猪戊型肝炎(5)

研究生论文

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.

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%.

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.

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.

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%.