H5N1亚型禽流感病毒的分离与鉴定及其对鸡的致病性研究
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摘要
禽流感(avian influenza，AI)是由正粘病毒科A型流感病毒引起家禽和野禽感染的一种病毒性传染病。本实验中，从病死鸡组织病料中分离到一株H5N1亚型AIV，鸡胚半数致死量(50％egg lethal dose, ELD_(50))滴度为10~(-7.67)／0.1ml。用此鸡源毒株和另外两株鸭源、鹅源H5N1亚型AIV的尿囊液进行1∶10稀释后，分别鼻腔接种8周龄的SPF鸡，0.2ml／只。通过观察人工感染鸡的发病情况和临床表现，定时采血、剖检和取材，进行眼观病变和组织学病变的检查，检测病毒在肺脏、肾脏、肝脏、脾脏、肌肉和脑组织动态复制情况，以及检测血清中抗体水平和13项生化指标的变化情况等实验方法来研究分离于不同禽源的三株H5N1亚型AIV对SPF鸡的致病性。实验结果表明：鸡源毒株感染病例的临床症状轻微，到36h全部死亡；眼观病变有脚鳞轻度出血，头面部轻度肿胀，腺胃乳头、腺胃和肌胃连接处严重出血，心脏冠状沟脂肪出血，肾脏肿大、色彩红白相间，肝脏肿大、表面有土黄色条纹，胰腺和脾脏表面有米粒大坏死灶等；组织学病变为非化脓性脑炎，胸腺、法氏囊、脾脏、盲肠扁桃体、气管、肺脏、十二指肠、胰腺、腺胃、肝脏、肾脏和心脏等组织器官均受到严重损害，其病变以其大量细胞成分的变性、坏死为主，伴有充血、出血和轻度的炎性细胞浸润和浆液性渗出；在肺脏、肾脏、肝脏、脾脏、肌肉和脑组织中较早的检测到高滴度病毒；感染后机体始终没有产生相应的抗体，一些尘化指标在死亡时出现显著变化。表明此毒株对鸡的致病性较强，属于泛嗜性病毒。鸭源毒株与鸡源毒株在致病性和致死率上相似，鹅源毒株在致死率上明显低于鸡源和鸭源毒株，表明这三株不同禽源的H5N1亚型AIV均为泛嗜性病毒，但是在致病性上存在一定程度的差异。
Avian influenza (AI) is a kind of viral and infectious disease by A type influenza virus of orthomyxovirus family, which happens in poultry and wild avian usually. In the experiment, an avian H5N1 influenza A virus was isolated from chicken tissues infected. The 50% egg lethal dose (ELD50)titer of this AIV is 10-7.67 /0.lml. Eight-week-old chickens were inoculated by intranasal route with 0.2ml per chicken of the 1:10 dilution allantotic fluid of three H5N1 AIVs isolated from chicken (above mentioned) and duck and goose, respectively. In laboratory infectious, three chickens were taken out random on hours 12, 24, 36, 48, 72..and l68 postinoculation. Through observing clinical signs, examining gross and histopathological changes, examining the kinetics of virus replication in brain, lung, kidney, liver, spleen and muscles, and detecting the changes of antibody liter and 13 item serum biochemical markers, we study the pathogenicity of this three H5N1 AIVs.Although no clinical signs of disease were observed in H5N1 virus-inoculated chicken that died, gross and histopathological changes are more typical, such as hemorrhage in the squama of foot, gland-stomach ripple and the joint of two stomach, and degeneration and necrosis of cell components in the spleen, thymus, bursa, mecum tonsil, trachea, lung, duodenum pancreas, gland-stomach, liver, kidney and heart. Infectious virus could be detected in six tissues of above mentioned. In addition to some serum biochemical markers denote significant changes. The results indicate that this AIV is highly pathogenic and replicated to higher liters in chicken, which belong to extensive indulged virus. As with this H5N1 AIV isolated from chicken, the two H5N1 AIVs isolated from duck and goose produced similar systemic lesions in chicken following i.n. inculation, but the three H5N1 AIVs exist differences of pathogenicity in certain degree!
Avian influenza (AI) is a kind of viral and infectious disease by A type influenza virus of orthomyxovirus family, which happens in poultry and wild avian usually. In the experiment, an avian H5N1 influenza A virus was isolated from chicken tissues infected. The 50% egg lethal dose (ELD50)titer of this AIV is 10-7.67 /0.lml. Eight-week-old chickens were inoculated by intranasal route with 0.2ml per chicken of the 1:10 dilution allantotic fluid of three H5N1 AIVs isolated from chicken (above mentioned) and duck and goose, respectively. In laboratory infectious, three chickens were taken out random on hours 12, 24, 36, 48, 72..and l68 postinoculation. Through observing clinical signs, examining gross and histopathological changes, examining the kinetics of virus replication in brain, lung, kidney, liver, spleen and muscles, and detecting the changes of antibody liter and 13 item serum biochemical markers, we study the pathogenicity of this three H5N1 AIVs.Although no clinical signs of disease were observed in H5N1 virus-inoculated chicken that died, gross and histopathological changes are more typical, such as hemorrhage in the squama of foot, gland-stomach ripple and the joint of two stomach, and degeneration and necrosis of cell components in the spleen, thymus, bursa, mecum tonsil, trachea, lung, duodenum pancreas, gland-stomach, liver, kidney and heart. Infectious virus could be detected in six tissues of above mentioned. In addition to some serum biochemical markers denote significant changes. The results indicate that this AIV is highly pathogenic and replicated to higher liters in chicken, which belong to extensive indulged virus. As with this H5N1 AIV isolated from chicken, the two H5N1 AIVs isolated from duck and goose produced similar systemic lesions in chicken following i.n. inculation, but the three H5N1 AIVs exist differences of pathogenicity in certain degree!
引文
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34 Y.Kobayashi Et Al. Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses. Avian Pathology, 1996, 25: 284-304.
35 P.T.Hooper et al. Observation on the relationship in chickens between the virulence of some avian influenza viruses and the pathogenicity for various organs. Avian Disease, 1995, 39:458-446.
36 皇甫和平，杨俊兴，牛明福等．鸭流感病毒对鸡的人工感染试验[C]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集，101～102
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2 Mukaigawa, J, and D. P. Nayak. Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2[J]. Virol, 1991,65:245～253
3 Alexander D. J, Gough R. E. Isolations of avian influenza virus from birds in Great Britain [J]. Vet Res.1986, 118:537～538
4 Alexander D.J. A review of avian influenza in different bird species [J]. Vet Microbiol. 2000,74(1-2): 3-13
5 于康震．禽流感与养禽业发展和人类健康[J]．中国预防兽医学报，1998，20(1)：1～6
6 郭元吉，程小雯．流行性感冒病毒及其实验技术[M]．中国三峡出版社，1997
7 甘孟侯．禽流感[M]．北京农业大学出版社．1995
8 Sugimura T, Murakami Y, et al. The susceptibility of culture cells to avian influenza viruses [J]. Vet Med Sci,2000,62(6): 659～660
9 Klingebory, B., Englund L., et al. 1985. An avian influenza A virus killing a mammalina species-the mink [J]. Arch Virol,86:347～351
10 Murphy B. R. and R. G. Webster: Influenza Viruses. In B. Fields (ed.)[M]. Virology. Reven Press New York, 1985:1179～1240
11 O'Neill R. E., J. Talon, et al. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins[J]. EMBO J. 1998,17:288～296
12 Steinhauer, D.A.Role of hemagglution cleavage for the pathogenicity of influenza virus [J]. Virology, 1999,258:1～20
13 Kida H., R.Yangawa. Duck influenza lacking evidence of disease signs and immune response [J]. Infect.Immune. 1980, 30:547～533
14 Webster R.G., M.A.Yakhwo. Intestinal influenza replication and characterization of influenza viruses in ducks [J]. Virology, 1978,84:268～278
15 Donis R. O., W.J.Bean, et al. Distinct lineages of influenza virus H4 hemagglutinin genes in different regions of the world [J]. Viro-logy, 1989,169:408～417
16 Ito T., K.Okazaki.Perpectutation of influenza A virus in Alaskan waterfowl reservoir [J]. Arch Virol, 1995,140:1163～1172
17 Subbarao K., Klimov A., et al. Characterization of an avian influenza A (H5N1) viuse isolated from a child with a fatal respiratory illness [J]. Science. 1998,297:393～396
18 B．W．卡尔尼克主编，高福，苏敬良主译．禽病学(第十版)[M]．北京，中国农业大学出版社，1999
19 范根成，张怀宝．禽流感的临床特征[J]．中国家禽，2000，Vol 22，No12：42～43
20 李大刚，衣翠玲．禽流感的诊断与防治[J]．临床实践，2002，32(1)：25～26
21 许金俊，陶建平．禽流感的发病特点及其防制[J]．畜禽业，2000，NO．9：12～14
22 刘月焕，姜北宇．H5N1亚型禽流感的发病特点及其防治[C]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集，131～132
23 祁保民，黄瑜，李文扬等．鸭源H5N1亚型流感病毒人工感染鸡的病理学观察[C]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集，103～104
24 林祥梅．鸭源流感病毒人工感染鸡的病理学研究[D]．南京．南京农业大学，1998
25 林祥梅，赵增连，陈万芳．鸭源流感病毒人工感染鸡的病毒抗原定位动态[C]．中国兽医学报．2001，Vol．21，No．5：466～470
26 罗开健，廖明，任涛等．一株鸭源H5亚型AIV致病性的测定[C]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集，97～100
27 黄淑坚，廖明，辛朝安．鹅源H5N1亚型AIV对鸡的致病性研究[C]．中国畜牧兽医学会兽医病理学分会学术讨论会论文集，55～57
28 黄兴国，马春全，卢玉葵等．鹅禽流感的病理组织学变化的观察[C]．中国畜牧兽医学会兽医病理学分会学术讨论会论文集，65～66
29 罗玲，胡薛英，程国富等．H9N2亚型AIV人工感染蛋鸡的组织病理学研究[C]．中国畜牧兽医学会兽医病理学分会学术讨论会论文集，57～61
30 Terrence M.Tumpey, David L.Suarez.Characterization of a highly pathogenic H5N1 avian influenza a virus isolated from duck meat[J].Journal of virology,2002,(6):6344～6355
31 D.E.Swayne, M.L.Perdue, M.Garcia. Pathogenicity and diagnosis of H5N2 mexican avian influenza viruses in chickrns.Avian Diseases 41:335～346
32 Alexander, D.J., G.Parsons, R.J.Manvell.Experiment assessment of the pathogenicity of eight influenza a viruse of H5 subtype for chickens, turkeys, ducks and quail. Avian Pathol. 1986,15:647～662
33 Swayne,D.E., And R.D.Slemons.Comparative pathology of a chicken-origin and two duck-origin influenza virus isolates in chickens: the effect of route of inoculation.Vet.Pathol. 1994, 31:237～245
34 Y.Kobayashi Et Al. Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses. Avian Pathology, 1996, 25: 284-304.
35 P.T.Hooper et al. Observation on the relationship in chickens between the virulence of some avian influenza viruses and the pathogenicity for various organs. Avian Disease, 1995, 39:458-446.
36 皇甫和平，杨俊兴，牛明福等．鸭流感病毒对鸡的人工感染试验[C]．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集，101～102
37 Gambaryan A.S., Yamnikova S.S., et al. Comparison of receptor specificity for influenza A viruses isolated from duck, chicken and human. Mol. Biol.2002, 36:1～9.
38 郭玉璞．家禽传染病诊断与防治[M]．北京农业大学出版社．1994
39 王兰芳，李嘉，杜金龙等．禽流感的诊断技术及研究进展．当代畜牧，2003，5：15～16
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