猪流行性腹泻病毒分离鉴定及其灭活疫苗的研制

旨在对疑似猪流行性腹泻(PED)患猪的病料进行病毒分离,并制备灭活疫苗.从因腹泻病死的7日龄内仔猪的小肠组织及内容物中,分离出1株猪流行性腹泻病毒(PEDV),命名为PEDV-GS.用Vero细胞对病毒进行扩增,病毒液中加入终浓度为0.7 mg/mL甲醛,28 ℃灭活48 h,加入ISA 61 VG佐剂进行乳化,经稳定...     

猪流行性腹泻病毒分离鉴定和灭活疫苗免疫保护效果探讨

猪流行性腹泻病毒是诱发猪流行性腹泻的关键因素,而猪流行性腹泻容易造成养殖猪,尤其是猪仔的死亡,进而给养猪业带来严重的经济损失.基于此,本文首先阐述了猪流行性腹泻的流行病学,紧接着分析了猪流行性腹泻的临床症状与病理变化,然后分析了猪流行性腹泻病毒的分离鉴定,并进行灭活免疫实验探究,希望能够为今后相关问题的研究提供一定的参考依据.     

猪流行性腹泻灭活疫苗佐剂筛选

为探究不同佐剂对猪流行性腹泻灭活疫苗免疫效果的影响,选用水包油佐剂A(O/W)、油包水佐剂B(W/O)和双相佐剂C(W/O/W)3种不同佐剂配制疫苗,分别接种3～5日龄仔猪后攻毒,综合评价不同佐剂的免疫效果。结果显示：A、B佐剂组疫苗免疫猪的抗体水平低于C佐剂组;攻毒后进行猪流行性腹泻病毒检测发现,仅C佐剂组试验猪全部呈阴性;剖检所有疫苗免疫组试验猪均未发现脏器病变。结果说明：双相佐剂制备的灭活疫苗免疫效果明显优于水包油和油包水佐剂疫苗,且安全性较高,可优先选择双相佐剂制备猪流行性腹泻灭活疫苗。本研究为猪流行性腹泻疫苗制备、生产提供了技术支撑。     

猪流行性腹泻病毒分子生物学研究进展

猪流行性腹泻病毒在Vero细胞上培养成功后,病毒全基因组的序列和分子结构特征,结构蛋白和非结构蛋白的生物学特性和功能,病原学、血清学和分子生物学的诊断技术,体液免疫、细胞免疫和局部黏膜免疫的机理,疫苗和病毒的细胞受体等方面得到了广泛的研究。但是,与其他冠状病毒相比,猪流行腹泻病毒的研究还比较滞后,许多研究还没有开展或不够深入,为了能加快对猪流行性腹泻病毒研究进程,对其最近的分子生物学研究进展做了综述,为猪流行性腹泻病毒的深入研究提供参考。     

一株变异型猪流行性腹泻病毒的分离鉴定及其灭活疫苗的免疫效力试验

本研究旨在分离猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）变异株,通过悬浮培养工艺制备成高效价的PEDV灭活疫苗。2017年从中国多个规模化猪场采集腹泻病死仔猪的小肠及其内容物200份,通过RT-PCR方法进行PEDV检测并测序,筛选一株PEDV变异株,将其在2 L反应器里悬浮培养的Vero细胞上进行病毒分离与传代培养,收获的病毒液鉴定后测定TCID₅₀,经甲醛灭活后加入氢氧化铝胶佐剂配制成PEDV灭活疫苗,对其物理性状、稳定性、黏度、无菌等进行检验,检验合格后免疫妊娠母猪及所产仔猪,对其安全性和免疫效力进行研究。结果显示,200份病料中有86份为PEDV阳性,将筛选的PEDV变异株病料在Vero细胞上传至第5代时出现细胞病变,传至第10代收获病毒液,经鉴定后确定为PEDV变异毒株,并命名为PEDV-GF10株。收获的病毒液浓缩后测得病毒滴度可达1×10^8.0 TCID₅₀/mL。疫苗检验合格后在母猪产前40和25 d时试验组后海穴肌内注射4 mL疫苗,空白组不免疫,结果显示试验组与空白组母猪的生产情况无明显差异,所产3日龄仔猪分别免疫不同剂量后体温无显著差异,表明该疫苗对母猪和仔猪均安全性良好。随机挑选试验组与空白组母猪所产3日龄仔猪各20头,分别口服4 mL PEDV-F10病毒培养物,空白组母猪所产仔猪在攻毒24 h后PEDV发病率为100%,抗体均为阴性;试验组母猪所产仔猪只有10%出现了轻微的腹泻症状,仔猪获得了高达90%保护率,且仔猪被动免疫后抗体能持续至35 d以上。以上结果表明,PEDV-GF10变异株通过悬浮细胞培养后病毒滴度显著提高,研制的PEDV-GF10株灭活疫苗安全有效,能够对中国的PEDV变异株达到有效防控,为国内PED防控提供了理论依据。     

猪流行性腹泻病毒研究现状

概括了猪流行性腹泻病毒的基因分型和流行病学,阐述了猪流行性腹泻病毒与机体免疫系统的相互作用,在哺乳动物细胞中的增殖特点,以及毒力相关基因的预测,分析了病毒的遗传变异特点,讨论了控制猪流行性腹泻所面临的问题.     

猪流行性腹泻病毒抗体ELISA检测方法的建立

为检测猪血清中猪流行性腹泻病毒(PEDV)抗体水平,以纯化的PEDV作为包被抗原,优化ELISA反应条件,建立了检测PEDV血清IgG抗体的诊断方法:当血清OD_(450nm)值0.31时,判定阳性;当OD_(450nm)值小于0.26时,判定阴性;当OD_(450nm)值介于两者之间判定可疑。结果表明,试验建立的ELISA抗体检测方法可用于检测猪血清中猪流行腹泻病毒抗体、监测猪流行腹泻病的流行情况和评价相关疫苗的免疫效果。     

猪流行性腹泻的诊断

猪流行性腹泻在仔猪中以高发病率、高死亡率为特征,已经逐渐成为影响猪养殖业的一个非常严重的疾病。因此,在养殖过程中如何快速而准确地诊断猪流行性腹泻尤为重要。目前,许多有效的诊断方法已经被应用于该病的诊断中。本文旨在系统地阐述猪流行性腹泻的临床、病理和实验室学的诊断方法,为养殖场对于该病的诊断和后续的预防提供一定的理论参考。     

猪流行性腹泻病毒的特性研究

猪流行性腹泻(porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)引起的一种严重的病毒性传染病。对于大多数的病毒性腹泻疾病来说,防治措施一般选择疫苗免疫。近年来,有关猪流行性腹泻病毒疫苗的研究已经取得了一定的进展,包括灭活疫苗、弱毒疫苗、转基因植物疫苗、乳酸杆菌疫苗、亚单位疫苗和联合疫苗等。实验参照2010年版《中华人民共和国兽药典》第3部的要求对该病毒的特性进行了系统研究与记录,为PEDV HLJBX株可作为弱毒疫苗候选株的研究提供了进一步的参考资料。     

Isolation and Identification of a Variant Porcine Epidemic Diarrhea Virus and the Immune Efficacy of Its Inactivated Vaccine

This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.     

Research Progress on Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a contagious acute gastrointestinal infection, which has brought great economic losses to the pig industry in recent years.The article reviewed advances in molecular biology, methods of detection, genetic variation and vaccine development to provide references for further study in preventing porcine epidemic diarrhea.     

猪流行性腹泻病毒研究进展

猪流行性腹泻病毒是引起猪腹泻的重要病原体之一,广泛流行于世界各地,造成了重大的经济损失。文章阐述了猪流行性腹泻病毒的分子生物学特点,概括了病毒的检测方法,并对近年来流行病学及疫苗的研发情况进行了综述,以期对猪流行性腹泻病毒研究提供帮助。     

桃金娘鞣质抗猪流行性腹泻病毒作用研究

为测试桃金娘鞣质对猪流行性腹泻病毒(PEDV)的影响,体外试验以PEDV感染PK细胞为模型,采用细胞病变(CPE)效应法、MTT比色法检测细胞存活率,测定桃金娘鞣质抑制PEDV增殖的半数有效浓度(EC₅₀)和治疗指数(TI),同时设利巴韦林对照组;体内试验以PEDV感染昆明种小鼠为模型,60只昆明种小鼠随机均分为6组,设桃金娘鞣质高剂量组、中剂量组、低剂量组、利巴韦林对照组、病毒对照组、健康对照组,采用实时荧光定量PCR法检测肠系膜淋巴结PEDV含量,观察桃金娘鞣质抗PEDV增殖能力。结果显示,桃金娘鞣质能明显抑制PEDV引起的细胞病变,半数有效浓度为0.021 mg/mL,不如利巴韦林0.0098 mg/mL(P<0.05);治疗指数为361.9,优于利巴韦林214.28(P<0.05);桃金娘鞣质能影响PEDV在小鼠体内的增殖数量,并呈现一定量效关系;桃金娘鞣质各剂量组小鼠肠系膜淋巴结PEDV含量均低于病毒对照组(P<0.05),高、中剂量组小鼠肠系膜淋巴结PEDV含量均低于利巴韦林对照组(P<0.05),小鼠肠系膜淋巴结PEDV含量在...     

Research Advances on Porcine Epidemic Diarrhea and New Vaccine

Porcine epidemic diarrhea (PED) is one of the important diseases that endanger the healthy development of the pig industry.It has acute and highly infectious characteristics,causing severe economic losses to the pig industry.Porcine epidemic diarrhea virus (PEDV) infection could destroy the animal's digestive system,cause the appetite of sick pigs to decline,vomit,and severe diarrhea.And the piglets cured by drugs will also have a serious impact on production due to factors such as poor growth and development,which will bring huge problems to the farmers.The outbreak of PEDV highly pathogenic mutant strains in China in 2010 led to a significant increase in the incidence and mortality of PED,which severely affected pig production in China.The outbreak of PED has drawn close attention from the pig industry.Scholars in related research fields have conducted more in-depth research on the pathogenic mechanism of PEDV and the new PED vaccine.Five new types of PED vaccines,including subunit vaccine,virus live vector vaccine,bacterial live vector vaccine,transgenic plant vaccine and nucleic acid vaccine,have been developed successively,and new breakthroughs and progress have been made.Compared with the traditional PED inactivated vaccine and PED attenuated vaccine,the new PED genetically engineered vaccine has the advantages of good safety,simple preparation,and good immune effect.This article focuses on the pathogenesis of PEDV and the research progress of five new PED vaccines,so as to deepen our understanding of new PEDV and PED vaccines,in order to provide references for prevention and control measures of PEDV infection.     

猪流行性腹泻病毒SD株的分离与鉴定

利用Vero细胞自安徽合肥腹泻仔猪小肠内容物中分离到一株病毒,经胶体金、PCR方法和攻毒试验,确定为猪流行性腹泻病毒,命名为SD株。SD株在Vero细胞上传代,第8代(F8)开始出现稳定的细胞病变(CPE),F8代的毒价(TCID50)为10-6.25/0.1 m L,F8代细胞培养物接种未吃初乳仔猪,接种仔猪均出现典型腹泻症状,死亡仔猪3头(50%),未死亡猪生长严重发育不良,说明SD株具有较强的致病性。     

猪流行性腹泻病病毒的分离鉴定

猪流行性腹泻病,是造成养猪业经济损失严重的传染病之一。本文是针对某养猪场的病死猪进行临床症状观察、病理剖检,通过病毒的分离培养及RT-PCR鉴定,确诊病死猪病原为猪流行性腹泻病毒应加强对猪流行性腹泻病的诊断及监控。     

猪流行性腹泻及新型疫苗研究进展

猪流行性腹泻（porcine epidemic diarrhea,PED）是危害养猪业健康发展的重要疾病之一,具有急性、高度传染性的特征,给养猪业造成了严重的经济损失。猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）感染会破坏动物机体的消化系统,造成患病猪食欲下降、呕吐以及严重腹泻等,且药物治愈后的仔猪也会因为生长发育不良等因素严重影响生产,给养殖户带来巨大的困扰。2010年PEDV高致病变异毒株在中国暴发流行,导致PED的发病率和死亡率大幅升高,严重影响中国养猪生产。此次PED的暴发流行引起了养猪行业的密切关注,相关研究领域的学者对PEDV致病机制和PED新型疫苗进行了更加深入的研究,亚单位疫苗、病毒活载体疫苗、细菌活载体疫苗、转基因植物疫苗和核酸疫苗5种PED新型疫苗相继被开发,并取得全新的突破和进展。与传统的PED灭活苗和PED弱毒疫苗相比,PED新型基因工程疫苗具有安全性好、制备简单、免疫效果好等优点。文章着重对PEDV发病机理和5种PED新型疫苗的研究进展展开综述,从而加深对PEDV和PED新型疫苗的了解,以期为PEDV感染的预防和控制措施提供参考。