应用嵌套式RT-PCR技术快速诊断猪流行性腹泻

根据文献,设计和合成内外两对扩增猪流行性腹泻病毒M基因的引物,应用嵌套式RT-PCR技术检测病猪群的粪便。扩增出854bp和412bp的目的基因片段。表明导致猪群发病的病毒为PEDV。     

Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets

Porcine epidemic diarrhea virus (PEDV) is the causative agent of neonatal diarrhea in piglets, which causes high mortality rates. In this study, the immunoprophylactic effects of chicken egg yolk immunoglobulin (Ig Y) against PEDV were investigated in neonatal pigs. Ig Y was found to reduce the mortality in piglets after challenge exposures. The field application of Ig Y also revealed significant differences in survival rates of piglets given Ig Y, as compared with placebo or control. The results in this study indicated that Ig Y against PEDV could be an alternative way of supplementing prophylactic measures like colostral antibodies from sows.     

Impact of porcine group A rotavirus co-infection on porcine epidemic diarrhea virus pathogenicity in piglets

Porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus (PGAR) are the main causative agents of acute diarrhea in piglets. In South Korea, PGAR is prevalent in piglets naturally infected with PEDV. Piglets naturally co-infected with PEDV and PGAR appeared to have severe and prolonged diarrhea that was distinct from that commonly observed. The aim of this study was to determine the impact of PGAR co-infection on PEDV pathogenicity in piglets. Thirty-six colostrum-deprived, one-day old, Large White-Duroc crossbred pigs were randomly divided into four equal groups: PEDV, PEDV/PGAR, PGAR, and control groups. The piglets were euthanized at 1, 2, or 3 days post-inoculation (DPI) to measure the villous height:crypt depth (VH:CD) ratio and to collect fecal samples for RT-PCR and virus isolation. No significant differences in mean VH:CD ratio and clinical symptoms (diarrhea, vomiting, dehydration, and anorexia) were observed between the PEDV/PGAR-infected and PEDV-infected groups of piglets at 1, 2 and 3 DPI; however, at 2 and 3 DPI, PGAR was detected in all fecal samples by RT-PCR and virus isolation. These findings failed to detect any interaction between PEDV and porcine rotavirus in the small intestines of piglets, suggesting that concurrent infection of PGAR may not synergistically enhance intestinal villous atrophy of piglets with PEDV disease. We propose that the severe diarrhea exhibited in PEDV and PGAR co-infected piglets may be more associated with the immunity level of the host rather than to any synergistic effect of PGAR on PEDV enteritis.     

检测猪流行性腹泻病毒的R-PCR方法的建立

根据猪流行性腹泻病毒 (PEDV)的N基因自行设计和合成了一对可扩增长度为 641bp目的片段的引物 ,成功地建立了检测的猪流行性腹泻病毒的RT PCR方法。对猪轮状病毒 (PRV)、猪传染性胃肠炎病毒 (TGEV)的RT PCR检测结果均呈阴性。对PEDV JS株的RT PCR产物的序列分析表明 ,与CV777株的同源性为 97 3 %。     

Genetic analysis of reproductive performance in sows during porcine reproductive and respiratory syndrome(PRRS) and porcine epidemic diarrhea(PED)outbreaks

Background: Porcine reproductive and respiratory syndrome(PRRS) is one of the most infectious swine diseases in the world, resulting in over 600 million dollars of economic loss in the USA alone. More recently, the USA swine industry has been having additional major economic losses due to the spread of porcine epidemic diarrhea(PED).However, information regarding the amount of genetic variation for response to diseases in reproductive sows is still very limited. The objectives of this study were to identify periods of infection with of PRRS virus(PRRSV) and/or PED virus(PEDV), and to estimate the impact their impact on the phenotypic and genetic reproductive performance of commercial sows.Results: Disease(PRRS or PED) was significant(P 0.05) for all traits analyzed except for total piglets born.Heritability estimates for traits during Clean(without any disease), PRRS, and PED ranged from 0.01(number of mummies; Clean and PED) to 0.41(abortion; PED). Genetic correlations between traits within disease statuses ranged from-0.99(proportion born dead with number weaned; PRRS) to 0.99(number born dead with born alive;Clean). Within trait, between disease statuses, estimates ranged from-0.17(number weaned between PRRS and PED) to 0.99(abortion between Clean and PRRS).Conclusion: Results indicate that selection for improved performance during PRRS and PED in commercial sows is possible and would not negatively impact performance in Clean environments.     

The effect of porcine epidemic diarrhea (PED) on ovarian function and reproductive performance after weaning in Berkshire sows

Tropical Animal Health and Production - The purpose of this study was to investigate the ovarian condition at weaning and subsequent reproductive performance of Berkshire sows following an outbreak...     

抗猪流行性腹泻病毒纳米单链抗体CNC-CBD-scFv的制备及鉴定

为防治猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV),本文制备纳米化抗PEDV单链抗体(single-chain fragment,scFv)并验证其生物学特性。通过PCR方法扩增含有纤维素结合结构域(cellulose binding domain,CBD)的单链抗体基因CBD-scFv,构建抗PEDV的原核重组表达载体pET19b-CBD-scFv,鉴定正确后进行蛋白表达与鉴定。经硫酸水解法制备纳米纤维素(cellulose nanocrystal,CNC)后,将纯化复性后的CBD-scFv蛋白与CNC孵育结合得到纳米抗体CNC-CBD-scFv,通过SDS-PAGE和透射电镜鉴定复合物的结合效果。SDS-PAGE与Western blot结果表明抗体蛋白CBD-scFv正确表达;制备的纳米纤维素粒度均达到纳米材料级别;CNC与CBD-scFv结合后SDS-PAGE与透射电镜结果说明CNC与CBD-scFv在室温条件下可高效结合。本试验在高效表达合成复合纳米抗体材料的同时为PEDV的防治及应用奠定了一定的试验基础。     

猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定

为制备猪流行性腹泻病毒(PEDV)抗M蛋白单克隆抗体(MAb),本研究以截短表达的His-M重组蛋白免疫BALB/c小鼠;以截短表达的GST-M重组蛋白作为包被抗原,采用常规的淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA进行筛选,得到一株稳定分泌抗M蛋白MAb.MAb亚类鉴定为IgG2b型,轻链为к链,杂交瘤细胞培养上清和诱导的小鼠腹水抗体效价分别为1:3000和1:2×105.Western blot试验表明该MAb能够识别重组及天然的PEDV M蛋白.间接免疫荧光试验表明该MAb能够与PEDV感染的Vero E6细胞产生特异性免疫荧光.     

猪流行性腹泻病毒(PEDV)辽宁株序列测定及分析

为弄清2014年沈阳某猪场腹泻原因,应用RT-PCR方法对采自该场样品进行猪流行性腹泻病毒(PEDV)S1基因扩增,并将产物纯化后测序、分析。结果得到长为2 376 bp的S1基因序列;本病毒与2013年美国暴发毒株以及我国2010年毒株核苷酸和氨基酸同源性较高,而与疫苗株同源性较低;同时存在氨基酸的插入、缺失及糖基化位点的改变;在进化树上,与2010-2011年在我国暴发的猪流行性腹泻病毒为与同一亚群,与2013年美国毒株也有较近的亲缘关系。表明分离到猪流行性腹泻病毒,此病毒已发生部分变异,需给予密切重视。     

Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV isolation from rectal swab samples. The sera containing antibodies induced by the complete S protein or the amino terminal half of the S protein showed protective properties, indicated by delayed onset of clinical signs and virus shedding, similar to the TGEV hyperimmune serum. Those immune sera containing antibodies induced by shorter recombinant proteins were not protective.     

2011年河南省猪流行性腹泻病毒流行株的遗传进化分析

2011年前后,猪流行性腹泻(PED)在河南省爆发流行.为探明该病再次流行的原因,本研究对2011年收集的7份来自河南省不同地区PED病毒(PEDV)流行病毒株M基因进行RT-PCR扩增、克隆、测序及分析.序列分析显示,7株PEDV河南流行株M基因部分核苷酸及氨基酸发生变异,不同于以往参考病毒株.基于M基因的遗传进化分析显示,PEDV河南流行株和参考株可以分为3个群(G1、G2、G3),7株PEDV河南流行株均属于第3群,与2010年中国BJ2010株、2007年韩国PFF188株及2008年泰国08RB03株亲缘关系密切,而与欧洲病毒株(CV777、Br1/87)、1995年和2004年泰国株(M_NIAH1795_04、M_NIAH2013_95)及2006年中国LZC株亲缘关系较远.结果表明,2011年在中国河南省及其它地区流行的PEDV是一个新的基因型,可能起源于韩国和泰国.     

猪流行性腹泻病毒及其卵黄抗体研究进展

猪流行性腹泻病毒是引起仔猪病毒性腹泻甚至死亡的关键诱因,而安全高效的抗猪流行性腹泻病毒卵黄抗体具有降低仔猪腹泻率、死亡率等优点已应用于生猪养殖。本文总结了近年来猪流行性腹泻病毒的致病机理及其卵黄抗体的制备与应用效果的研究进展,为卵黄抗体在防治猪流行性腹泻上的应用提供理论基础和技术指导。     

猪流行性腹泻病毒N蛋白单克隆抗体的制备与鉴定

采用RT-PCR扩增猪流行性腹泻病毒(PEDV)N蛋白基因并克隆至原核表达载体pET-28a,转化到宿主表达菌BL21后经IPTG诱导表达和Ni柱纯化,SDS-PAGE和Western Blot鉴定重组N蛋白,其大小约为58 ku。将纯化重组N蛋白免疫BALB/c小鼠,通过杂交瘤细胞融合,PEDV间接ELISA方法筛选,制备获得4株能稳定分泌PEDV抗体的杂交瘤细胞株1C9、4C8、4F8和6A11。Western blot和间接免疫荧光试验证明其与PEDV均有特异性反应,单抗亚类鉴定均属于IgG1,轻链为κ型。杂交瘤细胞培养上清ELISA抗体效价为1∶1 600~6 400,腹水ELISA抗体效价达1∶1.024×105以上;4株细胞连续培养20代,其ELISA抗体效价基本一致。本研究为PEDV感染的诊断和致病机制研究提供了有用工具。     

2011年猪流行性腹泻病毒的遗传变异分析

为分析猪流行性腹泻病毒(PEDV)的遗传变异情况,本研究设计合成2对扩增S1基因的引物,利用套式RT-PCR方法,对2011年分离的17个PEDV S1基因进行扩增、克隆和序列测定;并将其进行比对和遗传演化分析。结果表明:17个PEDV S1基因序列与参考病毒株S1基因核苷酸序列同源性为90.54%～99.96%,与经典病毒株CV777相比,其中有6个S1基因在453 bp～454 bp处存在3个核苷酸的缺失;一个在453 bp～454 bp处缺失6个核苷酸;其它10个S1基因在173 bp～186 bp之间存在12个核苷酸的插入,413 bp～417 bp之间有3个核苷酸的插入,467 bp～468 bp处缺失6个核苷酸,插入和缺失位点与韩国病毒株KNU-0901、KNU-0905、KNU-0801相似。S1基因系统进化树分析表明,PEDV S1基因分为3群,其中7个S1基因属于PEDVⅠ群,另外10个属于PEDVⅢ群。     

Passive protection of piglets by monoclonal antibodies against experimental infection with Actinobacillus pleuropneumoniae

Mouse monoclonal antibodies 11C11 (an IgG) and 4A9 (an IgM), which combine with a superficial component of cells belonging, respectively, to serovars 1 or 3 of Actinobacillus pleuropneumoniae, were given intraperitoneally 24 hours before and intranasally one hour before two-week-old, colostrum-deprived piglets were exposed by the intranasal route to 10(9) viable cells of either strain Shope 4074 (serovar 1) or 2/10 (serovar 3). Compared with control piglets given phosphate buffered saline or the heterologous monoclonal antibody, this procedure conferred substantial protection against the development of peracute or acute pleuropneumonia. Protection against the experimental disease was somewhat less in other piglets to which monoclonal antibody 4A9 was given only by the intranasal route one hour before the organism was administered than in those given the antibody intraperitoneally 24 hours beforehand, although its effect was still significantly greater than in piglets given phosphate buffered saline only. These two monoclonal antibodies consequently offer means of investigating at the molecular level the pathogenesis of the disease associated with A pleuropneumoniae and the potential value of anti-idiotypes as immunising agents.     

断奶前仔猪经胎盘感染猪圆环病毒2型对猪流行性腹泻病毒引起的肠炎的影响

本文报道了经胎盘感染猪圆环病毒2型对猪流行性腹泻病毒引起新生仔猪肠炎的影响。6头怀孕母猪被随机分为感染组和对照组,每组3头。感染组3头怀孕母猪在分娩前3周鼻内接种6mLPCV2组织培养毒(病毒含量为1.2×105TCID50/mL,毒株为SNUVR000470);对照组3头怀孕母猪同时接种正常细胞培养上清液。PCV2感染母猪所产的30头仔猪被随机分为A、B两组,每组15头;对照组非感染母猪所产的30头仔猪被随机分为C、D两组,每组15头。A和C组仔猪在3日龄口服2mLPEDV第3次传代病毒液(病毒含量为1×106.5TCID50/mL,毒株为SNUVR971496)。在感染后36、48和72h测量绒毛高度与隐窝深度的平均比值,A组PCV2感染母猪所产的仔猪感染PEDV与C组PCV2阴性母猪所产的仔猪感染PEDV差异显著。在感染后24h,A组PEDV感染仔猪的空肠组织中比C组检测到更多的PEDV核酸。此后,在感染后36、48、60和70h,C组PCV2阴性母猪所产的PEDV感染仔猪的空肠组织中比A组检测到更多的PEDV核酸。由此推断,经胎盘感染PCV2显著影响PEDV所致疾病的临床进程。