CN103721253A - Live avian encephalomylitis and henpox combined vaccine - Google Patents
Live avian encephalomylitis and henpox combined vaccine Download PDFInfo
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Abstract
The invention provides a live avian encephalomylitis and henpox combined vaccine. The combined vaccine consists of an antigen and a protective agent, wherein the antigen comprises henpox virus and avian encephalomylitis virus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.8505. The combined vaccine prepared by the invention can be used for preventing avian encephalomylitis and henpox at the same time to achieve the aim of preventing two viruses with one injection. Furthermore, The avian encephalomylitis virus screened by the invention is an attenuated virus, performs serial passage in an avian body through horizontal transmitted infection, does not have the phenomenon of virulence return, is stable in heredity, and accords with the standard of no virulence return of an avian encephalomylitis attenuated vaccine strain; the prepared vaccine can provide effective immune protection, and has good commercial development prospect.
Description
Technical field
The invention belongs to poultry vaccine preparing technical field, be specifically related to a kind of avian encephalomyelitis and fowlpox bigeminal live vaccine.
Background technology
Avian encephalomyelitis (AE) is a kind of main harm following chickling in 4 week age being caused by avian encephalomyclitis virus (AEV), take infringement, central nervous system causes the viral infectious of apyetous encephalitis as main pathological characters, show as that movement disorder, head and neck tremble and the nervous symptoms such as hallux paralysis, particularly head and cervical region trembles.Therefore, used is called " epidemic tremor ".After 4 week age following Chickens Infected AEV, sickness rate is generally 20%～60%, and mortality rate average out to 25% also can exceed 50%; Adult Chicken infects and does not show nervous symptoms, but can cause that one crosses property egg drop reduction, and fall is 16%～43%, have up to more than 60%, but there is not nervous symptoms, after approximately two weeks, lay eggs and can recover normal.But the hatching egg altitudinal belt poison producing this period presents two kinds of trend when hatching: a part of Embryo Gallus domesticus is in hatching lat theta-dead; Though another part can hatch shell, when going out shell or go out after shell in a couple of days can reveal any symptoms, in the feces of these chickling, with a large amount of virus, can cause the infection of other chickling.Avian encephalomyelitis all has generation in the whole world, susceptible chicken group all can fall ill at all seasons, and Winter-Spring occurs more.Nearly all chicken group finally can be by viral infection, but clinical onset rate is lower.
Nineteen thirty, Jones is the red chicken discovery AE on island, 2 week commercial generation in age Lip river first, shows as and trembles.Within 1931, observe twice and break out, occur in 1 week age and the 4 week age chicken group of different chicken houses, these two groups come from same breeder flock.Between afterwards 2 years, in New England Region, breaking out of AE all found in multiple states, is therefore called again " New England's disease ".1934, the cerebral tissue filtrate that Jones gets natural occurrence chicken copied primary disease through intracranial inoculation susceptible chicken.1938, Van Roekel etc. points out: be not can observe to tremble, when occurring trembling, there will be subsequently ataxia, cause for this reason disease is infectious and mainly encroaches on central nervous system, so propose to be called " infectiousness avian encephalomyelitis ", be condensed to afterwards " avian encephalomyelitis " and adopted by committee of U.S. Veterinary Medical Association in nineteen thirty-nine.1958, Schaaf reported for the first time by immunity and has successfully controlled primary disease.Nineteen sixty Calnek etc. has illustrated the epidemiology of AE, has developed subsequently oral vaccine, becomes the basis of world today's commercial chicken group control AE.This disease has now spread all over all over the world, and there is this pathogenetic report a lot of countries and regions.
, all there is the report that primary disease occurs in the many provinces and cities of China, and to aviculture, cause many-sided loss.Within 1980, first Zhang Zeji finds doubtful AE in the chickling group in Guangdong.Nineteen eighty-two Li Xinping makes a definite diagnosis this disease by histopathology.Nineteen eighty-three Bi Yingzuo makes a definite diagnosis this disease by epidemiology, histopathology.Yao great Ming, Qin Aijian, Zhao Zhenhua, Yao Yongxiu etc. have carried out the separation of AEV and development and the application test of evaluation work and vaccine in succession.There is successively the report of AE in the ground such as Liaoning, Jiangsu, Heilungkiang, Hebei, Shandong, the Inner Mongol, Fujian, Shanghai, Henan, Jiangxi, Shaanxi in recent years.The use Medulla Gallus domesticus sensitization test such as Huang Junming have detected the laying hen group of 17 of Harbin Cities, find that these laying hen groups are 95.1% to the pollution rate of AE.As can be seen here, this disease is extensively to exist in China.
Fowlpox (Fowl Pox) is that the one of the chicken that caused by bird pox virus and turkey is acute, contagious disease, all has generation all over the world.This sick spread speed is slow, and feature is that skin has proliferative lesion incrustation, upper digestive tract and respiratory tract diphtheria shape pathological changes.Skin-type case mortality is conventionally lower, than the easy rehabilitation of diphtheria type case.Diphtheria type is at nasal meatus, pharynx or larynx generation proliferative lesion, thereby causes dyspnea, dies of asphyxiation.Can make hen lay eggs and temporarily reduce, young chicken growth is slow.Now existingly commercially by bird pox virus, cause weak live vaccine, by Embryo Gallus domesticus or birds cell culture, manufacture.In fowlpox Prevalent district or diagnosis have fowlpox infect chicken house can use vaccine.
The chickling of the acute AE of breaking out is not had to effective Therapeutic Method.In finishing period vaccination, can control breeder flock and no longer infect after sexual maturity, also can prevent that virus from spreading through egg approach simultaneously.In addition, maternal antibody can protect chickling to infect at 2～3 weeks opposing AEV of susceptible.Commodity laying hen group also can carry out immunity inoculation, with a space egg drop reduction that prevents from being caused by AE.Many countries have been used for live vaccine the most chicken groups of immunity inoculation in the world, the domestic inactivated vaccine of having succeeded in developing, the area that can be used for out producing chicken or restriction use live vaccine, the current domestic live vaccine product that also do not have.
Summary of the invention
The object of this invention is to provide a kind of avian encephalomyelitis and fowlpox bigeminal live vaccine (hereinafter to be referred as bigeminy Seedling); the vaccine of being prepared as antigen by avian encephalomyelitis attenuated vaccine strain and attenuated fowlpox quail strain, the attack of vaccine of the present invention to current Chinese Poultry encephalomyelitis virus and the attack of bird pox virus provide good immune protection effectiveness.
Avian encephalomyelitis of the present invention and fowlpox bigeminal live vaccine, be comprised of antigen and protective agent, and wherein antigen includes bird pox virus and deposit number is the avian encephalomyclitis virus of CGMCC No.8505.
Avian encephalomyclitis virus YBF02 used in the present invention strain (avian encephalomyclitis virus Avian Encephalomyelitis virus), this Strain is deposited in China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, and deposit number is: CGMCC No.8505.
Protective agent wherein can be any viral vaccine protective agent using at present, for example sucrose and gelatin, and its mass percent final concentration in vaccine is respectively 5% and 1%;
In order to improve the protectant heat resistance of vaccine, in protective agent, be also added with sodium caseinate, the mass percent concentration of interpolation is 0.5%, makes vaccine after the long-term preservation of freezing conditions, effect still can reach standard.
In above-mentioned vaccine, added antibiotic, penicillin, streptomycin final concentration are 200 units/ml;
The preparation method of above-mentioned bigeminy Seedling, is by avian encephalomyclitis virus inoculated into chick embryo, and results blastochyle and Embryo Gallus domesticus brain, stomach, intestinal, pancreas, are got supernatant after centrifugal and obtained virus liquid mixed grinding, freeze thawing; Bird pox virus inoculation SPF Embryo Gallus domesticus or chick embryo fibroblast are cultivated, and results viral cultures, after then avian encephalomyclitis virus liquid and bird pox virus culture fluid mix, adds protective agent to make.
Bigeminy Seedling prepared by the present invention can prevent avian encephalomyelitis and fowlpox simultaneously, has reached the anti-object of a pin two.And; the avian encephalomyclitis virus of composition the present invention screening is low virulent strain; by horizontal transmission, infect in the continuous passage of chicken body; be showed no virulence and return strong phenomenon; genetic stability; meet avian encephalomyelitis attenuated vaccine strain avirulence and return strong standard, the vaccine of making can provide effective immunoprotection, has good commercialized development prospect.
The specific embodiment
Below in conjunction with specific embodiment, further describe the present invention, those of ordinary skill in the art, on the basis of technical solution of the present invention, can select the conventional method step in this area, and is not limited only to the concrete record of description embodiment of the present invention.
Embodiment 1: the selection-breeding of avian encephalomyclitis virus strain
Get the brain of suffering from avian encephalomyelitis chickling, pancreas and duodenum, under aseptic condition, grind, with sterile saline, making 1:5 dilutes, multigelation 3 times, centrifugal 30 minutes of 4000rpm, get after the filtration of supernatant filter, 10 of intracranial inoculation 1 age in days SPF chickling, 0.1ml/ only, movement disorder to be occurred, after the symptoms such as head and neck trembles, take a blood sample lethal, the aseptic brain of getting disease chickling, pancreas and duodenum, under aseptic condition, grind, with sterile saline, making 1:5 dilutes, multigelation 3 times, centrifugal 30 minutes of 4000rpm, getting supernatant saves backup as 1st generation separation poison (F1).Get the same method intracranial inoculation 1 age in days SPF chickling of F1 generation and go down to posterity, preparation 2nd generation separates poison (F2).So connect and passed for 4 generations.It is the weak poison of the adaptive avian encephalomyclitis virus of the non-Embryo Gallus domesticus of a strain that Embryo Gallus domesticus pathogenicity shows to separate poison.Get F4 that 1 age in days SPF chickling goes down to posterity for kind of a poison, after 10 times of dilutions, yolk sac inoculation 6 age in days SPF Embryo Gallus domesticus, continuation is hatched to 16 ages in days receipts poison at 37～38 ℃: draw blastochyle, observe idiosome activity and developmental state, claim idiosome to weigh and brain weight, check idiosome, embryo cerebral lesion (hemorrhage, hydrops, atrophy etc.); And get embryo brain, and stomach, intestinal, pancreas, does 1:5 dilution by blastochyle, adds dual anti-ly, grind, multigelation 3 times, centrifugal 30 minutes of 4000rpm, makes suspension standby.Reached for the 18th generation continuously like this.Choose the candidate's strain AEV YBF02 strain as vaccine development, and being deposited in China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, deposit number is: CGMCC No.8505.
AEV YBF02 strain seed culture of viruses safety testing: respectively by the 5th, 7, the 10 generations seed culture of viruses dilution of basic bacteria, get each 10 of SPF chicken in 1～10 week age, every thorn kind or oral 10
4eID
50yBF02 strain virus, observe 28, and record result.Result shows: AEV YBF02 strain basic bacteria the 5th, 7,10 generation seed culture of viruses to 1～10 week age SPF chicken be safe, without any avian encephalomyelitis specific symptom, do not affect growth promoter.
Table 1:AEV YBF02 strain safety testing result
Virulence is returned strong test: YBF02 strain F5 is connected and passed for 5 generations by the SPF chicken cohabitation infection approach in 3～8 week age for basic seed culture of viruses, get the viral intracranial inoculation 1 age in days SPF chickling respectively of each generation, thorn kind of inoculation 28 age in days SPF chickling and a peak laying hen, the each generation poison after observation is gone down to posterity is pathogenic to 1,28 age in days SPF chickens and laying hen.Result shows: the virulence of each generation is basic identical, and virulence is not returned by force, is still the weak poison that non-Embryo Gallus domesticus adapts to.Meet avian encephalomyelitis attenuated vaccine strain avirulence and return strong standard, can be used as vaccine strain and carry out commercialized development.
The avian encephalomyclitis virus attenuated vaccine strain YBF02 strain that above-mentioned seed culture of viruses safety testing result and virulence are returned strong result of the test explanation the present invention screening is safe, can be used for preparing vaccine.
The immune efficacy of AEV YBF02 strain detects
Respectively by the the the 1st, 7,10,14, the 18 generations seed culture of viruses dilution of AEV YBF02 strain, get 1～10 age each 10 of SPF chicken, every thorn kinds 10
3.0eID
50virus, after 28 days, with 10 same ages in days, contrast chicken with the SPF raising under condition, intracranial inoculation VR strain avian encephalomyelitis is poison by force, 10
3.0eID
50/ only, observe 28.Result shows: the immune efficacy difference of AEV YBF02 strain the 1st, 7,10,14,18 generation seed culture of viruses is not remarkable, has good immune protective, and 10 immune chickens are all protected at least 9, and 9 morbidities of 10 contrast chickens.Illustrate that AEV YBF02 strain seed culture of viruses its immunogenicity within 18 generations is basically identical, there is good immune protective.
Table 2:AEV YBF02 strain immune efficacy detects result of the test
The actual immune effect of the live vaccine made from YBF02 strain shows, with YBF02 strain, inoculates latter 14 days and ELISA antibody detected, and antibody 100% positive on the 28th, 80% above antibody positive can be maintained to 53 weeks.Simultaneously within 14,21,28 after exempting from, with the strong malicious VR strain of avian encephalomyclitis virus, carry out counteracting toxic substances in brain, result protective rate reaches 100%, proves that YBF02 strain can effectively carry out immunity to chickling, has the application prospect of preparing vaccine.And, when the bigeminal live vaccine of preparing the productions such as avian encephalomyelitis, the same Intervet of fowlpox bigeminal live vaccine, Italy with separation YBF02 strain carries out immune effect contrast test, with liking that moral scholar ELISA test kit detects serum antibody, the bigeminal live vaccine effect that positive rate is respectively 100%, 90%, 80%, prepare with YBF02 strain is best.Molecular biological analysis shows that the avian encephalomyclitis virus of screening and known avian encephalomyclitis virus exist the difference in sequence, is a novel Strain.
Preparation and the application of embodiment 2 avian encephalomyelitises, fowlpox bigeminal live vaccine
1 material
The bird pox virus Carnis Coturnicis japonicaeization low virulent strain that 1.1 seeds culture of viruses manufacture avian encephalomyelitises, fowlpox bigeminal live vaccine are used is identified, is preserved and supply by China Veterinery Drug Inspection Office; Also can be with other bird pox virus low virulent strain, but avian encephalomyclitis virus selects the deposit number of separation to be: the weak malicious YBF02 strain of CGMCC No.8505.
1.2 hatching egg and test chicken
1.2.1SPF hatching egg is purchased from the logical laboratory animal technology of Beijing Cimmeria dimension company limited.
1.2.27～14 age in days SPF chicken SPF hatching egg are purchased Beijing logical laboratory animal technology of Cimmeria dimension company limited, hatching voluntarily after buying back, and after hatching, negative pressure isolator is raised.
1.3 Other Instruments for seedling, reagent provide by YEBIO Bioengineering Co., Ltd of Qingdao.
2 methods
2.1 seedling materials are selected well-developed 6 age in days SPF Embryo Gallus domesticus; Select well-developed 11～12 age in days SPF Embryo Gallus domesticus or chick embryo fibroblast as seedling material.
The preparation of venom for 2.2 seedlings
2.2.1 seed culture of viruses is got in the preparation of avian encephalomyclitis virus liquid, with physiological saline solution or 100 times of dilutions of PBS do, yolk sac inoculation 6 age in days SPF Embryo Gallus domesticus, and every embryo 0.2ml, sealing pin hole after inoculation, puts 37 ℃ and continues to hatch to 16 ages in days receipts malicious.Before receipts poison, Embryo Gallus domesticus is put to 2～8 ℃ and freeze to death, check Embryo Gallus domesticus pathological changes, collect fluid of chick embryo, brain, gastrointestinal, pancreas, after weighing, with blastochyle 1:5, dilute homogenate freeze thawing 3 times, centrifugal 30 minutes of 4000r/min, get supernatant mixing and be sub-packed in sterile chamber, put 2～8 ℃ of preservations, be no more than 20.The inspection of semifinished product is carried out in sampling simultaneously.
2.2.2 the preparation of fowl pos disease venom
2.2.2.1 the inoculation of Embryo Gallus domesticus, results are got seed culture of viruses, with 100～1000 times of sterile saline dilutions, are inoculated in allantocherion 0.2ml or are inoculated in 0.1m1 in allantoic cavity, inoculation rear enclosed pin hole, put 35～37 ℃ and continue to hatch, needn't egg-turning, per sunshine egg 1 time.Embryo Gallus domesticus dead before 96 hours is discarded, get 96～120 hours dead germs and 120 hours embryos alive, every several embryos are one group, aseptic collection has the chick chorioallantoic membrane of edema or pox speckle, put the preservation of freezing in sterile chamber, get every embryo allantoic liquid mixed in equal amounts simultaneously, do steriling test and chicken red blood cell agglutination test.
2.2.2.2 the inoculation of cell, results are chosen well-grown cell monolayer bottle, by 0.1%～0.3% access seed culture of viruses of cultivating liquid measure, pH value are adjusted to 7.2, continue to cultivate.Observation of cell pathological changes after inoculation, when there is specific lesions in more than 75% cell, cell culture fluid is all discarded, by 10000ml cell bottle, add the fresh newborn Chinese liquid 150～200m1 containing 3%～5% serum or original fluid is left to 150～200ml or all stays, adding bead turns down cell monolayer, be harvested from sterilization container ,-15 ℃ of following preservations, should be no more than 1 month.
2.3 the inspection of semifinished product
2.3.1 steriling test is tested by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
2.3.2 viral level is measured avian encephalomyclitis virus liquid physiological saline solution or PBS is made to 10 times of serial dilutions, gets 10
-4, 10
-5, 10
-6, 10
-74 dilution factors, 10 pieces of each yolk sac inoculation 6 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml, separately gets 10 pieces and does not inoculate in contrast with a batch SPF Embryo Gallus domesticus.After 3 days that 37 ℃ were continued to hatch to normal hatching day, add up the chickling number that it does not go out shell embryo number, the dead young number of specificity and has avian encephalomyelitis specificity clinical symptoms, according to Reed-Muench method, calculate EID
50.Viral level answers>=10
5.5eID
50/ 0.2ml, contrast Embryo Gallus domesticus should at least 8 pieces of hatchings on time, and go out chickling all without the specificity clinical symptoms of avian encephalomyelitis.
2.3.3 fowlpox cell venom poison valency is measured the cell toxicant of results is mixed, and with sterile saline or 10 times of serial dilutions of PBS work, gets 10
-5, 10
-6, 10
-73 dilution factors, 5 pieces of each allantocherion vaccination instar chicken embryos on the 11st, 0.2ml/ embryo, after inoculation 96～120 hours, chick chorioallantoic membrane edema, thicken or have pox speckle, be judged to infection, according to Reed-Muench method, calculate EID
50.Viral level answers>=10
6.0eID
50/ 0.2ml, can be used for joining Seedling.
2.4 join Seedling and subpackage is mixed in the two-strain liquid being up to the standards in same container by a certain percentage, plumage part adds 5% sucrose gelatin used as stabilizers in accordance with regulations, final cane sugar content is 5%, gelatine content is 1%, add suitable antibiotic (penicillin, streptomycin final concentration are 200 units/ml) simultaneously, fully shake up quantitative separating.Every plumage part viral level: avian encephalomyclitis virus YBF02 strain>=10
3.5eID
50; Bird pox virus>=10
3.5eID
50.
In order to improve the protectant heat resistance of vaccine, in protective agent, be also added with 0.5% sodium caseinate.
For the ratio of avian encephalomyelitis and fowl pos disease venom, as long as can guarantee, these two kinds of diseases are produced to immune effect.
After 2.5 lyophilizing subpackages, carry out rapidly lyophilisation.
2.6 product inspection
2.6.1 the appearance color of character observation vaccine, character, depart from situation and add diluent with bottle wall after dissolving situation.
2.6.2 steriling test is tested by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
2.6.3 mycoplasma check is tested by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
2.6.4 exogenous virus check is tested by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
2.6.6 safety verification is with 10 of 7～14 age in days SPF chickens, and each intramuscular injection vaccine 0.2ml (containing 10 plumage parts) observes 21, should all be good for work, without any part or systemic adverse reactions.
2.6.7 the following method of efficacy test is appointed and is selected one.
2.6.7.1 avian encephalomyelitis part
(1) filter the check of fowlpox method and press the dated plumage part of label, vaccine is diluted to l plumage part/0.2ml with sterile saline or PBS, 2～8 ℃ of 12000g are centrifugal 15 minutes, through 0.22um filter, filter 1 time, 0.10um filter filters 2 times, remakes 10 times of serial dilutions, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, respectively 10 pieces of inoculation instar chicken embryos on the 6th in yolk sac, every embryo 0.2m1, separately gets 10 pieces with batch SPF Embryo Gallus domesticus in contrast.Hatch (died discards in 48h, does not enter record) to 3 days of normal hatching day, add up it and do not go out shell embryo number, the dead young number of specificity and have avian encephalomyelitis specificity clinical symptoms chickling number, by Reed-Muench method calculating EID for 37 ℃
50, every plumage part answers>=10
3.0eID
50.Contrast Embryo Gallus domesticus should at least 8 pieces of hatchings on time, and go out chickling all without the specificity clinical symptoms of avian encephalomyelitis.
(2) chloroform is pressed label except the check of fowlpox method and is indicated plumage part, vaccine is diluted to 1 plumage part/0.2ml with sterile saline or PBS, get 10ml and add equal amounts of chloroform, on impeller, concussion mixes twice, every minor tick 30 seconds, centrifugal 10 minutes of 600g, continue again to do 10 times of serial dilutions, get 3 suitable dilution factors, inoculate respectively 12 piece of 6 age in days SPF Embryo Gallus domesticus, every embryo AS approach inoculation 0.2ml, separately establish 20 pieces of contrasts, in 48 hours, dead Embryo Gallus domesticus is disregarded, record chicken embryo death, situation, each dilution factor Embryo Gallus domesticus is hatched respectively, after hatching the 3rd day (latter 18～19 days of inoculation), Embryo Gallus domesticus do not hatched in record.Dead Embryo Gallus domesticus, paralysis chicken and ataxia chicken are judged to the positive.Matched group at least 75% hatching, every plumage part vaccine>=10
2.5eID
50.
2.3.7.2 fowlpox part
(1) viral level is measured and is pressed the dated plumage part of label, vaccine is diluted to 1 plumage part/0.2ml with sterile saline or PBS, continue again to do 10 times of serial dilutions, get 3 suitable dilution factors, through allantocherion, inoculate respectively 5 pieces of 11～12 age in days SPF Embryo Gallus domesticus, every embryo 0.2m1, puts 37 ℃ and hatches 96～120 hours, chick chorioallantoic membrane edema thickens or occurs that pox speckle is judged to infection, calculates EID
50, every plumage part viral level should be not less than 10
3.0eID
50.
(2) with Embryo Gallus domesticus check, press label and indicate plumage part, vaccine is diluted to 0.01 plumage part/0.2ml with sterile saline, through 10 pieces of allantocherion vaccination 11～12 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml, put 37 ℃ and hatch 96～120 hours, all chick chorioallantoic membranes are answered edema to thicken or are occurred pox speckle.
2.6.8 residual moisture is measured and is undertaken by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
2.6.9 vacuum is measured and is undertaken by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010.
3 results
3.1 inspection of semifinished product results
3.1.1 steriling test is tested by existing < < Chinese veterinary pharmacopoeia > > appendix method, all asepsis growth.
3.1.2 viral level mensuration avian encephalomyelitis venom viral level is 10
5.5eID
50/ 0.2ml; Bird pox virus venom viral level is 10
6.0eID
50/ 0.2ml.
3.2 product inspection results
3.2.1 the micro-yellow Sponge Porosity agglomerate of character, during dandle, sample is easy to depart from bottle wall.After adding diluent, all dissolve rapidly.
3.2.2 10 bottles of steriling test vaccine randomizations, with physiological saline solution or PBS, recover commercial weight respectively, by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010, test respectively for every bottle.All without antibacterial, fungus growth.
3.2.3 5 bottles of mycoplasma check vaccine randomizations, with physiological saline solution or PBS, recover commercial weight and mix respectively, by version < < Chinese veterinary pharmacopoeia > > appendix method in 2010, testing.Vaccine is all grown without mycoplasma.
3.2.4 exogenous virus check
3.2.4.1 3 bottles of Embryo Gallus domesticus inspection technique vaccine samplings, recover to do to mix after suitably dilution after commercial weight with physiological saline solution or PBS, mixes with the anti-avian encephalomyclitis virus specific serum of equivalent, in 37 ℃ with l hour.Inoculated into chick embryo is tested.Result shows, every group of equal 10/10 survival of check Embryo Gallus domesticus, and fetus all grows normally, and allantocherion is all without pathological changes, and Embryo Gallus domesticus liquid is all negative to 1% chicken red blood cell agglutination test.
3.2.4.2 cytoscopy method
3.2.4.2.1 get the each 0.2ml of chick embryo fibroblast that 2 bottles of the each inoculations of the sample of processing have grown up to good monolayer, observe 7.All acellular pathological changes of check, and without erythrocyte adsorption phenomena.
3.2.4.2.2 the each 2ml of chick embryo fibroblast that 2 bottles of the each inoculations of the sample of processing have grown up to good monolayer is got in avian leukosis virus check, establish negative control (normal cell) and positive-virus compares simultaneously, with sample, connect and passed for 3 generations, get 3 generation cell culture (comprising sample and all matched groups) freeze thawing 3 times, and mark, carry out ELISA detection with avian leukosis virus test kit.Sample survey result is all negative.
3.2.4.3 chicken inspection technique sample is done suitably dilution with physiological saline solution or PBS, inoculate each 20 of the SPF chicken of 14 ages in days, every eye dripping, collunarium are inoculated 10 plumage parts simultaneously, intramuscular injection 100 plumage parts, exempt from latter 21 days, with dosage repeated inoculation 1 time, the 1st inoculation blood sampling in latter 42 days, carries out the Serum Antibody Detection about cause of disease as stated above.Result demonstration, after check vaccination chicken, in 42 days, chicken is all healthy, without the part or General Symptoms and untoward reaction or the death that cause because of vaccine.While carrying out Serum Antibody Detection, except the specific antibody that this vaccine produces, without the antibody of other cause of diseases, exist.The results are shown in Table 3.
Table 3: the testing result of the exogenous virus of vaccine prepared by the present invention
In sum, Embryo Gallus domesticus inspection technique, cytoscopy method and chicken inspection technique result all illustrate that vaccine exogenous virus is up to the standards.
3.2.5 3 bottles of safety verification vaccine samplings, recover to do suitably dilution after commercial weight with physiological saline solution or PBS respectively, 10 of intramuscular injection 10 age in days SPF chickens, and every 0.2m1 (containing 10 plumage parts), observes 21.Result shows, inoculates chicken all without any untoward reaction, and 10/10 strong living.
3.2.6 efficacy test
3.2.6.1 avian encephalomyelitis part
(1) filter the check of fowlpox method and press the dated plumage part of label, vaccine is diluted to l plumage part/0.2ml with sterile saline or PBS, 2～8 ℃ of 12000g are centrifugal 15 minutes, through 0.22um filter, filter 1 time, 0.10um filter filters 2 times, remakes 10 times of serial dilutions, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, respectively 10 pieces of inoculation instar chicken embryos on the 6th in yolk sac, every embryo 0.2m1, separately gets 10 pieces with batch SPF Embryo Gallus domesticus in contrast.Hatch (died discards in 48h, does not enter record) to 3 days of normal hatching day, add up it and do not go out shell embryo number, the dead young number of specificity and have avian encephalomyelitis specificity clinical symptoms chickling number, by Reed-Muench method calculating EID for 37 ℃
50, every plumage part answers>=10
3.0eID
50.Contrast Embryo Gallus domesticus should at least 8 pieces of hatchings on time, and go out chickling all without the specificity clinical symptoms of avian encephalomyelitis.Assay: every plumage part viral level is 10
3.1eID
50, vaccine potency is up to the standards.
(2) chloroform is pressed label except the check of fowlpox method and is indicated plumage part, vaccine is diluted to 1 plumage part/0.2ml with sterile saline or PBS, get 10ml and add equal amounts of chloroform, on impeller, concussion mixes twice, every minor tick 30 seconds, centrifugal 10 minutes of 600g, continue again to do 10 times of serial dilutions, get 3 suitable dilution factors, inoculate respectively 12 piece of 6 age in days SPF Embryo Gallus domesticus, every embryo AS approach inoculation 0.2ml, separately establish 20 pieces of contrasts, in 48 hours, dead Embryo Gallus domesticus is disregarded, record chicken embryo death, situation, each dilution factor Embryo Gallus domesticus is hatched respectively, after hatching the 3rd day (latter 18～19 days of inoculation), Embryo Gallus domesticus do not hatched in record.Dead Embryo Gallus domesticus, paralysis chicken and ataxia chicken are judged to the positive.Matched group at least 75% hatching, every plumage part vaccine>=10
2.5eID
50.Assay: every plumage part viral level is 10
2.9eID
50, vaccine potency is up to the standards.
3.2.6.1 fowlpox part
(1) viral level is measured and is pressed the dated plumage part of label, vaccine is diluted to 1 plumage part/0.2ml with sterile saline or PBS, continue again to do 10 times of serial dilutions, get 3 suitable dilution factors, through allantocherion, inoculate respectively 5 pieces of 11～12 age in days SPF Embryo Gallus domesticus, every embryo 0.2m1, puts 37 ℃ and hatches 96～120 hours, chick chorioallantoic membrane edema thickens or occurs that pox speckle is judged to infection, calculates EID
50, every plumage part viral level should be not less than 10
3.0eID
50.Assay: every plumage part viral level is 10
3.7eID
50, vaccine potency is up to the standards.
(2) with Embryo Gallus domesticus check, press label and indicate plumage part, vaccine is diluted to 0.01 plumage part/0.2ml with sterile saline, through 10 pieces of allantocherion vaccination 11～12 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml, put 37 ℃ and hatch 96～120 hours, all chick chorioallantoic membranes are answered edema to thicken or are occurred pox speckle.Assay: 10/10 chick chorioallantoic membrane edema also occurs pox speckle, and vaccine potency is up to the standards.
3.2.7 residual moisture is measured 4 bottles of vaccine samplings and is tested with boulton process.Product test sample residual moisture content is 2.0%～2.7%, all≤4%.Illustrate that vaccine residual moisture is measured check all qualified.
3.2.8 vacuum mensuration vaccine is tested with vacuum leak detector respectively.Product test sample is all purple aura.Illustrate that vaccine vacuum is measured check all qualified.
For the holding time of vaccine is provided, the application is optimized the protective agent of vaccine, includes mass percent concentration and be the sodium caseinate (1ml vaccine is considered as to 1g) of 5% sucrose, 1% gelatin and 0.5% in the vaccine of finally making.By adding above-mentioned component, can make vaccine after long-term preservation, can also maintain vigour.Under 2～8 ℃ of conditions, place 180, its antibody titer is substantially constant, places 360, and its antibody blocking rate decline 2-3 percentage point, still higher than specified standard; And with the product of common protective agent lyophilizing, under 2～8 ℃ of conditions, place 180, its antibody titer can reach specified standard substantially, but it is very fast to place after 360 days and 720 days antibody titer decline, especially after 720 days, can not re-use, and can only be by scrap processing.
Claims (7)
1. avian encephalomyelitis and a fowlpox bigeminy vaccine, is characterized in that, described vaccine is comprised of antigen and protective agent, and wherein antigen includes bird pox virus and deposit number is the avian encephalomyclitis virus of CGMCC No.8505.
2. bigeminy vaccine as claimed in claim 1, is characterized in that described avian encephalomyclitis virus content>=10
3.5eID
50; Bird pox virus content>=10
3.5eID
50.
3. bigeminy vaccine as claimed in claim 1, is characterized in that described protective agent is sucrose and gelatin, and its mass percent final concentration in vaccine is respectively 5% and 1%.
4. bigeminy vaccine as claimed in claim 1, is characterized in that being added with sodium caseinate in described protective agent, and the mass percent concentration of interpolation is 0.5%.
5. bigeminy vaccine as claimed in claim 1, is characterized in that adding antibiotic in described protective agent.
6. bigeminy vaccine as claimed in claim 5, is characterized in that described antibiotic is penicillin and streptomycin, and final concentration is 200 units/ml.
7. the preparation method of bigeminy vaccine claimed in claim 1, is by avian encephalomyclitis virus inoculated into chick embryo, and results blastochyle and Embryo Gallus domesticus brain, stomach, intestinal, pancreas, are got supernatant after centrifugal and obtained virus liquid mixed grinding, freeze thawing; Bird pox virus inoculation SPF Embryo Gallus domesticus or chick embryo fibroblast are cultivated, and results viral cultures, after then avian encephalomyclitis virus liquid and bird pox virus culture fluid mix, adds protective agent to make.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106177937A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | Chicken egg drop syndrome and avian encephalomyelitis bivalent inactivated vaccine and preparation method thereof |
| CN109718371A (en) * | 2018-12-20 | 2019-05-07 | 天津瑞普生物技术股份有限公司 | A kind of newcastle disease, the preparation method of avian encephalomyelitis bivalent inactivated vaccine |
| JP2023503430A (en) * | 2019-11-29 | 2023-01-30 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハー | Triple vaccine against Avibacterium paragallinarum, avian encephalomyelitis virus and fowlpox virus |
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| CN102427827A (en) * | 2009-05-14 | 2012-04-25 | 拜尔动物保健有限责任公司 | Enhanced immune response in avian species |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106177937A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | Chicken egg drop syndrome and avian encephalomyelitis bivalent inactivated vaccine and preparation method thereof |
| CN109718371A (en) * | 2018-12-20 | 2019-05-07 | 天津瑞普生物技术股份有限公司 | A kind of newcastle disease, the preparation method of avian encephalomyelitis bivalent inactivated vaccine |
| JP2023503430A (en) * | 2019-11-29 | 2023-01-30 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハー | Triple vaccine against Avibacterium paragallinarum, avian encephalomyelitis virus and fowlpox virus |
| JP7342264B2 (en) | 2019-11-29 | 2023-09-11 | ベーリンガー インゲルハイム フェトメディカ ゲーエムベーハー | Triple vaccine against Avibacterium paragarinarum, avian encephalomyelitis virus and fowlpox virus |
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| CN103721253B (en) | 2015-06-10 |
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