CN103013930B - Culture method for same-embryo proliferation of new castle disease and infectious bronchitis viruses - Google Patents
Culture method for same-embryo proliferation of new castle disease and infectious bronchitis viruses Download PDFInfo
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- CN103013930B CN103013930B CN201210545895.8A CN201210545895A CN103013930B CN 103013930 B CN103013930 B CN 103013930B CN 201210545895 A CN201210545895 A CN 201210545895A CN 103013930 B CN103013930 B CN 103013930B
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Abstract
The invention discloses a method for same-embryo proliferation cultivation of new castle disease and infectious bronchitis viruses. The culture method comprises the followings steps: inoculating the new castle disease and infectious bronchitis viruses at the same embryo; symmetrically adjusting the hatching temperature, inoculation age in days, embryo collecting time, and the concentration and proportion of the new castle disease and infectious bronchitis viruses to reach the optimal combination; and collecting allantoic fluid antigen from the chick embryo through the processes such as hatching, cooling, disinfecting and the like. The culture method provided by the invention increases the yield of the antigen, simplifies the production steps, saves the time and materials, reduces the production cost, is quicker to operate, has better economical benefits and social benefits, and is convenient to carry out, popularize and apply.
Description
Technical field
The present invention relates to belong to veterinary biologics and cultivate technical field, be specifically related to the cultural method of a kind of newcastle disease, infectious bronchitis virus homeomorphism propagation.
Background technology
Newcastle disease (Newcastledisease) is by Avian pneumo-encephalitis virus (Newcastledisease virus, NDV) cause a kind of chicken acute, height contagious disease, often be septic process, having difficulty in breathing, diarrhea, nervous function disorder, mucous membrane and serous coat hemorrhage as principal character.This disease morbidity is anxious, and infection rate and mortality ratio are all higher, sometimes even up to 100%, causes immeasurable financial loss to aviculture, especially poultry husbandry, is therefore decided to be a class transmissible disease by China, and International Office of Epizootics is decided to be category-A transmissible disease.
NDV has blood clotting (HA), the fine hemagglutinin on prominent in NDV cyst membrane surface can with the receptors bind of erythrocyte surface, cause red cell agglutination.NDV can all batrachianss of aggegation, the red corpuscle of reptiles, bird and mouse, cavy, but the ability of aggegation ox, goat, sheep, pig, horse and people's 0 type erythrocyte is different with the difference of strain.This virus can be cultivated on the chick chorioallantoic membrane of 9-11 age in days He in allantoic cavity.
Chicken infectious bronchitis (Avian Infectious Bronchitis, AIB) is that the one being caused by infectious bronchitis virus (Infectious Bronchitis Virus, IBV) is acute, height contagiousness respiratory tract disease.This disease was found in U.S.'s dakota state first in nineteen thirty, by Schalk and Hawn(1931) reported first.Beach and Schalm(1936) studies have shown that by the cross immunity of chicken infectious bronchitis virus is different from infectious laryngotracheitis virus, reconfirm the filterability of IBV, determine that its cause of disease is for virus.Airway epithelial cell be IBV former with main propagation position, the chicken of infection shows as tracheae rale, coughs, sneezes etc.; But part strain also can copy and breed in the epithelial cell of the histoorgans such as the kidney of infected chicken, uterine tube and enteron aisle, cause ureterectasia, kidney enlargement, pale, present typical " piebald kidney ", cause the egg productivity decline of infected chicken generation death or bird inlay and the decline of Egg Quality.The financial loss that chicken infectious bronchitis causes is very serious, mainly comprise: the price of deed declines, weightening finish reduces, accompanying infection causes that airsacculitis is eliminated broiler chicken in the course of processing, the financial loss that under egg drop reduction and Egg Quality, degradation causes is often far longer than by the directly lethal loss causing of this disease, can cause great financial loss to aviculture equally.
Along with the continuous expansion of livestock and poultry cultivation scale, biological products industry has also obtained developing rapidly, and especially the continuous renewal of biotechnology significantly increases the kind of vaccine product, and numerous in variety, uses vaccine can effectively prevent the generation of these two kinds of poultry dieases.The wherein bigeminy seedling of newcastle disease and infectious bronchitis, its effect and single seedling are basic identical, have the advantages such as easy to use, vaccine program is simple, the multi-joint seedling of this class develop into a kind of trend.Conventionally the method for preparing at present newcastle disease and infectious bronchitis vaccine is first to prepare respectively this two-strain, after then mixing by a certain percentage, makes seedling, so not only time-consuming but also effort, and also production cost is also higher.Therefore, cultivate respectively for existing newcastle disease and infectious bronchitis, time-consuming, effort, the present situation of take material, we will improve newcastle disease, infectious bronchitis training method at urgent need, reach the object of once cultivating two kinds of antigens.
Summary of the invention
The object of the invention is, improve for the deficiencies in the prior art, the cultural method of a kind of newcastle disease, infectious bronchitis virus homeomorphism propagation has been proposed, the present invention passes through lot of experiment validation, optimum concn ratio that creationary discovery newcastle disease, infectious bronchitis two-strain coexist, connect embryo age in days, incubation temperature and receive the embryo time, make newcastle disease, infectious bronchitis two-strain can produce best coexisting, in homeomorphism, inoculate and produce two-strain.
Technical scheme of the present invention realizes as follows:
(1) newcastle disease seed culture of viruses LaSota strain seed culture of viruses is diluted to 10
6eID
50, infectious bronchitis strain M41 strain is diluted to 10 with PBS liquid
3eID
50, after dilution, two-strain liquid is mixed with the ratio of 1:1.
(2) seed culture of viruses after mixing is seeded in 11 age in days susceptible chick embryo allantois, every embryonic breeding kind allantoic cavity 0.15ml, inoculates hole and pore with sealing with wax after inoculation;
(3) chicken embryo after inoculation is put into incubator, at 36.5 DEG C, hatches;
Before (4) 72 hours, every 24 hours photograph embryos once, after 72 hours, every 6 hours photograph embryos once, discard the dead embryo before 72h, leave the dead embryo of 72-96h time period and the embryo alive of 96h, embryo cooling 12h under 2-8 DEG C of environment will live, cooling chicken embryo is taken out, put into and receive embryo chamber, by the air chamber position of eggshell with after iodine disinfection, peel off the eggshell at air chamber position with aseptic operation, by egg shell membrane, chorioallantoic membrane and amnion are torn with tip pincet, check that whether blastochyle is muddy or have foreign matter, if, do not extract blastochyle, collect, obtain newcastle disease, infectious bronchitis antigen.
Wherein newcastle disease seed culture of viruses LaSota strain and infectious bronchitis strain M41 strain are preserved by China Veterinery Drug Inspection Office's qualification, and LaSota strain preserving number is CVCC AV1615; M41 strain preserving number is CVCC AV1511.
The beneficial effect of technical solutions according to the invention is: cultural method of the present invention passes through newcastle disease, the concentration of infectious bronchitis kind poison, connect embryo age in days, incubation temperature, carry out the adjustment of system with parameters such as receiving the embryo time, make newcastle disease, infectious bronchitis two-strain can produce the best and coexist, in homeomorphism, inoculate and produce two-strain, improve antigen receipts amount, simplify production stage, time and starting material are saved, reduce production cost, and operate more quick, there is good economic benefit and social benefit, be convenient to implement and apply.
Embodiment
In order to make technical scheme of the present invention clearer, below in conjunction with specific embodiment, the present invention is further illustrated.
In the embodiment of the present invention, unless otherwise indicated, all umbers and per-cent all by volume calculate.
Embodiment: the cultural method of homeomorphism propagation newcastle disease and infectious bronchitis virus
Newcastle disease seed culture of viruses LaSota strain for the present embodiment, infectious bronchitis strain are that M41 strain is purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
1, cultural method and step
(1) newcastle disease seed culture of viruses LaSota strain seed culture of viruses is diluted to 10
5eID
50, 10
6eID
50, 10
7eID
50three groups, infectious bronchitis strain M41 strain is diluted to 10
3eID
50, 10
4eID
50, 10
5eID
50, after dilution, two-strain liquid is mixed with the ratio of 1:1;
(2) seed culture of viruses after mixing is seeded in 9,10,11 age in days susceptible chick embryo allantois, every embryonic breeding kind allantoic cavity 0.15ml, inoculates hole and pore with sealing with wax after inoculation;
(3) chicken embryo after inoculation is put into incubator, arranges respectively at 36 DEG C, 36.5 DEG C, 37 DEG C and hatch;
(4) discard the dead embryo before 72h, the dead embryo of collection 72,76,80,86,90,96h time period and the embryo of living, embryo cooling 12h under 2-8 DEG C of environment will live, cooling chicken embryo is taken out, put into receipts embryo indoor, by the air chamber position of eggshell with after iodine disinfection, peel off the eggshell at air chamber position with aseptic operation, by egg shell membrane, chorioallantoic membrane and amnion are torn with tip pincet, check that whether blastochyle is muddy or have foreign matter, if not, extract blastochyle, collect, obtain newcastle disease, infectious bronchitis antigen.
2, malicious valency decision data
The impact of the different extent of dilution of table 1 newcastle disease, infectious bronchitis strain on viral titer
From implementation data in table 1, newcastle disease seed culture of viruses LaSota strain seed culture of viruses is diluted to 10
6eID
50, infectious bronchitis strain M41 strain is diluted to 10
3eID
50time, NDV poison valency is respectively 10
8.4eID
50, 10
8.6eID
50, 10
8.5eID
50, 10
8.6eID
50, 10
8., 5eID
50, be all greater than 10
7.0eID
50, and being greater than the malicious valency that other extension rates obtain, IBV poison valency is respectively 10
7.2eID
50, 10
6.8eID
50, 10
7.0eID
50, 10
6.7eID
50, 10
7.0eID
50, be all greater than 10
6.0eID
50, and be greater than the malicious valency that other extension rates obtain, therefore newcastle disease seed culture of viruses LaSota strain seed culture of viruses is diluted to 10
6eID
50, infectious bronchitis strain M41 strain is diluted to 10
3eID
50time, effect is best.
Table 2 newcastle disease, the different inoculation age in days comparison of infectious bronchitis strain
From implementation data in table 2, when the LaSota strain of newcastle disease seed culture of viruses and infectious bronchitis strain M41 strain seed culture of viruses inoculation age in days are 11d, antigen receipts amount is respectively 14.3ml, 14.6ml, 13.5ml, and all far more than other inoculations day, NDV poison valency is respectively 10
8.5eID
50, 10
8.6eID
50, 10
8.6eID
50, be all greater than 10
7.0eID
50, and being greater than the malicious valency that other inoculation ages in days obtain, IBV poison valency is respectively 10
6.8eID
50, 10
7.0eID
50, 10
6.8eID
50, be all greater than 10
6.0eID
50, and be greater than other malicious valencys of obtaining of inoculation ages in days, when therefore the LaSota strain of newcastle disease seed culture of viruses and infectious bronchitis strain M41 strain seed culture of viruses being inoculated to age in days and being 11d, effect is best.
Table 3 newcastle disease, the different incubation temperature comparison of infectious bronchitis strain
From implementation data in table 3, when the LaSota strain of newcastle disease seed culture of viruses and the postvaccinal incubation temperature of infectious bronchitis strain M41 strain seed culture of viruses are 36.5 DEG C, NDV poison valency is respectively 10
8.8eID
50, 10
8.6eID
50, 10
8.6eID
50, all much larger than 10
7.0eID
50, and being greater than the malicious valency that other incubation temperatures obtain, IBV poison valency is respectively 10
7.0eID
50, 10
6.8eID
50, 10
7.2eID
50, all much larger than 10
6.0eID
50, and be greater than the malicious valency that other incubation temperatures obtain, while therefore the LaSota strain of newcastle disease seed culture of viruses and the postvaccinal incubation temperature of infectious bronchitis strain M41 strain seed culture of viruses being made as to 36.5 DEG C, effect is best.
Table 4 newcastle disease, the different receipts embryo time comparison of infectious bronchitis strain
From implementation data in table 4, the receipts embryo time that newcastle disease seed culture of viruses LaSota strain is different from infectious bronchitis strain M41 strain, in the antigen receipts amount obtaining and antigen, NDV poison valency and IBV poison valency are all different, in the time that the receipts embryo time is 96h, antigen receipts amount is 14.2ml, 14.3ml, 14.5ml, all receive much larger than other antigen that embryo time obtains, and NDV poison valency is respectively 10
8.5eID
50, 10
8.5eID
50, 10
8.6eID
50, all much larger than 10
7.0eID
50, and being greater than the malicious valency that other receipts embryo times obtain, IBV poison valency is respectively 10
7.2eID
50, 10
6.8eID
50, 10
7.0eID
50, all much larger than 10
6.0eID
50, and being greater than the malicious valency that other receipts embryo times obtain, when therefore the receipts embryo time of newcastle disease seed culture of viruses LaSota strain and infectious bronchitis strain M41 strain is 96h, effect is best.
In sum, cultural method of the present invention by the concentration to newcastle disease, infectious bronchitis kind poison, connect embryo age in days, incubation temperature and receive the parameters such as embryo time and carry out the adjustment of system, make newcastle disease, infectious bronchitis two-strain can produce the best and coexist, in homeomorphism, inoculate and produce two-strain, greatly improve antigen receipts amount, simplify production stage, saved time and starting material, reduced production cost.
Claims (1)
1. a cultural method for newcastle disease, infectious bronchitis virus homeomorphism propagation, is characterized in that cultural method and step are as follows:
(1) newcastle disease seed culture of viruses LaSota strain seed culture of viruses is diluted to 10
6eID
50, infectious bronchitis strain M41 strain is diluted to 10 with PBS liquid
3eID
50, after dilution, two-strain liquid is mixed with the ratio of 1:1;
(2) seed culture of viruses after mixing is seeded in 11 age in days susceptible chick embryo allantois, every embryonic breeding kind allantoic cavity 0.15ml, inoculates hole and pore with sealing with wax after inoculation;
(3) chicken embryo after inoculation is put into incubator, at 36.5 DEG C, hatches;
Before (4) 72 hours, every 24 hours photograph embryos once, after 72 hours, every 6 hours photograph embryos once, discard the dead embryo before 72h, leave the dead embryo of 72-96h time period and the embryo alive of 96h, cooling 12h under 2-8 DEG C of environment, cooling chicken embryo is taken out, put into and receive embryo chamber, by the air chamber position of eggshell with after iodine disinfection, peel off the eggshell at air chamber position with aseptic operation, by egg shell membrane, chorioallantoic membrane and amnion are torn with tip pincet, check that whether blastochyle is muddy or have foreign matter, if, do not extract blastochyle, collect, obtain newcastle disease, infectious bronchitis antigen.
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| CN103599533A (en) * | 2013-09-23 | 2014-02-26 | 天津瑞普生物技术股份有限公司 | Chicken new castle disease-infectious bronchitis trivalent combined vaccine and preparation method thereof |
| CN105368789A (en) * | 2015-10-23 | 2016-03-02 | 浙江美保龙生物技术有限公司 | Newcastle disease and bird flu specificity virus diluent and preparation method thereof |
| CN105368787A (en) * | 2015-10-23 | 2016-03-02 | 浙江美保龙生物技术有限公司 | Newcastle disease and bird flu homeomorphic cultivation method |
| CN105219736A (en) * | 2015-10-23 | 2016-01-06 | 浙江美保龙生物技术有限公司 | A kind of method of newcastle disease, infectious bronchitis virus cultivation in the same enbryo |
| CN105441394A (en) * | 2015-12-29 | 2016-03-30 | 浙江美保龙生物技术有限公司 | Avian influenza virus diluent and preparation method thereof |
| CN109091669A (en) * | 2018-09-19 | 2018-12-28 | 天康生物股份有限公司 | Swine fever-annulus hybrid antigen preparation method and products thereof, swine fever-annulus bigeminy subunit vaccine and preparation method thereof |
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| CN101491674A (en) * | 2008-12-26 | 2009-07-29 | 天津瑞普生物技术股份有限公司 | Production technique of dual vaccine of chicken new castle disease and infectious bronchitis |
| CN102580080A (en) * | 2012-03-19 | 2012-07-18 | 天津瑞普高科生物药业有限公司 | Method for preparing newcastle disease and infectious bronchitis virus solution |
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Inventor after: Guo Xianpo Inventor after: Chen Shenmiao Inventor after: Niu Chengming Inventor after: Yang Lingzhi Inventor after: Dong Xinrong Inventor before: Meng Zhaojie Inventor before: Gong Yuzhen Inventor before: Yang Lingzhi |
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