CN103263667A - Chicken infectious bursal disease live vaccine and production method thereof - Google Patents
Chicken infectious bursal disease live vaccine and production method thereof Download PDFInfo
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- CN103263667A CN103263667A CN2013101691873A CN201310169187A CN103263667A CN 103263667 A CN103263667 A CN 103263667A CN 2013101691873 A CN2013101691873 A CN 2013101691873A CN 201310169187 A CN201310169187 A CN 201310169187A CN 103263667 A CN103263667 A CN 103263667A
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Abstract
The invention relates to a production method of a chicken infectious bursal disease live vaccine. A virulent strain having the characteristics of good immunogenicity, broad antigen spectrum and immune synergistic effect is screened out through the researches on the characteristics comprising the immunogenicity, the antigen spectrum, the pathogenicity to the bursa of Fabricius, the maternal antibody breakthrough capability and the like of different subtype chicken infectious bursal viruses to develop the researches on chicken infectious bursal disease live vaccines in order to produce the chicken infectious bursal disease trivalent live vaccine which has a good immunogenicity, can breakthrough high-level maternal antibodies, can continuously immunize for above 3 months and can well protect different virulent strains.
Description
Technical field
The present invention relates to a kind of infectious bursal disease live-vaccine and production method thereof.Belong to the veterinary biologics field.
Background technology
Infectious bursal disease be the chickling that caused by double-stranded rna virus a kind of acute, hyperinfection, kill the lymphatic disease, be to endanger one of the most serious infectious disease of poultry husbandry all over the world, preventing this sick major measure is to adopt vaccine to carry out immunity inoculation.Because maternal antibody is bigger to the interference of live vaccine, therefore, must determine just to exempt from the time according to the maternal antibody level, with the immune effect of guaranteeing vaccine in actual production, because also there is larger difference in its Different Individual maternal antibody level among the same chicken group, the suitableeest immunity time is difficult for determining, in case the immunity time is improper, wild poison is usually availed oneself of the opportunity to get in, and causes morbidity.In recent years, the IBD vaccine research is in the ascendant, and many new generation vaccines are also studied successfully in succession, comprising genetic engineering subunit vaccine, live recombinant vectors vaccine etc.The infectious bursal disease live-vaccine of this research invention utilizes IBDV and the anti-IBDV hyper-immune serum of a certain amount of pig specific bond, can postpone vaccine virus copying in the chicken body.Its immune mechanism is, when chicken body maternal antibody is reduced to certain level, virus begins to copy release, produce the active immunity protection, and the immunoreation of inducing is stronger than conventional live vaccine.Because it is very little that this vaccine is disturbed by maternal antibody, adopt the interior inoculation of instar chicken embryo embryo on the 18th or 1 Japanese instar chickling cervical region subcutaneous vaccination can produce good immunoprotection, avoid chicken group early infection.The present invention adopts the anti-IBDV hyper-immune serum of pig is the seedling material, has avoided adopting chicken serum and the fowl exogenous virus that causes pollutes, and vaccine is safer.
Summary of the invention
The object of the invention is to utilize the good IBDVCF-C strain of immunogenicity and the anti-IBDV hyper-immune serum of pig specific bond to prepare vaccine; adopt instar chicken embryo on the 18th to implement to inoculate in the embryo or 1 Japanese instar chickling is carried out the cervical region subcutaneous vaccination, once immunity can produce good immune protection power.Vaccine is the seedling material with the anti-IBDV hyper-immune serum of pig in addition, has avoided the employing chicken serum and causes the fowl exogenous virus to pollute, and the vaccine of preparation is safer.
Technology path
1. a kind of infectious bursal disease live-vaccine involved in the present invention is the live vaccine that contains chicken infectivity bursa of Fabricius virus CF-C strain and the anti-IBDV hyper-immune serum of pig, wherein chicken infectivity bursa of Fabricius virus (Infectiousbursaldiseasevirus) CF-C strain virus is delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on April 1st, 2013, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number are CGMCC No.7404.
2. the preparation method of the infectious bursal disease live-vaccine that the present invention relates to is:
(1) inoculation of Embryo Gallus domesticus, results: with sterile saline/or phosphate-buffered meat soup will produce to be diluted to seed culture of viruses CF-C strain chicken infectivity bursa of Fabricius virus and contain 1000～10000EID
50/ 0.1ml, through allantocherion inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml puts 37 ℃ and hatches to 96 hours, discards dead germ before 48 hours with the CF-C strain virus liquid after the dilution; 48～96 hours dead germs and survived in 96 hours embryo and chorioallantoic membrane are collected in grouping respectively, are loaded in the sterile chamber to shred, and put below-20 ℃ frozen; Freeze thawing 2～3 times is by centrifugal or cross and filter to remove sediment, and supernatant is seedling with CF-C strain semi-finished product, through steriling test qualified, viral level is every 0.2ml ≧ 10
4.5ELD
50, be stored in below-20 ℃;
(2) preparation of the anti-IBDV hyper-immune serum of pig:
Obtain the anti-IBDV hyper-immune serum of pig with CF-C strain chicken infectivity bursa of Fabricius virus immune health pig, 56 ℃ of deactivation 30min are stored in below-20 ℃.
(3) join Seedling and divide assembling Seedling and packing to learn from else's experience to be up to the standards and through sterile saline/or phosphate-buffered meat soup to be diluted to viral level be every 0.2ml ≧ 10
4.5ELD
50Viral liquid by volume 1:1 mix with the anti-IBDV hyper-immune serum of pig, add freeze drying protectant commonly used and antibiotic through abundant mixing, lyophilisation is carried out in quantitatively packing rapidly after the packing.
Embodiments of the present invention
1. Strain is originated and feature
(1) strain source: chicken infectivity bursa of Fabricius virus (Infectiousbursaldiseasevirus, IBDV) the CF95 strain is from China Veterinery Drug Inspection Office, a little less than being caused in Beijing's isolation identification and through manually repeatedly going down to posterity in nineteen ninety-five by China Veterinery Drug Inspection Office, after this inventor obtains the clone strain of higher virus titer by the terminal point dilution method, called after chicken infectivity bursa of Fabricius virus (Infectiousbursaldiseasevirus) CF-C strain, this strain virus is delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on April 1st, 2013, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number CF-C strain is CGMCCNo.7404.
1) be separated in and gather the fowl disease of dying of illness in the chicken house that the doubtful infectious bursal disease in Beijing infects and become fabricius bursa, after the antibiotic degerming is handled, inoculated into chick embryo, continuous passage, the propagation isolated viral through being accredited as IBDV virulent strain, is 10 to the fabricius bursa median infective dose of SPF chicken
4.0BID
50/ 0.1ml, called after CF95 strain.
2) cause weak adopt Embryo Gallus domesticus to go down to posterity to cause a little less than, when reaching for 90 generations, virus loses the pathogenicity to chicken, called after CF strain.After in 3～5 week ages SPF chicken bodies, passing for 5 generations by the CF strain continuously, get the basis plant poison with the 5th generation fabricius bursa carry out virulence relatively, show the CF strain in the susceptible chicken body after the continuous passage virulence stable, virulence do not occur and return strong phenomenon, each generation capsule poison inoculation chicken bursa does not all have obvious pathological changes, color and elasticity are normal, are slightly less than the normal control chicken bursa.Through terminal point dilution method clone, the clone strain viral level of acquisition is significantly improved the inventor, its viral level 〉=10 with the CF strain
5.0ELD
50/ 0.2ml, called after CF – C strain (or being called for short the CF strain).
(2) strain characteristics
1) virulence of Embryo Gallus domesticus is diluted CF-C strain seed culture of viruses with physiological saline solution, through 10 piece of 10～11 age in days SPF Embryo Gallus domesticus of allantocherion inoculation, every embryonic breeding kind 0.2ml(contains 1000 ELD
50), Embryo Gallus domesticus should be dead more than 8 in 48～168 hours after inoculation, cuts open obviously edema of inspection fetus whole body, brain hyperemia, and toe hyperemia, liver has pathological changes such as mottling.
2) safety of chickling is carried out safety testing with CF-C strain seed culture of viruses with 7～10 30 of age in days SPF chickling.Test chicken is divided into two groups, and first group 20, every eye dripping or collunarium inoculation 10
4.0ELD
50Viral liquid; Second group 10, not virus inoculation in contrast, two groups of isolated rearings.Inoculate back 72 hours, inoculation group is cutd open 10 of inspections, pathological changes such as that 5 of matched groups, chickling fabricius bursa all do not have is hemorrhage, downright bad, yellow mucus appearance.All the other chickling continue breeding observings to 21 day, should be good for work.
3) viral level is made 10 times of serial dilutions with CF-C strain seed culture of viruses with sterile saline, gets 10
-4, 10
-5With 10
-63 dilution factors, through 5 pieces of allantocherion inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml puts 37 ℃ and hatched 168 hours, calculates ELD
50, viral level answers 〉=10
4.5ELD
50/ 0.2ml.
4) specificity is diluted to 10 with CF-C strain seed culture of viruses with sterilization Hank ' s liquid
3.0ELD
50/ 0.1ml with infectious bursal disease positive serum mixed in equal amounts, in 37 ℃ and 60 minutes, inoculates 5 pieces of 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml on allantocherion; Establish 5 of virus control simultaneously, every embryonic breeding kind virus liquid 0.2ml(contains 10
3.0ELD
50), observe cultivation 168 hours with condition.Should all be good for work with the group Embryo Gallus domesticus in the serum, the virus control group should have 4 pieces of death at least, and chick embryo allantoic liquid should be negative to chicken red blood cell agglutination test (HA).
5) immunogenicity with CF-C strain seed culture of viruses with 2～4 age in week 20 of SPF chickling carry out the immunogenicity test.Wherein 10 each collunariums or eye dripping virus inoculation liquid 0.03ml contain 50ELD approximately
50, in addition do not inoculate and compare isolated rearing for 10.After 14 days, immune chicken is all used the strong malicious BC6-85 strain eye dripping 0.1ml of 10 fabricius bursa Minimum Infective Doses together with 5 of contrast chickens, after 72 hours, all chickens is slaughtered, and cuts open inspection.The fabricius bursa of counteracting toxic substances contrast chicken should at least 4 obvious pathological changes occur, and the fabricius bursa of immune chicken should have 8 no pathological changes at least, and the fabricius bursa of 5 chickens of normal healthy controls group all should not have any variation.
6) inhibitive ability of immunity carries out the inhibitive ability of immunity test with CF-C strain seed culture of viruses with 20 1 age in days SPF chickens.Test chicken is divided into 2 groups, 10 every group, the viral liquid of 1 using dosage of 1 winding kind wherein, in addition 10 in contrast, isolated rearing.The newcastle disease vaccine of 1 using dosage of every inoculation of every group of chicken is observed with isolated rearing under the condition after 14 days.The HI antibody titer of newcastle disease is measured in immunity blood sampling in back 14 days, between immune group and the newcastle disease matched group geometric mean titer (GMT) of newcastle disease antibody titer by statistics credit analyse, answer there was no significant difference.
2 vaccine manufacturing and the inspections of semifinished product
(1) production prepares with seed culture of viruses
CF-C strain seed culture of viruses is diluted to 1000～10000EID with sterile saline or phosphate-buffered meat soup
50/ 0.1ml, through allantocherion or allantoic cavity inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml puts 37 ℃ and hatches to 96 hours, discards dead germ before 48 hours.Collect 48～96 hours dead germs and 96 hours live embryos and chorioallantoic membrane, be loaded in the sterile chamber and shred, steriling test is made in sampling, blastochyle put-30 ℃ frozen.The Embryo Gallus domesticus liquid freeze thawing that check is aseptic 2～3 times, the centrifugal sediment that goes of Embryo Gallus domesticus liquid, supernatant add an amount of antibiotic mixing packing, put-30 ℃ of preservations.Indicate harvest date, seed culture of viruses generation and loading amount.Through identifying that standard compliant seed culture of viruses is as the production seed.Seed culture of viruses is kept at below-20 ℃ and preserves, and should be no more than 12 months, and the seed culture of viruses subculture should be no more than for 3 generations.
(2) preparation of seedling virus liquid
Select well-developed 10～11 age in days SPF Embryo Gallus domesticus or chick embryo fibroblast as the seedling material.Get to produce and use seed culture of viruses, be diluted to sterile saline or phosphate-buffered meat soup and contain 1000～10000EID
50/ 0.1ml, through allantocherion inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml puts 37 ℃ and hatches to 96 hours, discards dead germ before 48 hours.48～96 hours dead germs and survived in 96 hours embryo and chorioallantoic membrane are collected in grouping respectively, are loaded in the sterile chamber to shred, and put below-20 ℃ frozen.Freeze thawing 2～3 times is by centrifugal or cross and filter to remove sediment, and supernatant is stored in below-20 ℃ to be checked.
(3) preparation of seedling hyper-immune serum
With chicken infectivity bursa of Fabricius virus repeatedly the immune health pig obtain the anti-IBDV hyper-immune serum of pig, 56 ℃ of deactivation 30min are stored in below-20 ℃ to be checked.
(4) inspection of semifinished product
1) steriling test extracts Embryo Gallus domesticus virus liquid and the anti-IBDV hyper-immune serum of pig, is undertaken by existing " Chinese veterinary drug allusion quotation " appendix, answers asepsis growth.
2) viral level is measured the CF-C strain is made 10 times of serial dilutions with sterile saline, gets 10
-4, 10
-5With 10
-63 dilution factors, through 5 pieces of allantocherion inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml puts 37 ℃ and hatched 168 hours, calculates ELD
50, viral level Ying ≧ 10
4.5ELD
50/ 0.2ml.
3) joining Seedling and packing gets and is up to the standards and is diluted to viral level Ying ≧ 10 with sterile saline
4.5ELD
50The CF-C strain virus liquid of/0.2ml and pig hyper-immune serum by volume 1:1 mix, and add the protective agent of proper proportion, add the abundant mixing of an amount of antibiotic simultaneously, quantitatively packing.
(5) carry out lyophilisation after the lyophilizing packing rapidly.
3. product inspection
(1) the spongy loose agglomerate of character milky is easy to bottle wall and breaks away from, and adds dissolving rapidly behind the diluent.
(2) steriling test is tested by existing " Chinese veterinary drug allusion quotation " appendix method, answers asepsis growth.
(3) the mycoplasma check is tested by existing " Chinese veterinary drug allusion quotation " appendix method, should not have the mycoplasma growth.
(4) the exogenous virus check is tested by existing " Chinese veterinary drug allusion quotation " appendix method, should be up to specification.
(5) safety verification with vaccine with diluted to the 0.1ml/10 plumage, in embryo, inoculate 18 age in days SPF Embryo Gallus domesticus (30 pieces) or cervical region subcutaneous vaccination 1 age in days SPF chicken (20) then respectively, chickling 8 ages in days, each immune group is got 10 chickens, contrast is got 5 chickens and is cutd open inspection, the observation fabricius bursa changes, pathological change appearance such as that the chickling fabricius bursa does not all have is hemorrhage, downright bad, yellow mucus.All the other chickens continue to raise to 21 ages in days, observe the growing state of chicken, should be good for work.In the embryo immunoprophylaxis group also should be more on the same group incubation rate, incubation rate should not have significant difference between immune group and the matched group.
(6) potency test is diluted to the 0.1ml/0.1 plumage with vaccine, and then through 10 SPF chickens of 1 age in days subcutaneous vaccination, immunity blood sampling in back 14 days is surveyed antibody and used the BC6/85 strong virus attack simultaneously.Attack back 72h and cut open the inspection fabricius bursa, pathological changes such as that the immune group fabricius bursa should not have is hemorrhage, downright bad, yellow mucus are protected more than 8/10, and not immune counteracting toxic substances group fabricius bursa is answered 4/5 pathological changes, and it is 5/5 normal that blank group fabricius bursa is answered.
(7) residual moisture is measured and is measured by existing " Chinese veterinary drug allusion quotation " appendix method, should be up to specification.
(8) vacuum is measured and is measured by existing " Chinese veterinary drug allusion quotation " appendix method, should be up to specification.
The microbial resources information that the present invention relates to
The microorganism that the present invention relates to is chicken infectivity bursa of Fabricius virus CF-C strain, by China Veterinery Drug Inspection Office nineteen ninety-five in Beijing's isolation identification, and by Embryo Gallus domesticus repeatedly go down to posterity cause a little less than, called after chicken infectivity bursa of Fabricius virus (Infectious bursaldiseasevirus) CF strain, the inventor is further purified the clone with the CF strain and obtains this strain virus of the higher chicken infectivity bursa of Fabricius virus of virus titer (Infectiousbursaldiseasevirus) CF-C strain and deliver the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on April 1st, 2013, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number CF-C strain is CGMCCNo.7404.
Positive effect of the present invention
The present invention utilizes the good IBDVCF-C strain of immunogenicity and the anti-IBDV hyper-immune serum of pig specific bond to prepare vaccine, can adopt the interior inoculation of instar chicken embryo embryo on the 18th or 1 Japanese instar chickling is carried out the cervical region subcutaneous vaccination, vaccine is not disturbed by maternal antibody, and once immunity can produce good immune protection power; Vaccine safety is effective, and the immunity back does not all have obvious influence to Embryo Gallus domesticus incubation rate and chick growth.Adopt the anti-IBDV hyper-immune serum of pig as the seedling material in addition, avoided the employing chicken serum and cause the birds exogenous virus to pollute, vaccine is safer.
Description of drawings
Fig. 1: vaccine production process chart.
Embodiment
Following examples are for further setting forth the present invention, but these embodiment are not construed as limiting the invention.
Embodiment 1
---the vaccine manufacturing
1. material
(1) chicken infectivity bursa of Fabricius virus CF-C strain virus content 10
5.25ELD
50/ 0.2ml is provided by inventor's preparation, preservation.
(2) the anti-IBDV hyper-immune serum of pig is prepared by the inventor.
(3) check is provided by China Veterinery Drug Inspection Office with strong malicious infectious bursal disease BC6-85 virulent strain.
(4) the SPF Embryo Gallus domesticus is available from the logical laboratory animal technology company limited of Beijing Cimmeria dimension.
(5) the SPF chicken is available from the logical laboratory animal technology company limited of Beijing Cimmeria dimension.
2. produce with kind of a poison preparation
(1) chicken infectivity bursa of Fabricius virus CF-C strain
1) inoculation and results are diluted to 100 times (volume ratios) with CF-C strain seed disease venom, are inoculated in the allantocherion of instar chicken embryo on the 10th, every embryo 0.2ml, and totally 45,37 ℃, 65% humidity hatching observation, 48h～96h receives 40, and results are organized 300g altogether; To gather in the crops tissue and be loaded in the sterile chamber and shred, put below-20 ℃ frozen.Freeze thawing 2～3 times is by centrifugal or cross and filter to remove sediment, and supernatant is stored in below-20 ℃ to be checked.
2) the steriling test steriling test is qualified.
3) viral level is measured above-mentioned kind of venom by 10
-3, 10
-4, 10
-5Dilution, 5 10 age in days SPF Embryo Gallus domesticus of each titre inoculation, every embryo 0.2ml, through the allantocherion inoculation, 37 ℃, 65% humidity cultivation 168h judge that viral level is 10
5.17ELD
50/ 0.2ml is for qualified.
4) the specificity check is 10 with the venom dilution
3.0ELD
50/ 0.1ml, seed culture of viruses and serum 1:1 neutralization, in 37 ℃ and 60 fens kinds, allantocherion inoculation every embryo 0.2ml of instar chicken embryo (SPF) on the 10th, 5 of neutralization groups, 5 of matched groups, 37 ℃, 65% humidity hatching observation 168h, neutralization group 5/5 is lived, and virus control group 0/5 is lived, and it is qualified to be judged to.
5) safety examination with 10 times of dilutions of venom after eye dripping inoculate 10 of 7 age in days SPF chickling, 0.2ml/ only stays 10 not inoculate and do matched group, isolated rearing was observed 21 days.Inoculation back 72h cuts open inspection (5/group), inoculation chicken and contrast chicken bursa and all do not have variations such as hemorrhage, downright bad, yellow mucus sample.In 21 day observation period, inoculation chicken and contrast chicken equal 5/5 strong living.
6) the mycoplasma check is inoculated in the 5ml seed culture of viruses in the improvement FreyShi culture medium, transplants 4 times, observed 28 days for 37 ℃, and little Huang, pH value is 7.6, mycoplasma verifies as feminine gender.
7) the every embryo SPF of the virulence of Embryo Gallus domesticus egg inoculation 0.2ml contains 1000 ELD
50, instar chicken embryo was 20 on 11st, the allantocherion inoculation, and every embryo 0.2ml observed 7 days, and dead 20 of accumulative total is cutd open inspection fetus hydrosarca, brain hyperemia, liver has the mottling pathological changes, and heart is cold cuts shape canescence.It is qualified to be judged to.
3. viral liquid preparation
(1) preparation of chicken infectivity bursa of Fabricius virus CF-C strain virus liquid
1) inoculation and results are with 100 times (volume ratio) of above CF-C strain seed disease venom dilution, and allantocherion is inoculated 100 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml.37 ℃, 65% humidity hatching observation, 48～96h, 48h early dead germ is 8 pieces, discarding need not.48～96h results are spent the night in Cool Room 4 for 92 pieces, and the results Embryo Gallus domesticus is organized 720g; To gather in the crops tissue and be loaded in the sterile chamber and shred, put below-20 ℃ frozen.Freeze thawing 2～3 times is by centrifugal or cross and filter to remove sediment, and supernatant is stored in below-20 ℃ to be checked.2) steriling test, no antibacterial, fungus growth are carried out in the steriling test sampling.
3) viral level is measured viral liquid is carried out 10 times with 10 times of serial dilutions of sterile saline work, gets 10
-3, 10
-4, 10
-5Three dilution factors, allantocherion is inoculated each 5 of 10 age in days SPF Embryo Gallus domesticus respectively, every embryo 0.2ml, 37 ℃, 65% humidity observation 168h, the viral level of the viral liquid of this batch is 10
5.32ELD
50/ 0.2ml.
4. finished product is made the preparation (500 plumages part/bottle) of 201301 batches of infectious bursal disease antigen-antibody vaccines
(1) joining Seedling will be up to the standards to get and be up to the standards and be diluted to viral level 10 with sterile saline
5.32ELD
50The anti-IBDV hyper-immune serum of the CF-C strain semi-finished product of/0.2ml and pig is joined Seedling for the 1:1 ratio by volume, and 500ml adds the 500ml protective agent altogether, and 1000ml adds a certain amount of antibiotic altogether.
(2) the packing lyophilizing is divided into and adorns 500 bottles, every bottle of 2ml, and the vacuum freeze drier of packing into carries out lyophilizing.
Embodiment 2
---product inspection (be example with 1301 batches of vaccines)
1. the character vaccine is the spongy loose agglomerate of milky, is easy to bottle wall and breaks away from, and adds dissolving rapidly behind the diluent.
2. the steriling test vaccine carries out no antibacterial, fungus growth by existing " Chinese veterinary drug allusion quotation " appendix.
3. mycoplasma check vaccine is undertaken by existing " Chinese veterinary drug allusion quotation " appendix, no mycoplasma growth.
4. exogenous virus check vaccine is tested by existing " Chinese veterinary drug allusion quotation " appendix, and no exogenous virus pollutes.
5. safety verification is 10 plumage part/0.1ml with the vaccine dilution, in embryo, inoculate 18 age in days SPF Embryo Gallus domesticus (30 pieces) and cervical region subcutaneous vaccination 1 age in days SPF chicken (20) respectively, stay 30 pieces of instar chicken embryos on the 18th and 20 1 age in days SPF chickens not to inoculate as blank, isolate hatching and raising with condition, chickling is in the time of 8 days, and each immune group is got 10 chickens, and contrast is got 5 chickens and cutd open inspection, the observation fabricius bursa changes, pathological change appearance such as that the chickling fabricius bursa does not all have is hemorrhage, downright bad, yellow mucus.All the other chickens continue to raise to 21 ages in days, observe the growing state of chicken, all strong living.Immunoprophylaxis group and matched group incubation rate are 97% in the embryo.
6. the efficacy test vaccine is diluted to the 0.1ml/0.1 plumage, then through 10 SPF chickens of 1 age in days subcutaneous vaccination, stays 10 not inoculate and do blank, and 5 blood sampling surveys of the back 14 days whole immune group chickens in sky and blank group chicken of immunity antibody is used the BC6/85 strong virus attack simultaneously.Attack back 72h and cut open the inspection fabricius bursa, immune group fabricius bursa 10/10 is normal, not immune counteracting toxic substances group fabricius bursa 5/5 yellow gelatin pathological changes, and blank group fabricius bursa 5/5 is normal.Immune group 10/10 antibody positive, matched group 5/5 negative antibody.
7. residual moisture is measured and is undertaken by existing " Chinese veterinary drug allusion quotation " appendix, and 4 bottles of vaccine residual moistures of sampling observation are respectively 2.33%, 1.95%, 2.02%, 2.08%.
8. vacuum is measured and is undertaken by existing " Chinese veterinary drug allusion quotation " appendix, and 35/35 the purple aura occurs.
Embodiment 3
---with the like product comparative test
Get with external infectious bursal disease (2512 strain) single Seedling of the identical blood serum subtype of CF-C strain and 201201 batches of vaccines of Laboratory Production and carry out safety and immune efficacy contrast test.
The broiler of the different maternal antibody levels of 1 age in days is divided into 3 groups at random, 30 every group, label, antibody titer is surveyed in blood sampling.201201 batches of vaccines of laboratory are diluted to 1 plumage/0.1ml, and each is through 30 of cervical region subcutaneous vaccination 1 age in days broiler, and 0.1ml/ only.Single Seedling by specification is diluted to 1 plumage/0.1ml, collunarium eye dripping, 0.1ml/ only, stay 30 not immune as contrast, the equal conditions isolated rearing.
Got 10 of each immune group, 5 blood sampling surveys of matched group antibody in back 14 days, 21,28 in immunity, attack with the strong poison of BC6/85 (dosage: 0.1ml/ only contains 100BID, the collunarium eye dripping) simultaneously.Observed 72 hours behind the counteracting toxic substances, cut open inspection, observe fabricius bursa pathological changes situation.
The result shows, immunity back 21 days and 28 days, and laboratory products counteracting toxic substances protective rate and antibody positive rate are more than 90%, and external single Seedling counteracting toxic substances protective rate and antibody positive rate are below 10%.
Claims (3)
1. infectious bursal disease live-vaccine, it is characterized in that this live vaccine contains chicken infectivity bursa of Fabricius virus CF-C strain and the anti-IBDV hyper-immune serum of pig, infections chicken cloacal bursa virus (Infectious bursal disease virus) CF-C strain virus is delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on April 1st, 2013, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number CF-C strain is CGMCCNo.7404.
2. the preparation method of the described a kind of infectious bursal disease live-vaccine of claim 1 is characterized in that
(1) inoculation of Embryo Gallus domesticus, results: with sterile saline/or phosphate-buffered meat soup will produce to be diluted to seed culture of viruses CF-C strain chicken infectivity bursa of Fabricius virus and contain 1000～10000EID
50/ 0.1ml, through allantocherion inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml puts 37 ℃ and hatches to 96 hours, discards dead germ before 48 hours with the CF-C strain virus liquid after the dilution; 48～96 hours dead germs and survived in 96 hours embryo and chorioallantoic membrane are collected in grouping respectively, are loaded in the sterile chamber to shred, and put below-20 ℃ frozen; Freeze thawing 2～3 times is by centrifugal or cross and filter to remove sediment, and supernatant is seedling with CF-C strain semi-finished product, through steriling test qualified, viral level is every 0.2ml ≧ 10
4.5ELD
50, be stored in below-20 ℃;
(2) join Seedling and packing learnt from else's experience be up to the standards and through sterile saline/or phosphate-buffered meat soup to be diluted to viral level be every 0.2ml ≧ 10
4.5ELD
50Viral liquid by volume 1:1 mix with the anti-IBDV hyper-immune serum of pig, add freeze drying protectant commonly used and antibiotic through abundant mixing, lyophilisation is carried out in quantitatively packing rapidly after the packing.
3. as the preparation method of a kind of infectious bursal disease live-vaccine as described in the claim 2, it is characterized in that wherein the preparation method of the anti-IBDV hyper-immune serum of pig is: obtain the anti-IBDV hyper-immune serum of pig, 56 ℃ of deactivation 30min with CF-C strain chicken infectivity bursa of Fabricius virus immune health pig.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310169187.3A CN103263667B (en) | 2013-05-09 | 2013-05-09 | Chicken infectious bursal disease live vaccine and production method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310169187.3A CN103263667B (en) | 2013-05-09 | 2013-05-09 | Chicken infectious bursal disease live vaccine and production method thereof |
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| Publication Number | Publication Date |
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| CN103263667A true CN103263667A (en) | 2013-08-28 |
| CN103263667B CN103263667B (en) | 2014-10-22 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103923885A (en) * | 2014-04-16 | 2014-07-16 | 江苏省农业科学院 | Infectious bursal disease virus Vero cell-adapted strain and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103923885A (en) * | 2014-04-16 | 2014-07-16 | 江苏省农业科学院 | Infectious bursal disease virus Vero cell-adapted strain and application thereof |
| CN103923885B (en) * | 2014-04-16 | 2016-05-04 | 江苏省农业科学院 | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof |
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