猪伪狂犬病的抗体检测及分析

伪狂犬病是由伪狂犬病病毒引起的多种家畜的一种传染病。随着我国养猪规模的扩大．猪群流动和种猪调运异常频繁,猪伪狂犬病日趋成为严重危害猪群的原发性疾病之一。它的控制与净化关系到猪群其他疫病的发生与防控,天系到规模化养猪场的经济效益。2007年8月及11月我们对邯郸市某猪场同时进行了猪伪狂病、猪瘟及猪蓝耳病跟踪监测,现将有关情况报道如下：     

Dot-PPA-ELISA检测猪血清伪狂犬病抗体的研究

本研究建立了Dot-PPA-ELISA检测猪伪狂犬病抗体的方法.制备的诊断膜片PRV点样抗原含量为0.5μg,可以检测的最低猪IgG含量为1.398×10-8g,灵敏性高.不与猪瘟、猪细小病毒病、猪衣原体病、布氏杆菌病、日本乙型脑炎、口蹄疫阳性血清发生交叉反应,特异性好.研究初步确定检测猪伪狂犬病抗体的阳性标准为血清效价1:64以上.试验结果表明:该法具有操作方便、快速,结果客观,易于判读等优点,可应用于猪场伪狂犬病的诊断和抗体检测.     

双抗体夹心ELISA检测伪狂犬病病毒的研究

建立了检测伪狂犬病病毒（ＰＲＶ）抗原的双抗体夹心ＥＬＩＳＡ,试验方法的最佳工作条件为：抗体包被量为５μｇ／孔,酶标抗体的浓度为１：２００。对人工感染兔和自然感染猪的组织脏器检测,结果发现扁桃体,脑和肺的检出率最高,其次为心,肝,脾,肾等组织。     

猪伪狂犬病概述

文章对猪伪狂犬病的病原、检测方法、疫苗研究、国内外净化措施进行概述,为猪伪狂犬病的深入研究和防控净化提供参考。     

苏州一规模猪场猪伪狂犬病抗体监测及结果分析

猪伪狂犬病是严重危害规模猪场的主要传染病之一,猪是伪狂犬病病毒(PRV)的天然宿主和感染后唯一的散毒动物并终身带毒排毒〔1〕。据报道,PRV会侵害免疫系统〔2〕,导致猪的免疫应答低甚至失败,严重影响猪群免疫效果,同时也是造成猪生殖-呼吸综合征的原因之一〔3〕,因此猪伪狂犬病的控制和净化关系到整个猪群其     

规模猪场猪伪狂犬病的净化研究

猪伪狂犬病是由伪狂犬病病毒引起的多种动物共患传染病,可引起仔猪大量发病死亡,母猪流产、死胎等繁殖障碍,育肥猪生长发育缓慢,经济损失惨重。为净化规模猪场的猪伪狂犬病,试验采用综合性的净化措施,除淘汰病猪及临床症状猪、加强饲养管理等技术措施外,采用猪伪狂犬病弱毒疫苗加强免疫,结合野毒抗体监测,成功的根除了伪狂犬的感染。     

伪狂犬病病毒通用转移载体的构建及应用

将来自质粒pFSV40的300bpBamHI/PstI片段[其中含有SV40poly(A)和部分多克隆位点]插入到质粒pUSK相应的酶切位点中，获得重组质粒USKSV40。该重组质粒中gG基因5’端编码区缺失了428bp。将来自质粒pcDNA3.1( )的946bpBglⅡEcoRI片段（其中含CMV启动子及部分多克隆位点）插入到质粒pUSKSV40的BamHIEcoRI位点，构建了通用载体pPRVCMV-uni，其中含有CMV启动子，SV40poly(A)以及NheI,Pme1,BamHI,BstXI,EcoRI,StuI,XbaI等7个单一克隆位点，将eGFP基因插入到该通用载体的BamHI和EcoRI之间，用所获得的转移载体与TK/gG-/LacZ^ PRV基因组共转染PK-15细胞，经检测eGFP基因在重组伪狂犬病病毒中获得表达，从而证实该通用载体的构建是可行的。本研究研制以伪狂犬病病毒为载体的二价或多价基因工程疫苗奠定了物质基础。     

大理地区散养户猪伪狂犬病病毒野毒株感染的血清学分析

为了解云南大理地区散养户猪伪狂犬病病毒(PRV)野毒感染的情况,从大理地区30多个散养户6月龄及6月龄以上猪群中随机采集了共112份血清,采用间接ELISA方法对血清样本进行PRVgE抗体检测。结果显示,112份血清中抗PRVgE抗体阳性率为8.04%。结果表明,大理地区散养户6月龄及6月龄以上猪只猪伪狂犬病病毒野毒株感染率较低。     

猪细小病毒和猪伪狂犬病病毒混合感染的诊治

2009年9月,贵州省贵阳市某养猪场暴发了以母猪流产和仔猪发热、神经症状为特征的疾病,经过流行病学调查、临床症状观察、病理解剖及实验室诊断,诊断为猪细小病毒、猪伪狂犬病病毒混合感染。通过采取综合有效的防制措施,使猪场疫情得到及时、有效的控制。     

Establishment and Application of Duplex RT-PCR Assay for Detection of Porcine Epidemic Diarrhea Virus and Pseudorabies Virus

In order to establish an assay for detecting porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV),two pairs of primers were designed basing on the M gene of PEDV and gE gene of PRV,respectively.The total RNA of standard PEDV and PRV strains were used as templates to establish the duplex RT-PCR assay.The specificity,sensitivity,repetition and clinic detection of the established assay were tested.The result revealed that the threshold of duplex RT-PCR was 10 TCID₅₀/mL of PEDV and PRV,and no products were amplified from the cell or the nucleic acid of other 7 kinds of pathogenic viral or bacterial microorganism.The detection results for 26 clinical suspicious PEDV or PRV infected pigs were consistent with the results tested by sequencing.This study suggested that the duplex RT-PCR method was highly specific,repeatable and sensitive,and was suitable for clinic rapid differential diagnosis of PEDV and PRV.     

猪流行性腹泻病毒与猪伪狂犬病病毒二重RT-PCR检测方法的建立及应用

为建立猪流行性腹泻病毒（PEDV）与猪伪狂犬病病毒（PRV）的快速鉴别检测方法,本研究根据GenBank已登录的PEDV膜蛋白M基因和PRV gE基因保守区域序列设计了2对特异性引物,以PRV和PEDV混合总RNA为反转录模板,初步建立了PRV和PEDV的二重RT-PCR检测方法,并进行了特异性、敏感性、重复性验证和临床应用检测。结果显示,该方法对两种病毒的最低检测限均为10 TCID₅₀/mL病毒含量,重复性好,特异性强,可特异性地扩增PEDV和PRV细胞培养物,但对其他7种病原对照扩增不出任何条带,对26份临床疑似PEDV和PRV感染样品检测结果与测序鉴定结果完全一致。本研究成功建立了PEDV和PRV的二重RT-PCR检测方法,为临床上猪流行性腹泻和猪伪狂犬病的快速鉴别诊断提供了方法。     

Investigation and Analysis of Porcine Epidemic Diarrhea Virus,Transmissible Gastroenteritis Virus and Pseudorabies Virus of Swine Diarrhea Cases in Shandong Province

To understand porcine viral diarrhea prevalence in the large-scale pig farms of Shandong province, a total of 3 035 clinical samples were detected by PCR from January, 2014 to December, 2016.Those samples were detected for porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and pseudorabies virus (PRV). The results showed that the detection rate of PEDV, PRV and TGEV were 67.49%, 9.33% and 3.29%, respectively.During the past three years, the lowest detection rate of PEDV was 48.15% in the fourth quarter of 2014,and the highest was 88.57% in the fourth quarter of 2015.In 2016,the detection rate represented fluctuate declining compared with 2015.The highest positive rate of TGEV was 18.52% in the fourth quarter of 2014,and in the third quarter of 2015 was 15.38%.The lowest positive rate of TGEV was 6.67% in the first quarter of 2016 and TGEV was not detected in the other quarters. The highest detection rate of RPV was 15.68% in the second quarter of 2016,and the lowest was 2.56% in the second quarter of 2014,except the first quarter of 2014 that the PRV was 0. By detecting three kinds of viruses in 69 clinical samples collected passively, the results showed that the detection rate of PEDV,TGEV and PRV were 86.96%,5.80% and 37.68%,respectively. The total single infection rate was 69.57%,the single infection rates of PEDV,TGEV and PRV were 57.97%,1.45% and 10.14%, respectively;The total mixed infection rate was 30.43%,the mixed infection rates of PEDV/PRV,PEDV/TGEV and TGEV/PRV were 26.09%,2.90% and 1.45%, respectively;Obviously, the total single infection rate was higher than the total mixed infection rate. The results showed that the PEDV, PRV and TGEV were prevailing in Shandong province. There were PEDV/TGEV, TGEV/PRV, PEDV/PRV mixed infection, and the number of PEDV/PRV mixed infection was in the majority. However, there was no PEDV/PRV/TGEV mixed type infection. At present, PEDV was the major pathogen of porcine viral diarrhea and the test results could provide the reference to the diagnosis of porcine viral diarrhea.     

山东省猪腹泻病例中PEDV、TGEV和PRV的感染调查与分析

为了解山东省规模化猪场由猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）和猪伪狂犬病毒（PRV）引起猪病毒性腹泻的流行情况,自2014年1月至2016年12月,对来自山东省各地规模化猪场的猪腹泻病料（共3 035份）进行PCR检测。结果显示,PEDV、PRV和TGEV阳性率分别为67.49%、9.33%和3.29%;3年间,PEDV阳性率在2014年第四季度最低,为48.15%,2015年第四季度阳性率最高,为88.57%,2016年各季度阳性率相对2015年呈波动下降趋势;TGEV阳性率在2014年第四季度最高,为18.52%,2015年第三季度阳性率为15.38%,2016年第一季度阳性率为6.67%,其他季度未检测出阳性病料;PRV阳性率在2016年第二季度最高,为15.68%,除2014年第一季度未检出阳性病料外,2014年第二季度阳性率最低,为2.56%。通过对69份被动送检的病料进行PEDV、TGEV和PRV混合感染检测发现,这部分病料中PEDV、TGEV、PRV阳性率分别为86.96%、5.80%和37.68%;总单独感染率为69.57%,PEDV、TGEV和PRV单独感染率分别为57.97%、1.45%和10.14%;总混合感染率为30.43%,PEDV/PRV、PEDV/TGEV和TGEV/PRV混合感染率分别为26.09%、2.90%和1.45%;总单独感染率比总混合感染率高。结果表明,山东省存在PEDV、PRV和TGEV 3种病毒流行,存在PEDV/TGEV、TGEV/PRV和PEDV/PRV的混合感染,混合感染中主要为PEDV/PRV混合感染,不存在PEDV/PRV/TGEV的混合感染。目前PEDV是引起山东省猪病毒性腹泻的主要病因,本试验结果可为山东省猪病毒性腹泻的诊断和控制提供参考。     

一例猪伪狂犬病病毒的分离鉴定

从临床疑似猪伪狂犬病发病仔猪的脑组织等病料中,经PCR扩增出大小为217 bp的伪狂犬病病毒gp50的基因片段,结果证实为猪伪狂犬病病毒(porcine pseudorabies virus,PRV)感染。随后采用BHK-21细胞进行猪伪狂犬病病毒的分离培养,该分离株经细胞传代培养5代后,能够产生典型的细胞病变,经PCR鉴定为伪狂犬病病毒,其病毒感染力达10^8.68 TCID₅₀/0.1mL。最后用10^7.0 TCID₅₀/mL病毒培养物接种家兔,48 h后注射部位出现典型瘙痒、皮肤破损等症状,于72 h后全部死亡。结果表明,该伪狂犬病病毒分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫及诊断研究奠定了基础。     

Expression and Immunogenicity Analysis of Porcine Pseudorabies Virus gB Protein

In order to study the expression and immunogenicity of porcine pseudorabies virus (PRV) gB protein,the specific primers were designed with the template of PRV preserved in the laboratory, and the 612 bp conserved gene fragments were amplified and sequenced, then it was cloned into the expression vector pET-28a and transformed into E.coli BL21 (DE3), the target protein was obtained after induced expression and purification.Western blotting was performed to analyse its immunogenicity. The results showed that gB protein was 30 ku, which mainly expressed in the form of inclusion body, and the concentration of the protein was 106 μg/mL, with well reactogenicity. 13 PRV positive serum and 16 negative serum in the samples were detected using ELISA Kit on sale, using positive serum, the PRV antibody detection method was initially established with the PRV gB protein as antigen package.     

猪伪狂犬病病毒gB蛋白表达及免疫原性分析

本试验旨在研究猪伪狂犬病病毒（pseudorabies virus,PRV） gB蛋白的表达并分析其免疫原性,以PRV病毒液为模板,设计特异性引物,扩增大小为612 bp的保守片段并测序,将其克隆到表达载体pET-28a中,转化表达菌BL21（DE3）,经诱导表达、纯化得到目的蛋白,进行Western blotting分析验证并分析免疫原性。结果表明,表达的gB蛋白大小为30 ku,主要以包涵体形式存在,复性后浓度为106 μg/mL,且具有良好的反应原性。应用市售试剂盒检测到样品中含13份PRV阳性血清和16份阴性血清,利用检出的阳性血清,初步可建立以PRV gB蛋白为包被抗原的PRV抗体ELISA检测方法。     

大中型猪场猪伪狂犬病抗体检测与分析

为了解大中型猪场猪伪狂犬病免疫抗体水平,采用ELISA方法对辽宁3个区域大中型猪场共7个场采集猪血清样品共270份进行猪伪狂犬病gE抗体和gB抗体检测。结果显示:猪伪狂犬病gE抗体和gB抗体的总阳性率分别为15.9%和85.9%。其中区域一、区域二、区域三猪伪狂犬病gE抗体阳性率分别为21.8%、0和23.8%,猪伪狂犬病g B抗体阳性率分别为85.5%、91.3%和81.3%。结果表明:大中型猪场的免疫措施得当,免疫效果明显,但还存在感染的猪只。区域一和区域三所采样的大中型场中均有不同程度的野毒感染,区域二的野毒感染率和免疫抗体水平均优于其它两个地区。