CN103599533A - Chicken new castle disease-infectious bronchitis trivalent combined vaccine and preparation method thereof - Google Patents
Chicken new castle disease-infectious bronchitis trivalent combined vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chicken new castle disease-infectious bronchitis trivalent combined vaccine and a preparation method thereof. The trivalent combined vaccine comprises antigens and a freeze-drying protective agent. The antigens comprise a chicken kidney-type infectious bronchitis virus K136 strain having an accession number of CCTCC-V201320, a chicken breath-type infectious bronchitis virus H120 strain, and a chicken new castle disease virus La Sota strain. A mode of inoculating the K136 strain and the La Sota strain in a homeomorphism manner and inoculating the H120 strain individually is adopted for preparing the antigens. Based on the mode, after culture products are mixed according to a ratio to prepare the antigens, a product of the vaccine is obtained by adding the freeze-drying protective agent. The trivalent combined vaccine can prevent the chicken new castle disease and the chicken infectious bronchitis simultaneously, thus solving effectively a problem that vaccines at present can only provide a part of protection or cannot provide protection for the kidney-type IB, and achieving an objective that one injection can be used for preventing two diseases.
Description
Technical field
The invention belongs to biological new medicine technical field for poultry, especially relate to a kind of infectious bronchitis of chicken and newcastle disease bigeminy trivalent live vaccine and preparation method thereof.
Background technology
Infectious bronchitis virus (IBV) belongs to coronaviridae coronavirus genus, can cause that a kind of chicken is acute, height contact respiratory infectious disease, its clinical symptoms main manifestations is disease chicken dyspnea, send rale, cough, mouth breathing, sneeze etc., mortality rate is generally very low, but to the equal easy infection of the chicken of various ages in days, sex.After children's Chickens Infected, often cause the permanent degeneration of fallopian tube; Grow up and conventionally can cause that the price of deed reduces, death rate rises after broiler infects; After laying hen infects, table can cause the quality of egg production reduction, egg to decline conventionally.In addition, this disease is concurrent or secondary escherichia coli staphylococcus or Mycoplasma Mycoides Infection often, and mortality rate is increased greatly, and poultry husbandry is caused to serious harm.
Infectious bronchitis of chicken is worldwide distribution, but the division of IBV pathological form is not still had to clear and definite standard at present, mainly according to virus, close preferendum, the major organs of infringement and the clinical symptoms causing of tissue to be determined, comprising breathing pattern, reproductive tract type, kidney type, visible peristalsis visible intestinal peristalsis, Glandular Stomach Type and anomaly etc., country variant and different regions IBV have comparatively significant areal variation, the infectious bronchitis of chicken of China is mainly breathing pattern and kidney type, is used for preventing the vaccine strain of breathing pattern IBV mainly to comprise H120 strain, M41 strain etc. at present in China; For preventing the vaccine strain of kidney type IBV mainly to comprise W93 strain, Ma5 strain etc.
Newcastle disease virus (NDV) belongs to Paramyxoviridae paramyxovirus and belongs to, and can cause the height contact of a kind of chicken, acute, deadly infectious disease.In its course of disease, often present septicemia, main clinical characteristics shows as disease chicken dyspnea, dysentery, and nervous function is disorderly, and mucosa and serous coat are hemorrhage etc.The chicken at each kind chicken and various ages can infect, and young chicken and middle chicken be easy infection more, and mortality rate is high, often poultry breeding industry is caused to crushing blow.Avian pneumo-encephalitis virus only has a serotype, but different subgroups and strain exist obvious difference in virulence, antigenicity and the aspect such as pathogenic, although this species diversity is not enough to cause immuning failure, but likely make some pathogenic strains at vaccinated flock sustainable existence, bring potential safety hazard to chicken aquaculture, the long-term popular newcastle disease virus of production practices proof newcastle La Sota strain vaccine Dui China has prevention effect special, wide spectrum.
Along with to the deepening continuously of this two kinds of disease researches, prevention and control measure also obtains constantly perfect, and the particularly successful utilization of corresponding vaccine makes these two kinds of diseases obtain to a certain extent effective control.But, in actual production process, IB is not effectively controlled, the phenomenon of immuning failure happens occasionally, its main cause is the easy origination point sudden change of IBV genome, gene delection, insert and restructuring, cause new serotype and genotype virus constantly to occur, it is reported, the whole world has more than 30 serotype, infectious bronchitis virus serotype is numerous, this has brought very large difficulty to immunity, single serogroup vaccine can only produce immunity to the infectious bronchitis virus of homotype, and can only provide part protection or not protection to the infectious bronchitis virus of its alloytype, as traditional H52, H120, vaccine prepared by the strains such as M41 can not provide protective effect to a lot of endemy infectious bronchitis virus.Up to now; never have on the market the good infectious bronchitis vaccine of cross-protection; kidney type IB almost can not get controlling; at present both at home and abroad for preventing the research of renal type infectious bronchitis vaccine more; but what obtain take W93 strain as representing that kidney type classical strains is also along with the protection to kidney type IB has been lost in viral variation gradually; and the side reaction of these strains is larger; be not suitable for the control to the epidemic strain of China; therefore screening obtains the new strain that can effectively prevent and treat kidney type IB that has, and for the preparation of multiple vaccines, just seems particularly important.Different from IBV is that NDV serotype is relatively fixing, the newcastle disease vaccines such as the conventional I of China's newcastle prevention at present system, II system, III system, wherein La Sota strain vaccine is relatively better to the prevention effect of ND, on broad spectrum activity, possesses certain advantage, especially La Sota strain vaccine is subject to the impact of maternal antibody lower when inoculation, when mixed infection, be not generally subject to the interference of IBV or other virus, therefore often become the optimum selection in multiple vaccines preparation process simultaneously.
Existing in a large number about the research report of IBV and NDV bigeminy vaccine in China, but wherein rarely about the research of the preparation method of bigeminy trivalent vaccine, few especially for the research of kidney type IBV and NDV bigeminy trivalent vaccine.2006, Cui Baoan (number of patent application: 200610018069.2) utilize the La Sota strain of breathing pattern M41 strain, the HN99 strain of kidney type and the NDV of IBV successfully to prepare IB/ND bigeminy tervalence inactivated vaccine, but it is the inactivated vaccine that utilizes formalin-inactivated to produce, its preparation exists the intrinsic deficiency of traditional inactivated vaccine, and immune effect is poor, 2012, Song Xinyu (number of patent application: 201210594755.X) utilize the Glandular Stomach Type YB160 strain of IBV, IB/ND bigeminy trivalent live vaccine has been prepared in the La Sota strain of breathing pattern H120 strain and NDV, its goods have been given full play to the advantage of live vaccine, successfully to the popular Glandular Stomach Type of current China, the attack of breathing pattern avian infectious bronchitis virus and the attack of Avian pneumo-encephalitis virus provide good immune protective efficiency, but because infectious bronchitis of chicken exists the poor characteristic of cross-protection between different shaped vaccine, so these goods are still helpless to the protective capability of Testis et penis Gallus domesticus type IB.For meeting the needs of China to kidney type IB control, screening obtains the weak poison of the new kidney type IBV with good protection power and just becomes the task of top priority for the preparation of multi-joint live vaccine.
In the preparation process of traditional multiple vaccines, normal employing is inoculated different virus respectively and the pattern of amplification culture is prepared antigen, in the preparation technology of existing IB/ND bigeminy trivalent vaccine, continued to use this antigen preparation mode, it often exists, and technique is loaded down with trivial details, cost is high, the problem such as unstable of tiring between easy pollution, different batches.In the present invention, we adopt same chick embryos inoculation to breed IBV, NDV, can reach easy and simple to handle, and pollution rate is low, saves the objects such as labour force.
Summary of the invention
The object of this invention is to provide a kind of bigeminy trivalent live vaccine that prevents newcastle disease, kidney type and respiratory infectious bronchitis, utilize the separated kidney type infectious bronchitis of chicken attenuated vaccine strain K136 strain of classical Avian pneumo-encephalitis virus La Sota strain, the H120 strain of infectious bronchitis of chicken breathing pattern and the inventor oneself to prepare live vaccine as antigen, vaccine of the present invention can effectively be prevented and treated China's popular kidney type, breathing pattern infectious bronchitis of chicken and newcastle at present, realizes a pin two anti-.
The present invention also aims to provide above-mentioned a kind of preparation method of preventing the bigeminy trivalent live vaccine of newcastle disease, kidney type and respiratory infectious bronchitis; be about to newcastle disease virus La Sota strain and avian infectious bronchitis virus K136 strain same chick embryos inoculation SPF Embryo Gallus domesticus; the independent inoculated into chick embryo of avian infectious bronchitis virus H120 strain, adds freeze drying protectant to make after three kinds of virus liquids of amplification are in a large number mixed in proportion.
For realizing above-mentioned purpose of the present invention, the technical scheme adopting is as follows:
Newcastle disease, an infectiousness bronchitis bigeminy trivalent live vaccine, this live vaccine is comprised of antigen and freeze drying protectant, and described antigen comprises Nephropathogenic infectious bronchitis virus, chicken respiratory infectious bronchitis is viral and newcastle disease virus.Its composition of described freeze drying protectant comprises 10% sucrose and 5% skimmed milk.
In above-mentioned bigeminy trivalent live vaccine, the Nephropathogenic infectious bronchitis virus that described antigen comprises refers to that deposit number is the K136 strain of CCTCC-V201320; The chicken respiratory infectious bronchitis virus that described antigen comprises refers to H120 strain; The newcastle disease virus that described antigen comprises refers to La Sota strain.
Newcastle disease virus La Sota strain and avian infectious bronchitis virus H120 strain are all classical vaccine antigens, and the vaccine of preparing with it has good prevention effect to the popular newcastle of China and chicken respiratory infectious bronchitis.The K136 strain virus that antigen comprises be by inventor for China at present popular Nephropathogenic infectious bronchitis virus in Embryo Gallus domesticus 136 generations of going down to posterity, cause weak continuously, it has effectively overcome the shortcoming that classical kidney type strain W93 strain side reaction in vaccine preparation process is large, be not suitable for China's epidemic strain control, does antigen prepare vaccine and still belong to the first time in the present invention with K136 strain.
The present invention also provides the preparation method of a kind of newcastle disease, infectiousness bronchitis bigeminy trivalent live vaccine; the feature of described preparation method is to be after newcastle disease La Sota strain and infectious bronchitis K136 strain or infectious bronchitis H120 strain homeomorphism amplification culture; be mixed in proportion and be prepared into antigen with the infectious bronchitis H12O strain of independent amplification culture or infectious bronchitis K136 strain, then antigen and freeze drying protectant are mixed in proportion and carry out being prepared into vaccine after lyophilization.In preparing the process of antigen, preferably newcastle disease La Sota strain and infectious bronchitis K136 strain same chick embryos inoculation Embryo Gallus domesticus are cultivated, by the independent inoculated into chick embryo amplification culture of infectious bronchitis H120 strain.
A preparation method for above-mentioned bigeminy trivalent live vaccine, comprising following steps:
1), by NDV-La Sota strain and IBV-K136 or IBV-H120 is long-pending mixes through allantoic cavity inoculated into chick embryo, carry out viral amplification culture;
2) by IBV-H120 strain or IBV-K136 strain through the independent inoculated into chick embryo of allantoic cavity, carry out viral amplification culture;
3) gather in the crops respectively above-mentioned two-strain scale-up medium, through aseptic process and carry out after viral level mensuration the antigen of equal-volume mix homogeneously preparation cost invention vaccine;
4) vaccine antigen is mixed homogeneously with freeze drying protectant equal-volume after lyophilizing, the vaccine in preparation cost invention.
Above-mentioned preparation method specifically comprises the following steps:
1) after La Sota strain and K136 strain or H120 strain are mixed with 100-1000 times of equal-volume of normal saline dilution respectively, inoculate instar chicken embryo on the 10th, collect fluid of chick embryo dead after 72 hours as viral scale-up medium I;
2) use normal saline dilution 50-100 doubly H120 strain or K136 strain simultaneously, inoculate separately instar chicken embryo on the 10th, hatch after 50-60 hour for 37 ℃ and gather in the crops whole fluid of chick embryo as viral scale-up medium II;
3) secondly the viral scale-up medium I of results and II carried out respectively aseptic process and surveyed respectively after viral level, two-strain scale-up medium I and II are mixed to the antigen as vaccine of the present invention according to volume ratio 1:1, being semi-finished product;
4) vaccine antigen and freeze drying protectant are carried out to proportioning according to volume ratio 1:1, after lyophilization, after 36-48h, obtain vaccine, be finished product.
Bigeminy trivalent vaccine prepared by the present invention can prevent newcastle disease and infectious bronchitis simultaneously, realized the anti-object of a pin two, especially successful separation, attenuation and the use of IBV kidney type strain K136 strain, significantly promoted the control probability of China's Nephropathogenic infectious bronchitis, meet the demand of preventing and treating of current China Nephropathogenic infectious bronchitis, and effectively overcome the side effect that vaccine product that existing Nephropathogenic infectious bronchitis virus produces as antigen produces.
The specific embodiment
For further illustrating content of the present invention, Characteristic, hereby exemplify following examples and further describe the present invention.It should be appreciated by those skilled in the art, under prerequisite without departing from the spirit and scope of the present invention, can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall into protection scope of the present invention.
Embodiment 1: the screening of infectious bronchitis of chicken kidney type strain K136 strain
Our company gathered doubtful avian infectious bronchitis virus isolated strain at the different chicken houses of China respectively from 2003-2005.Sick chicken main manifestations is lassitude, cough, often hear snore, pathology cuts open inspection and finds that degasification pipe ring has kidney outside little petechia to also have obvious pathological changes, through Preliminary Identification, obtain 4 strain renal type infectious bronchitis virus stains, called after CK/IBV/QD03 strain respectively, CK/IBV/ZC03 strain, CK/IBV/YT04 strain and CK/IBV/SD05 strain.A little less than using 10 age in days SPF Embryo Gallus domesticus that four strain strains are carried out continuous passage and caused, in per generation, is inoculated 4 pieces of Embryo Gallus domesticus by allantoic cavity, hatch for 37 ℃, and the 36h Embryo Gallus domesticus of freezing to death after inoculation, results allantoic fluid, for inoculation of future generation, connects and reached for 136 generations.Cause during this time weak evaluation, its evaluation methodology is to get the virus strain infection in the 2nd, 20,40,50,60,70,80,90,100,110,120,124,128,132,134,136,140 generations 1 Japanese instar chickling, observes chickling incidence.
The results are shown in Table 1:
The pathogenic result of the different generations of table 14 kind of separated strain to SPF chicken
Table 1 is continuous
Result shows to only have CK/IBV/SD05 strain to start without dead from reaching the tested chicken of the 80th generation Strain, and the tested chicken of the 100th generation Strain starts without observing pathological changes, the 120th generation Strain start tested chicken and start inorganization and learn pathological changes.Other 3 strains its viral titer a little less than not high this shows that virus can not well adapt to, cause on Embryo Gallus domesticus always in the process of going down to posterity; M & M is not all 0, a little less than proving and not causing after these three kinds of strains go down to posterity on Embryo Gallus domesticus, the in the situation that even some strain being a little less than causing, occurred again returning by force, these all illustrate the vaccine strain use that low virulent strain is all not suitable as live vaccine that causes of CK/IBV/QD03 strain, CK/IBV/ZC03 strain and CK/IBV/YT04 strain.
And a little less than CK/IBV/SD05 strain causes completely in the 120th generation, tested chicken is had to good safety.Simultaneously with the immune 3 age in days SPF chickens of the 124th, 128,132,134,136 generation poison difference; carry out immunoprotection evaluation; homology strong virus attack is used in immunity for latter 14 days; immune component poison rate is 0/10; contrast component poison rate is 10/10; illustrate that strain of the present invention has good immune protective; for ease of distinguishing with former strain; the CK/IBV/SD05 strain that is passaged to the reduction of 136 generations is decided to be to original species poison E1 generation; the called after coronavirus genus avian infectious bronchitis virus avian infectious bronchitis virus K136 of section strain, candidate's strain of preparing as vaccine in the present invention.Avian infectious bronchitis virus K136 of the present invention strain is preserved in Wuhan University's Chinese Typical Representative culture collection center on June 28th, 2013, and deposit number is: CCTCC-V201320.
Embodiment 2:K136 strain safety detects
1, the malicious valency of different generation K136 strains on Embryo Gallus domesticus measured
Get E2, E4, E7, E10, the E15 of K136 strain for seed culture of viruses, do suitable multiple dilution, through allantoic cavity, inoculate 10 age in days SPF Embryo Gallus domesticus, every embryo 0.1mL arranges blank group simultaneously, and every embryo, through allantoic cavity inoculation normal saline 0.1ml, is hatched cultivation for 37 ℃.Discard dead Embryo Gallus domesticus in 24h, hatch after 144h, take out the chick embryo development situation of observing.According to Reed-Muench method, calculate EID
50, E2, E4, E7, E10, E15 are respectively for the malicious valency of seed culture of viruses: 10
6.7, 10
6.8, 10
6.5, 10
6.6, 10
6.8eID
50/ 0.1ml.Result of the test explanation K136 strain is bred stable on Embryo Gallus domesticus, and avirulence is returned strong phenomenon.
2, the pathogenicity of different generation K136 strains on chickling
Choose 10 age in days SPF chickling, be divided at random 6 groups, every group of 10 chickens, respectively with 10
5.0eID
50collunarium inoculation is got E2, E4, E7, E10, the E15 of K136 strain for seed culture of viruses 0.1ml, last group is usingd normal saline collunarium 0.1ml as blank group, each organizes all separately isolated rearings of tested chicken, after Continuous Observation 20 days, slaughter, cut open inspection and respectively organize tested chicken and whether occur the characteristics of lesion such as tracheorrhagia, secretions increase, renomegaly, urate deposition, the results are shown in Table 2.
The safety testing result of the different generation K136 strains of table 2 on chickling
Above-mentioned result of the test shows, respectively organizes all strong living of tested chicken after off-test, without any abnormal response, cuts open inspection inorganization and learns pathological changes, and this shows that K136 strain virulence on chickling is stablized and virulence does not occur returns by force.
Embodiment 3: different generation K136 strain immune efficacies are evaluated
With E2, E4, E7, E10, E15 generation K136 strain seed culture of viruses chick embryo allantoic liquid, with 5 * 10
2.0eID
50dosage, to 1 age in days SPF chicken collunarium, latter 14 days of immunity, together with contrast chicken, every chicken is with 10
3.68eID
50the strong malicious CK/IBV/SD05 strain eye dripping of separated strain of/0.1ml, each 0.05ml of collunarium, after counteracting toxic substances the 5th day, gather the trachea swab of every chicken, through allantoic cavity, inoculate 5 pieces of 10 age in days SPF Embryo Gallus domesticus, 0.1ml/ embryo, hatches and observes to 144 hours.Result demonstration, immune component poison rate is between 0/10-1/10, and protective rate is between 9/10-10/10; Contrast component poison rate is 10/10.Illustrate that IBV-K136 strain seed culture of viruses is basically identical with interior its immunogenicity in 15 generations, all there is good immunogenicity, illustrate that IBV-K136 strain has good immune protective.
Embodiment 4: the preparation and determination methods of newcastle disease and infectiousness bronchitis bigeminy trivalent live vaccine
1 material
1.1 produce with seed culture of viruses newcastle La Sota strain purchased from China Veterinery Drug Inspection Office; Avian infectious bronchitis virus H120 strain is purchased from China Veterinery Drug Inspection Office; Avian infectious bronchitis virus K136 strain, seed culture of viruses preserving number is CCTCC-V201320.
1.2 detect with the strong malicious Beijing Strain of newcastle disease (CVCC AV1611 strain), purchased from China Veterinery Drug Inspection Office; Avian infectious bronchitis virus M41 strain, purchased from China Veterinery Drug Inspection Office; The strong poison of separated strain CK/IBV/SD05 is by inventor's isolation identification, preservation.
1.3 hatching egg and test chicken
SPF hatching egg is purchased from 61 farms, Shandong, and SPF test chicken is purchased from 61 farms, Shandong.
2 methods
The preparation of 2.1 newcastle disease virus La Sota strains and avian infectious bronchitis virus K136 strain
Select well-developed 10 breeding of age in days SPF Embryo Gallus domesticus ND, IB viruses.1:100-1000 NDV doubly and the IBV-K136 strain mixing of 1:100-1000 times of sterile saline dilution for inoculation, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1m1.Sealing pin hole after inoculation, 37 ℃ are continued to hatch, dead discarding in 24 hours, every 6 hours photograph embryos once, during take out at any time dead Embryo Gallus domesticus, put 4 ℃ temporary, finally collect 72 hours dead chick embryo allantoic liquids.
The preparation of 2.2 avian infectious bronchus virus H120 strains
Inoculation is got and is produced with seed culture of viruses IBV-H120 strain with sterile saline dilution 50-100 doubly, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1m1.After inoculation, sealing pin hole, hatches for 37 ℃, discards Embryo Gallus domesticus dead in 24 hours, takes out at any time dead Embryo Gallus domesticus, and gathered in the crops whole chick embryo allantoic liquids in the time of 50-60 hour.
2.3 results allantoic fluids
Cooling Embryo Gallus domesticus is taken out, with iodine tincture disinfection air chamber position, then with aseptic operation, divest air chamber portion chorion, throw off membrana putaminis, break allantocherion and amniotic membrane (not making yolk break), draw Embryo Gallus domesticus liquid, the blastochyle of every some pieces of Embryo Gallus domesticus is mixed into one group.In results blastochyle, should check one by one Embryo Gallus domesticus, as fetus is corrupt, blastochyle is muddy and have the suspicious person of any pollution to be discarded.Blastochyle after results is placed in sterilizing bottle, and 2-8 ℃ of preservation, should be no more than 5.The inspection of semifinished product is carried out in sampling simultaneously.
2.4 the inspection of semifinished product
2.4.1 the blastochyle of the treated mistake of steriling test, every group samples respectively, by existing < < Chinese veterinary pharmacopoeia > > appendix method, tests.
2.4.2 viral level is measured
Sota strain) and the mensuration of (K136 strain) content 2.4.2.1(La
By the blastochyle after collecting with normal saline dilution to 100-1000EID
50, being divided into two parts, a copy of it mixes with equivalent anti-newcastle disease specific antibody, in room temperature, with l hour, gets three suitable dilution factor blastochyles and inoculates each 5 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml through allantoic cavity.Putting 37 ℃ hatches, in 24 hours, death is disregarded, and takes out at any time afterwards dead Embryo Gallus domesticus, to 144 hours, discard all dead Embryo Gallus domesticus, according to there is dehydration in not dead Embryo Gallus domesticus, the summation of rolling up, growing the specific lesions Embryo Gallus domesticus such as little calculates EID by Reed-Muench method
50.
Second part is mixed with the anti-IBV-K136 strain of equivalent specific antibody, in room temperature and 1 hour, get three suitable dilution factor blastochyles and in allantoic cavity, inoculate each 5 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml, hatch for 37 ℃, in 48 hours, death is disregarded, take out at any time Embryo Gallus domesticus dead in 48-120 hour, results Embryo Gallus domesticus liquid, with dilution Embryo Gallus domesticus mixed in equal amounts, took out all embryos alive to 120 hours, gather in the crops one by one blastochyle, with hemagglutination test, measure blood clotting valency respectively, tiring reaches 1:28 and is judged to be above infection, by Reed-Muench method, calculates EID
50.
2.4.2.2H120 the mensuration of strain content
By 47 pages-48 pages, existing < < Chinese veterinary pharmacopoeia > > text, undertaken
2.5 vaccine preparations
By the H120 strain of the newcastle disease La Sota strain being up to the standards and infectious bronchitis K136 strain hybrid virus scale-up medium and amplification culture separately by volume 1:1 be mixed with into vaccine antigen; again by vaccine antigen and freeze drying protectant by volume 1:1 mix homogeneously, the mixture of having prepared carries out obtaining vaccine finished product after lyophilization.
2.6 product inspection
Get the lot number of preparing respectively according to the method described above and be respectively 1001,1002 and 1003 vaccine finished products for check.
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 existing < < Chinese veterinary pharmacopoeia > > appendix method.
2.6.3 mycoplasma check is tested by existing < < Chinese veterinary pharmacopoeia > > appendix method.
2.6.4 diagnostic test is done suitable dilution (0.1 plumage part/0.1m1) by vaccine with normal saline, mix with equivalent anti-newcastle disease, infectious bronchitis virus H120 strain specific serum and IBV-K136 strain specific antibody, in room temperature and l hour, in allantoic cavity, inoculation 10 age in days SPF Embryo Gallus domesticus is 10 pieces, every embryo 0.2ml.Put 37 ℃ and hatch observation 144 hours.
2.6.5 safety verification
2.6.5.1 single dose inoculation safety testing
With 70 3 age in days SPF chickling, SPF chickling is divided into one group of 3 groups of every batch of vaccine at random, every group of 20 chickens, separately establish 10 of matched group chickens, test group collunarium vaccination 1 plumage part, the normal saline of matched group collunarium equivalent, observes 14; Cut open inspection.Observe and whether occur newcastle disease and the sick characteristic pathological changes of infectious bronchitis.
2.6.5.2 single dose repeated inoculation safety testing
With 70 3 Japanese instar chicklings, every batch of vaccine is used 20, separately establishes 10 of matched group chickens, test group collunarium vaccination l plumage part, and every chicken was repeated the vaccine of collunarium inoculation same dose afterwards in 14 days.The normal saline of matched group collunarium equivalent, observes 14; Cut open inspection, observe and whether occur newcastle disease, the special pathological changes of infectious bronchitis of chicken.
A 2.6.5.3 overdose inoculation safety testing
One time overdose inoculation safety testing shares 70 3 age in days SPF chickling, and every batch of vaccine is used 20, separately establishes 10 of matched group chickens, vaccine is diluted to the dosage of 10 plumage part/0.1ml, the vaccine 0.1ml that the inoculation of test group collunarium has been diluted, the normal saline of matched group injection equivalent, observes 14.Cut open inspection, observe and whether occur newcastle disease, the special pathological changes of infectious bronchitis of chicken.
2.5.6 efficacy test
2.5.6.1 newcastle disease part
With 10 of the SPF chickens of 3 ages in days, every newcastle disease and infectiousness bronchitis bigeminy trivalent live vaccine prepared by collunarium inoculation the present invention, exempts from latter 14 days, together with 10 of the identical immunity contrast chickens of condition, every chicken respectively intramuscular injection containing 10
4.0eID
50the strong malicious lml of newcastle disease Beijing Strain, observe 14.Record Mortality situation.
2.5.6.2 infectious bronchitis of chicken K136 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, together with contrast chicken, every chicken attacks 10 by nasal drip
3.68eID
50separated virulent strain observe 10.Record incidence.
2.5.6.3 infectious bronchitis of chicken H120 strain part
With 10 of 3 age in days SPF chickens, the bigeminy trivalent Seedling of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, strong each 0.1ml of malicious collunarium of IBV M41 strain together with every chicken of contrast chicken with 10 times of dilutions, observes 10.Record incidence.
2.5.7 residual moisture is measured and is undertaken by existing < < Chinese veterinary pharmacopoeia > > appendix method
2.5.8 vacuum is measured and is undertaken by existing < < Chinese veterinary pharmacopoeia > > appendix method.
3 results
3.1 the inspection of semifinished product
Steriling test carries out according to existing < < Chinese veterinary pharmacopoeia > > appendix method, all without bacterial growth.Its viral level is in Table 3.
Table 3: each batch of seed culture of viruses viral level and steriling test result
3.2 product inspection
3.2.1 character and outward appearance
3 batches of bigeminy trivalent Seedling sample survey outward appearances are micro-yellow Sponge Porosity agglomerate, and during dandle, sample is easy to depart from bottle wall.3 batches of bigeminy trivalent Seedlings all dissolve rapidly after adding respectively diluent.
3.2.2 steriling test
3 batches of every batch of bigeminy trivalent Seedlings are taken out 5 bottles and with physiological saline solution, are returned to 2.5ml respectively, by existing < < Chinese veterinary pharmacopoeia > > appendix method, are undertaken.Result demonstration, all without antibacterial, fungus growth, 3 batches of bigeminy trivalent Seedling steriling tests are all qualified.
3.2.3 mycoplasma check
3 batches of every batch of bigeminy trivalent Seedlings are taken out 5 bottles and with physiological saline solution, are returned to 2.5ml and mix respectively, by existing < < Chinese veterinary pharmacopoeia > > appendix method, test.Result demonstration, 3 batches of bigeminy trivalent Seedlings are all without mycoplasma growth, and mycoplasma check is all qualified.
3.2.4 diagnostic test
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 vaccine diagnostic test is qualified.
3.2.5 safety examination
3.2.5.1 single dose is inoculated safety testing result
Test chicken adopts after collunarium approach vaccination 1 plumage part, observes 14.Result demonstration, the spirit of test chicken, appetite, feces, growth promoter are all normal, the results are shown in Table 4.
Table 4: single dose safety testing result
3.2.5.2 single dose repeated inoculation safety testing result
Collunarium vaccination l plumage part, every chicken was repeated the vaccine of collunarium inoculation same dose afterwards in 14 days.The normal saline of matched group collunarium equivalent, observes 14; Cut open inspection, observe and whether occur newcastle disease, the special pathological changes of infectious bronchitis of chicken.Result shows that each test group chicken, all without abnormal, the results are shown in Table 5.
Table 5: single dose repeated inoculation safety testing
3.2.5.3 an overdose is inoculated safety testing result
Single dose (1 plumage part/only) inoculate after SPF chicken safety testing result test chicken employing collunarium approach vaccination 10 plumage parts, observe 14.Result demonstration, the spirit of test chicken, appetite, feces, growth promoter are all normal, the results are shown in Table 6.
Table 6: overdose inoculation safety testing
Bigeminy trivalent vaccine prepared by above result of the test explanation the present invention is safe.
3.2.6 efficacy test test
3.2.6.1 newcastle disease part
With 10 of the SPF chickens of 3 ages in days, every newcastle disease and infectiousness bronchitis bigeminy trivalent live vaccine prepared by collunarium inoculation the present invention, exempts from latter 14 days, together with 10 of the identical immunity contrast chickens of condition, every chicken respectively intramuscular injection containing 10
4.0eLD
50the strong malicious lml of newcastle disease Beijing Strain, observe 14.Record Mortality situation, the results are shown in Table 7.
Table 7: newcastle disease efficacy test test
3.2.6.2 avian infectious bronchitis virus K136 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, together with contrast chicken, every chicken attacks 10 by nasal drip
3.68eID
50separated virulent strain observe 10.Record incidence, the results are shown in Table 8.
The test of table 8:K136 strain efficacy test
3.2.6.3 infectious bronchitis of chicken H120 strain part
With 10 of 3 age in days SPF chickens, the bigeminy trivalent Seedling of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, strong each 0.1ml of malicious collunarium of IBV M41 strain together with every chicken of contrast chicken with 10 times of dilutions, observes 10.Record incidence, the results are shown in Table 9.
The test of table 9:H120 strain efficacy test
To sum up result is known; after the immunity of this product; the strong malicious counteracting toxic substances protective rate of three kinds of virus correspondences, all more than 90%, is met to the relevant regulations of < < Chinese veterinary pharmacopoeia > > 2010 editions.
3.2.6.4 residual moisture and vacuum testing result
By < < Chinese veterinary pharmacopoeia > > appendix method, detect.Result is as following table:
Table 10: moisture and vacuum detect
Embodiment 5: the homeomorphism amplification culture of newcastle La Sota strain and infectious bronchitis of chicken K136 strain
5.1 inoculations and cultivation
Select well-developed 10 breeding of age in days SPF Embryo Gallus domesticus ND, IB viruses.1:100-1000 NDV doubly and the IBV-K136 strain mixing of 1:100-1000 times of sterile saline dilution for inoculation, inoculation 10 age in days SPF Embryo Gallus domesticus in allantoic cavity, every embryo 0.1m1.Sealing pin hole after inoculation, 37 ℃ are continued to hatch, dead discarding in 24 hours, every 6 hours photograph embryos once, during take out at any time dead Embryo Gallus domesticus, put 4 ℃ temporary, finally collect 72 hours dead chick embryo allantoic liquids.
The collection of 5.2 viral scale-up mediums
Cooling Embryo Gallus domesticus is taken out, with iodine tincture disinfection air chamber position, then with aseptic operation, divest air chamber portion chorion, throw off membrana putaminis, break allantocherion and amniotic membrane (not making yolk break), draw Embryo Gallus domesticus liquid, the blastochyle of every some pieces of Embryo Gallus domesticus is mixed into one group.In results blastochyle, should check one by one Embryo Gallus domesticus, as fetus is corrupt, blastochyle is muddy and have the suspicious person of any pollution to be discarded.Blastochyle after results is placed in sterilizing bottle.
5.3 cultured products viral levels detect
By 10000 times of dilutions of normal saline for the blastochyle after collecting, be divided into two parts, a copy of it mixes with equivalent anti-newcastle disease specific antibody, in room temperature and l hour, get three suitable dilution factor blastochyles and inoculate each 5 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml through allantoic cavity.Putting 37 ℃ hatches, in 24 hours, death is disregarded, and takes out at any time afterwards dead Embryo Gallus domesticus, to 144 hours, discard all dead Embryo Gallus domesticus, according to there is dehydration in not dead Embryo Gallus domesticus, the summation of rolling up, growing the specific lesions Embryo Gallus domesticus such as little calculates EID50 by Reed-Muench method.
Second part is mixed with the anti-IBV-K136 strain of equivalent specific antibody, in room temperature and 1 hour, get three suitable dilution factor blastochyles and in allantoic cavity, inoculate each 5 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2ml, hatch for 37 ℃, in 48 hours, death is disregarded, take out at any time Embryo Gallus domesticus dead in 48-120 hour, results Embryo Gallus domesticus liquid, with dilution Embryo Gallus domesticus mixed in equal amounts, took out all embryos alive to 120 hours, gather in the crops one by one blastochyle, with hemagglutination test, measure blood clotting valency respectively, tiring reaches 1:28 and is judged to be above infection, by Reed-Muench method, calculates EID50.
5.4 cultured products viral level testing results
According to different dilution ratios, the malicious valency of its same chick embryos inoculation the results are shown in Table 11.
Table 11: cultured products viral level testing result
The malicious valency that when result shows that proportioning when ND-La Sota strain and IBV-K136 strain is at 1:10-100, its same chick embryos inoculation obtains is the highest.
Claims (10)
1. a newcastle disease, infectiousness bronchitis bigeminy trivalent live vaccine; this live vaccine is comprised of antigen and freeze drying protectant, it is characterized in that described antigen comprises Nephropathogenic infectious bronchitis virus, chicken respiratory infectious bronchitis is viral and newcastle disease virus.
2. a kind of newcastle disease according to claim 1, infectiousness bronchitis bigeminy trivalent live vaccine, is characterized in that the Nephropathogenic infectious bronchitis virus that described antigen comprises refers to that deposit number is the K136 strain of CCTCC-V201320; The chicken respiratory infectious bronchitis virus that described antigen comprises refers to H120 strain; The newcastle disease virus that described antigen comprises refers to La Sota strain.
3. a kind of newcastle disease according to claim 1 and 2, infectiousness bronchitis bigeminy trivalent live vaccine, is characterized in that K136 strain virus that described antigen comprises is that Embryo Gallus domesticus 136 generations of going down to posterity cause weak acquisition continuously for the at present popular Nephropathogenic infectious bronchitis virus of China by inventor.
4. according to a kind of infectious bronchitis of chicken bivalent vaccine described in claim 1 or 2 or 3, it is characterized in that K136 strain virus that described antigen comprises is in Attenuation is evaluated, reaching the tested chicken of the 80th generation Strain starts without dead, the tested chicken of the 100th generation Strain starts without observing pathological changes, and the tested chicken of the 120th generation Strain starts inorganization and learns pathological changes.
5. according to a kind of infectious bronchitis of chicken bivalent vaccine described in claim 1 or 2 or 3, it is characterized in that K136 strain virus that described antigen comprises is in immunity evaluation, reaching the malicious rate of the 136th generation Strain immune component is 0/10, and contrast component poison rate is 10/10.
6. the preparation method of a newcastle disease, infectiousness bronchitis bigeminy trivalent live vaccine; it is characterized in that being by after newcastle disease La Sota strain and infectious bronchitis K136 strain or infectious bronchitis H120 strain same chick embryos inoculation Embryo Gallus domesticus amplification culture; be mixed in proportion and be prepared into antigen with the infectious bronchitis H12O strain of independent inoculated into chick embryo amplification culture or infectious bronchitis K136 strain, then antigen and freeze drying protectant are mixed in proportion and carry out being prepared into vaccine after lyophilization.
7. the preparation method of a kind of newcastle disease according to claim 6, infectiousness bronchitis bigeminy trivalent live vaccine, is characterized in that comprising the following steps:
1), by NDV-La Sota strain and IBV-K136 or IBV-H120 is long-pending mixes through allantoic cavity inoculated into chick embryo, carry out viral amplification culture;
2) by IBV-H120 strain or IBV-K136 strain through the independent inoculated into chick embryo of allantoic cavity, carry out viral amplification culture;
3) gather in the crops respectively above-mentioned two-strain scale-up medium, through aseptic process and carry out after viral level mensuration the antigen of equal-volume mix homogeneously preparation cost invention vaccine;
4) vaccine antigen is mixed homogeneously with freeze drying protectant equal-volume after lyophilizing, the vaccine in preparation cost invention.
8. according to the preparation method of a kind of newcastle disease described in claim 6 or 7, infectiousness bronchitis bigeminy trivalent live vaccine, it is characterized in that in preparing the process of antigen, preferably newcastle disease La Sota strain and infectious bronchitis K136 strain same chick embryos inoculation Embryo Gallus domesticus are cultivated, by the independent inoculated into chick embryo amplification culture of infectious bronchitis H120 strain.
9. according to the preparation method of a kind of newcastle disease described in claim 6 or 7, infectiousness bronchitis bigeminy trivalent live vaccine, the scale-up medium that it is characterized in that two-strain in antigen preparation process is that equal-volume mixes.
10. according to the preparation method of a kind of newcastle disease described in claim 6 or 7, infectiousness bronchitis bigeminy trivalent live vaccine, it is characterized in that antigen and freeze drying protectant are that equal-volume mixes in vaccine preparation process.
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