猪戊型肝炎
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关键词：屠宰猪，戊型肝炎病毒，免疫组织化学，巢式 RT-PCR，ELISA，病理学
I
Abstract
The objective of this study was to investigate the current prevalence of Hepatitis E Virus (HEV) in swine livers in China. Five hundred and eighty one swine liver samples and 139 serum samples were collected from slaughter-houses, these swine were purchased from Heilongjiang, Jilin, Liaoning, Hebei, Beijing and Henan. Six experiments were designed to provide enough evidence.
2．H.E 染色切片观察结果表明，536 份 HEV 免疫组织化学染色阳性的肝组织都存在不同程度 的病理变化，病变率 100%。病变表现为肝细胞变性，散在单个核固缩性坏死，淋巴细胞等炎性细 胞浸润、汇管区胆管增生，肝细胞萎缩，纤维结缔组织增生等。
3.巢式 RT-PCR 从 114 份肝脏样品中扩增到 2 条产物，与预期片段 348 bp 大小基本一致，位 于 HEV ORF2 区，用 DNAMAN 和 MEGA4.0 进行序列分析、比较显示，扩增出的 2 个片段同源性为 99.2%， 与 HEV 基因Ⅰ、Ⅱ、Ⅲ和Ⅳ型的同源性分别为 74.9%～78.7%，73.9%～74.1%，74.6%～77%和 81.2%～ 93.7%。用扩增的核苷酸片段绘制的基因进化树显示，2 个分离株与 HEV 基因Ⅳ型 AJ344171、 AJ344172 和 AJ344183 位于同一分支，属于 HEV 基因Ⅳ型。
独创性声明
本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成 果。尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发 表或撰写过的研究成果，也不包含为获得中国农业大学或其它教育机构的学位或证书 而使用过的材料。与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明 确的说明并表示了谢意。
5. One hundred and thirty nine slaughtered swine serum samples collected from Beijing several butchery were tested for the level of ALT and AST in serum. The result showed that the abnormality rate of ALT was 76.98%、the abnormality rate of AST was 79.10% and the simultaneous abnormality rate of ALT and AST was 69.40%.
研究生签名：
时间：
年月日
关于论文使用授权的说明
本人完全了解中国农业大学有关保留、使用学位论文的规定，即：学校有权保留 送交论文的复印件和磁盘，允许论文被查阅和借阅，可以采用影印、缩印或扫描等复 制手段保存、汇编学位论文。同意中国农业大学可以用不同方式在不同媒体上发表、 传播学位论文的全部或部分内容。
检
测
及
研
究
生： 王英华
肝
脏
指 导 教 师： 佘锐萍 教授
的
申 请 学 位 门 类 级 别： 农学硕士
病
理
专 业 名 称： 兽医公共卫生学
学
研 究 方 向： 动物性食品卫生学
观
所 在 学 院： 动物医学院
察
王 英 华
2008 年 5 月
本研究由国家自然科学基金提供资助 （项目编号：30771588）
2. Histopathologic observation on positive tissues for immunohistochemical staining approached dependability between pathological changes and HEV Ag distributed in liver. The results showed that positive tissues for HEV Ag immunohistochemical staining presented different pathological changes : sporadic denaturation and karyopyknosis of liver cell, infiltration of lymphocytes, hyperplasy of bile canaliculus and fibrous tissue in the portal area. It was found that total 581 liver samples have been affected. The incidence pathological changes were 100%.
3. RNA extracted and a RT-nPCR was performed using primers designed based on the HEV open reading from 2 (ORF2) sequences. The RT-nPCR products were then sequenced. The resulting nucleotide sequences were compared with the representative strains of HEV genotypes I-IV and a phylogenetic tree was constructed. Two nucleotide sequences were detected from the 114 HEV positive liver samples and were used for nucleotide sequence analysis. Sequence comparison using the DNAMAN and MEGA4.0 softwares showed that the two sequences shared a homogeneity of 99.2% and shared 74.9%~78.7%, 73.9%~73.9%，74.6%~77% and 81.2%~93.7% homogeneity with HEV genotypes Ⅰ,Ⅱ,Ⅲ, and Ⅳ, respectively. Phylogenetic tree analyses revealed that the two sequences detected were closely related to HEV 87, 277 and 292 strains and were grouped into genotype Ⅳ.
(保密的学位论文在解密后应遵守此协议)
研究生签名：
时间：
年月日
导师签名：
时间：
年月日
摘要
为了解屠宰猪体内戊型肝炎的感染情况，本研究从北京几个大型的生猪屠宰加工厂采集了 581 份屠宰猪肝脏样品和 139 份猪血清样品，采用免疫组织化学方法检测肝脏样品中 HEV 相关抗 原的分布情况；针对 HEV ORF2 区设计的简并引物，用巢式 RT-PCR 方法检测肝脏样品中 HEV 核酸， 对扩增产物测序，用 DNAMAN 和 MEGA4.0 进行序列分析并绘制遗传进化树；同时，采用 HEV-Ab ELISA 诊断试剂盒检测了血清中的 HEV 抗体水平；并采用 H.E 染色方法观察了 HEV 免疫组织化学阳性肝 组织的病理学变化，对 HEV 免疫组织化学强阳性的肝组织进行了超微结构观察。研究结果如下：
分类号： 密 级：
单位代码：10019 学 号：S05040584
屠
宰
猪
戊
硕士学位论文
型
肝
屠宰猪戊型肝炎病毒的检测及
炎
肝脏的病理学观察
病
毒
Detection of the Hepatitis E and Histopathologic
的
Observation in Liver of Slaughtered Swine
1.免疫组织化学观察结果表明，在 581 份屠宰猪肝脏中检出了 HEV 抗原阳性样品 536 份，检 出率高达 92.25%。显微镜下观察可见，HEV 阳性反应物主要呈胞浆弥漫型、胞核型两种类型分布。 在 HEV 免疫组化呈阳性反应的肝脏，具有共同的反应特点，即位于肝被膜附近的肝组织中的阳性 反应信号常散在分布，远离被膜的肝组织，阳性反应信号常成片分布，小叶间胆管的上皮细胞呈 阳性反应。
1. Immunohistochemical staining was carried out to detect HEV Ag in Slaughtered swine liver. The results showed that positive material sporadicly distributed in liver cells with liver cytoplasm presented negative reaction at the marginal of liver. Away from the marginal of liver positive objects extensively distributed in liver cells with little cell nucleus presented positive reaction. Inflammatory cells showed negative reaction in interstitial. Endothelial cells of interlobular bile duct veins and arteries showed positive reaction in the portal area of liver. Negative control doesn’t present positive reaction. It was found that 536 out of 581 liver samples were positive for HEV Ag. The positive rate was 92.25%.
4. The purpose of the present study was to determine the prevalence of swine hepatitis E virus （HEV）infection in the slaughtered swine. One hundred and five swine serum samples collected from Beijing several butchery were tested by ELISA for the presence of total antibodies against HEV. The result showed that anti-HEV total antibody were detected in 93 out of 105 pigs. The seropositivity rate was 88.57%. It suggested that slaughtered swine HEV was widespread.
4.ELISA 检测结果：105 份屠宰猪血清中有 93 份为阳性，阳性率为 88.57%。 5．对 139 份屠宰猪血清 ALT 和 AST 的检测结果表明，76.98%的样品血清 ALT 异常升高；79.10% 的样品血清 AST 异常升高；而且有 69.40%的血清样品中 ALT 和 AST 同时异常升高，超出正常值。 6.肝脏的超微结构观察发现，肝细胞胞浆空亮，细胞器减少；线粒体肿胀，嵴断裂、缺失， 甚至空化。免疫电镜观察发现肝细胞胞浆内有 HEV 病毒样粒子。 上述研究结果从 HEV 蛋白和基因两个角度都证明了屠宰猪肝脏中 HEV 的存在，并且证明猪的 HEV 与人的 HEV 有很大的相关性。这表明在我国的猪群中，HEV 的感染率很高。应引起公共卫生 部门的重视。
I
Abstract
The objective of this study was to investigate the current prevalence of Hepatitis E Virus (HEV) in swine livers in China. Five hundred and eighty one swine liver samples and 139 serum samples were collected from slaughter-houses, these swine were purchased from Heilongjiang, Jilin, Liaoning, Hebei, Beijing and Henan. Six experiments were designed to provide enough evidence.
2．H.E 染色切片观察结果表明，536 份 HEV 免疫组织化学染色阳性的肝组织都存在不同程度 的病理变化，病变率 100%。病变表现为肝细胞变性，散在单个核固缩性坏死，淋巴细胞等炎性细 胞浸润、汇管区胆管增生，肝细胞萎缩，纤维结缔组织增生等。
3.巢式 RT-PCR 从 114 份肝脏样品中扩增到 2 条产物，与预期片段 348 bp 大小基本一致，位 于 HEV ORF2 区，用 DNAMAN 和 MEGA4.0 进行序列分析、比较显示，扩增出的 2 个片段同源性为 99.2%， 与 HEV 基因Ⅰ、Ⅱ、Ⅲ和Ⅳ型的同源性分别为 74.9%～78.7%，73.9%～74.1%，74.6%～77%和 81.2%～ 93.7%。用扩增的核苷酸片段绘制的基因进化树显示，2 个分离株与 HEV 基因Ⅳ型 AJ344171、 AJ344172 和 AJ344183 位于同一分支，属于 HEV 基因Ⅳ型。
独创性声明
本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成 果。尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发 表或撰写过的研究成果，也不包含为获得中国农业大学或其它教育机构的学位或证书 而使用过的材料。与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明 确的说明并表示了谢意。
5. One hundred and thirty nine slaughtered swine serum samples collected from Beijing several butchery were tested for the level of ALT and AST in serum. The result showed that the abnormality rate of ALT was 76.98%、the abnormality rate of AST was 79.10% and the simultaneous abnormality rate of ALT and AST was 69.40%.
研究生签名：
时间：
年月日
关于论文使用授权的说明
本人完全了解中国农业大学有关保留、使用学位论文的规定，即：学校有权保留 送交论文的复印件和磁盘，允许论文被查阅和借阅，可以采用影印、缩印或扫描等复 制手段保存、汇编学位论文。同意中国农业大学可以用不同方式在不同媒体上发表、 传播学位论文的全部或部分内容。
检
测
及
研
究
生： 王英华
肝
脏
指 导 教 师： 佘锐萍 教授
的
申 请 学 位 门 类 级 别： 农学硕士
病
理
专 业 名 称： 兽医公共卫生学
学
研 究 方 向： 动物性食品卫生学
观
所 在 学 院： 动物医学院
察
王 英 华
2008 年 5 月
本研究由国家自然科学基金提供资助 （项目编号：30771588）
2. Histopathologic observation on positive tissues for immunohistochemical staining approached dependability between pathological changes and HEV Ag distributed in liver. The results showed that positive tissues for HEV Ag immunohistochemical staining presented different pathological changes : sporadic denaturation and karyopyknosis of liver cell, infiltration of lymphocytes, hyperplasy of bile canaliculus and fibrous tissue in the portal area. It was found that total 581 liver samples have been affected. The incidence pathological changes were 100%.
3. RNA extracted and a RT-nPCR was performed using primers designed based on the HEV open reading from 2 (ORF2) sequences. The RT-nPCR products were then sequenced. The resulting nucleotide sequences were compared with the representative strains of HEV genotypes I-IV and a phylogenetic tree was constructed. Two nucleotide sequences were detected from the 114 HEV positive liver samples and were used for nucleotide sequence analysis. Sequence comparison using the DNAMAN and MEGA4.0 softwares showed that the two sequences shared a homogeneity of 99.2% and shared 74.9%~78.7%, 73.9%~73.9%，74.6%~77% and 81.2%~93.7% homogeneity with HEV genotypes Ⅰ,Ⅱ,Ⅲ, and Ⅳ, respectively. Phylogenetic tree analyses revealed that the two sequences detected were closely related to HEV 87, 277 and 292 strains and were grouped into genotype Ⅳ.
(保密的学位论文在解密后应遵守此协议)
研究生签名：
时间：
年月日
导师签名：
时间：
年月日
摘要
为了解屠宰猪体内戊型肝炎的感染情况，本研究从北京几个大型的生猪屠宰加工厂采集了 581 份屠宰猪肝脏样品和 139 份猪血清样品，采用免疫组织化学方法检测肝脏样品中 HEV 相关抗 原的分布情况；针对 HEV ORF2 区设计的简并引物，用巢式 RT-PCR 方法检测肝脏样品中 HEV 核酸， 对扩增产物测序，用 DNAMAN 和 MEGA4.0 进行序列分析并绘制遗传进化树；同时，采用 HEV-Ab ELISA 诊断试剂盒检测了血清中的 HEV 抗体水平；并采用 H.E 染色方法观察了 HEV 免疫组织化学阳性肝 组织的病理学变化，对 HEV 免疫组织化学强阳性的肝组织进行了超微结构观察。研究结果如下：
分类号： 密 级：
单位代码：10019 学 号：S05040584
屠
宰
猪
戊
硕士学位论文
型
肝
屠宰猪戊型肝炎病毒的检测及
炎
肝脏的病理学观察
病
毒
Detection of the Hepatitis E and Histopathologic
的
Observation in Liver of Slaughtered Swine
1.免疫组织化学观察结果表明，在 581 份屠宰猪肝脏中检出了 HEV 抗原阳性样品 536 份，检 出率高达 92.25%。显微镜下观察可见，HEV 阳性反应物主要呈胞浆弥漫型、胞核型两种类型分布。 在 HEV 免疫组化呈阳性反应的肝脏，具有共同的反应特点，即位于肝被膜附近的肝组织中的阳性 反应信号常散在分布，远离被膜的肝组织，阳性反应信号常成片分布，小叶间胆管的上皮细胞呈 阳性反应。
1. Immunohistochemical staining was carried out to detect HEV Ag in Slaughtered swine liver. The results showed that positive material sporadicly distributed in liver cells with liver cytoplasm presented negative reaction at the marginal of liver. Away from the marginal of liver positive objects extensively distributed in liver cells with little cell nucleus presented positive reaction. Inflammatory cells showed negative reaction in interstitial. Endothelial cells of interlobular bile duct veins and arteries showed positive reaction in the portal area of liver. Negative control doesn’t present positive reaction. It was found that 536 out of 581 liver samples were positive for HEV Ag. The positive rate was 92.25%.
4. The purpose of the present study was to determine the prevalence of swine hepatitis E virus （HEV）infection in the slaughtered swine. One hundred and five swine serum samples collected from Beijing several butchery were tested by ELISA for the presence of total antibodies against HEV. The result showed that anti-HEV total antibody were detected in 93 out of 105 pigs. The seropositivity rate was 88.57%. It suggested that slaughtered swine HEV was widespread.
4.ELISA 检测结果：105 份屠宰猪血清中有 93 份为阳性，阳性率为 88.57%。 5．对 139 份屠宰猪血清 ALT 和 AST 的检测结果表明，76.98%的样品血清 ALT 异常升高；79.10% 的样品血清 AST 异常升高；而且有 69.40%的血清样品中 ALT 和 AST 同时异常升高，超出正常值。 6.肝脏的超微结构观察发现，肝细胞胞浆空亮，细胞器减少；线粒体肿胀，嵴断裂、缺失， 甚至空化。免疫电镜观察发现肝细胞胞浆内有 HEV 病毒样粒子。 上述研究结果从 HEV 蛋白和基因两个角度都证明了屠宰猪肝脏中 HEV 的存在，并且证明猪的 HEV 与人的 HEV 有很大的相关性。这表明在我国的猪群中，HEV 的感染率很高。应引起公共卫生 部门的重视。