CN103143010A - Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof - Google Patents
Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof Download PDFInfo
- Publication number
- CN103143010A CN103143010A CN2013100424714A CN201310042471A CN103143010A CN 103143010 A CN103143010 A CN 103143010A CN 2013100424714 A CN2013100424714 A CN 2013100424714A CN 201310042471 A CN201310042471 A CN 201310042471A CN 103143010 A CN103143010 A CN 103143010A
- Authority
- CN
- China
- Prior art keywords
- strain
- newcastle disease
- vaccine
- virus
- deactivation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to triad inactivated vaccine for newcastle disease, bird flu (H9 subtype) and escherichia coli disease and a preparation method thereof. The vaccine is an effective coping strategy aiming at a prevalence situation of mixed infection of the newcastle disease, the H9 subtype bird flu and the escherichia coli, and two kinds of disease can be controlled at the same by means of immunization at one time. According to the vaccine, a La Sota strain of a newcastle disease virus, a LG1 strain of an H9 subtype bird flu virus, a QL1 strain (O1) of bird source escherichia coli, QL2 strain (O2) of the bird source escherichia coli, QL78 strain (O78) of the bird source escherichia coli, and QL36 strain (O36) of the bird source escherichia coli serve as vaccine reserve virus strains, and the vaccine is formed through antigen liquid preparation, inactivation, concentration and mixing according to a certain proportion. The vaccine and the preparation method are related with the La Sota strain of the newcastle disease virus, the H9 subtype bird flu virus and the bird source escherichia coli which are all domestic prevalence virus strains, and current prevalence disease can be effectively prevented.
Description
Technical field
The present invention relates to newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine, belong to the veterinary biologics field.
Background technology
Newcastle (Newcastle disease, ND) is that a kind of birds that is caused by Avian pneumo-encephalitis virus (Newcastle disease virus, NDV) is acute, the height contagious disease.Newcastle often is the septicemia process clinically, principal character is dyspnea, dysentery-discharge green or yellow loose stool, nervous function disorder-clonospasm, head and neck torsion, opisthotonus, movement disorder and numb symptom occur, mucosa and serous coat are hemorrhage and downright bad.This disease morbidity is anxious, fatality rate is high, and the serious threat of development formation to aviculture is decided to be the category-A deadly infectious disease by World Organization for Animal Health, is one of main disease that affects world's poultry husbandry always.
the fowl colibacillosis claims again avian colibacillosis (Avian Colibacillosis), refer to partly or entirely by colon bacillus (Escherichiacoli, E.coli) disease of caused part or systemic infection, can cause embryonic death, umbilicus is scorching, septicemia, granuloma, yolk peritonitis and panophthalmitis (Calnek BW chief editor, Gao Fu, Su Jingliang master translates). disease of poultry. the 10th edition. Beijing: Chinese agriculture publishing house, 1999, a series of symptom such as 158-171), it is a kind of common transmittable disease of chicken and the turkey in 2 to 12 ages in week, cause serious economic loss to aviculture.E. coli serotype is extremely many, and known escherichia coli have 73 kinds of thalline (O) antigen 1s, 03 kind of surface (K) antigen 1,60 kinds of flagellar antigens.Many serotypes of pathogenic Escherichia coli can cause the many animals morbidity.World many countries has been done probe, determines that the most normal serotype relevant with fowl diseases is O1, O2, O36 and O78, seldom is separated to other serotype.Domestic scholars continue Hua Bin, Huo Longfei and Liu Ruitian etc. also must appear same conclusions (Japanese plum is rich. the epidemic characteristic of current avian colibacillosis and prevention and control measure. modern farming enterprise's veterinary .2011.12,39-40).
Low pathogenicity bird flu (Mildly pathogenic avian influenzavirus, MPAIV) under laboratory condition, infected chicken death rate is very low, but during with E.coli mixed infection, mortality rate has raising in various degree, and laying rate of chicken falls sharply, and broiler growth reduces (Swayne D E, Pathobiology of H5N2Mexican avian infiuenza virus infections of chickens.Vet Pathel.1997,34 (6): 557-567).Wherein, the H9 subtype avian influenza virus be cause that the low pathogenicity bird flu occurs, outbalance is a kind of in popular factors.
Along with the fast development of poultry husbandry and the pollution of breeding environment, in recent years the laying hen disease especially infectious disease be on the increase with complicated.Chicken group mixed infection is serious, and M ﹠ M is all higher, has caused huge economic loss to aquaculture.Wherein, newcastle disease, low pathogenicity bird flu and three kinds of disease mixed infections of fowl colibacillosis are comparatively common, lose also comparatively serious.Under this background, develop a kind of vaccine of these three kinds of diseases that can prevent simultaneously and seem particularly important.
Summary of the invention
The objective of the invention is by a strain H9 subtype avian influenza virus strain of Avian pneumo-encephalitis virus La Sota strain, separation voluntarily, the four strain fowl source colon bacillus bacterial strains (serotype is respectively: O1, O2, O78, O36) that separate voluntarily, a kind of vaccine that can prevent simultaneously newcastle disease, H9 subtype avian influenza and three kinds of diseases of fowl colibacillosis is provided, and primary immune response can prevent three kinds of diseases.
For solving the problems of the technologies described above, technical program of the present invention lies in:
1. newcastle disease, bird flu (H9 hypotype), the colibacillosis triple inactivated vaccine contains NDV La Sota Strain virus LaSota strain, the QL1 strain of the LG1 strain of H9 subtype avian influenza virus and fowl source colon bacillus bacterial strain serotype O1, the QL2 strain of serotype O2, the QL36 strain of serotype O36, the QL78 strain of serotype O78, above bacterial strain was delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 with on 01 31st, 2013, the center preservation of China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms, deposit number: the QL1 strain is CGMCC No.7241, the QL2 strain is CGMCC No.7242, the QL36 strain is that CGMCC No.7243 and QL78 strain are CGMCC No.7244.
2. the preparation method of newcastle disease of the present invention, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine:
(1) breed with Embryo Gallus domesticus that newcastle disease virus, H9 subtype avian influenza virus are prepared into deactivation according to a conventional method respectively and through concentrated virus liquid; Cultivate respectively that four type fowl source colon bacillus bacterial strains are prepared into deactivation and through concentrated bacterium liquid with the martin's bouillon that contains 0.1% trace salt solution, then the volume ratio mixing of press 3:3:2:2;
(3) deactivation with above preparation also is prepared into water after the concentrated mixed bacteria liquid of concentrated Newcastle disease venom, H9 subtype avian influenza virus liquid and four type fowl source colon bacillus deactivations mixes by the 1:1:1 volume ratio, then prepares according to a conventional method the deactivation Adjuvanted vaccines.
3. can make the inoculation chicken obtain newcastle disease virus, H9 subtype avian influenza virus and the domestic popular bacterium strain of fowl source colon bacillus are produced the effectively immunity of prevention for newcastle disease, bird flu (H9 hypotype), the colibacillosis triple inactivated vaccine of chicken inoculation effective dose.
The specific embodiment
1. production of vaccine bacterium strain
(1) bacterium strain source
Vaccine preparation of the present invention is Avian pneumo-encephalitis virus La Sota strain with strain, identifies, takes care of and supply by China Veterinery Drug Inspection Office.
It is a strain H9 subtype avian influenza virus strain LG1 strain that the present invention produces with strain, for my unit separates voluntarily, preserves, and commercialization, name of product: newcastle disease, bird flu (H9 hypotype) bivalent inactivated vaccine, product authentication code: veterinary drug new word (2010) 150252152.
It is four strain fowl source coli strain QL1 strains, QL2 strain, QL78 strain, QL36 that the present invention produces with bacterial strain, is respectively four serotype: O1, O2, O36, O78, is to be separated by the field by the inventor to obtain.Above bacterial strain was delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 with on 01 31st, 2013, the center preservation of China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms, deposit number: the QL1 strain is CGMCC No.7241, the QL2 strain is CGMCC No.7242, and the QL36 strain is that CGMCC No.7243 and QL78 strain are CGMCC No.7244.
(2) bacterium strain characteristic
1) immunogenicity
Avian pneumo-encephalitis virus La Sota strain is to the chickling collunarium immunity of 1 monthly age, and minimum immunity amount is answered≤5000EID
50, immunogenicity is good.
H9 subtype avian influenza virus strain LG1 strain immunity chicken HI antibody geometric mean titer is answered 〉=6.5log2, and immunogenicity is good.
Fowl source colon bacillus bacterial strain is with 10 of 1 monthly age SPF chickens, every cervical region subcutaneous injection vaccine 0.3ml, after 21 days together with 10 of the identical contrast chickens of condition, the mixed bacteria liquid 0.5ml(of each muscle or lumbar injection virulent strain QL1 strain (O1), QL2 strain (O2), QL78 strain (O78), QL36 strain (O36) counts 1:1:1:1 according to bacterium and mixes, and contains altogether viable bacteria 2～500,000,000).Observed 10, the contrast chicken should all fall ill or be dead, cut open and all have typical cytopathic after inspection (reaction exists ++ more than); The immunity chicken is protected 8 at least.
Criterion:
± slight clinical response is arranged, subtract food, the white feces of row 1～3 day, internal organs are without the visible pathological changes of naked eyes.
+ clinical subtracting eats more than 4 days or stops eating more than 1 day, and row's yellow-white feces cutd open inspection and sees that heart or liver have slight cellulose exudative inflammation more than 4 day.
++ clinical in (+), visible obviously pericardial effusion and cellulose exudative inflammation.
+++clinical has the severe cardiac chitonitis, and sticks together with liver or pleura with (+).
++ ++ test chicken is dead, and other are with (+++).
2) virulence
Newcastle disease virus La Sota strain is done 50～150 times of dilutions with seed culture of viruses with sterile saline, in allantoic cavity, inoculation 10 age in days SPF Embryo Gallus domesticus is 10, every embryo 0.1～0.3ml, putting 36～37 ℃ continues to hatch, Embryo Gallus domesticus in the inoculation after 24～120 hours dead more than 70%, fetus has obvious hyperemia, hemorrhage lesion.
H9 subtype avian influenza virus strain LG1 strain is done 5～15 times of dilutions with seed culture of viruses with sterile saline, and in allantoic cavity, inoculation 10 age in days SPF Embryo Gallus domesticus is 20, and every embryo 0.1～0.3ml puts 37 ℃ and hatches, and in 48～96 hours, has 15 chicken embryo deaths at least after inoculation.
Fowl source colon bacillus QL1 strain (O1), QL2 strain (O2), QL36 strain (O36), QL78 strain (O78) bacterial strain contain 0.1% trace salt solution, and (manufacture method is seen People's Republic of China's regulations, two 〇 〇 〇 versions, 81 pages) martin's bouillon 20～24 hours cultivate bacterium liquid 0.3～0.6ml(and contain viable bacteria 2～500,000,000), muscle or 1 monthly age of lumbar injection 5 of SPF chickens, obvious clinical symptoms should appear, death or live chickens internal organs have obvious pathological changes in 14 days, should be more than (++).
3) fowl source colon bacillus serotype
According to domestic present fowl source colibacillosis popularity, the present invention has chosen bacterial strain that in four serotypes, immunogenicity is good as the seedling bacterial strain, is respectively O1, O2, O36, the QL1 strain in O78 serotype, QL2 strain, QL36 strain, QL78 strain.
2. newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine
(1) antigen liquid preparation
1) Newcastle disease venom preparation
1. the seed culture of viruses breeding is diluted 5000～15000 times with seed culture of viruses with sterile saline, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1～0.3ml, death in 72～96 hours and the obvious Embryo Gallus domesticus of lesion after the choosing inoculation, gather in the crops respectively Embryo Gallus domesticus liquid (allantoic cavity liquid and amniotic fluid), be loaded in sterilization container, Embryo Gallus domesticus liquid red cell agglutination valency is not less than 1:512.
2. inoculation is got and is produced with kind of a poison, does 5000～15000 times of dilutions with sterile saline, inoculation 9～11 age in days susceptible Embryo Gallus domesticus in allantoic cavity, and every embryo 0.1～0.3ml, after inoculation, the sealing pin hole, put 36～37 ℃ and continue to hatch, needn't egg-turning.
3. hatch and observe after egg inoculation egg 1 time at per sunshine, dead Embryo Gallus domesticus before 60 hours is discarded.The photograph egg was 1 time in every 4～6 hours later on, and dead Embryo Gallus domesticus takes out at any time, until 96 hours, no matter death whether, is all taken out, air chamber is upwards upright, put 2～8 ℃ cooling 4～24 hours.
4. the Embryo Gallus domesticus that results will be cooling takes out, and with iodine tincture disinfection air chamber position, then divests air chamber section chorion with aseptic method, throws off membrana putaminis, breaks allantocherion and amniotic membrane (guarding against yolk breaks), the absorption blastochyle.Tackle each Embryo Gallus domesticus and carefully check, all fetuses are corrupt, blastochyle is muddy and the suspicious person of any pollution is arranged, and discarding need not.Dead germ and the embryo of living are gathered in the crops respectively, and often several Embryo Gallus domesticus are divided into one group, draw blastochyle and put into the container of same sterilization, and the blood clotting valency is measured in every group of sampling, and the blood clotting valency is lower than the 1:256(micromethod) person should discard.Steriling test is made in the blastochyle sampling of results, answers asepsis growth, puts below-15 ℃ and preserves, and should be no more than 6 months.
2) H9 subtype avian influenza virus liquid preparation
1. the seed culture of viruses breeding is produced H9 subtype avian influenza virus strain LG1 strain with seed culture of viruses and is done 500～1500 times of dilutions with sterile saline, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1～0.3ml, gather in the crops Embryo Gallus domesticus liquid dead in 24～96 hours, be loaded in sterilization container, the red cell agglutination valency is not less than 1:256.
2. inoculation is got to produce and is used seed culture of viruses, does 500～1500 times of dilutions with sterile saline, the healthy susceptible Embryo Gallus domesticus of inoculation 9～11 ages in days in allantoic cavity, every embryo 0.1ml.After inoculation, the sealing pin hole, put 36～37 ℃ and continue to hatch, needn't egg-turning.
3. after hatching and observing egg inoculation, the photograph egg was 1 time in 24 hours, discarded dead germ.The photograph egg was 1 time in every 4～8 hours later on, and dead Embryo Gallus domesticus takes out at any time, until 96 hours, no matter death whether, is all taken out, air chamber is upwards upright, put 2～8 ℃ cooling 4～24 hours.
4. the Embryo Gallus domesticus that results will be cooling takes out, and with iodine tincture disinfection air chamber position, then divests air chamber section chorion with aseptic method, throws off membrana putaminis, breaks allantocherion and amniotic membrane (yolk is broken), absorption Embryo Gallus domesticus liquid.In the results blastochyle, should check one by one Embryo Gallus domesticus, as corrupt in fetus, blastochyle is muddy and have the suspicious person of any pollution to discard need not.Dead germ and the embryo of living are gathered in the crops respectively, and often the blastochyle of several Embryo Gallus domesticus is mixed into one group, draw blastochyle and put into the container of same sterilization, and the blood clotting valency is measured in every group of sampling, and dead germ liquid blood clotting valency should be not less than the 1:256(micromethod), the blastochyle of living blood clotting valency should be not less than 1:64.Steriling test is made in the blastochyle sampling of results, answers asepsis growth, puts below-15 ℃ and preserves, and should be no more than 3 months.
3) fowl source colon bacillus bacterium solution preparation
with four fowl source colon bacillus bacterial strain QL1 strains (O1), QL2 strain (O2), QL78 strain (O78), the martin's bouillon that contains 0.1% trace salt solution is inoculated respectively in QL36 strain (O36), and (manufacture method is seen People's Republic of China's regulations, two 〇 〇 〇 versions, 339 pages of appendix), cultivated 20～24 hours for 35～36 ℃, gather in the crops respectively that the volume ratio according to 3:3:2:2 mixes after bacterium liquid, again mixed bacterium liquid is contained the martin's bouillon of 0.1% trace salt solution according to 2%～4% inoculum concentration inoculation, 35～36 ℃ of aerobic culture 10～14 were as a child gathered in the crops bacterium liquid afterwards.
(3) deactivation
The deactivation of Newcastle disease venom adds formalin by 0.1～0.2%, through 37 ℃ of deactivations 16 hours, therebetween every jolting in 4～6 hours 1 time.
The deactivation of H9 subtype avian influenza virus liquid adds formalin by 0.1～0.2%, through 37 ℃ of deactivations 48 hours, therebetween every jolting in 4～6 hours 1 time.
The colon bacillus bacterium liquid deactivation of fowl source adds formalin by 0.4%～0.8%, through 38 ℃ of deactivations 48～72 hours, during every jolting in 4～6 hours once.
(4) concentrated
The Newcastle disease venom is concentrated, and virus liquid after with deactivation is condensed into 30%～50% of full dose with hollow fiber membrane ultrafiltration device.
H9 subtype avian influenza virus liquid is concentrated, and virus liquid after with deactivation is condensed into 40～60% of full dose with hollow fiber membrane ultrafiltration device.
Fowl source colon bacillus bacterium liquid concentrated with inactivated bacterial liquid in 2～8 ℃ standing 2～3 days, take out and abandon supernatant, be condensed into 50%～70% of full dose.
(5) emulsifying
1) oil phase preparation
Get 94 parts of injection white oils, Jia Siben-806 part after mixing, add 2 parts of aluminium stearate, with add be stirred to transparent till, 121 ℃ of autoclavings 30 minutes are put under room temperature and are saved backup.
2) water preparation
Get 4 parts of tween 80s of sterilization, add 96 parts of deactivation blastochyles (newcastle disease inactivation of viruses liquid, H9 subtype avian influenza inactivation of viruses liquid mix in the ratio of 1:1:1 with fowl source colon bacillus inactivated bacterial liquid), shake well dissolves tween 80 fully.
3) emulsifying
Get 2 parts of oil phases and be put in colloid mill or Emulsion cylinder, start the motor slow rotation and stir, slowly add simultaneously 1 part of water, stirred 2～5 minutes with 10000r/min after adding again, add 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%.After emulsifying, get 3～5ml with 3000r/min centrifugal 15 minutes, if lamination is arranged, should repeat to stir once.
3. newcastle disease, bird flu (H9 hypotype), the product inspection of colibacillosis triple inactivated vaccine
(1) character
Outward appearance should be milky Emulsion.
The dosage form water-in-oil type.Get 1 cleaning suction pipe, draw a small amount of vaccine and drip in cold water, except the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, and through the centrifugal 15min of 3000r/min, managing the water of separating out at the end should no more than 0.5ml.
Viscosity by existing " Chinese veterinary pharmacopoeia " (Chinese veterinary pharmacopoeia committee. People's Republic of China's veterinary drug allusion quotation, two three ones of 〇 one 〇 versions. Chinese agriculture publishing house, 2011, the present invention is referred to as " existing " Chinese veterinary pharmacopoeia " ") prescriptive procedure tests, and should be up to specification.
(2) steriling test
Test by existing " Chinese veterinary pharmacopoeia " prescriptive procedure, answer asepsis growth.
(3) safety verification
With 3～5 age in week 10 of SPF chickens, the subcutaneous or intramuscular injection vaccine 2ml of each cervical region observed 14, any part and the systemic adverse reactions that are caused by vaccine injection should not occur.
(4) efficacy test
1) newcastle part
With 15 of 30～60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l(1/25 plumage parts), another 5 compare.Rear 21～28 days of inoculation, (the CVCCAV1611 strain) 10 of every strong poison of each intramuscular injection newcastle disease virus Beijing Strain of chicken
5.0ELD
50, observed 14.Matched group should be all dead, and immune group should be protected 7 at least.
2) bird flu part
With 15 of 7～10 age in days SPF chickens, wherein 10 each muscle or cervical region subcutaneous injection vaccine 0.3ml, do not inoculate in contrast for another 5.After 21 days, blood sampling, separation of serum is with bird flu virus H9 hypotype antigen measuring HI antibody.Immunity chicken HI antibody geometric mean titer is answered 〉=6.5log2, and the contrast chicken should be negative.
3) fowl source colon bacillus part
With 10 of 1 monthly age SPF chickens, every cervical region subcutaneous injection vaccine 0.3ml, after 21 days together with 8 of the identical contrast chickens of condition, the mixed bacteria liquid 0.5ml(of each muscle or lumbar injection fowl source colon bacillus bacterial strain QL1 strain (O1), QL2 strain (O2), QL36 strain (O36), QL78 strain (O78) counts 1:1:1:1 according to bacterium and mixes, and contains altogether viable bacteria 2～500,000,000 CFU).Observed 10, the contrast chicken should all fall ill or be dead, cut open and all have typical cytopathic after inspection (reaction exists ++ more than); The immunity chicken is protected 8 at least.
(5) formaldehyde, the antiseptic mercurials determination of residual amount
Measure by existing " Chinese veterinary pharmacopoeia " prescriptive procedure, should be up to specification.
The microorganism fungus kind that the present invention relates to
The Newcastle disease strain La Sota strain that the present invention relates to is identified, is taken care of and supply by China Veterinery Drug Inspection Office.
The H9 subtype avian influenza virus strain LG1 strain that the present invention relates to is for the inventor separates voluntarily, preserves.And commercialization, name of product: newcastle disease, bird flu (H9 hypotype) bivalent inactivated vaccine, product authentication code: veterinary drug new word (2010) 150252152.
the fowl source colon bacillus (Escherichia coli) that the present invention relates to has four bacterial strains, QL1 strain (O1), QL2 strain (O2), QL78 strain (O78), QL36 strain (O36), for the inventor separates voluntarily, above bacterial strain was delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 with on 01 31st, 2013, the center preservation of China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms, deposit number: the QL1 strain is CGMCCNo.7241, the QL2 strain is CGMCCNo.7242, the QL36 strain is that CGMCCNo.7243 and QL78 strain are CGMCCNo.7244.
Positive effect of the present invention
The present invention relates to a kind of newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine and preparation method thereof.This vaccine is in domestic birds epidemic disease, and the popular situation of newcastle, H9 subtype avian influenza and fowl source many serotype of colon bacillus mixed infection has proposed one and successfully managed strategy, and accomplishes two kinds of diseases of primary immune response prevention and control simultaneously.This vaccine utilizes Avian pneumo-encephalitis virus La Sota strain, the LG1 strain of H9 subtype avian influenza virus and fowl source colon bacillus QL1 strain (O1), QL2 strain (O2), QL36 strain (O36), QL78 strain (O78) as vaccine preparation bacterium strain, through antigen liquid preparation, deactivation, concentrate, be mixed with according to a certain percentage and form.The Avian pneumo-encephalitis virus that the present invention relates to, H9 subtype avian influenza strain, fowl source colon bacillus bacterial strain is domestic popular bacterium strain, can effectively prevent present pandemic.
Embodiment
Following examples further illustrate the present invention.
Embodiment 1
---production prepares with the bacterium kind
(1) preparation with seed culture of viruses is produced in newcastle disease virus La Sota strain
Seed culture of viruses with 10000 times of sterile saline dilutions, is inoculated 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1ml, death in 72～96 hours and the obvious Embryo Gallus domesticus of lesion after the choosing inoculation are gathered in the crops respectively Embryo Gallus domesticus liquid (allantoic cavity liquid and amniotic fluid), are loaded in sterilization container.To check aseptic and red cell agglutination valency to be not less than the 1:512(micromethod) Embryo Gallus domesticus liquid mix, quantitative separating, harvest date, Virus passages etc. are indicated in freezing preservation.
(2) H9 subtype avian influenza virus LG1 strain is produced and is prepared with seed culture of viruses
Seed culture of viruses is done 1000 times of dilutions with sterile saline, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1ml gathers in the crops Embryo Gallus domesticus liquid dead in 24～96 hours, is loaded in sterilization container.The Embryo Gallus domesticus liquid that the aseptic and red cell agglutination valency of check is not less than 1:256 mixes, quantitative separating, freezing preservation.
(3) production of fowl source colon bacillus bacterial strain prepares with seed culture of viruses
The first order seed breeding is inoculated in respectively fowl source colon bacillus QL1 strain (O1), QL2 strain (O2), QL36 strain (O36), each strain freeze-drying lactobacillus of QL78 strain (O78) in martin's bouillon, cultivated 24 hours for 36 ℃, then cultivate on streak inoculation maconkey agar flat board, 10 of colonies typicals choosing pink or brick-red, neat in edge, smooth surface, bacterium colony protuberance are mixed in a small amount of martin's bouillon, inoculate some of Martin's agar slants, put 36 ℃ of cultivations 24 hours, as first order seed.
The secondary seed breeding is got first order seed and is inoculated in martin's bouillon, cultivates 24 hours for 36 ℃, as secondary seed.
Embodiment 2
---the vaccine preparation
(1) antigen liquid preparation
1) Newcastle disease venom preparation
1. inoculation is got and is produced with kind of a poison, does 10000 times of dilutions with sterile saline, inoculation 9～11 age in days susceptible Embryo Gallus domesticus in allantoic cavity, and every embryo 0.1～0.3ml, after inoculation, the sealing pin hole, put 36～37 ℃ and continue to hatch, needn't egg-turning.
2. hatch and observe after egg inoculation egg 1 time at per sunshine, dead Embryo Gallus domesticus before 60 hours is discarded.The photograph egg was 1 time in every 4～6 hours later on, and dead Embryo Gallus domesticus takes out at any time, until 96 hours, no matter death whether, is all taken out, air chamber is upwards upright, put 2～8 ℃ cooling 4～24 hours.
3. results, deactivation and concentrated Embryo Gallus domesticus that will be cooling take out, and with iodine tincture disinfection air chamber position, then divest air chamber section chorion with aseptic method, throw off membrana putaminis, break allantocherion and amniotic membrane (guarding against yolk breaks), the absorption blastochyle.Tackle each Embryo Gallus domesticus and carefully check, all fetuses is corrupt, it is muddy and the suspicious person of any pollution arranged to discard blastochyle.Dead germ and the embryo of living are gathered in the crops respectively, and often several Embryo Gallus domesticus are divided into one group, draw blastochyle and put into the container of same sterilization, and the blood clotting valency is measured in every group of sampling, discards the blood clotting valency lower than the 1:256(micromethod) blastochyle.Steriling test is made in the blastochyle sampling of results, all asepsis growth; After virus liquid is mixed, then by 0.1%(V/V) add formalin, through 37 ℃ of deactivations 16 hours, therebetween every jolting in 4 hours 1 time; Virus liquid with hollow fiber membrane ultrafiltration device after with deactivation is condensed into the 40%(V/V of full dose).
2) H9 subtype avian influenza virus liquid preparation
1. inoculation is got to produce and is used seed culture of viruses, does 1000 times of dilutions with sterile saline, the healthy susceptible Embryo Gallus domesticus of inoculation 10 ages in days in allantoic cavity, every embryo 0.1ml.After inoculation, the sealing pin hole, put 37 ℃ and continue to hatch, needn't egg-turning.
2. after hatching and observing egg inoculation, the photograph egg was 1 time in 24 hours, discarded dead germ.The photograph egg was 1 time in every 4 hours later on, and dead Embryo Gallus domesticus takes out at any time, until 96 hours, no matter death whether, is all taken out, air chamber is upwards upright, put 2～8 ℃ cooling.
3. results, deactivation and concentrated Embryo Gallus domesticus that will cooling 4～24 hours take out, and with iodine tincture disinfection air chamber position, then divest air chamber section chorion with aseptic operation, throw off membrana putaminis, break allantocherion and amniotic membrane (yolk is broken), absorption Embryo Gallus domesticus liquid.In the results blastochyle, should check one by one Embryo Gallus domesticus, as corrupt in fetus, blastochyle is muddy and have the suspicious person of any pollution to discard need not.
Dead germ and the embryo of living are gathered in the crops respectively, and often the blastochyle of several Embryo Gallus domesticus is mixed into one group, draw blastochyle and are put in the container of same sterilization, and the blood clotting valency is measured in every group of sampling, dead germ liquid blood clotting valency 〉=1:256, the blastochyle of living blood clotting valency 〉=1:64; Virus liquid is mixed rear by 0.1%(V/V) add formalin, through 37 ℃ of deactivations 48 hours, therebetween every jolting in 4 hours 1 time; Virus liquid with hollow fiber membrane ultrafiltration device after with deactivation concentrated 2 times (V/V).
3) fowl source colon bacillus bacterium solution preparation
The secondary seed solution of fowl source colon bacillus QL1 strain (O1), QL2 strain (O2), QL36 strain (O36), each bacterial strain of QL78 strain (O78) is mixed according to the volume ratio of 3:3:2:2, (manufacture method is seen People's Republic of China's regulations to inoculate the martin's bouillon that contains 0.1% trace salt solution, two 〇 〇 〇 versions, 339 pages of appendix), inoculum concentration is for cultivating 3% of base unit weight, cultivated 12 hours results bacterium liquid for 36 ℃; By 0.6%(V/V) add formalin, through 38 ℃ of deactivations 60 hours, during every jolting in 4 hours once; Again with inactivated bacterial liquid in 2～8 ℃ standing 3 days, take out and abandon supernatant, be concentrated into the 60%(V/V of full dose).
(2) emulsifying
1) oil phase preparation
Get 94 parts of injection white oils (stereometer), Jia Siben-806 part (stereometer) after mixing, adds 2 parts of aluminium stearate (quality meter), with add be stirred to transparent till, 121 ℃ of autoclavings 30 minutes.
2) water preparation
Get sterilization 4 parts of tween 80s (stereometer), add antigen liquid 96 parts (stereometer) (this antigen liquid is to be mixed by the 1:1:1 volume ratio with fowl source colon bacillus inactivated bacterial liquid by newcastle disease inactivation of viruses liquid, H9 subtype avian influenza inactivation of viruses liquid), shake well dissolves tween 80 fully.
3) emulsifying
Get 2 parts of oil phases and be put in colloid mill or Emulsion cylinder, start the motor slow rotation and stir, slowly add simultaneously 1 part of water, stirred 5 minutes with 10000r/min after adding again, add 1% thimerosal solution before stopping stirring, making its ultimate density is 0.01%.After emulsifying, get 3～5ml with 3000r/min centrifugal 15 minutes, without lamination.
Embodiment 3
---the vaccine product inspection
It is 6 batches of newcastle diseases, bird flu (H9 hypotype), the colibacillosis triple inactivated vaccine that the applicant prepares by technical scheme provided by the invention that the present embodiment uses vaccine, and lot number is 301,302,303,304,305 and 306 batches.
(1) character
Outward appearance should be milky Emulsion.
The dosage form water-in-oil type.Get 1 cleaning suction pipe, draw a small amount of vaccine and drip in cold water, except the 1st, be showed no diffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, through the centrifugal 15min of 3000r/min, the pipe end without water separate out.
Viscosity by existing " Chinese veterinary pharmacopoeia " (Chinese veterinary pharmacopoeia committee. People's Republic of China's veterinary drug allusion quotation, two three ones of 〇 one 〇 versions. Chinese agriculture publishing house, 2011, the present invention is referred to as " existing " Chinese veterinary pharmacopoeia " ") prescriptive procedure tests, result is 38cP, and is up to specification.
(2) steriling test
Test by existing " Chinese veterinary pharmacopoeia " prescriptive procedure, all asepsis growth.
(3) safety verification
With 3～5 age in week 10 of SPF chickens, the subcutaneous or intramuscular injection vaccine 2ml of each cervical region observed 14, any part and the systemic adverse reactions (seeing Table 1) that are caused by vaccine injection did not all occur.
Table 1 newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine finished product safety verification result
| The vaccine lot number | Injected dose | Chicken quantity | General reaction | The injection site |
[0135]?
| 301 | 1ml | 10 | Nothing | Absorb good |
| 302 | 1ml | 10 | Nothing | Absorb good |
| 303 | 1ml | 10 | Nothing | Absorb good |
| 304 | 1ml | 10 | Nothing | Absorb good |
| 305 | 1ml | 10 | Nothing | Absorb good |
| 306 | 1ml | 10 | Nothing | Absorb good |
(4) efficacy test
1) newcastle part
With 15 of 30～60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l(1/25 plumage parts), another 5 compare.Rear 21～28 days of inoculation, (the CVCC AV1611 strain) 10 of every strong poison of each intramuscular injection newcastle disease virus Beijing Strain of chicken
5.0ELD
50(available from China Veterinery Drug Inspection Office) observed 14.Matched group all dead (5/5), each immune group are all protected 10/10 protection (seeing Table 2).
Table 2 newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine finished product efficacy test result (1)
| The vaccine lot number | Injected dose | Chicken quantity | The injection site | Observe natural law | The protection number |
| 301 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| 302 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| 303 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| 304 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| 305 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| 306 | 20μl | 10 | Intramuscular injection | 14 | 10/10 |
| Matched group | - | 5 | - | 14 | 0/5 |
2) bird flu part
With 15 of 7～10 age in days SPF chickens, wherein 10 each muscle or cervical region subcutaneous injection vaccine 0.3ml, do not inoculate in contrast for another 5.After 21 days, blood sampling, separation of serum is with bird flu virus H9 hypotype antigen measuring HI antibody.Immunity chicken HI antibody geometric mean titer all 〉=1:90.5, be respectively: 1:137.2,1:147,1:181,1:181,1:238.9 and 1:208, matched group negative (seeing Table 3).
Table 3 newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine finished product efficacy test result (2)
| The vaccine lot number | Injected dose | Chicken quantity | The injection site | Observe natural law | HI average antibody level |
| 301 | 0.3ml | 10 | Intramuscular injection | 21 | 1:137.2 |
| 302 | 0.3ml | 10 | Intramuscular injection | 21 | 1:147 |
| 303 | 0.3ml | 10 | Intramuscular injection | 21 | 1:181 |
| 304 | 0.3ml | 10 | Cervical region is subcutaneous | 21 | 1:181 |
| 305 | 0.3ml | 10 | Intramuscular injection | 21 | 1:238.9 |
| 306 | 0.3ml | 10 | Intramuscular injection | 21 | 1:208 |
| Matched group | - | 5 | - | 21 | 0 |
3) fowl source colon bacillus part
With 8 of 1 monthly age SPF chickens, every cervical region subcutaneous injection vaccine 0.3ml, after 21 days together with 8 of the identical contrast chickens of condition, the mixed bacteria liquid 0.5ml(of each muscle or lumbar injection fowl source colon bacillus bacterial strain QL1 strain (O1), QL2 strain (O2), QL78 strain (O78), QL36 strain (O36) counts 1:1:1:1 according to bacterium and mixes, and contains altogether viable bacteria 2～500,000,000 CFU).Observed 10, the contrast whole Mortalities of chicken (5/5), cut open and all have typical cytopathic after inspection (reaction exists ++ more than); Each immune group chicken 01,04 and 05 group are 9/10 protection, and other 3 groups are 10/10 protection (seeing Table 4).
Table 4 newcastle disease, bird flu (H9 hypotype), colibacillosis bivalent inactivated vaccine finished product efficacy test result (3)
| The vaccine lot number | Injected dose | Chicken quantity | The injection site | Observe natural law | The protection number |
| 301 | 0.3ml | 10 | Intramuscular injection | 21 | 9/10 |
| 302 | 0.3ml | 10 | Intramuscular injection | 21 | 10/10 |
| 303 | 0.3ml | 10 | Intramuscular injection | 21 | 10/10 |
| 304 | 0.3ml | 10 | Intramuscular injection | 21 | 9/10 |
| 305 | 0.3ml | 10 | Intramuscular injection | 21 | 9/10 |
| 306 | 0.3ml | 10 | Intramuscular injection | 21 | 10/10 |
| Matched group | - | 5 | - | 21 | 0/5 |
(5) formaldehyde, the antiseptic mercurials determination of residual amount
Measure content of formaldehyde all less than 0.2% by existing " Chinese veterinary pharmacopoeia " prescriptive procedure, up to specification.
Claims (3)
1. a newcastle disease, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine, it is characterized in that this vaccine contains the newcastle disease virus of deactivation, the H9 subtype avian influenza virus of deactivation, the O1 of deactivation, O2, O78 and O36 four type fowl source colon bacillus.
2. the preparation method of a newcastle disease claimed in claim 1, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine is characterized in that:
(1) breed with Embryo Gallus domesticus that newcastle disease virus, H9 subtype avian influenza virus are prepared into deactivation according to a conventional method respectively and through concentrated virus liquid; Four type fowl source colon bacillus bacterium liquid with the martin's bouillon that contains 0.1% trace salt solution is cultivated respectively through deactivation and concentrated, then are mixed into the concentrated bacterium liquid of four type fowl source colon bacillus deactivations by the volume ratio of 3:3:2:2;
(2) deactivation with above preparation also is prepared into water after the concentrated mixed bacteria liquid of concentrated Newcastle disease venom, H9 subtype avian influenza virus liquid and four type fowl source colon bacillus deactivations mixes by the 1:1:1 volume ratio, then prepares according to a conventional method the deactivation Adjuvanted vaccines.
3. the application of a newcastle disease claimed in claim 1, bird flu (H9 hypotype), colibacillosis triple inactivated vaccine is characterized in that newcastle disease, bird flu (H9 hypotype), the colibacillosis triple inactivated vaccine to the chicken inoculation effective dose can make the inoculation chicken obtain newcastle disease virus, H9 subtype avian influenza virus and the domestic popular bacterium strain of fowl source colon bacillus are produced the effectively immunity of prevention.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310042471.4A CN103143010B (en) | 2013-02-04 | 2013-02-04 | Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310042471.4A CN103143010B (en) | 2013-02-04 | 2013-02-04 | Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103143010A true CN103143010A (en) | 2013-06-12 |
| CN103143010B CN103143010B (en) | 2014-11-26 |
Family
ID=48541517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310042471.4A Active CN103143010B (en) | 2013-02-04 | 2013-02-04 | Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103143010B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484411A (en) * | 2013-10-10 | 2014-01-01 | 南京天邦生物科技有限公司 | Avian Escherichia coli vaccine strain (NN strain) and application thereof |
| CN103768591A (en) * | 2014-02-17 | 2014-05-07 | 齐鲁动物保健品有限公司 | Duck hemorrhagic ovaritis and bird flu (H9 subtype) duplex inactivated vaccine and preparation method thereof |
| CN104774796A (en) * | 2015-04-29 | 2015-07-15 | 中国农业科学院上海兽医研究所 | Inactivated vaccine for poultry pathogenic escherichia coli and preparation method thereof |
-
2013
- 2013-02-04 CN CN201310042471.4A patent/CN103143010B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 中国兽药典委员会: "《中华人民共和国兽药典:兽药使用指南.生物制品卷》", 31 March 2006 * |
| 刘明生等: "蛋鸡新城疫、H9亚型禽流感和大肠杆菌混合感染的诊治", 《中国动物检疫》 * |
| 庄向生等: "鸡大肠杆菌多价灭活疫苗的研制", 《福建农业学报》 * |
| 戚建刚，王道坤: "肉鸡新城疫、H9亚型禽流感与大肠杆菌混感物药物防制", 《兽药市场指南》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484411A (en) * | 2013-10-10 | 2014-01-01 | 南京天邦生物科技有限公司 | Avian Escherichia coli vaccine strain (NN strain) and application thereof |
| CN103768591A (en) * | 2014-02-17 | 2014-05-07 | 齐鲁动物保健品有限公司 | Duck hemorrhagic ovaritis and bird flu (H9 subtype) duplex inactivated vaccine and preparation method thereof |
| CN104774796A (en) * | 2015-04-29 | 2015-07-15 | 中国农业科学院上海兽医研究所 | Inactivated vaccine for poultry pathogenic escherichia coli and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103143010B (en) | 2014-11-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104530232B (en) | The preparation method of duck virus hepatitis refined yolk antibody | |
| CN104922663B (en) | A kind of newcastle disease and H9 subtype avian influenza bigeminy vaccines | |
| CN102492661B (en) | Preparation method and application of immunization preparation for controlling novel duck reovirus | |
| CN110251671B (en) | Preparation method of goose astrovirus egg yolk antigen-antibody complex | |
| CN105031638B (en) | A kind of newcastle disease, bird flu and infectious bursa of Fabricius triple inactivated vaccine | |
| CN103820397A (en) | Muscovy duck parvovirus and application thereof | |
| CN102068695B (en) | Method for producing quadruple inactivated vaccine for newcastle disease, infectious bronchitis, avian influenza (H9 subtype) and infectious bursal disease | |
| CN105949307B (en) | It is a kind of for preventing and treating a kind Yolk antibody for duck source gosling plague | |
| CN105950564A (en) | Muscovy duck hepatitis virus and method for preparing muscovy duck hepatitis virus refined egg yolk antibody by adopting same | |
| CN102816740A (en) | Avian influenza virus, inactivated vaccine and method for preparing same | |
| CN102125687B (en) | Production method for bivalent inactivated vaccine for infectious serositis of ducks | |
| CN103833848A (en) | Egg yolk antibody for preventing and curing muscovy duck parvovirus diseases | |
| CN104258389B (en) | A kind of vaccine combination and its preparation method and application | |
| CN101475639A (en) | Mixed yolk antibody for preventing and treating duck infectious serositis and preparation thereof | |
| CN103495167B (en) | A kind of preparation method of chicken infection bursal disease composite live vaccine | |
| CN103143010B (en) | Triad inactivated vaccine for newcastle disease and bird flu (H9 subtype) and escherichia coli disease and preparation method thereof | |
| CN102743751A (en) | Preparation method of newcastle diseases, infectious bursal disease bigeminy high immunity-yolk-antibody lyophilized powder | |
| CN105920596B (en) | Muscovy duck parvovirus disease and gosling plague bivalent vaccine | |
| CN102600463A (en) | Production method of trivalent inactivated vaccine preventing duck infectious serositis | |
| CN104922665A (en) | Triple inactivated vaccine for newcastle disease, infectious bronchitis and H9 subtype avian influenza | |
| CN109207436A (en) | One plant of 4 type aviadenovirus strain of I group and its application | |
| CN102166355B (en) | Quadruple inactivated vaccine for preventing chicken diseases | |
| CN103055310B (en) | Bigeminal inactivated vaccine for avian influenza (H9 sub type) and Escherichia coli and preparation method thereof | |
| CN103007266B (en) | Predominant serum type vaccines in duck colibacillosis area, and preparation method and application thereof | |
| CN102688487A (en) | Production method of newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease virus (IBD) and viral arthritis (VA) four-joint inactivated vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |