bacterium antigen of eggs crack detection C48-2 strain of deactivation, described newcastle antigen is the Avian pneumo-encephalitis virus LaSota strain totivirus antigen of deactivation, and described H9 subtype avian influenza antigen is the H9 subtype avian influenza antigen HL strain totivirus antigen of deactivation.
4. vaccine combination according to claim 1, wherein, described adjuvant comprises one or more in aqueous adjuvants, propolis adjuvant; Preferably, described aqueous adjuvants is Gel adjuvant.
5. vaccine combination according to claim 1, wherein, described adjuvant is the 5%-25%V/V of vaccine combination, is preferably 10%-15%V/V.
6. vaccine combination according to claim 1, wherein, described eggs crack detection antigenic content is>=5 × 10
9cFU/ml; Described newcastle antigenic content is>=6 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is>=6 × 10
8.5eID
50/ ml.
7. vaccine combination according to claim 6, wherein, described eggs crack detection antigenic content is 1 × 10
11cFU/ml; Described newcastle antigenic content is 7.5 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is 7.5 × 10
8.5eID
50/ ml.
8. prepare a method for vaccine combination described in any one of claim 1 ~ 7, described method comprises:
(1) propagation described eggs crack detection, described Avian pneumo-encephalitis virus, described H9 subtype avian influenza virus is cultivated respectively, deactivation respectively;
(2) be mixed in proportion described eggs crack detection full bacterium antigen, described newcastle totivirus antigen, described H9 subtype avian influenza totivirus antigen, and add adjuvant.
9. method according to claim 8, wherein, described antigen ratio is eggs crack detection antigenic content is>=5 × 10
9cFU/ml; Described newcastle antigenic content is>=6 × 10
8.5eID
50/ ml; Described H9 subtype avian influenza antigenic content is>=6 × 10
8.5eID
50/ ml.
10. the vaccine combination described in claim 1 ~ 7 prevents and/or treats the application in the medicine of eggs crack detection, newcastle, the infection of H9 subtype avian influenza in preparation.
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| CN109651489B (en) * | 2018-12-13 | 2019-09-06 | 广东渔跃生物技术有限公司 | A kind of preparation method of A groups of Yolk antibodies of avian pasteurella multocida capsular antigen |
| CN114288286A (en) * | 2022-01-11 | 2022-04-08 | 湖北省农业科学院畜牧兽医研究所 | Application of theaflavin in preparation of medicine for resisting avian pathogenic bacteria |
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