新城疫病毒玉林株的分离鉴定与油乳剂灭活疫苗的研制
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摘要
新城疫是由新城疫病毒引起禽类的急性、高度接触性、烈性传染病，对养禽业危害极大。我国对鸡新城疫的主要防制措施是用活疫苗和LaSota油苗进行免疫，产生ND抗体而抵御鸡新城疫的发生。据资料表明，现在许多地方都有非典型新城疫发生，这除了一部分是由于免疫方法、免疫程序不够科学合理外，其他多数可能是由于疫苗毒株与当地流行毒株的基因差异导致免疫保护不够充分而引起的。本研究就本地流行毒株进行分离鉴定和油乳剂灭活疫苗的研制工作。
本研究从广西玉林市某养鸡场分离到四株新城疫毒株，通过对这四株分离株进行F基因克隆和序列分析、动物致病性试验、分子生物学特性鉴定，测定YL03的ELD_(50)为10~(-8.5)／0.1mL；MDT为42h；ICPI为1.89；对鸡、鸭、鹅等家禽和部分野禽有100％的致死率；YL02、YL03、YL04的F基因裂解位点氨基酸序列为~(112)R-R-Q-K-R-F~(117)，与新城疫强毒株F基因裂解位点氨基酸序列~(112)R／K-R-Q-K／R-R-F~(117)的特征相符；且这三个分离株101位为K和121位为Ⅴ这两个特征性氨基酸，综合以上数据表明这三个分离株为新城疫F基因Ⅶ型强毒株，这说明本地已有Ⅶ型新城疫强毒株的流行。通过F基因核苷酸和氨基酸序列分析，YL02、YL03、YL04株F基因核苷酸、氨基酸序列与标淮毒株F_(48)E_9、LaSota疫苗株的同源性都较低，这个结果提示我们应该针对当地流行ND毒株进行更多的研究，找出防制非典型新城疫的措施。
以分离到的YL03株作为种毒进行疫苗的研制，获得一个油乳剂灭活疫苗。经检验该疫苗安全、稳定性较好。以该疫苗对本地三黄鸡、番鸭进行免疫接种，接种3周后分别以YL03毒株和F_(48)E_9株攻毒试验，免疫动物获得100％的保护，而对照组动物则100％死亡，免疫动物可抵御不同类型强毒株的侵袭；以四种不同的免疫方法和程序对鸡进行免疫对比试验表明，YL03株油乳剂灭活苗与新城疫Ⅳ系弱毒苗联合使用，使鸡获得充分的粘膜局部免疫和机体抗体保护作用，相对于常规疫苗免疫可抵御新城疫强毒的侵袭。经在本地田间试验农明YL03株油乳剂灭活苗是安全、有效的，可以为我国研制ND新疫苗提供一些材料和经验。
Newcastle disease is a highly fatal and contagious avaian disease caused by Newcastledisease virus. The main measures of prevent poultry from ND is vaccinatted them with vaccine.According to the material indicastd, The non-model Newcastle disease is in popular in manypalcesOutside this unreasonable immunity vaccination, It maby was caused by the diffrentgenetype NDV with vaccine swain.
Four Newcastle disease virus strains was isolated、identified from four local poultryfarms, named YL01、YL02、YL03、YL04. Result of identification shows YL03 the ELD_(50)is 10~(-8.5)/0.1mL; MDT is 42 hours and ICPI is 1.89. all the character of the isolate indicates strainYL03 is strong virulent one. The F gene of the isolates NDV was amplified by RT-PCR, theresult of nucleotide and deduced animo-acid sequence shows the cleavage site of the peptidewas ~(112)R-R-Q-K-R-F~(117). All the results shows the YL02、YL03、YL04 belongs to gene FtypeⅦin Avian paramyxovirus 1 of NDV.
The oil-adjuvant invactive vaccine with strain YL03 was preparated. The vaccineimmunized local poultry, after immunization 14 day, the chickens was inooalated with the YL03and F48E9 virus, the incidence of protection is 100% in the immunized group, the mortality is100% in the control group. It shows that the oil-adjuvant vaccine is effective.
本研究从广西玉林市某养鸡场分离到四株新城疫毒株，通过对这四株分离株进行F基因克隆和序列分析、动物致病性试验、分子生物学特性鉴定，测定YL03的ELD_(50)为10~(-8.5)／0.1mL；MDT为42h；ICPI为1.89；对鸡、鸭、鹅等家禽和部分野禽有100％的致死率；YL02、YL03、YL04的F基因裂解位点氨基酸序列为~(112)R-R-Q-K-R-F~(117)，与新城疫强毒株F基因裂解位点氨基酸序列~(112)R／K-R-Q-K／R-R-F~(117)的特征相符；且这三个分离株101位为K和121位为Ⅴ这两个特征性氨基酸，综合以上数据表明这三个分离株为新城疫F基因Ⅶ型强毒株，这说明本地已有Ⅶ型新城疫强毒株的流行。通过F基因核苷酸和氨基酸序列分析，YL02、YL03、YL04株F基因核苷酸、氨基酸序列与标淮毒株F_(48)E_9、LaSota疫苗株的同源性都较低，这个结果提示我们应该针对当地流行ND毒株进行更多的研究，找出防制非典型新城疫的措施。
以分离到的YL03株作为种毒进行疫苗的研制，获得一个油乳剂灭活疫苗。经检验该疫苗安全、稳定性较好。以该疫苗对本地三黄鸡、番鸭进行免疫接种，接种3周后分别以YL03毒株和F_(48)E_9株攻毒试验，免疫动物获得100％的保护，而对照组动物则100％死亡，免疫动物可抵御不同类型强毒株的侵袭；以四种不同的免疫方法和程序对鸡进行免疫对比试验表明，YL03株油乳剂灭活苗与新城疫Ⅳ系弱毒苗联合使用，使鸡获得充分的粘膜局部免疫和机体抗体保护作用，相对于常规疫苗免疫可抵御新城疫强毒的侵袭。经在本地田间试验农明YL03株油乳剂灭活苗是安全、有效的，可以为我国研制ND新疫苗提供一些材料和经验。
Newcastle disease is a highly fatal and contagious avaian disease caused by Newcastledisease virus. The main measures of prevent poultry from ND is vaccinatted them with vaccine.According to the material indicastd, The non-model Newcastle disease is in popular in manypalcesOutside this unreasonable immunity vaccination, It maby was caused by the diffrentgenetype NDV with vaccine swain.
Four Newcastle disease virus strains was isolated、identified from four local poultryfarms, named YL01、YL02、YL03、YL04. Result of identification shows YL03 the ELD_(50)is 10~(-8.5)/0.1mL; MDT is 42 hours and ICPI is 1.89. all the character of the isolate indicates strainYL03 is strong virulent one. The F gene of the isolates NDV was amplified by RT-PCR, theresult of nucleotide and deduced animo-acid sequence shows the cleavage site of the peptidewas ~(112)R-R-Q-K-R-F~(117). All the results shows the YL02、YL03、YL04 belongs to gene FtypeⅦin Avian paramyxovirus 1 of NDV.
The oil-adjuvant invactive vaccine with strain YL03 was preparated. The vaccineimmunized local poultry, after immunization 14 day, the chickens was inooalated with the YL03and F48E9 virus, the incidence of protection is 100% in the immunized group, the mortality is100% in the control group. It shows that the oil-adjuvant vaccine is effective.
引文
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2．殷震，刘景华。动物病毒学[M]。北京：北京科学出版社，1997，743．
3．殷震，刘景华。动物病毒学[M]。北京：北京科学出版社，1997，744．
4. Huang, Z., High-level expression of a foreign gone from the wost 3 '-Proximal locus of a recombinant Newcastle disease virus[J]. J Gen Vol., 2001, 82(pt7): 1729～1736.
5. Calnek B W, John Barnes H, Charles Beard W, et al disease of poultry(tenth edition) [M], 1997, 541～562.
6．殷震，刘景华。动物病毒学[MI。北京：北京科学出版社，1997，745。
7．陆承平。兽医微生物学[M]。北京：中国农业出版社，2001，523～524。
8．候云德。分子病毒学[M]。北京：北京学苑出版社，1990，310～311。
9. Dolye, T.M., Ahitherto unrecorded diseade of fowls due to a filter-passing vorus [J]. J Comp pathol therap, 1927 (40): 144～169.
10．马旭平，李继连。鸡非典型性新城疫流行特点及防治[J]。河北畜牧兽医，2002(11)：33～34．
11．陈杰：几株新城疫地方分离株的生物学特性鉴定和遗传变异分析。西北农林科技大学硕士论文，2000．
12．李英霞，杨林，杨丽丽等。黑龙江省鸡传染流行的新特点及对策[J]。黑龙江畜牧兽医杂志，2002，(9)：33～34。
13．王红宁。禽呼吸系统疾病[M]。北京：中国农业出版社，2002，63～79。
14．殷震，刘景华。动物病毒学[M]。北京：北京科技出版社，1997，747。
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