CN102352346A - Novel-pathotype duck reovirus and attenuated vaccine thereof - Google Patents
Novel-pathotype duck reovirus and attenuated vaccine thereof Download PDFInfo
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- CN102352346A CN102352346A CN2011103255545A CN201110325554A CN102352346A CN 102352346 A CN102352346 A CN 102352346A CN 2011103255545 A CN2011103255545 A CN 2011103255545A CN 201110325554 A CN201110325554 A CN 201110325554A CN 102352346 A CN102352346 A CN 102352346A
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Abstract
The invention discloses a novel-pathotype duck reovirus (NDRV), which has the microbial preservation number: CGMCCNO.5198. The vaccine strains prepared from the attenuated reovirus have stable heredity and good safety, are safe to young moscovey ducks and have no pathogenicity. The immune young moscovey ducks by using the vaccine strains can effectively resist against the attack of the virulent strains of the novel-pathotype duck reovirus.
Description
Technical field
The present invention relates to a kind of weak and attenuated vaccine strain of causing of virus, relate in particular to the development of new pathotype reovirus of a kind of duck and attenuated vaccine thereof, belong to the veterinary biological product technical field.
Background technology
Since winter in 2005; The young kind duck of province such as Fujian, Zhejiang 4-25 age in days, young half kind of duck, sheldrake take place a kind of with the irregular necrosis of liver, hemorrhage and myocardium, cloacal bursa is hemorrhage is the new eqpidemic disease of principal character; Be commonly called as " the new hepatopathy of duck ", " duck hepatitis sequestrans " etc.; Sickness rate 5 %-32.5 %; Case fatality rate differs greatly; Be 4 %-20 %, but the duck age in days is littler, sickness rate, case fatality rate are higher.We pass through Study on etiology; Separate and evaluation through virus; Confirm that its cause of disease is the new pathotype reovirus of duck (the Novel-pathotype Duck Reovirus that is different from domestic and international report; NDRV) (Huang Yu etc. the isolation identification of new pathotype duck reovirus. Chinese animal doctor's magazine; 2009,45 (12): 29-31.).
Summary of the invention
The object of the present invention is to provide new pathotype reovirus of a kind of duck and attenuated vaccine thereof, this attenuated vaccine inheritance stability, security are good, and the new pathotype reovirus of duck is had good immunoprotective effect.
Technical problem to be solved by this invention is to realize through following technological approaches:
Virus provided by the invention is the new pathotype reovirus of duck (Novel-pathotype Duck Reovirus; NDRV); Strain is LH13; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 6th, 2011; It is called for short CGMCC; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC NO. 5198.
Above-mentioned virus is fastened 28-30 acquisition of continuous passage in chick embryo fibroblast cause low virulent strain, virus titer is 1 * 10
6-6.25TCID
50/ 0.1ml processes attenuated vaccine after the freeze-drying of adding lyophilized vaccine, and virus titer should be 5 * 10
5.5More than the TCID50/0.1ml.
The application of described attenuated vaccine in the new pathotype reovirus of the young kind duck of anti-system disease.
The preparation method of the new pathotype reovirus of duck attenuated vaccine is specific as follows:
(1) a little less than the causing of the new pathotype reovirus of duck: isolated viral is a little less than chick embryo fibroblast system carries out continuous passage and causes.Observe the pathogenic of viral pair cell, and get its TCID of poison mensuration that goes down to posterity of different generations
50With virus titer is 1 * 10
6-6.25TCID
50The low virulent strain of/0.1ml is malicious with planting as vaccine.
(2) preparation of the new pathotype reovirus vaccine of duck: vaccine is bred with kind of a malicious inoculated into chick embryo inoblast in a large number, measure behind the malicious valency, be prepared into vaccine with lyophilized vaccine and viral cultures mixing and packing such as gelatin.
The toxicity test test-results shows that attenuated vaccine of the present invention is to young kind duck safety, had no pathogenicity.
Remarkable advantage of the present invention:
New pathotype reovirus low virulent strain involved in the present invention; Be new pathotype reovirus (the Novel-pathotype Duck Reovirus that strong malicious type is arranged; NDRV) low virulent strain through obtaining after the chick embryo fibroblast continuous passage; The distinctive important biomolecule activity of this low virulent strain is to have lost pathogenic to the young kind duck of its former host animal; Cause the attack that is highly resistant to the strong poison of the new pathotype reovirus of duck of current popular behind the young kind duck of attenuated vaccine immunity of weak virus strain development with this, and genetic stability is good.
Embodiment
In order further to set forth the present invention, provide a series of application implementation examples below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
1, Bing Du separation and Culture with cause a little less than
The liver of the clinical young kind duck of 11 ages in days that dies of illness of aseptic collection censorship; With the aseptic PBS that contains two anti-(wherein blue or green, each 100u/ml of Streptomycin sulphate); After carrying out homogenate in the ratio (W/V) of liver and aseptic PBS=1:3-5; African green monkey kidney cell (the Vero cell of individual layer has been cultivated in the inoculation of centrifuging and taking 0.5ml supernatant liquor; ATCC:CCL-81); Behind 37 ℃ of absorption 60min; Clean 3 times with containing two anti-aseptic PBS; Add the high sugared DMEM nutrient solution (Gibco company) that contains 10% tire serum (Gibco company), put 37 ℃, 5% CO
2Incubator is cultivated and is observed 48-72h.First culture does not cause obvious cytopathy; Behind the 72h with cell multigelation 3 times; Reclaim toxic enchylema; After continuation inoculation Vero cell and continuous passage cultivated for 4 generations according to the method described above; Viral cultures can cause obvious cytopathy, and cell is visible to be heaped, merge, and occurs obviously drawing in the net, coming off; Collect the viral liquid of cultivating 48-72h and obtain the new pathotype reovirus of duck LH13 strain, preserving number is CGMCC NO. 5198.
Get and separate the new pathotype reovirus of the duck LH13 strain that obtains, inoculum size inoculation 75ml by 5%
2The individual layer chick embryo fibroblast, 45min is made in sense, and after aseptic PBS cleaning 2 times, adding 15ml contains 2% foetal calf serum and 1% couple of anti-high sugared DMEM cultivates based on 5%CO
2, 37 ℃ of incubators are cultivated and are observed.When treating that pathology appears in 75% cell, with cell multigelation 3 times, reclaim toxic enchylema, the part poison that goes down to posterity is frozen, and another part is malicious as follow-on kinds, goes down to posterity so repeatedly.
Get the malicious inoculated into chick embryo inoblast of going down to posterity of the 15th, 20,24,28 and 30 generations, observe different generations go down to posterity the poison virus titer and produce the cytopathic time.Mensuration result shows that the virus titer of the poison that goes down to posterity in the 15th generation is 10
-4.75, the viral disease poison titre that goes down to posterity subsequently progressively increases, and 28-30 is basicly stable at 1x10 for the virus titer of the poison that goes down to posterity
-6.25TCID
50About; The poison that goes down to posterity of different generations causes that the cytopathic time also shortens along with the increase of passage number; Occurring circle during the 15th generation behind the virus inoculation about 120h contracts, comes off or cytopathy such as wire drawing; During to 28 generations; Promptly visible significantly cytopathy about 72h behind the virus inoculation, virus titer and the cytopathogenic effect time of poison on chick embryo fibroblast of going down to posterity of different generations is seen table 1.
Get the poison that goes down to posterity in the 30th generation and make kind of a poison; The inoculated into chick embryo inoblast is cultivated virus in batches; The virus of results is after steriling test and exogenous virus detection; Ratio in 1.5% adds the aseptic freeze-dried bottle of gelatin mixing packing; Every bottle 2.5 ml; Attenuated vaccine is processed with vacuum-freeze-dry machine freeze-drying 22h in-20 ℃ of frozen backs of spending the night, and virus titer should be 5 * 10
5.5TCID
50More than/the 0.1ml.
The different generations of table 1 poison pathogenic to the DF1 cell that go down to posterity
2, optimum immuning dose and attack malicious protection test
Young kind duck is this viral susceptible animal, and the young kind duck of the health of 50 plumages, 1 age in days is divided into 5 groups, every group 10 plumage at random.Wherein immunity is with the attenuated vaccine of 5,10,100 and 1000 times of dilutions of sterile saline respectively for 4 groups, and every young bird kind duck is all through neck subcutaneous vaccination 0.1mL, and the 5th group of injecting normal saline is as control group.14d exhales the lonely strong malicious intramuscular injection of intestines, every plumage 0.1ml (10 with the new pathotype of duck after immunity
3.5TCID
50).After observing 20d, 10 times of dilutions (5 * 10
4.5TCID
50) dosage immune group duck all healthy, 100 times of dilutions (5 * 10
3.5TCID
50) 1 morbidity of dosage immune group duck, gradually recover one's health to the later stage, protection ratio is more than 90%, to be minimum immune dosage.Attack poison protection experiment detailed data and see table 2.
Table 2 optimum immuning dose and attack malicious protection test
3, vaccine safety test
With 100 times of minimum immune dosage, 10 1 young kind ducks of age in days test of muscle or neck subcutaneous vaccination are observed the reaction of the young kind duck of inoculation, observation periods 15 d.Do not have morbidity and dead behind the young kind duck immune vaccine of 1 age in days as a result, cut open inspection and do not see macroscopic cardinal principle pathology.
4, preservation period test
Going bail for is stored under-20 ℃ of conditions 6,12,15,18,24 and 28 months attenuated vaccine, by the young kind duck of confirming of optimum immuning dose immunity 1 age in days, sets not immune control group simultaneously, and 14 ages in days are attacked poison.-20 ℃ of vaccines of preserving after 6,12,15 months of result's demonstration all can be protected the attack of young kind of duck opposing with the source strength poison fully; The vaccine protection ratio of preserving after 18 months only is 80%, and the vaccine protection ratio of preserving 24,28 months is respectively 60%, 20%.It is thus clear that the longest effective preservation period of this vaccine is 18 months.
5, virulence is returned strong mensuration
Attenuated vaccine is inoculated the young kind duck of non-immune 1 age in days; Cut open behind the 3d and get liver, spleen etc. extremely and be organized under the aseptic condition in behind ratio homogenate, the multigelation of tissue and PBS=1:3 (W/V) 3 times; 4 ℃ of following centrifuging and taking supernatant liquors 5 generations of blind passage continuously in young kind duck body; Observe of the reaction of young kind duck to the vaccine live virus; The result is inoculated a young kind duck and is grown all good; Do not see that virulence returns strong phenomenon, show this low virulent strain genetic stability.
Claims (3)
1. the new pathotype reovirus of duck (Novel-pathotype Duck Reovirus), strain is LH13; Preserving number is: CGMCC NO. 5198 was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 6th, 2011.
2. the attenuated vaccine of the new pathotype reovirus of duck, it is characterized in that: the said virus of claim 1 is fastened 28-30 acquisition of continuous passage in chick embryo fibroblast cause low virulent strain, virus titer is 1 * 10
6-6.25TCID
50/ 0.1ml processes attenuated vaccine after the freeze-drying of adding lyophilized vaccine.
3. the application of attenuated vaccine as claimed in claim 3 in the new pathotype reovirus of the young kind duck of anti-system disease.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611441A (en) * | 2018-05-22 | 2018-10-02 | 山东农业大学 | It is a kind of detection novel duck reovirus PCR kit for fluorescence quantitative and application |
| CN108913666A (en) * | 2018-08-10 | 2018-11-30 | 山东农业大学 | A kind of duck reovirus leading to duck spleen necrosis and its inactivated vaccine and application |
| CN108977579A (en) * | 2018-08-10 | 2018-12-11 | 山东农业大学 | It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis |
| CN109295007A (en) * | 2018-09-19 | 2019-02-01 | 福建省农业科学院畜牧兽医研究所 | The selection of NDRV low virulent strain and its preparing the application in NDRV live vaccine |
| CN111172122A (en) * | 2020-01-07 | 2020-05-19 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
| CN113832113A (en) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
| CN114891753A (en) * | 2021-11-30 | 2022-08-12 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Novel duck reovirus passage attenuated strain and application thereof |
| CN114958781A (en) * | 2022-07-28 | 2022-08-30 | 佛山科学技术学院 | NDRV low virulent strain and breeding method and application thereof |
| CN116478937A (en) * | 2023-06-12 | 2023-07-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Gene III avian reovirus variant and application thereof in preparing vaccine |
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| 张宝来: "鸭源呼肠孤病毒的分离与鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》, 15 July 2010 (2010-07-15) * |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611441A (en) * | 2018-05-22 | 2018-10-02 | 山东农业大学 | It is a kind of detection novel duck reovirus PCR kit for fluorescence quantitative and application |
| CN108913666A (en) * | 2018-08-10 | 2018-11-30 | 山东农业大学 | A kind of duck reovirus leading to duck spleen necrosis and its inactivated vaccine and application |
| CN108977579A (en) * | 2018-08-10 | 2018-12-11 | 山东农业大学 | It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis |
| CN108913666B (en) * | 2018-08-10 | 2020-10-16 | 山东农业大学 | Duck reovirus causing duck spleen necrosis and inactivated vaccine and application thereof |
| CN109295007A (en) * | 2018-09-19 | 2019-02-01 | 福建省农业科学院畜牧兽医研究所 | The selection of NDRV low virulent strain and its preparing the application in NDRV live vaccine |
| CN109295007B (en) * | 2018-09-19 | 2022-03-15 | 福建省农业科学院畜牧兽医研究所 | Breeding method of NDRV low virulent strain and application thereof in preparation of NDRV live vaccine |
| CN111172122B (en) * | 2020-01-07 | 2022-07-12 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
| CN111172122A (en) * | 2020-01-07 | 2020-05-19 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
| CN113832113A (en) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
| CN113832113B (en) * | 2021-09-14 | 2023-09-22 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
| CN114891753A (en) * | 2021-11-30 | 2022-08-12 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Novel duck reovirus passage attenuated strain and application thereof |
| CN114891753B (en) * | 2021-11-30 | 2024-04-02 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | Novel duck reovirus passaging attenuated strain and application thereof |
| CN114958781A (en) * | 2022-07-28 | 2022-08-30 | 佛山科学技术学院 | NDRV low virulent strain and breeding method and application thereof |
| CN116478937A (en) * | 2023-06-12 | 2023-07-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Gene III avian reovirus variant and application thereof in preparing vaccine |
| CN116478937B (en) * | 2023-06-12 | 2023-08-18 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Gene III avian reovirus variant and application thereof in preparing vaccine |
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