CN103028112A - Newcastle disease and infectious bronchitis bivalent live vaccine - Google Patents
Newcastle disease and infectious bronchitis bivalent live vaccine Download PDFInfo
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Abstract
The invention relates to a newcastle disease and infectious bronchitis bivalent live vaccine which comprises antigens and vaccine assistants, wherein the antigens are an infectious bronchitis virus with a preservation serial number of CGMCC (China General Microbiological Culture Collection Center) No.6752 and a newcastle disease virus. The prepared bivalent vaccine can prevent newcastle diseases and infectious bronchitis, and achieves dual purposes. In addition, the screened infectious bronchitis virus forming the bivalent vaccine is a low virulent strain obtained through passage for many times, and a side effect arising from the existing infectious bronchitis virus as an antigen is reduced.
Description
Technical field
The invention belongs to the poultry vaccine technical field, be specifically related to a kind of newcastle disease and infectiousness bronchitis bigeminy live vaccine.
Background technology
Infectious bronchitis of chicken (IB) is a kind of acute height contact respiratory tract disease that is caused by infectious bronchitis virus (IBV), with trachea rale, cough, sneeze; Children chicken rhinorrhea, the laying hen egg production reduces and Egg―qualites drops to feature; Ill chicken often causes death because of respiratory tract, kidney or digestive tract infection.Primary disease at first was reported in the U.S. by Schalk etc. in 1931, determined by Beach and Hudson that the cause of disease of primary disease was virus in 1936.So far primary disease worldwide is widely current, and brings serious harm to poultry husbandry.In China, Kuang Ronglu (1972) finds primary disease first in Guangdong; To the later stage seventies in last century, Zheng Furong etc. respectively from plant with chicken group in isolate IBV at Beijing, Xin Chaoan etc. in Guangzhou at Shanghai, Ai Guoguang etc. at Hangzhou, Wu Zhida etc.The Rong Jun bow waits (1997) to be separated to Glandular Stomach Type avian infectious bronchitis virus D971 strain in suffering from the sick chicken of glandular stomach enlargement, return the SPF chickling with this strain and cause typical nephritis and proventriculitis pathological changes, and from artificial challenge SPF chickling, be recovered to avian infectious bronchitis virus.Wang Yudong, Fan Gencheng etc. (1998) suffer among the glandular stomach enlargement morbidity chicken group in the Shandong Province and gather pathological material of disease; passed continuously for 5～6 generations by Embryo Gallus domesticus; test, carry out physicochemical property mensuration, malicious valency mensuration, neutralization test etc. with annulus trachealis tissue culture through electron microscopic observation, animal recurrence and immunoprotection; the cause of disease of the infectious disease take the glandular stomach enlargement as principal character that proof is at present popular is coronavirus, with the scorching viral QX strain of the avian infectious arm of this strain called after Glandular Stomach Type.
The foreign scholar is in Europe, at first respectively at calendar year 2001 the Russia east and Russian western part in 2002 be separated to IBV class QX strain (Bochkov et al, 2006), this strain can cause that chicken suffers from proventriculitis, nephritis, and is similar to the QX strain.Many European countries in succession were separated to IBV class QX strain and comprise Germany, Holland, Belgium, France, Italy (Worthington et al, 2008 from 2003; Monne et al, 2008), Poland (Domanska-Blicharz et al, 2006), Britain (Gough et al, 2008) and Spain (Dolz et al, 2009).Various correlational study reports show that whole European Region has the existence of class QX strain virus.Obviously, it is very large threat that IB class QX strain cultivates for fowl, and there is no at present special vaccine for IB class QX strain.The foreign scholar makes the live vaccine of IB class QX strain, the effectiveness of the line correlation of going forward side by side and safety testing with the continuous passage more than 80 on Embryo Gallus domesticus of class QX strain for causing weak IB L1148 strain.For the SPF chicken also or carry the chicken of maternal antibody show, after the Seedling alive that IB L1148 strain is made is exempted from, can both provide effective protection to the counteracting toxic substances of class QX strain D388.
Newcastle disease (newcastle disease, ND) is the birds acute infectious disease that is caused by newcastle disease virus (NDV), and spread speed is fast, has brought huge threat for for a long time China's aviculture.Although chicken Newcastle disease live vaccine is effective, cheap, easy to use, but its duration of immunity is short, immunity is subject to maternal antibody and disturbs, and cause easily the respiratory tract side effect, interference experiment chamber diagnosis virus is separated, therefore, since 1980, used the newcastle disease inactivated vaccine to become gradually China's chicken house and prevented and treated one of conventional method of newcastle disease.
Immunity inoculation is the effective means of control infectious bronchitis of chicken.At present, the research that is used for prevention Glandular Stomach Type Infections Bronchitis vaccine both at home and abroad is more, but all not yet commercializations.Because IBV serotype is numerous, the good low virulent strain of cross-protection is not arranged always on the market simultaneously, Glandular Stomach Type IB can not get effective control.Therefore, the new IB attenuated vaccine with better protection of screening acquisition just seems particularly important.
Summary of the invention
The purpose of this invention is to provide a kind of newcastle disease and infectiousness bronchitis bigeminy live vaccine (hereinafter to be referred as the bigeminy Seedling); the vaccine strain that is namely prepared as antigen by new Glandular Stomach Type infectious bronchitis of chicken attenuated vaccine strain and Avian pneumo-encephalitis virus La Sota strain, vaccine of the present invention can provide good immune protection effectiveness to the attack of the popular Glandular Stomach Type avian infectious bronchitis virus of present China and the attack of Avian pneumo-encephalitis virus.
Bigeminy Seedling of the present invention is comprised of antigen and vaccine adjuvant, and wherein antigen is that deposit number is avian infectious bronchitis virus and the newcastle disease virus of CGMCCNo.6752.
The preparation method of above-mentioned bigeminy Seedling is that the results blastochyle obtains viral scale-up medium with behind newcastle disease virus and the avian infectious bronchitis virus difference inoculated into chick embryo; Then with after Avian pneumo-encephalitis virus and the mixing of avian infectious bronchitis virus scale-up medium, the adding protective agent is made.
Described protective agent is sucrose and gelatin, and its final concentration in vaccine is respectively 5% and 1%.
The bigeminy Seedling of the present invention's preparation can prevent newcastle disease and infectious bronchitis simultaneously, has reached the purpose that a pin two is prevented.And, form the avian infectious bronchitis virus of the present invention's screening for the low virulent strain of the acquisition of repeatedly going down to posterity, reduced the side effect that existing infectious bronchitis virus brings as antigen.
Description of drawings
Fig. 1: the antibody Fluctuation of bigeminy Seedling of the present invention.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment.What it should be appreciated by those skilled in the art is, under the prerequisite that does not depart from spirit and scope of the invention, can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replace and all fall into protection scope of the present invention.
One, the screening of the weak malicious YB160 strain of infectious bronchitis of chicken of the present invention
Infectious bronchitis is malicious by force.A little less than using 10 ~ 11 age in days SPF Embryo Gallus domesticus that the strong poison of this infectious bronchitis is carried out continuous passage and causes, per generation is by 4 pieces of SPF Embryo Gallus domesticus of allantoic cavity inoculation, connects to reach for 160 generations, causes weak evaluation.Get 10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160 Strain that go down to posterity inoculation and infect the SPF chicken of 1 age in days.The result since the 80th generation Strain without dead chicken, the 90th generation strain inoculation chicken begins without observing pathological changes, the 130th pickup kind chicken begins inorganization and learns pathological changes.A little less than illustrating that the 130th generation of IBV caused fully, chicken had good safety.Simultaneously with strain 130 of the present invention, 140,150 160 generations poison immune 3 age in days SPF chickens respectively, in immunity rear 14 days, use the homology strong virus attack.Immune component poison rate 0/10～1/10, protective rate 9/10～10/10, contrast component poison rate 10/10.Presentation of results strain of the present invention has good immune protective.For the ease of distinguishing with former strain, the embryo toxicity that 160 generations of going down to posterity are weakened is decided to be primordial seed E1 generation, and called after YB160 strain.Choose IBV YB160 strain as candidate's strain of vaccine development.Avian infectious bronchitis virus of the present invention (Avian infectious bronchitis virus) YB160 strain, this Strain is deposited in the China Committee for Culture Collection of Microorganisms common micro-organisms center of Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica on October 31st, 2012, and deposit number is: CGMCC No.6752.
Two, the safety of IBVYB160 strain detects
Virulence is returned strong test and is utilized IBV virulent strain to pass continuously for 160 generations on Embryo Gallus domesticus, cultivates the good low virulent strain IBV-YB160 strain of immunogenicity.In this low virulent strain E1 generation,, artificial challenge and cohabitation infection on SPF chicken body passed respectively for 5 generations continuously, and all are normal for test chicken, inoculates rear 5 days and cut open detector tube, glandular stomach, kidney etc. to be showed no the naked eyes pathological change; Test finds that virus separates trachea cotton swab and the 4th generation of glandular stomach and rise minute less than virus, and minute less than virus, liver divides less than virus from 2nd generation kidney from the 3rd generation.Minute malicious obstructed organ virus isolated rate is different in the tissue homogenates such as test chicken trachea, glandular stomach, kidney, and trachea, glandular stomach virus isolated rate are higher, illustrate that this strain is mainly in trachea, two organs propagation of glandular stomach.Along with the increase virus of generation is outwards drained also gradually minimizing, a minute malicious rate reduces gradually.The same rule of artificial challenge also appears in the level infection test, and just cohabitation infection divides the relative artificial challenge of malicious rate low with generation.This strain of above experiment results proved is artificial challenge or horizontal transmission infects in the continuous passage of chicken body no matter, be showed no virulence and return strong phenomenon, genetic stability meets infectious bronchitis of chicken attenuated vaccine strain avirulence and returns strong standard, can be used as vaccine strain and carries out commercialized development.
IBV YB160 strain seed culture of viruses safety testing get YB160 strain E2, E4, E7, E10, E13 for seed culture of viruses with 10
5.0EID
501 of collunarium (about 0.03ml) is inoculated respectively 1,3 age in days SPF chickens, Continuous Observation 10 days.The result inoculates all strong living of chicken, all without any abnormal response.The results are shown in Table 1,2.
The safety testing result of table 1 IBV YB160 strain seed culture of viruses
The safety testing result of table 2 IBV YB160 strain seed culture of viruses
The chicken infectivity bronchitis virus attenuated vaccine strain YB160 strain that above-mentioned IBV YB160 strain virulence is returned strong result of the test and the present invention of seed culture of viruses safety testing presentation of results screening is safe, can be used for preparing vaccine.
Three, the immune efficacy of IBV YB160 strain detects
With E2, E4, E7, E10, E13 generation YB160 strain seed culture of viruses chick embryo allantoic liquid, with 5 * 10
2.0EID
50Dosage, to 1 age in days SPF chicken collunarium, rear 14 days of immunity, together with the contrast chicken, respectively blood sampling, separation of serum carries out IB HI antibody titer and measures.Simultaneously every chicken is with each 1 of the strong malicious eye dripping of IB separated strain, the collunarium of 10 times of dilutions, and behind the counteracting toxic substances the 5th day, gather the trachea swab of every chicken, inoculate 5 pieces of 10 age in days SPF Embryo Gallus domesticus through allantoic cavity, the 0.1ml/ embryo was hatched observation to 144 hours.The result shows that the geometrical mean of the serum HI antibody titer of immune group chicken is between 1:32～1:39.4, and is equal 〉=the 1:16(micromethod); The serum HI antibody titer of matched group chicken all≤the 1:8(micromethod).Immune component poison rate is between 0/10～1/10 simultaneously, and protective rate is between 9/10～10/10; Contrast component poison rate is 10/10.Illustrate that IBV YB160 strain seed culture of viruses is basically identical with interior its immunogenicity in 13 generations, all have good immunogenicity, illustrate that IBV YB160 strain has good immune protective.
Four, newcastle disease and infectiousness bronchitis bigeminy live vaccine
1 material
1.1 producing with seed culture of viruses manufacturing this product uses newcastle disease virus La Sota strain available from China Veterinery Drug Inspection Office; Avian infectious bronchitis virus YB160 strain deposit number is: CGMCC No.6752.
1.2 check is with malicious Virulent Newcastle Disease Virus Beijing Strain (CVCC AV1611 strain), available from China Veterinery Drug Inspection Office.The IB separated strain is poison by force, by YEBIO Bioengineering Co., Ltd of Qingdao's isolation identification, preservation.
1.3 hatching egg and test chicken
1.3.1SPF hatching egg is available from the logical laboratory animal technology company limited of Beijing Cimmeria dimension.
1.3.2 1 age in days, 10 ages in days, 35 age in days SPF chicken SPF hatching egg are purchased the logical laboratory animal technology company limited of Beijing Cimmeria dimension, voluntarily hatching after buying back, and negative pressure isolator is raised after the hatching.
1.4 test suppresses antigen with antigen newcastle disease blood clotting, is provided by YEBIO Bioengineering Co., Ltd of Qingdao; IB separated strain blood clotting suppresses antigen, the laboratory preparation.
1.5 seedling provides by YEBIO Bioengineering Co., Ltd of Qingdao with Other Instruments, reagent.
2 methods
2.1 the seedling material is selected the well-developed breeding of 10 age in days SPF Embryo Gallus domesticus ND, IB virus.
2.2 the preparation of newcastle disease virus
2.2.1 getting to produce with seed culture of viruses, inoculation dilutes 10000 times with sterile saline, inoculation 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity, every embryo 0.1ml.The sealing pin hole is put 36～37 ℃ and is continued to hatch after the inoculation.
2.2.2 after hatching and observing egg inoculation, per sunshine, embryo was 1 time, will discard by dead Embryo Gallus domesticus before 60 hours.After 60 hours, the photograph embryo was 1 time in per 4～8 hours, and dead Embryo Gallus domesticus takes out at any time.Until 96 hours, no matter death whether, is all taken out, air chamber upwards places 2～8 ℃ of coolings 12～24 hours.
Take out 2.2.3 results will be cooled off Embryo Gallus domesticus, 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), draw Embryo Gallus domesticus liquid, the blastochyle of every some pieces of Embryo Gallus domesticus is mixed into one group.In the results blastochyle, should check one by one Embryo Gallus domesticus, corrupt such as fetus, blastochyle is muddy and have the suspicious person of any pollution to discard need not.Blastochyle after the results places the sterilization bottle, and 2～8 ℃ of preservations should be no more than 5.The inspection of semifinished product is carried out in simultaneously sampling.
2.3 the preparation of avian infectious bronchitis virus liquid
2.3.1 getting to produce, inoculation uses seed culture of viruses, with 100 times of physiological saline solution dilutions, and inoculation 10 age in days SPF Embryo Gallus domesticus in the allantoic cavity, every embryo 0.1ml, the sealing pin hole is put 37 ℃ and is continued to hatch after the inoculation.
2.3.2 after hatching and observing egg inoculation, the photograph embryo was 1 time in 24 hours, discarded dead germ, with the embryo alive of 24～36 hours dead germs and 36 hours, air chamber is upwards upright, places 2～8 ℃ to cool off 12～24 hours.
2.3.3 results are taken out the Embryo Gallus domesticus of cooling, 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), draw Embryo Gallus domesticus liquid, whenever the blastochyle of several Embryo Gallus domesticus is mixed into one group.In the results blastochyle, should check one by one Embryo Gallus domesticus, corrupt such as fetus, blastochyle is muddy and have the suspicious person of any pollution to discard need not.Blastochyle after the results places the sterilization bottle, and 2～8 ℃ of preservations are no more than 5.The inspection of semifinished product is carried out in simultaneously sampling.
2.4 the inspection of semifinished product
2.4.1 the blastochyle of the treated mistake of steriling test is taken a sample respectively for every group, tests by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
2.4.2 viral level is measured
2.4.2.1 the newcastle disease virus assay is made 10 times of serial dilutions with toxic Embryo Gallus domesticus liquid with sterile saline, gets 10
-7, 10
-8, 10
-93 dilution factors, inoculation 10 age in days SPF Embryo Gallus domesticus are 5 pieces in each allantoic cavity, and every embryo 0.1ml puts 36～37 ℃ and continues to hatch, dead Embryo Gallus domesticus discards and disregards before 48 hours, dead Embryo Gallus domesticus took out at any time and puts 2～8 ℃ in 48～120 hours, took out the embryo of living to 120 hours, the Embryo Gallus domesticus of 48～120 hours dead Embryo Gallus domesticus and survival in 120 hours, collect one by one Embryo Gallus domesticus liquid, measuring respectively the red cell agglutination valency, agglutination titer 〉=1:128(micromethod) person is judged to infection, calculates EID
50
2.4.2.2 the avian infectious bronchitis virus assay is made 10 times of serial dilutions with toxic Embryo Gallus domesticus liquid with sterile saline, gets 10
-5, 10
-6, 10
-7, 10
-8Inoculation 10 age in days SPF Embryo Gallus domesticus are 5 pieces in 4 dilution factors, each allantoic cavity, and every embryo 0.1ml puts 36～37 ℃ and continues to hatch.Dead Embryo Gallus domesticus discards and disregards before 24 hours, and Embryo Gallus domesticus dead in 24～144 hours takes out at any time, to 144 hours, takes out all embryos of living, and collects one by one Embryo Gallus domesticus liquid.Embryo Gallus domesticus dehydration occurs the Embryo Gallus domesticus fetus of 24～144 hours dead germs and survival in 144 hours, rolls up, grows specificity lesions such as little (more than the low 2g of the inoculation the lightest fetal weight of fetus contrast) and be judged to infection, calculating EID after the inoculation
50
2.5 join Seedling and packing Newcastle disease venom and the infectious bronchitis virus liquid that is up to the standards is mixed in the same container by equal proportion, an amount of 5% sucrose, 1% gelatin of plumage part adding made freeze drying protectant in accordance with regulations, fully shakes up quantitative separating.Every plumage part viral level: newcastle disease virus La Sota strain 〉=10
7.0EID
50Avian infectious bronchitis virus YB160 strain 〉=10
4.0EID
50
For the ratio of Newcastle disease venom and infectious bronchitis virus liquid, as long as can guarantee these two kinds of diseases are produced immune effect.
2.6 carry out rapidly lyophilisation after the lyophilizing packing, lot number is respectively 0801,0802,0803.
2.7 product inspection
2.7.1 the appearance color of character observation vaccine, character, break away from situation and add diluent with the bottle wall after the dissolving situation.
2.7.2 steriling test is tested by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
2.7.3 the mycoplasma check is tested by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
2.7.4 diagnostic test is done suitable dilution (0.1 plumage part/0.1ml) with vaccine with normal saline, mix with equivalent anti-newcastle disease, infectious bronchitis virus YB160 strain specific serum, with 1 hour, inoculation 10 age in days SPF Embryo Gallus domesticus were 10 pieces in the allantoic cavity, every embryo 0.2ml in the room temperature.Put 37 ℃ and hatch observation 144 hours.
2.7.5 the exogenous virus check is tested by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
2.7.6 safety verification with vaccine with normal saline dilution after collunarium inoculate 10 of 10 age in days SPF chickens, every 0.03ml (containing 10 plumage parts) observed 14.
2.7.7 efficacy test
2.7.7.1 with Embryo Gallus domesticus check with vaccine with normal saline dilution to 2 plumage part/ml, be respectively charged in two test tubes every pipe 1ml.The first pipe adds the anti-newcastle disease virus specific serum of equivalent, and the second pipe adds the anti-avian infectious bronchitis virus YB160 strain specific serum of equivalent.With 1 hour (shake 1～2 time the centre), this moment, viral level was 0.1 plumage part/0.1ml in the room temperature.The first pipe continues 10 times of serial dilutions, gets and inoculates 5 pieces of 10 age in days SPF Embryo Gallus domesticus in 3 each allantoic cavitys of suitable dilution factor, and every embryo 0.1ml puts 37 ℃ and hatches, and observes 144 hours.Dehydration occurs in the Embryo Gallus domesticus according to 24～144 hours dead germs after the inoculation and survival in 144 hours, roll up, grow the summation of specific lesions embryos such as little (more than the low 2g of the inoculation the lightest fetal weight of fetus contrast), calculating EID
50The second pipe continues 10 times of serial dilutions, get and inoculate 5 pieces of 10 age in days SPF Embryo Gallus domesticus in 3 each allantoic cavitys of suitable dilution factor, every embryo 0.1ml, Embryo Gallus domesticus dead before 48 hours is disregarded, and dead Embryo Gallus domesticus took out at any time and puts 2～8 ℃ in 48～120 hours, took out the embryo of living to 120 hours, the Embryo Gallus domesticus of 48～120 hours dead Embryo Gallus domesticus and survival in 120 hours is gathered in the crops blastochyle one by one, measures respectively 1% chicken red blood cell agglutination titer, agglutination titer 〉=1:128 (micromethod) is judged to infection, calculates EID
50
2.7.7.2 check with chicken
2.7.7.2.1 newcastle disease is partly used 10 of the SPF chickens of 35 ages in days, every collunarium is inoculated 1/100 plumage part bigeminy Seedling, exempts from rear 14 days, and together with 5 of the identical immunity contrast chickens of condition, every chicken respectively intramuscular injection contains 10
4.0ELD
50The strong malicious 1ml of newcastle disease Beijing Strain, observed 14.Record Mortality situation.
2.7.7.2.2 infectious bronchitis of chicken is partly used 10 of 3 age in days SPF chickens, the bigeminy Seedling of every collunarium 1 plumage part.Establish simultaneously 10 not immune comparing.Rear 14 days of immunity is taken a blood sample respectively together with every chicken of contrast chicken, and separation of serum carries out the HI antibody titer by appendix and measures.Simultaneously every chicken is with each 1 of the strong malicious eye dripping of IB separated strain, the collunarium of 10 times of dilutions, and behind the counteracting toxic substances the 5th day, gather the trachea swab of every chicken, inoculate 10 age in days SPF Embryo Gallus domesticus through allantoic cavity, the 0.1ml/ embryo is hatched observation to 144 hours for 37 ℃.Add up viral separation case.
2.7.8 measuring, residual moisture undertaken by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
2.7.9 measuring, vacuum undertaken by version " Chinese veterinary pharmacopoeia " appendix method in 2005.
3 results
3.1 semi-finished product production and assay
3.1.1 steriling test is tested by existing " Chinese veterinary pharmacopoeia " appendix method, all without bacterial growth.The results are shown in Table 1.
3.1.2 viral level is measured
3 batches of NDV virus liquid viral levels are respectively 10
8.7EID
50/ 0.1ml, 10
8.7EID
50/ 0.1ml, 10
8.9EID
50/ 0.1ml, 3 batches of IBV virus liquid viral levels are respectively 10
6.9EID
50/ 0.1ml, 10
6.7EID
50/ 0.1ml, 10
6.5EID
50/ 0.1m.The results are shown in Table 3.
Table 3 inspection of semifinished product result
3.2 product inspection result
3.2.1 character
3 batches of bigeminy Seedlings of outward appearance sample survey outward appearance is little yellow Sponge Porosity agglomerate, and during dandle, sample is easy to break away from the bottle wall.3 batches of bigeminy Seedlings all dissolve rapidly after adding respectively diluent.The results are shown in Table 4.
Table 4 bigeminy seedlings assay
3.2.2 3 batches of bigeminy Seedlings of steriling test respectively every batch appoint and to take out 5 bottles and return to 2ml with physiological saline solution respectively, undertaken by version " Chinese veterinary pharmacopoeia " appendix method in 2005.The result shows that all without antibacterial, fungus growth, 3 batches of bigeminy Seedling steriling tests are all qualified, the results detailed in Table 5.
Table 5 bigeminy Seedling steriling test result
3.2.3 every batch of 3 batches of bigeminy Seedling of mycoplasma check are taken out 5 bottles and are returned to 2ml and mixing with physiological saline solution respectively, test by version " Chinese veterinary pharmacopoeia " appendix method in 2005.The result shows that 3 batches of bigeminy Seedlings are all without the mycoplasma growth, and the mycoplasma check is all qualified.The results detailed in Table 6.
Table 6 bigeminy Seedling mycoplasma assay
3.2.4 3 batches of bigeminy Seedlings of diagnostic test are done suitable dilution (0.1 plumage part/0.1ml) with physiological saline solution, mix with equivalent anti-newcastle disease, infectious bronchitis virus YB160 strain specific serum respectively, in the room temperature and l hour, inoculation 10 age in days SPF Embryo Gallus domesticus are 10 pieces in the allantoic cavity, every embryo 0.2ml, put 37 ℃ and hatch, observed 144 hours.The result shows that inoculated into chick embryo is 10/10 strong living, and Embryo Gallus domesticus liquid is all negative to 1% chicken red blood cell agglutination test, illustrates that the vaccine diagnostic test is qualified.The results are shown in Table 7.
Table 7 bigeminy Seedling diagnostic test result
3.2.5 exogenous virus check
3.2.5.1 3 batches of bigeminy Seedlings of Embryo Gallus domesticus inspection technique are taken a sample respectively 3 bottles, do suitable dilution with physiological saline solution after mixing with batch sample, mixes with equivalent anti-newcastle disease, infectious bronchitis virus YB 160 strain specific serums, in the room temperature with 1 hour.Inoculated into chick embryo is tested.The 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.The results are shown in Table 8.
Table 8 bigeminy Seedling exogenous virus check (chick embryo method) result
3.2.5.2 cytoscopy method
3.2.5.2.1 3 batches of bigeminy Seedlings are taken a sample respectively 3 bottles, do suitable dilution with physiological saline solution after mixing with batch sample, mix with equivalent anti-newcastle disease, infectious bronchitis virus YB160 strain specific serum, at room temperature with 1 hour.Get the sample of processing and respectively inoculate 2 bottles of each 0.2ml of chick embryo fibroblast that grown up to good monolayer, observed 7.Check is acellular pathological changes all, and without the erythrocyte adsorption phenomena.
3.2.5.2.2 3 batches of bigeminy Seedlings of avian leukosis virus check are taken a sample respectively 3 bottles, with doing suitable dilution with physiological saline solution after the batch sample mixing, mix with equivalent anti-newcastle disease, infectious bronchitis virus YB160 strain specific serum, at room temperature with 1 hour.Get the sample of processing and respectively inoculate 2 bottles of each 2ml of chick embryo fibroblast that grown up to good monolayer, establishing simultaneously negative control (normal cell) and positive-virus compares, even passed for 3 generations with sample, get 3 generation cell culture (comprising sample and all matched groups) freeze thawing 3 times, and mark, carry out ELISA with the avian leukosis virus test kit and detect.The sample survey result is all negative.The results are shown in Table 9.
Table 9 bigeminy Seedling exogenous virus check (cell method) result
3.2.5.3 3 batches of bigeminy Seedlings of chicken inspection technique are taken a sample respectively 3 bottles, after doing suitable dilution with physiological saline solution after the sample mix, inoculate 20 ages in days the SPF chicken each 20, wherein collunarium, eye dripping are respectively inoculated 10 plumage parts, and intramuscular injection 100 plumage parts were exempted from rear 21 days, repeated inoculation is 1 time again, in the 1st inoculation blood sampling in rear 42 days, separation of serum carries out the Serum Antibody Detection of relevant cause of disease and records clinical symptoms.The result shows, behind the check vaccination chicken 42 days, chicken was all healthy, apnea road symptom and clinical response.And serum only reacts with newcastle disease antigen and biography Zhi Kangyuan, and does not all react with other pathogen antigens.The results are shown in Table 10.
Table 10 bigeminy Seedling exogenous virus check (chicken inspection technique) result
In sum, Embryo Gallus domesticus inspection method result, cell inspection method and chicken inspection method result illustrate that all 3 batches of bigeminy Seedling exogenous viruses are up to the standards.
Take out 3 bottles respectively with 10 10 age in days SPF chickens of collunarium inoculation after the suitable dilution of physiological saline solution 3.2.6 every batch of 3 batches of bigeminy Seedling of safety verification are appointed, every 0.03ml (containing 10 plumage parts) observed 14.The result shows that the inoculation chicken is all without any untoward reaction, and 10/10 strong living.The results are shown in Table 11.
Table 11 bigeminy Seedling safety verification result
3.2.7 efficacy test
3.2.7.1 every batch of 3 batches of bigeminy Seedling of Embryo Gallus domesticus inspection technique are taken out 1 bottle and are diluted to 2 plumage part/1ml with physiological saline solution respectively, are respectively charged in two test tubes every pipe 1ml.Test by the 2.7.7.1 method.The result shows that the every plumage part of newcastle disease part viral level is respectively 10
6.7EID
50, 10
6.9EID
50With 10
6.9EID
50, all 〉=10
6.7EID
50The every plumage part of infectious bronchitis of chicken part viral level is respectively 10
3.9EID
50, 10
3.7EID
50With 10
3.7EID
50, all 〉=10
3.7EID
50Illustrate that bigeminy Seedling efficacy test (chick embryo method) is up to the standards.The results are shown in Table 12.
Table 12 bigeminy Seedling efficacy test (chick embryo method) result
3.2.7.2 every batch of 3 batches of bigeminy Seedling of chicken inspection technique are taken out 1 bottle and are diluted with physiological saline solution respectively.
(1) newcastle disease is partly used 10 of the SPF chickens of 35 ages in days, and every collunarium is inoculated 1/100 plumage part, establishes simultaneously 5 not immune in contrast chickens, exempts from rear 14 days, and ND HI antibody titer is surveyed in blood sampling.The result shows that behind the strong malicious counteracting toxic substances of newcastle disease Beijing Strain, immune group equal 10/10 is protected matched group 5/5 Mortality.The results are shown in Table 13.
Table 13 bigeminy Seedling efficacy test (chicken inspection technique) newcastle partial results
(2) infectious bronchitis of chicken is partly used 10 of 3 age in days SPF chickens, and the vaccine of every collunarium 1 plumage part is established 10 not immune comparing simultaneously.Rear 14 days of immunity, the IBV-HI antibody titer is surveyed in blood sampling; Simultaneously with the 10 strong malicious counteracting toxic substances of IB separated strain that dilute.The result shows that immune group IBV-HI antibody titer geometrical mean is 1:36.8～1:52, matched group IBV-HI antibody titer all≤1:8; Behind the strong malicious counteracting toxic substances of IB separated strain, immune component poison rate is 0/10～1/10, and protective rate is 9/10～10/10, and contrast component poison rate is 8/10.The results are shown in Table 14.
Table 14 bigeminy Seedling efficacy test (chicken inspection technique) passes a part result
3.2.8 every batch of 3 batches of bigeminy Seedling of residual moisture mensuration are taken out 4 bottles and are tested with boulton process.Product test sample residual moisture content is between 2.0～2.7%, all≤4%.It is all qualified to illustrate that bigeminy Seedling residual moisture is measured check.The results are shown in Table 15.
3.2.9 vacuum is measured 3 batches of bigeminy Seedlings and is tested with vacuum leak detector respectively.The product test sample all is the purple aura.It is all qualified to illustrate that bigeminy Seedling vacuum is measured check.The results are shown in Table 15.
Table 15 bigeminy Seedling residual moisture and vacuum assay
The newcastle disease of the present invention's preparation and the inoculation of infectiousness bronchitis bigeminy live vaccine single dose, single dose repeated inoculation and an overdose inoculation safety testing
1 material
1.110,70,180 age in days SPF chicken SPF hatching egg are available from from the logical laboratory animal technology company limited of Beijing Cimmeria dimension, voluntarily hatching is raised in the negative pressure isolator after the hatching after buying back.
1.2 vaccine bigeminy Seedling, lot number are the trial-production of 0801,0802,0803 laboratory.
2 methods
Mix after 3 bottles of redissolution 2.1 sampling method is taken a sample 3 batches of bigeminy Seedlings respectively, do suitable dilution with physiological saline solution again.
2.2 single dose inoculation SPF chicken safety testing is got 30 of 10 age in days SPF chickens, be divided at random 3 groups of A, B, C, every group 10,0801 batch of vaccine of A group collunarium inoculation, 0801 batch of vaccine of B group eye dripping inoculation, 1 plumage part/only, C group is not inoculated and is compared group, with the condition isolated rearing, examines searching for food after the test chicken immunity, drinks water, spirit, growth promoter situation, Continuous Observation is 14 after the immunity, record test chicken result.
2.3 single dose repeated inoculation SPF chicken safety testing is got 30 of 10 age in days SPF chickens, be divided at random 3 groups of A, B, C, every group 10,0801 batch of vaccine of A group collunarium inoculation, 0801 batch of vaccine of B group eye dripping inoculation, 1 plumage part/only, respectively at rear 7 days of inoculation, carry out repeated inoculation by above-mentioned identical method system with dosage, C group is not inoculated and is compared group, and every group with respectively isolated rearing under the condition, examines searching for food after the immunity of test chicken, drinks water, spirit, growth promoter situation, the rear Continuous Observation of for the second time immunity 14 days, the record result.
2.4 one time overdose inoculation SPF chicken safety testing is got 70 of 10 age in days SPF chickens, be divided at random 7 groups of A, B, C, D, E, F, G etc., every group 10, A, B, C group be 0801,0802,0803 batch of vaccine of collunarium inoculation respectively, D, E, F group be 0801,0802,0803 batch of vaccine of eye dripping inoculation respectively, 10 plumage parts/only, the G group is not inoculated and is compared group, every group with respectively isolated rearing under the condition, examine test chicken postvaccinally search for food, drink water, spirit, growth promoter situation, Continuous Observation 14 days.Cut open inspection inoculation chicken after observing end, aseptic collection glandular stomach, kidney are done pathology section examination, and the record result.
2.5 an overdose is inoculated 70 age in days SPF laying hen safety testings and is got 70 of 70 age in days SPF chickens, be divided at random 7 groups of A, B, C, D, E, F, G etc., every group 10, A, B, C group be 0801,0802,0803 batch of vaccine of collunarium inoculation respectively, D, E, F group be 0801,0802,0803 batch of vaccine of eye dripping inoculation respectively, 10 plumage parts/only, the G group is not inoculated and is compared group, every group with respectively isolated rearing under the condition, examine test chicken postvaccinally search for food, drink water, spirit, growth promoter situation, Continuous Observation 30 days.Cut open inspection inoculation chicken after observe finishing, observe glandular stomach, kidney and have or not pathological changes and ovary, Oviduct Development situation, and the record result.
2.6 an overdose is inoculated 180 age in days SPF laying hen safety testings and is got 70 of 180 age in days commodity eggs, be divided into two groups of A, B, 0801 batch of vaccine of 50 collunarium inoculations of A group, 10 plumage parts/only, 20 of B groups are not inoculated and are compared group, and every group with respectively isolated rearing under the condition, examine test chicken postvaccinally search for food, drink water, spirit, test group and matched group laying rate changed and the quality of laying eggs in statistics was inoculated rear 30 days, and the record result.
3 results
3.1 single dose (1 plumage part/only) inoculation SPF chicken safety testing after test chicken adopts collunarium, two kinds of approach of eye dripping to inoculate respectively 0801 batch of vaccine, 1 plumage part as a result, was observed 14.The result shows that the spirit of test chicken, appetite, feces, growth promoter are all normal.The results are shown in Table 16.
Table 16 single dose inoculation SPF chicken safety testing result
3.2 the safety testing of single dose (1 plumage part/only) repeated inoculation SPF chicken as a result test chicken adopts collunarium, two kinds of approach of eye dripping to inoculate respectively 0801 batch of vaccine, 1 plumage part/only, is inoculating rear 7 days first and carry out repeated inoculation 1 plumage part/only, observed 14.The result shows, test chicken is searched for food, drunk water, spirit, growth promoter situation are all good.The results are shown in Table 17.
The safety testing result of table 17 single dose repeated inoculation SPF chicken
3.3 overdose (10 plumage parts/only) inoculation SPF chicken safety testing as a result test chicken adopts collunarium, 0801,0802,0803 batch of vaccine 10 plumage part of two kinds of approach inoculations of eye dripping/only, observed 14, the spirit of test chicken, appetite, feces, growth promoter are all normal.The results are shown in Table 18.Observe after 14 days, test chicken is cutd open inspection observe no abnormality seen, gather glandular stomach, kidney preparation section, histological observation glandular stomach, kidney are showed no unusually.
An overdose inoculation of table 18 SPF chicken safety testing result
3.4 0801 batch of vaccine 10 plumage part of as a result test chicken employing of an overdose (10 plumage part) inoculation 70 age in days SPF laying hen safety testings collunarium approach inoculation/only, observed 30.The result shows that the spirit of test chicken, appetite, feces, growth promoter are all normal.Observe after 14 days, test chicken is cutd open inspection observe glandular stomach, kidney without pathological changes, ovary, Oviduct Development are all normal.The results are shown in Table 19.
Overdose of table 19 (10 plumage part) inoculation 70 age in days SPF laying hen safety testing results
3.5 overdose inoculate 180 age in days SPF laying hen safety testings knot as a result test chicken adopt 0801 batch of vaccine 10 plumage part of collunarium approach inoculation/only, observed 30.The result shows that the spirit of test chicken, appetite, feces are all normal, and the test chicken laying rate is 86.0%, and contrast laying rate of chicken 85.0% is showed no abnormal-shape egg, soft-shelled egg, husky shell egg etc.The results are shown in Table 20.
Overdose of table 20 (10 plumage part) inoculation 180 age in days SPF laying hen safety testing results
4 conclusions
4.1 the bigeminy Seedling repeats with single dose, single dose respectively with the collunarium approach and disposable overdose is inoculated respectively the SPF chicken of 10 ages in days, Continuous Observation 14 days.After inoculation in the observation period, search for food, drink water, spirit, growth promoter situation is all normal.
Whether 4.2 laying hen is Oviduct Development period of maturation at 70 ~ 120 ages in days generally, it is influential to the laying hen Oviduct Development in order to observe lower overdose inoculation therefore to select 70 age in days laying hens to carry out disposable overdose test.In the test Continuous Observation test chicken on the 30th search for food, drink water, spirit is all normal, cuts open inspection glandular stomach, kidney, ovary, fallopian tube no abnormality seen, illustrate that the overdose inoculation is to laying hen Oviduct Development generation harmful effect.。
4.3 because large age in days SPF laying hen cost is high, it is many that raising accounts for isolator, the difficult raising.Therefore the bigeminy Seedling has only been used a kind of approach to the safety testing of laying hen, and a batch of vaccine carries out.Inoculate 180 age in days SPF laying hens with the disposable overdose of collunarium approach, Continuous Observation 30 days, test chicken is searched for food, is drunk water, spirit is all normal, the test chicken laying rate is 86.0%, the contrast laying rate of chicken is 85.0%, be showed no abnormal-shape egg, soft shell list, husky shell egg etc., illustrate that the overdose immunity does not affect the chicken egg laying performance.
Above result of the test explanation bigeminy Seedling is safe.
The newcastle disease of the present invention's preparation and the test of infectiousness bronchitis bigeminy live vaccine antibody Fluctuation and immune duration
1 material
1.1 vaccine newcastle disease, infectiousness bronchitis bigeminy live vaccine (La Sota strain+YB160 strain), laboratory trial-production, lot number is 0801,0802,0803.
1.21 age in days SPF chicken hatching egg is available from the logical laboratory animal technology company limited of Beijing Cimmeria dimension, voluntarily hatching is raised in the negative pressure isolator after the hatching after buying back.
1.3 check is with strong malicious Virulent Newcastle Disease Virus Beijing Strain (CVCC AV1611 strain), available from China Veterinery Drug Inspection Office; The IB separated strain is poison by force, is separated, identifies, is preserved by YEBIO Bioengineering Co., Ltd of Qingdao.
1.4 test suppresses antigen, infectious bronchitis YB160 strain blood clotting inhibition antigen with antigen newcastle disease blood clotting, provides by YEBIO Bioengineering Co., Ltd of Qingdao.
2 methods with 3 batches of bigeminy Seedlings respectively by indicate plumage part with the physiological saline solution dilution after, get 175 of 1 age in days SPF chickens for every batch, 100 immune vaccines wherein, collunarium 1 plumage part/only (0.03ml), 75 immune comparing not in addition are with isolated rearing under the condition.
2.1 above-mentioned immune chicken was randomly drawed 10 on 4th, 7,14,21,28,42,56,70,84,98,112,126,140 after immunity, blood sampling, and separation of serum is measured ND and IB HI antibody titer.
2.2 with above-mentioned 3 batches of bigeminy Seedlings immunity chicken after inoculation 7,90,120,150 days, randomly draw 20 immune chickens for every batch, wherein 10 together with 5 of matched groups, each intramuscular injection 10
4.0ELD
50The strong malicious Beijing Strain 1.0ml of newcastle disease, observed 14.In addition 10 together with each 1 of each eye dripping of the strong poison of IB separated strains, the collunarium of 10 times of dilutions of 10 usefulness of matched group, and behind the counteracting toxic substances the 5th day, gathers the trachea swab of every chicken, inoculate 10 age in days SPF Embryo Gallus domesticus through allantoic cavity, the 0.1ml/ embryo was hatched observation to 144 hours.Embryo Gallus domesticus is death in 24～144 hours after the inoculation, and survival Embryo Gallus domesticus fetus dehydration occurs, rolls up, grows specificity lesions such as little (inoculating more than the low 2g of the lightest fetal weight of fetus contrast) and be judged to infection.There is one piece of Embryo Gallus domesticus above-mentioned case feature to occur if every group of cotton wiped away sample, can declares virus and separate positive.
3 results
3.1 after ND and IB HI antibody titer testing result were inoculated with the bigeminy Seedling after the immunity of bigeminy Seedling, the newcastle disease part produced antibody on 4th, the geometrical mean of antibody titer reaches 3.1log
2, the geometrical mean of inoculating rear 7 days antibody titers reaches 4.0; The geometrical mean of the inoculating rear 14 days antibody titers 5.8log that peaks
2The infectious bronchitis of chicken part produced antibody after 4 days, and the geometrical mean of antibody titer reaches 3.0log
2, the geometrical mean of inoculating rear 7 days antibody titers reaches 4.1; The geometrical mean of the inoculating rear 14 days antibody titers 5.7log that peaks
2ND and IB antibody begin slow decreasing after 70 days.The results are shown in Table 21.
Table 21 bigeminy Seedling ND and IB HI antibody titer testing result
3.2 with 3 batches of immune 1 age in days SPF chickens of bigeminy Seedlings difference, when exempting from rear 7,90,120,150 days, extract respectively test chicken and carry out protest test.The bigeminy Seedling reaches more than 9/10 ND strong virus attack protective rate 7 days the time after exempting from, and dividing malicious rate to IB separated strain strong virus attack is 1/10～2/10, and protective rate reaches 8/10 ~ 9/10; In the time of 120 days ND strong virus attack protective rate is reached 9/10, dividing malicious rate to IB separated strain strong virus attack is 0/10～2/10, and protective rate reaches 8/10 ~ 10/10; And only be 6/10 to ND strong virus attack protective rate 150 days the time, dividing malicious rate to IB separated strain strong virus attack is 3/10～5/10, protective rate only is 5/10～7/10.The results are shown in Table 22.
Protest test result after the immunity of table 22 bigeminy Seedling
3 batches of newcastle diseases of 4 conclusion laboratorys trial-production, infectiousness bronchitis bigeminy live vaccine (La Sota strain+YB160 strain) inoculation can produce antibody after rear 4 days, and antibody peaked on 14th, keep and reduce gently afterwards in 70th; ND strong virus attack protective rate is reached more than 9,/10 7 days the time after exempting from simultaneously, dividing malicious rate to IB separated strain strong virus attack is 1/10～2/10, and protective rate reaches 8/10 ~ 9/10; In the time of 120 days ND strong virus attack protective rate is reached 9/10, dividing malicious rate to IB separated strain strong virus attack is 0/10～2/10, and protective rate reaches 8/10～10/10; And only be 6/10 to ND strong virus attack protective rate 150 days the time, dividing malicious rate to IB separated strain strong virus attack is 3/10～5/10, protective rate only is 5/10～7/10.So still can reach desirable protection effect to the SPF chicken in inoculation in rear 120 days.In order to ensure the immune effect of vaccine, newcastle disease, infectiousness bronchitis bigeminy live vaccine (La Sota strain+YB160 strain) duration of immunity are defined as 3 months.The prepared bigeminy Seedling of above presentation of results is safe and effective, can apply.
Claims (4)
1. a newcastle disease and infectiousness bronchitis bigeminy live vaccine are comprised of antigen and vaccine freeze-drying protective agent, and wherein antigen is that newcastle disease virus and deposit number are the avian infectious bronchitis virus of CGMCC No.6752.
2. the preparation method of bigeminy vaccine claimed in claim 1 is that the results blastochyle obtains viral scale-up medium with behind Avian pneumo-encephalitis virus and the avian infectious bronchitis virus difference inoculated into chick embryo; Then with after Avian pneumo-encephalitis virus and the mixing of avian infectious bronchitis virus scale-up medium, the adding freeze drying protectant is made.
3. method as claimed in claim 2 is characterized in that described freeze drying protectant is sucrose and gelatin, and its final concentration in vaccine is respectively 5% and 1%.
4. bigeminy vaccine claimed in claim 1 is used for prevention newcastle disease and infectious bronchitis.
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