中国农业科学 ›› 2019, Vol. 52 ›› Issue (23): 4415-4422.doi: 10.3864/j.issn.0578-1752.2019.23.022
王小蕾1,刘月焕1,段会娟1,刘立新1,杨志远1,赵际成1,潘洁1,刘瑞华1,赵文奇2,田方杰2,吕金宝3,林健1()
收稿日期:
2019-04-23
接受日期:
2019-07-01
出版日期:
2019-12-01
发布日期:
2019-12-01
通讯作者:
林健
作者简介:
王小蕾,E-mail:wangxl227@126.com
基金资助:
WANG XiaoLei1,LIU YueHuan1,DUAN HuiJuan1,LIU LiXin1,YANG ZhiYuan1,ZHAO JiCheng1,PAN Jie1,LIU RuiHua1,ZHAO WenQi2,TIAN FangJie2,Lü JinBao3,LIN Jian1()
Received:
2019-04-23
Accepted:
2019-07-01
Online:
2019-12-01
Published:
2019-12-01
Contact:
Jian LIN
摘要:
【目的】研究鸭坦布苏病毒是否具有血凝性,确定其凝集的红细胞种类和血凝反应条件。【方法】鸭坦布苏病毒以不同稀释度经脑内途径接种1—3日龄的BALB/c乳鼠,观察乳鼠发病情况;收集发病乳鼠鼠脑,参照文献记载的方法用蔗糖-丙酮提纯病毒液;以适当灭活剂灭活病毒,观察灭活后的病毒液性状,并以6日龄SPF鸡胚进行灭活检验;参照文献记载的方法初步测定病毒液与鸡、鸭、鹅、鸽、猪红细胞悬液的反应性;比较并确定鸭坦布苏病毒进行血凝反应的适宜红细胞悬液浓度;比较pH6.0—7.0的反应体系对病毒血凝效价的影响;比较4℃、室温和37℃对病毒血凝反应结果的影响。【结果】乳鼠接种鸭坦布苏病毒后6日,1﹕10稀释接种组的乳鼠出现严重的临床症状,表现为四肢抽搐,瘫痪等。大部分乳鼠处于濒死状态,1﹕50和1﹕100稀释接种组的乳鼠症状稍轻于1﹕10稀释接种组乳鼠的症状;提纯后鸭坦布苏病毒液呈淡红色澄明液体,用甲醛灭活后病毒液性状无明显改变,但用β-丙内酯后产生大量絮状沉淀,性状发生明显改变;病毒液可以凝集鸡、鸭、鹅、鸽、猪红细胞悬液;不同浓度红细胞悬液的比较结果显示,病毒液与0.33%的红细胞悬液作用时凝集图形清晰稳定;通过不同pH反应体系的比较,发现病毒液在pH 6.0—6.8时均可发生血凝反应,当pH为6.0—6.2时凝集效价最高;通过不同反应温度的比较,证实病毒液在4℃、室温和37℃时均具有血凝性,但4℃时的血凝反应时间明显延长。【结论】乳鼠增殖的鸭坦布苏病毒提纯后具有广谱的血凝性,可以凝集鸡、鸭、鹅、鸽和猪的红细胞,血凝性稳定,在4℃、室温和37℃下均可以与鹅红细胞凝集,以0.33%浓度的红细胞悬液血凝为宜,反应最适pH为6.0—6.2。
王小蕾, 刘月焕, 段会娟, 刘立新, 杨志远, 赵际成, 潘洁, 刘瑞华, 赵文奇, 田方杰, 吕金宝, 林健. 鸭坦布苏病毒的血凝性[J]. 中国农业科学, 2019, 52(23): 4415-4422.
WANG XiaoLei, LIU YueHuan, DUAN HuiJuan, LIU LiXin, YANG ZhiYuan, ZHAO JiCheng, PAN Jie, LIU RuiHua, ZHAO WenQi, TIAN FangJie, Lü JinBao, LIN Jian. Hemagglutinating Activity of Duck Tembusu Virus[J]. Scientia Agricultura Sinica, 2019, 52(23): 4415-4422.
表3
不同动物红细胞的HA效价和凝集图形"
动物 Animal | 日龄 Age (d) | 性别 Gender | HA效价 HA titer | 凝集图形 Agglutination graphics |
---|---|---|---|---|
猪(长白猪) Swine (Landrace) | 60 | 公母混合(3头) Mixed of male and female | 1:256 | 红细胞对照不完全下沉,形成环状 The erythrocyte control sank not completely and formed a ring |
鸡(SPF) Chicken (SPF chicken) | 5 | 公(3只)Male | 1:256 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
360 | 公(3只)Male | 1:256 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination | |
鸭(北京鸭) Duck (Peking duck) | 60 | 母(3只)Female | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
鸽(信鸽) Pigeon (Carrier pigeon) | 360 | 公母混合(3只) Mixed of male and female | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
鹅(白鹅) Goose (White goose) | 180 | 公母混合(3只) Mixed of male and female | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
表4
不同浓度鹅红细胞的HA效价和凝集图形"
鹅红细胞浓度 Concentration of goose erythrocyte | HA效价 HA titer | 凝集图形 Agglutination graphics |
---|---|---|
0.2% | 1:1024 | 红细胞对照孔下沉点小,结果不易判定 The erythrocyte control sank as a tiny point so that the results were difficult to interpretation |
0.33% | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
0.5% | 1:256 | 均一薄膜状凝集,清晰,有解凝迹象 Clear homogeneous thin-film agglutination |
1% | 1:128 | 均一薄膜状凝集,解凝快,不稳定 Clear homogeneous thin-film agglutination with fast dissolving |
表5
不同pH值反应体系对HA效价的影响"
pH值 pH value | HA效价 HA titer | 凝集图形 Agglutination graphics |
---|---|---|
6.0 | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
6.2 | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
6.4 | 1:256 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
6.6 | 1:256 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
6.8 | 1:128 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
7.0 | 1:64 | 易解凝,结果不易判定 The thin-film agglutination was easy to dissolve so that the result was difficult to interpretation |
表6
不同反应温度对HA效价的影响"
反应温度 Reaction temperature | HA效价 HA titer | 凝集图形 Agglutination graphics |
---|---|---|
37℃ | 1:512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
室温Room temperature 4℃(60 min) | 1: 512 / | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination 红细胞对照沉降不完全,结果无法判定 The erythrocyte control sedimentated slowly so that the result was difficult to interpretation |
4℃(过夜作用)(overnight) | 1: 512 | 均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination |
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