| Border disease(BD) is a world-wide occurring viral disease of sheep flocks which is caused by Border disease virus(BDV). Clinical manifestations of the disease are characterized by many reproductive disturbance, such as barren ewes, abortion, stillbirth, and the birth of lambs with "hairy shaker". Border disease, first reported in 1959 from the border region of England and Wales and following spread to many countries such as UK、America, Canada、Spain、Netherlands、Sweden and Japan, lead to significant economic losses in the sheep industry. In China, until 2013, BDV was first identified and isolated from a goat suffered persistent diarrhea; which indicated the existence of BD in China. Then clinical monitoring of BDV infection in sheep flocks from different areas of Jiangsu Province was examined in the laboratory. After that, one sheep infected with BDV was screened preliminarily.For the suspected cases, viral isolation and test method were performed. The contents of the paper as follows:1 Identification and genome sequence analysis of the border disease virus from sheepIn this study, RT-PCR was used to detect the pestivirus in the sera from suspected sheep. Positive serum samples were inoculated onto MDBK cells for viral isolation and identification. RT-PCR amplification of Npro gene, electron microscopy detection of isolated virus, the complete genome sequences and phylogenetic analysis of the 5’-UTR and Npro gene were performed. The isolated virus culture was used to inoculate sheep in artificial infection test,in order to measure its virulence. The results displayed that one BDV strain was successfully isolated with noncytopathic on cell culture, and named as JSLS12-01. Amplification of complete genomic sequence showed that the viral near-full genome was about 12227 nucleotides in size and sent to Genbank with registered number KC963426.5’-UTR and 3’-UTR flanked by ORF are 278bp and 255bp, respectively. Based on comparative sequence analysis of full genome and the deduced amino acids between this isolate and BDV reference strains, there were 72.3%-80.4% and 80.1%-89.7% identities in the strains, respectively. Phylogenetic analysis using partial 5’-UTR nucleotide sequence and Npro sequence identified that the virus clustered within BDV 3 viruses and had the nearest genetic relationship with Germany isolation Gifhorn and China AH12-01 strain and AH12-02 strain isolate from goats. Clinical monitoring of the BDV positive sheep showed weak, grew slowly compared with the normal individuals in the same flock. At the same time, the animal was negative antibody against BDV in blood and positive in the viral RNA detection during the whole monitoring period. During 3-7 days post infection, viremia was appearance and followed specific antibody response. No obvious clinical symptoms were observed during the experimental infection. The result showed that there were comparatively large variability among JSLS12-01 strain and other foreign BDV isolates of the full genome sequence,5’-UTR and Npro gene. It hinted that BDV strains isolate from China might possess special evolution type and orientation.2 The establishment of an indirect ELISA detection method based on E2 proteinPrimers are designed through the E2 gene sequence alignment and analysis the antigenicity、hydrophobicity of this fragment. E2 gene from the isolate is amplified by RT-PCR and cloned into a pET-32α expression vector, generated the pET-32α-E2 construction. It is transformed into the E. coli BL21 competent cells. Recombinant protein is identified by SDS-PAGE and western-blot testing. With purified E2 protein as the coating antigen, an indirect ELISA method was established through the optimization of reaction conditions. Compared with the commercialization of BDV antibody detection kit and western-blot test, analysis the detection sensitivity and specificity of ELISA method. Then apply this indirect ELISA in detecting clinical serum samples.The results of SDS-PAGE analysis and Western-blot demonstrated that recombinant E2 protein of BDV was carried out and possessed antigenic activity. The working parameters of the indirect ELISA were determined for the optimization of reaction conditions and as follows:the antigen concentration was 4ug/ml; blocking solution as 20g/L BSA; testing sera dilution at 1:50 and reaction time for 1h; rabbit-anti-goat IgG-HRP dilution of 1:30000 and incubation time for 45min; TMB substrate reaction time at 10min. Compared with the BDV antibody detection kit and western-blot testing, the detection sensitivity of the method is 76.25%、61.54% and the specificity is 73.83%、87.10%. The technique was used to examine 307 serum samples collected from clinical sheep fields of Jiangsu province. The results displayed that the percentages of positive serum samples is 41.37%.The result showed that E2 recombination protein had good immunogenicity. The indirect ELISA method based on E2 protein showed better specificity and little variability. While in the compared test, the indirect ELISA had a higher agreement rate with BDV antibody detection kit and western-blot testing. The reports about BDV infection of goats and sheep in China and the results of antibody against BDV detection in this study indicated the prevalence of BD among domestic small ruminant. It reminds us need to realize about BDV infections in goats and sheep farms, monitor its prevalence trend and try to find efficient prevention and control technique for limitation of the harm to sheep flocks. |
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