安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。     

PRV、PCV2与PPV三重PCR检测方法的建立与初步应用

为建立可同时检测猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)与猪细小病毒(PPV)的三重PCR方法,根据Gen Bank登录的病毒相关基因序列,选择保守区域设计引物,经过反应条件的优化,建立了可同时检测以上3种病毒的PCR诊断方法,扩增的目的片段长度分别为192 bp(PRV)、255 bp(PCV2)和759 bp(PPV)。对PRV、PCV2和PPV的核酸检测最低浓度分别为:2.5×10-2,2.2×10-3和3.7×10-2ng,证明该方法具有良好的特异性和较高的敏感性。应用该方法对56份临床样品进行检测发现,PRV、PCV2和PPV的阳性率分别为16.1%、50.0%和19.6%,二重感染率为12.5%,三重感染阳性率为3.6%。该方法的成功建立为快速高效地检测以上3种病毒提供了有效手段。     

伪狂犬病病毒gB蛋白抗原表位基因串联构建及原核表达

本试验通过自行设计两段引物,利用重叠延伸PCR方法,将伪狂犬病病毒(PRV)gB基因两段优势抗原表位序列串联,克隆至pMD18-T载体,测序正确后双酶切连接至pET-32a(+)载体,构建重组质粒;重组质粒转化入大肠杆菌BL21(DE3)受体菌,经IPTG诱导后,通过SDS-PAGE和Western blotting检测融合蛋白表达情况。经检测,诱导后的重组蛋白获得表达,重组蛋白大小约为54ku,其中串联蛋白大小约为34ku。该重组蛋白可与伪狂犬病病毒gB蛋白单克隆抗体发生特异性反应,表明重组蛋白的抗原性良好。     

Bacteriophages as antimicrobial agents against major pathogens in swine: a review

In recent years, the development of antibiotic resistant bacteria has become a global concern which has prompted research into the development of alternative disease control strategies for the swine industry. Bacteriophages (viruses that infect bacteria) offer the prospect of a sustainable alternative approach against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes. This paper reviews the use of phages as an antimicrobial strategy for controlling critical pathogens including Salmonella and Escherichia coli with an emphasis on the application of phages for improving performance and nutrient digestibility in swine operations as well as in controlling zoonotic human diseases by reducing the bacterial load spread from pork products to humans through the meat.     

Detection of Novel Reassortant Influenza A (H3N2) and H1N1 2009 Pandemic Viruses in Swine in Hanoi,Vietnam

From May to September 2013, monthly samples were collected from swine in a Vietnamese slaughterhouse for influenza virus isolation and serological testing. A(H1N1)pdm09 viruses and a novel H3N2 originating from reassortment between A(H1N1)pdm09 and novel viruses of the North American triple reassortant lineage were isolated. Serological results showed low seroprevalence for the novel H3N2 virus and higher seroprevalence for A(H1N1)pdm09 viruses. In addition, serology suggested that other swine influenza viruses are also circulating in Vietnamese swine.     

Ethyl acetate fraction of flavonoids from Polygonum hydropiper L. modulates pseudorabies virus-induced inflammation in RAW264.7 cells via the nuclear factor-kappa B and mitogen-activated protein kinase pathways

Pseudorabies virus (PRV) infection leads to severe inflammatory responses and tissue damage, and many natural herbs exhibit protective effects against viral infection by modulating the inflammatory response. An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) was prepared through ethanol extraction and ethyl acetate fractional extraction. An inflammatory model was established in RAW264.7 cells with PRV infection to evaluate the anti-inflammatory activity of FEA by measuring cell viability, nitric oxide (NO) production, reactive oxygen species (ROS) release, and mRNA expression of inflammatory factors, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Its functional mechanism was investigated by analyzing the phosphorylation and nuclear translocation of key proteins in the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Our findings indicate that PRV induced inflammatory responses in RAW264.7 cells, and the responses were similar to that in lipopolysaccharide (LPS)-stimulated cells. FEA significantly suppressed NO synthesis and down-regulated both expression and secretion of COX-2, iNOS, and inflammatory cytokines (P<0.05 or P<0.01). FEA also reduced NF-κB p65 translocation into the nucleus and decreased MAPK phosphorylation, indicating that the NF-κB/MAPK signaling pathway may be closely related to the inflammatory response during viral infection. The findings suggested the potential pharmaceutical application of FEA as a natural product that can treat viral infections due to its ability to mitigate inflammatory responses.     

广东惠州生猪养殖现状调查及分析

惠州地处广东省珠江三角洲,近10多年城市化进程速度加快,城市人口增长迅速,随着人们生活水平不断提高,猪肉消费急速上升.同时,惠州也是广东省猪场最为集中的地区,是广东重要的肉猪、种猪生产基地.惠州地区规模生猪养殖发展之路更是广东省生猪养殖的一个缩影和风向标.通过深入基层调查,走访养殖户、屠宰场、大型养殖企业等,通过座谈交流、问卷调查和查阅生产记录的方式,对惠州不同规模生猪养殖发展现状及面临的主要问题进行了调查和分析.通过分析发现,中等规模的生猪养殖场（存栏500-2 999头）具有生产效益明显、环境容量合适、调整生产快、抗风险高和适合精细饲养等特点,比较适合目前惠州的生猪养殖发展.针对惠州面临的产业结构调整、划定动物禁养区等现实问题,本文提出应结合人文发展,完善行业标准,大力发展现代都市养殖模式-绿色生态园区的建议.     

旋毛虫肌幼虫排泄—分泌抗原诊断猪旋毛虫病的研究与应用

应用“ES”抗原和酶复合物对猪旋毛虫病进行了ELISA的诊断研究,其抗原是对Gamble(1984)的肌幼虫培养方法经过改进研制而成的.应用这种抗原和免疫制剂建立起来的酶联免疫吸附试验诊断方法,具有灵敏度高、特异性强、操作简便、容易判断等优点,改变了我国过去应用粗制虫体抗原诊断猪旋毛虫病的状况.通过对71头人工感染旋毛虫病猪血清的诊断,可获得100%的阳性检出率.对旋毛虫病流行区120万头猪的检测证明,患猪检出率达93—95%.对250头ELISA阳性猪与宰后检查对照,其诊断符合率为98%左右.     

猪舍夏季降温技术应用研究现状

叙述了国内外猪舍夏季降温技术研究与应用的现状及存在的问题，喷淋、湿垫—风机降温系统、细雾蒸发降温系统一般被应用于降低猪舍内的高温。对于夏季降温技术研究应继续完善已有的降温方法在猪舍的应用，同时要开展针对我国气候特点及猪舍建筑实际的夏季降温方式，特别是南方炎热地区开放型猪舍的降温技术措施的研究。     

Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States

Streptococcus suis is an important swine pathogen and a zoonotic agent. Differences in virulence have been noted among the 33 described serotypes, serotype 2 being considered the most virulent. In this study, we aimed at assessing the serotype distribution and the production of virulence-associated markers by strains recovered from diseased pigs in the United States (U.S.). Results showed that among the 100 strains evaluated, serotype 3 (20% of the isolates) and serotype 2 (17%) were the most prevalent. We then investigated the presence in these isolates of the genes sly, epf and mrp, encoding the virulence-associated markers suilysin (SLY), extracellular factor (EF) and muramidase-released (MRP) protein, respectively. The effective production of the markers by the strains was also verified. Results showed that the presence of the gene did not always correlate with actual expression of the respective protein. In the case of MRP, this was due, in most cases, to frameshift mutations at the 5′ end of the gene resulting in premature stop codons. The most prevalent phenotypes among U.S. strains were MRP⁺EF⁻SLY⁻ (40%) and MRP⁻EF⁻SLY⁺ (35%). Serotype distribution greatly differed from that reported in several European countries, as did the production of virulence markers, particularly for serotype 2. On the other hand, our results for the U.S. S. suis isolates are similar to those reported for Canadian strains, suggesting a common status in North America.