CN101792739A - Optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryo - Google Patents
Optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryo Download PDFInfo
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Abstract
The invention discloses an optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryos. The optimum condition is an orthogonal design combination of seed virus inoculation route, inoculated embryo day age, inoculation density, embryo gathering time and gathering part when the virus candidate strain i.e. Guangxi epidemically representative strains 040124, BH11 and TSC-2(9), which are designed by biostatistics software, are proliferated in chicken embryos, and an optimum production parameter is explored to supply useful data to the commercial production of vaccines. The invention has the advantages of exploring the optimum condition and supplying useful data for the commercial production of the vaccines.
Description
Technical field
The present invention the invention belongs to field of biology, specifically is the top condition of infections chicken cloacal bursa virus epidemic in Guangxi representative strains chicken embryo propagation.
Background technology
Infectious bursal disease (infectious bursal disease, IBD) be by infectious bursal disease virus (infectious bursal disease virus, acute, the height contact of a kind of main harm that IBDV) causes children chicken, molten lymphocyte sexually transmitted disease.It both can cause the clinical onset of young chicken, also can cause lower immune function because of subclinical infection makes the immunosuppression that causes, and make the immunne response level of multiple vaccine reduce and resistibility decline, thus cause the generation of multiple disease, result in greater loss.Therefore, effective prevention and control of IBD not only can exempt from the caused Direct Loss of IBD itself, and owing to avoided immunosuppression that IBD causes, thereby help the prevention and control of other diseases.
It also is the most effective prevention and control measure that vaccinoprophylaxis remains poultry husbandry important in current and even a very long time in the future.In production of vaccine, it then is a kind of generally the employing at present and the comparison effective means to produce abundant vaccine antigen that application chicken embryo comes virus of proliferation.The various proliferation conditions of vaccine virus strain in the chicken embryo promptly become factor the most key in the vaccine antigen production process.
Summary of the invention
The top condition that the purpose of this invention is to provide infections chicken cloacal bursa virus epidemic in Guangxi representative strains chicken embryo propagation.
The technical scheme that the present invention solves the problems of the technologies described above is:
The top condition of infections chicken cloacal bursa virus epidemic in Guangxi representative strains chicken embryo propagation, be that to design vaccine kind poison candidate strain by Biological Statistic Analysis Software be that epidemic in Guangxi representative strains 040124, BH11 and TSC-2 (9) are when breeding in the chicken embryo, the orthogonal design combination at the position of the concentration of the approach of kind of poison inoculation, the sky age that connects embryo, inoculation and the time of results chicken embryo and results, to explore best manufacturing parameter, for the commercial production of vaccine provides the data of usefulness, its experimental procedure is as follows:
1) groping of the best route of inoculation of each strain and each position virus multiplication output of chicken embryo:
Select well-developed 9 days each 8 pieces/strain of instar chicken embryo, 0.2mL/ embryo, carry out chorioallantoic membrane inoculation and allantoic cavity inoculation respectively, collect the dead germ of 24h-96h and IBDV feature pathology embryo alive occurs, gather in the crops chorioallantoic membrane respectively, idiosome and allantoic fluid, scissors grinds with mortar after cutting carefully again, centrifuging and taking supernatant after the freeze thawing 3 times, be loaded on respectively in the sterilization bottle, carry out sterility test with the blood agar plate, carry out hemagglutination test with 1% chicken red blood cell, after bacterium inspection and HA detected result are all negative, promptly can be used as the culture of each position propagation of strain, put-20 ℃ of refrigerators and preserve, the viral level that used the Reed-Muench method to calculate in each viral cultures in month is a chicken embryo median infective dose;
2) each strain the best connects embryo sky age, best inoculum density and bestly receives groping of embryo time:
Tabling look-up according to orthogonal experimental design draws 9 combinations, gets different dilution each viral liquid and inoculates the chicken embryo of 8d, 9d and 10d, 8 pieces of each combination inoculations, 0.2mL/ embryo through chorioallantoic membrane; Inoculation back sealing wax is put 37 ℃ of incubations, and every 12h according to the egg inspection once discards chicken embryo dead in the 24h; Postvaccinal respectively then 84h, 96h and 108h take out chicken embryo of each group respectively and put 4 ℃ and spend the night, and dissect chicken embryo and its allantoic fluid of aseptic collection, chorioallantoic membrane and idiosome.The viral level that used the Reed-Muench method to calculate in each viral cultures in one month is a chicken embryo median infective dose;
3) result judges:
(1) determining of the best route of inoculation of each strain and each position virus multiplication output of chicken embryo:
With the measurement result process statistics software analysis of each strain different vaccination approach at the viral yield of the culture of Different Chicken embryo position results, all there is significant difference between three combinations of each route of inoculation, can find out by size of data, the viral yield of inoculation of chorioallantoic membrane approach and chorioallantoic membrane position results is the highest, and all consistent from the result of two kinds of different approaches inoculations, each position viral level of chicken embryo is followed successively by from high to low: chorioallantoic membrane＞idiosome＞allantoic fluid;
(2) each strain the best connects embryo sky age, best inoculum density and the best measurement result of receiving the embryo time:
Three strains make up through chorioallantoic membrane approach inoculated into chick embryo according to orthogonal design, results embryo poison is surveyed EID50, the EID50 data all are the truth of a matter with Log10, utilize Biological Statistic Analysis Software that the data of quadrature composite design are analyzed, draw broken line graph, can find out that from broken line graph the optimum combination of each strain is respectively: BH11 inoculates at 9d age, viral liquid concentration is 10
4.80TCID
50/ 0.2mL/ embryo and inoculation back 96h results; TSC-2 (9) inoculates at 8d age, viral liquid concentration is 10
5.00TCID
50/ 0.2mL/ embryo and inoculation back 108h results; 040124 inoculate age at 9d, viral liquid concentration is 10
4.84TCID
50/ 0.2mL/ embryo and inoculation back 108h results.
The invention has the beneficial effects as follows: the present invention explores top condition, provides the data of usefulness for the commercial production of vaccine.
Embodiment
Below in conjunction with embodiment the present invention is further described.
The experimental procedure of the top condition of infections chicken cloacal bursa virus epidemic in Guangxi representative strains chicken embryo propagation is as follows:
One, the best route of inoculation of each strain and each position virus multiplication output of chicken embryo gropes
Select well-developed 9 days each 8 pieces/strain of instar chicken embryo, the 0.2mL/ embryo carries out chorioallantoic membrane inoculation and allantoic cavity inoculation respectively.Collect the dead germ of 24h-96h and the IBDV feature pathology embryo of living occurs, gather in the crops chorioallantoic membrane, idiosome and allantoic fluid respectively, scissors is cut and is ground with mortar after thin again, and centrifuging and taking supernatant after the freeze thawing 3 times is loaded on respectively in the sterilization bottle.Carry out sterility test with the blood agar plate, carry out hemagglutination test with 1% chicken red blood cell.After bacterium inspection and HA detected result are all negative, promptly can be used as the culture of each position propagation of strain, put-20 ℃ of refrigerators and preserve, the viral level that used the Reed-Muench method to calculate in each viral cultures in month is a chicken embryo median infective dose.
Chorioallantoic membrane, idiosome and the allantoic fluid of results are carried out 10 with sterile saline respectively
-3, 10
-4, 10
-5, 10
-6, 10
-7Times serial dilution, every extent of dilution is through the strong live chickens embryo of 59 ages in days of chorioallantoic membrane inoculation, 0.2mL/ embryo, the rearmounted 37 ℃ of incubators of sealing wax are cultured to 168h, dead chicken embryo is disregarded before discarding 24h, will take out at any time by dead chicken embryo in 24-168h, adds up dead embryo number, observe the pathology situation, to cultivate the chicken embryo that 168h also survives simultaneously and cut open inspection, and observe idiosome and have or not systemic bleeding, obviously pathology such as oedema, perform record, press the Reed-Muench method then and calculate chicken embryo median infective dose.
Distance than=be higher than the percentage ratio of 50% percentage ratio-50%/be higher than 50%-be lower than 50% percentage ratio
LgEID50=is higher than the logarithm of the logarithm+distance proportion * dilution factor of 50% viral dilution degree
Two, each strain the best connects embryo sky age, best inoculum density and bestly receives groping of embryo time
Orthogonal experimental design adopts the orthogonal table that is formed by the combinatorial theory derivation to arrange design experiment, and the result is carried out the multifactor experiment method of statistical study design according to the fraction principle of factor design.To implement in full test number (TN) too much in order to solve multiplefactor, and the unmanageable problem of condition is necessary to select incompatible the doing experiment of the very strong treatment group of part representativeness, and these representative partially disposed combinations generally can be determined by orthogonal table.
Inoculation age in days, inoculum density and these three factors of receipts embryo time respectively have three levels, table look-up according to orthogonal experimental design and to draw 9 combinations, get different dilution each viral liquid and inoculate the chicken embryo of 8d, 9d and 10d, 8 pieces of each combination inoculations, 0.2mL/ embryo through chorioallantoic membrane; Inoculation back sealing wax is put 37 ℃ of incubations, and every 12h according to the egg inspection once discards chicken embryo dead in the 24h; Postvaccinal respectively then 84h, 96h and 108h take out chicken embryo of each group respectively and put 4 ℃ and spend the night, and dissect chicken embryo and its allantoic fluid of aseptic collection, chorioallantoic membrane and idiosome.The viral level that used the Reed-Muench method to calculate in each viral cultures in one month is a chicken embryo median infective dose.
Three, the result judges
1. the best route of inoculation of each strain and each position virus multiplication output of chicken embryo determines
With the measurement result process statistics software analysis of each strain different vaccination approach, all there is significant difference between three combinations of each route of inoculation at the viral yield of the culture of Different Chicken embryo position results.Can find out by size of data, the viral yield of inoculation of chorioallantoic membrane approach and chorioallantoic membrane position results is the highest, and all consistent from the result of two kinds of different approaches inoculations, each position viral level of chicken embryo is followed successively by from high to low: chorioallantoic membrane＞idiosome＞allantoic fluid.
2. each strain the best connects embryo sky age, best inoculum density and best the determining of embryo time of receiving
Three strains make up through chorioallantoic membrane approach inoculated into chick embryo according to orthogonal design, and results embryo poison is surveyed EID50.The EID50 data are the truth of a matter with Log10 all, utilize Biological Statistic Analysis Software that the data of quadrature composite design are analyzed, and draw broken line graph.The optimum combination that can find out each strain from broken line graph is respectively: BH11 inoculates at 9d age, viral liquid concentration is 10
4.80TCID
50/ 0.2mL/ embryo and inoculation back 96h results; TSC-2 (9) inoculates at 8d age, viral liquid concentration is 10
5.00TCID
50/ 0.2mL/ embryo and inoculation back 108h results; 040124 inoculate age at 9d, viral liquid concentration is 10
4.84TCID
50/ 0.2mL/ embryo and inoculation back 108h results.
Claims (1)
1. the top condition of infections chicken cloacal bursa virus epidemic in Guangxi representative strains chicken embryo propagation, its spy is, designing vaccine kind poison candidate strain by Biological Statistic Analysis Software is that epidemic in Guangxi representative strains 040124, BH11 and TSC-2 (9) are when breeding in the chicken embryo, the orthogonal design combination at the position of the concentration of the approach of kind of poison inoculation, the sky age that connects embryo, inoculation and the time of results chicken embryo and results, to explore best manufacturing parameter, for the commercial production of vaccine provides the data of usefulness, its experimental procedure is as follows:
1) groping of the best route of inoculation of each strain and each position virus multiplication output of chicken embryo:
Select well-developed 9 days each 8 pieces/strain of instar chicken embryo, 0.2mL/ embryo, carry out chorioallantoic membrane inoculation and allantoic cavity inoculation respectively, collect the dead germ of 24h-96h and IBDV feature pathology embryo alive occurs, gather in the crops chorioallantoic membrane respectively, idiosome and allantoic fluid, scissors grinds with mortar after cutting carefully again, centrifuging and taking supernatant after the freeze thawing 3 times, be loaded on respectively in the sterilization bottle, carry out sterility test with the blood agar plate, carry out hemagglutination test with 1% chicken red blood cell, after bacterium inspection and HA detected result are all negative, promptly can be used as the culture of each position propagation of strain, put-20 ℃ of refrigerators and preserve, the viral level that used the Reed-Muench method to calculate in each viral cultures in month is a chicken embryo median infective dose;
2) each strain the best connects embryo sky age, best inoculum density and bestly receives groping of embryo time:
Tabling look-up according to orthogonal experimental design draws 9 combinations, gets different dilution each viral liquid and inoculates the chicken embryo of 8d, 9d and 10d, 8 pieces of each combination inoculations, 0.2mL/ embryo through chorioallantoic membrane; Inoculation back sealing wax is put 37 ℃ of incubations, and every 12h according to the egg inspection once discards chicken embryo dead in the 24h; Postvaccinal respectively then 84h, 96h and 108h take out chicken embryo of each group respectively and put 4 ℃ and spend the night, and dissect chicken embryo and its allantoic fluid of aseptic collection, chorioallantoic membrane and idiosome.The viral level that used the Reed-Muench method to calculate in each viral cultures in one month is a chicken embryo median infective dose;
3) result judges:
(1) determining of the best route of inoculation of each strain and each position virus multiplication output of chicken embryo:
With the measurement result process statistics software analysis of each strain different vaccination approach at the viral yield of the culture of Different Chicken embryo position results, all there is significant difference between three combinations of each route of inoculation, can find out by size of data, the viral yield of inoculation of chorioallantoic membrane approach and chorioallantoic membrane position results is the highest, and all consistent from the result of two kinds of different approaches inoculations, each position viral level of chicken embryo is followed successively by from high to low: chorioallantoic membrane＞idiosome＞allantoic fluid;
(2) each strain the best connects embryo sky age, best inoculum density and the best measurement result of receiving the embryo time:
Three strains make up through chorioallantoic membrane approach inoculated into chick embryo according to orthogonal design, results embryo poison is surveyed EID50, the EID50 data all are the truth of a matter with Log10, utilize Biological Statistic Analysis Software that the data of quadrature composite design are analyzed, draw broken line graph, can find out that from broken line graph the optimum combination of each strain is respectively: BH11 inoculates at 9d age, viral liquid concentration is 10
4.80TCID
50/ 0.2mL/ embryo and inoculation back 96h results; TSC-2 (9) inoculates at 8d age, viral liquid concentration is 10
5.00TCID
50/ 0.2mL/ embryo and inoculation back 108h results; 040124 inoculate age at 9d, viral liquid concentration is 10
4.84TCID
50/ 0.2mL/ embryo and inoculation back 108h results.
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| CN102260649A (en) * | 2011-06-30 | 2011-11-30 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN103143007A (en) * | 2013-02-27 | 2013-06-12 | 哈药集团生物疫苗有限公司 | Method for preparing infectious bursal disease virus live vaccine by utilizing passage chicken embryo fibroblast |
| CN103146653A (en) * | 2013-02-27 | 2013-06-12 | 哈药集团生物疫苗有限公司 | Infectious bursal disease virus chick embryo fibroblast adapted strain H11 and application thereof |
| CN103263667A (en) * | 2013-05-09 | 2013-08-28 | 北京中海生物科技有限公司 | Chicken infectious bursal disease live vaccine and production method thereof |
| CN104046596A (en) * | 2014-06-26 | 2014-09-17 | 中国科学院武汉病毒研究所 | Chick embryo allantois membrane separation method capable of enhancing separation rate of avian influenza virus |
| CN104046595A (en) * | 2014-06-26 | 2014-09-17 | 中国科学院武汉病毒研究所 | Chick embryo separating method for improving avian influenza virus separation rate |
| CN104087559A (en) * | 2014-07-09 | 2014-10-08 | 江苏省农业科学院 | Infectious bursal disease virus, inactivated vaccine for infectious bursal disease virus and preparation method of vaccine |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102260649B (en) * | 2011-06-30 | 2012-11-14 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN102260649A (en) * | 2011-06-30 | 2011-11-30 | 河南农业大学禽病研究所 | Infectious bursal disease virus (IBDV) and method for preparing inactivated vaccines and combined vaccines by breeding IBDV with chick-embryo cell system and bioreactor |
| CN103143007B (en) * | 2013-02-27 | 2014-10-01 | 哈药集团生物疫苗有限公司 | Method for preparing infectious bursal disease virus live vaccine by utilizing passage chicken embryo fibroblast |
| CN103143007A (en) * | 2013-02-27 | 2013-06-12 | 哈药集团生物疫苗有限公司 | Method for preparing infectious bursal disease virus live vaccine by utilizing passage chicken embryo fibroblast |
| CN103146653A (en) * | 2013-02-27 | 2013-06-12 | 哈药集团生物疫苗有限公司 | Infectious bursal disease virus chick embryo fibroblast adapted strain H11 and application thereof |
| CN103146653B (en) * | 2013-02-27 | 2015-04-01 | 哈药集团生物疫苗有限公司 | Infectious bursal disease virus chick embryo fibroblast adapted strain H11 and application thereof |
| CN103263667B (en) * | 2013-05-09 | 2014-10-22 | 北京中海生物科技有限公司 | Chicken infectious bursal disease live vaccine and production method thereof |
| CN103263667A (en) * | 2013-05-09 | 2013-08-28 | 北京中海生物科技有限公司 | Chicken infectious bursal disease live vaccine and production method thereof |
| CN104046595A (en) * | 2014-06-26 | 2014-09-17 | 中国科学院武汉病毒研究所 | Chick embryo separating method for improving avian influenza virus separation rate |
| CN104046596A (en) * | 2014-06-26 | 2014-09-17 | 中国科学院武汉病毒研究所 | Chick embryo allantois membrane separation method capable of enhancing separation rate of avian influenza virus |
| CN104087559A (en) * | 2014-07-09 | 2014-10-08 | 江苏省农业科学院 | Infectious bursal disease virus, inactivated vaccine for infectious bursal disease virus and preparation method of vaccine |
| CN104087559B (en) * | 2014-07-09 | 2016-03-23 | 江苏省农业科学院 | A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof |
| CN104523670A (en) * | 2014-12-23 | 2015-04-22 | 山东信得科技股份有限公司 | New method for verifying protocatechuic acid soluble powder in application of poultry disease |
| CN104523670B (en) * | 2014-12-23 | 2018-04-27 | 山东信得科技股份有限公司 | A kind of method for examining protocatechuic acid soluble powder to be applied in terms of poultry diease treatment |
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Open date: 20100804 |