雏鹅出血性坏死性肝炎病原鉴定及生物学特性的初步研究
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摘要
2001年初，江苏省一些鹅场发生了一种仅感染雏鹅的病毒性传染病，患病雏鹅以出血性坏死性肝炎为特征性的大体病理变化和显微病理变化。本教研室自发病鹅场中，采集患有“出血性坏死性肝炎”雏鹅的肝脾脏病料，分离到两株病毒，分别命名为WJ_(01-2)G和LY_(01-3)G。由于该病以前未见报道，为明确其病原，我们对其中一株病毒LY-(01-3)G进行了鉴定并对其特性作了初步研究。
实验证实该分离毒株通过绒毛尿囊膜途径接种适日龄鸡胚、鸭胚和鹅胚，均能引起规律性死亡和特征性病变，但病毒通过尿囊腔途径接种发育不良。分离株能适应鸡胚成纤维细胞、鸭胚成纤维细胞和鹅胚成纤维细胞，但初代无明显的细胞病变，在原代细胞上盲传5代后，鸡胚成纤维细胞和鹅胚成纤维细胞产生明显的细胞病变，感染细胞圆缩，胞膜折光度增强，细胞内颗粒增加；鸭胚成纤维细胞病变仍不明显。收集出现病变的鸡胚和鹅胚成纤维细胞冻融后，经绒尿膜接种鸡胚、鸭胚和鹅胚，均能致死并产生特征性病变。将适应细胞的病毒通过肌肉或爪垫注射雏鹅，复制到了与自然发病鹅相似的临床症状和病理变化，并回收到该病毒；接种1日龄雏鸡爪垫可引起注射部位炎性反应，但爪垫和肌肉注射雏鸡均不引起死亡；采取发病鹅肝脏、脾脏、胰腺、肾脏和脑等器官制作病理切片，对其微观病变进行了观察，发现这些器官的病变主要是组织局灶性坏死，其中以肝、脾尤为严重；耐过鹅血清可以抑制细胞病变的产生，与该毒株提纯抗原进行琼脂扩散试验，可产生清晰致密沉淀线。
感染细胞经吖啶橙染色，荧光显微镜下观察可见细胞浆内出现黄绿色荧光；而已知单股病毒对照呈现火红色荧光，双股病毒对照呈现绿色荧光。病毒经DNA代谢抑制物(5-溴脱氧尿嘧啶)处理后，感染力变化不明显。病毒耐酸耐热，对乙醚、氯仿、去氧胆酸盐等脂溶剂不敏感，胰蛋白酶处理后感染力增加，1M Mg~(2+)可提高病毒的热稳定性；对多种动物的红细胞无凝集作用。病毒在禽胚尿囊液中含量较低，而在胚体和尿囊膜中含量较高。血清中和试验表明该病毒不能被小鹅瘟、新城疫和禽流感抗血清所中和，只能被自身特异性抗血清中和；交叉血清学试验表明该分离株与鸡呼肠孤病毒有一定的抗原相关性。用离子捕捉法处理病毒尿囊液，负染后电镜观察发现病毒粒子呈球形，无囊膜，有可见的双层衣壳结构，外壳直径75nm左右，内核直径50nm左右，呈二十面体对称。胚肝和绒尿膜的超薄切片观察表明，病毒在细胞浆内复制，病毒粒子呈圆形，无囊膜，大小为80
扬州大学硕士论文
nm左右。
根据病毒分离、动物试验、病毒形态结构的观察以及核酸类型的鉴定，可以
判定该株病毒为呼肠孤病毒科成员。另外，理化特性和生物学特性表明该病毒具
有禽呼肠孤病毒的基本特性，分类学上应属于禽呼肠孤病毒。
在初步完成对该毒株病原鉴定基础上，建立了用琼脂扩散试验检测病毒抗原
及抗体的方法，运用该方法对感染死亡雏鹅肝脏样品及抗血清的阳性检出率分别
达到70%和65%，琼扩效价最高分别达到25和24，说明琼脂扩散试验检测鹅出
血性坏死性肝炎病毒抗原及抗体是一种简便、特异性较强、敏感性较高、易于判
断的诊断方法，可用于临床上对种鹅群免疫状态的监测以及对本病的初步诊断。
The disease is a new viral infectious disease which infects goslings in JiangSu Province in 2001. The diseased goslings showed characteristic hemorrhagic necrotic hepatitis. Two strains viruses were isolated from the livers and spleens with lots of gray-yellow local necroses of goslings by our laboratory, one of which was identified as avian reovirus.
The general hemorrhagic and gray-yellow necrotic foci were observed on the surface of liver and spleen of the dead embryos of chickens,ducks or geese inoculated by the way of allantoic membrane with the goose reovirus isolate LY01-3G However, the virus did not grow well when they were inoculated to the allantoic cavity of embryos. The LY01-3G isolated virus produced some cytopathic effects(CPE) on the chicken or goose embryo fibroblast cell which formed the rounded cells, granular degeneration, and then lysis. But in duck embryo fibroblast cell, the cytopathic changes were always less characteristic. The goslings could be infected with the reovirus isolate LY01-3G by intramuscular(i.m.) or pad injection. The pathologies of died goose were similar to those of field cases, and the virus was recovered. There were inflammatory responses caused by the pad inoculation to the one-day-old chickens, though the apparent clinical signs did not appear by i.m.injection on chickens. The liver, spleen, pancreas, kidney and
brain of died goose were made out pathologic slices. Antiserum from the geese infected by the virus specifically prohibited the production of CPE.
The viral nucleic acid of infected cells showed yellow-green fluorescence after
stained with acridine orange. The virus isolate was proved to be resistant to 5-BUDR, aether, chloroform, trypsin, heat and acid treatment, and failed to agglutinate red blood cells of chicken, duck, goose, rabbit and goat. There is thermostabilization effect in the presence of 1M Mg2+. According to neutralization tests, it showed that the virus was antigenically distinguishable from GPV(Goose Plague Virus), NDV(New Castle Disease), AIV(Avian Influenza Virus), and it had certain antigen correlation with avian reovirus by acrossing AGP.
The virus in the allantoic fluids dealt with the technology of ion exchange capture showed spherical, no envelope, having double-deck capsids, with outer-capsid of 75nm in diameter, with inner-capsid of 50nm in diameter. The ultrathin sections which were made out from the embryo livers were observed by TEM, the results suggested that the virus replicate in cytoplasm, no envelope, with diameter of approximately 80nm.
According to virus-isolation,experimental infection, electromicroscope observation and nucleic acid detection, the virus strain was identified as one number of the family reoviridae. In addition, physicochemical and biological properties showed that the isolated LYoi-aG has basic characters of avian reovirus , so the virus strain was categorized into avian reovirus.
On the basis of the identification of viral pathogen, we established a method of the agar gel precipitin(AGP) test to detect antigen or antibody of the gosling hemorrhagic necrotic hepatitis. The positive percentages in liver antigen or antiserum of infected goslings were 70% or 60% and the highest litres of AGP were 25 or 24. AGP test is a simple, higher specific and sensitive, easy to judge method for detecting antigen or antibody to gosling hemorrhagic necrotic hepatitis.
实验证实该分离毒株通过绒毛尿囊膜途径接种适日龄鸡胚、鸭胚和鹅胚，均能引起规律性死亡和特征性病变，但病毒通过尿囊腔途径接种发育不良。分离株能适应鸡胚成纤维细胞、鸭胚成纤维细胞和鹅胚成纤维细胞，但初代无明显的细胞病变，在原代细胞上盲传5代后，鸡胚成纤维细胞和鹅胚成纤维细胞产生明显的细胞病变，感染细胞圆缩，胞膜折光度增强，细胞内颗粒增加；鸭胚成纤维细胞病变仍不明显。收集出现病变的鸡胚和鹅胚成纤维细胞冻融后，经绒尿膜接种鸡胚、鸭胚和鹅胚，均能致死并产生特征性病变。将适应细胞的病毒通过肌肉或爪垫注射雏鹅，复制到了与自然发病鹅相似的临床症状和病理变化，并回收到该病毒；接种1日龄雏鸡爪垫可引起注射部位炎性反应，但爪垫和肌肉注射雏鸡均不引起死亡；采取发病鹅肝脏、脾脏、胰腺、肾脏和脑等器官制作病理切片，对其微观病变进行了观察，发现这些器官的病变主要是组织局灶性坏死，其中以肝、脾尤为严重；耐过鹅血清可以抑制细胞病变的产生，与该毒株提纯抗原进行琼脂扩散试验，可产生清晰致密沉淀线。
感染细胞经吖啶橙染色，荧光显微镜下观察可见细胞浆内出现黄绿色荧光；而已知单股病毒对照呈现火红色荧光，双股病毒对照呈现绿色荧光。病毒经DNA代谢抑制物(5-溴脱氧尿嘧啶)处理后，感染力变化不明显。病毒耐酸耐热，对乙醚、氯仿、去氧胆酸盐等脂溶剂不敏感，胰蛋白酶处理后感染力增加，1M Mg~(2+)可提高病毒的热稳定性；对多种动物的红细胞无凝集作用。病毒在禽胚尿囊液中含量较低，而在胚体和尿囊膜中含量较高。血清中和试验表明该病毒不能被小鹅瘟、新城疫和禽流感抗血清所中和，只能被自身特异性抗血清中和；交叉血清学试验表明该分离株与鸡呼肠孤病毒有一定的抗原相关性。用离子捕捉法处理病毒尿囊液，负染后电镜观察发现病毒粒子呈球形，无囊膜，有可见的双层衣壳结构，外壳直径75nm左右，内核直径50nm左右，呈二十面体对称。胚肝和绒尿膜的超薄切片观察表明，病毒在细胞浆内复制，病毒粒子呈圆形，无囊膜，大小为80
扬州大学硕士论文
nm左右。
根据病毒分离、动物试验、病毒形态结构的观察以及核酸类型的鉴定，可以
判定该株病毒为呼肠孤病毒科成员。另外，理化特性和生物学特性表明该病毒具
有禽呼肠孤病毒的基本特性，分类学上应属于禽呼肠孤病毒。
在初步完成对该毒株病原鉴定基础上，建立了用琼脂扩散试验检测病毒抗原
及抗体的方法，运用该方法对感染死亡雏鹅肝脏样品及抗血清的阳性检出率分别
达到70%和65%，琼扩效价最高分别达到25和24，说明琼脂扩散试验检测鹅出
血性坏死性肝炎病毒抗原及抗体是一种简便、特异性较强、敏感性较高、易于判
断的诊断方法，可用于临床上对种鹅群免疫状态的监测以及对本病的初步诊断。
The disease is a new viral infectious disease which infects goslings in JiangSu Province in 2001. The diseased goslings showed characteristic hemorrhagic necrotic hepatitis. Two strains viruses were isolated from the livers and spleens with lots of gray-yellow local necroses of goslings by our laboratory, one of which was identified as avian reovirus.
The general hemorrhagic and gray-yellow necrotic foci were observed on the surface of liver and spleen of the dead embryos of chickens,ducks or geese inoculated by the way of allantoic membrane with the goose reovirus isolate LY01-3G However, the virus did not grow well when they were inoculated to the allantoic cavity of embryos. The LY01-3G isolated virus produced some cytopathic effects(CPE) on the chicken or goose embryo fibroblast cell which formed the rounded cells, granular degeneration, and then lysis. But in duck embryo fibroblast cell, the cytopathic changes were always less characteristic. The goslings could be infected with the reovirus isolate LY01-3G by intramuscular(i.m.) or pad injection. The pathologies of died goose were similar to those of field cases, and the virus was recovered. There were inflammatory responses caused by the pad inoculation to the one-day-old chickens, though the apparent clinical signs did not appear by i.m.injection on chickens. The liver, spleen, pancreas, kidney and
brain of died goose were made out pathologic slices. Antiserum from the geese infected by the virus specifically prohibited the production of CPE.
The viral nucleic acid of infected cells showed yellow-green fluorescence after
stained with acridine orange. The virus isolate was proved to be resistant to 5-BUDR, aether, chloroform, trypsin, heat and acid treatment, and failed to agglutinate red blood cells of chicken, duck, goose, rabbit and goat. There is thermostabilization effect in the presence of 1M Mg2+. According to neutralization tests, it showed that the virus was antigenically distinguishable from GPV(Goose Plague Virus), NDV(New Castle Disease), AIV(Avian Influenza Virus), and it had certain antigen correlation with avian reovirus by acrossing AGP.
The virus in the allantoic fluids dealt with the technology of ion exchange capture showed spherical, no envelope, having double-deck capsids, with outer-capsid of 75nm in diameter, with inner-capsid of 50nm in diameter. The ultrathin sections which were made out from the embryo livers were observed by TEM, the results suggested that the virus replicate in cytoplasm, no envelope, with diameter of approximately 80nm.
According to virus-isolation,experimental infection, electromicroscope observation and nucleic acid detection, the virus strain was identified as one number of the family reoviridae. In addition, physicochemical and biological properties showed that the isolated LYoi-aG has basic characters of avian reovirus , so the virus strain was categorized into avian reovirus.
On the basis of the identification of viral pathogen, we established a method of the agar gel precipitin(AGP) test to detect antigen or antibody of the gosling hemorrhagic necrotic hepatitis. The positive percentages in liver antigen or antiserum of infected goslings were 70% or 60% and the highest litres of AGP were 25 or 24. AGP test is a simple, higher specific and sensitive, easy to judge method for detecting antigen or antibody to gosling hemorrhagic necrotic hepatitis.
引文
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