AbstractDuring the last decade, porcine epidemic diarrhea virus has detrimental consequences on swine industry, due to severe outbreaks especially in the suckling piglets. In March 2013, an outbreak was reported on a commercial swine farm in Guangdong Province, Southern China. A wild-type PEDV strain named as CHYJ130330 was identified, complete genome was sequenced and deposited in GenBank (accession no. KJ020932). The molecular epidemiological including evolutionary characteristics and pathogenicity assessment were explored during this study with particular interest and focus to develop this candidate strain for new vaccine. The isolates from China pre- and post-2013 shared 96.5–97.2% and 97–99% nt identity respectively with wild-type CHYJ130330 strain which during experimental studies has demonstrated high virulence and 100% mortality in 104 TCID50 group piglets within 5 days. The 22 reference strains selected from other parts of the world shared 98–99% identity with our sequence except Chinese (CV777) and S. Korean (vir.DR13, SM98 and atten.DR13) strains sharing 96.8, 97.6, 96.6 and 97.1% identity respectively. The phylogenetic tree revealed most strains reported after 2013 in GII genogroup while the prototype (CV777), S.korean and earlier Chinese (JS2008, 85-7mutant, Atten.vaccine, SD-M, LZC and CH/S) were GI Group. The amino acid sequence of CHYJ130330 E and M protein is highly conserved while ORF3 and N protein having 9 and 17 amino acid substitutions respectively in comparison to CV777 strain. The comparison of full length genome and the structural proteins revealed variations signifying that PEDV variant strains are still the main source of outbreaks in spite of continuous vaccination and also explain the variable trend of large scale outbreaks during this decade as compared to sporadic tendency of disease found before 2010. It is evident from this study that Chinese strains display significant level of mixing with the strains reported from other countries. The strain CHYJ130330 was also adapted successfully to Vero cell line and has shown high virulence in piglets. The information/findings will be helpful to develop a strategy for control of PEDV and have also shown that CHYJ130330 strain has strong virulence and is a more popular clinical strain in recent years, which has the potential to be developed into PEDV vaccine.

为了建立检测母猪初乳中猪流行性腹泻病毒分泌型免疫球蛋白A(SIgA)的方法,本研究将原核表达的刺突蛋白S1作为包被抗原,辣根过氧化物酶(HRP)标记的鼠抗猪免疫球蛋白A(IgA)的单克隆抗体作为二抗,并利用棋盘法确定了最优反应条件:S1蛋白的包被量为400 ng,二抗的稀释倍数为250倍,二抗的反应时间为45 min.对该检测方法的特异性、敏感性及重复性鉴定结果显示,母猪初乳中猪流行性腹泻病毒SIgA的检测方法具有特异性强、敏感性高、重复性好等特点.利用建立的ELISA方法对母猪分娩后不同时间采集的乳汁样品进行测定,发现不同时间点的乳汁样品中SIgA含量具有一定的消长规律,结果与其他文献报道的基本一致.该方法的建立为评价猪初乳中SIgA的抗体水平及PEDV疫苗的黏膜免疫效果提供了技术手段.

Prokaryotic expression vectors pET28a and pCold Ⅰ were used to construct recombinant expression plasmids with porcine parvovirus VP2 gene,and the recombinant protein of porcine par-vovirus VP2 was co-expressed with chaperone protein and induced to express at low temperature,and identified by electron microscope and hemagglutination titer determination.The results showed that the recombinant protein could be expressed soluble by the two expression vectors.The cold shock vector recombinant protein pCold Ⅰ-VP2 has a higher expression efficiency in the superna-tant and fewer heteroproteins.It did not need additional L-arabinose during induction,and the ex-pression process was relatively simple.The purified pCold Ⅰ-VP2 recombinant protein had good immune reactivity,and could be assembled into virus like particles(VLPs)in vitro,and had he-magglutination activity.After immunizing guinea pigs with 206 adjuvant mixed with the emulsified recombinant protein,high titer of hemagglutination inhibition antibody could be detected in the se-rum.The above results provide data support for further research on new safe and efficient porcine parvovirus vaccine.

串联表达猪瘟病毒(classical swine fever virus,CSFV)E0和E2蛋白,并制备多克隆抗体鉴定其免疫原性,为进一步研究亚单位疫苗和猪瘟ELISA抗体检测方法提供材料.应用PCR技术分别扩增CSFV E0和E2基因片段,运用重叠PCR将其串联扩增后克隆至pET-28a载体,构建重组表达质粒pET-28a-E0+E2.通过大肠杆菌表达系统表达重组蛋白E0+E2,切胶纯化后免疫昆明小鼠制备血清,通过间接ELISA方法测定抗体水平,间接免疫荧光(IFA)分析多克隆抗体的免疫原性和特异性.结果显示,37℃,1 mmol/LIPTG诱导条件下,E0+E2蛋白能够以包涵体形式表达,经纯化复性后免疫小鼠,制备的血清能与感染CSFV的细胞特异性反应.在大肠杆菌中高效表达了 E0+E2蛋白,制备的多克隆抗体具有较高的特异性和反应性.

为了确诊四川省某猪场1头25日龄左右的后腿跛行并急性死亡仔猪的病因,并制定对猪群其他猪只的预防和治疗方案.本试验剖检死亡仔猪,取脾脏、肝脏、肺脏组织和血液进行细菌分离培养,革兰染色镜检,PCR分型鉴定和药敏试验.结果显示,分离培养的细菌革兰染色为阳性;分离菌株经PCR检测为猪链球菌9型;药敏试验确定该分离菌株对氨苄西林、环丙沙星、氟苯尼考、青霉素、多西环素、头孢氨苄、氧氟沙星、头孢拉定、头孢呋辛和氯霉素敏感.结果表明,该猪场仔猪为感染猪链球菌9型引起的急性死亡,通过头孢类等药物可以有效的治疗发病仔猪.

为确定广东省英德市某规模化猪场传染性病原,对该猪场送检病料进行猪圆环病毒2型、猪繁殖与呼吸综合征病毒抗原检测,并进行细菌分离,对优势菌落经革兰氏染色、生化反应及16 S rRNA基因PCR检测测序,确定仔猪感染副猪嗜血杆菌;药敏试验结果表明,该分离株对恩诺沙星、环丙沙星较为敏感.PCR检测结果显示病料中猪圆环病毒2型、猪繁殖与呼吸综合征病毒抗原阴性.根据药敏试验结果,为规模猪场防控副猪嗜血杆菌感染提供用药指导与防控建议,实施效果较好.

炎症反应是机体对抗异物的一种先天性免疫反应,NLRP3(nod-like receptor family,pyrin domain-containing 3)炎症小体途径是炎症反应的主要途径之一,在机体对抗病原微生物反应中起着重要的作用.为了深入研究犬的NL-RP3炎症小体在犬感染H3N2犬流感病毒(CIV)后的变化,对未感染以及感染H3N2 CIV早期(3 d)和后期(7 d)的比格犬心脏、肝脏、脾脏、肺脏、肾脏、脑、气管和淋巴结组织进行病理学检测.结果发现,感染H3N2 CIV的比格犬,在早期没有明显的病理变化,炎症反应不明显,但后期炎症反应明显增强.通过荧光定量PCR检测比格犬感染H3N2 CIV后NLRP3、ASC、Caspase-1、IL-6、IL-1β、IL-18和TNF-a等炎症相关基因的转录水平,发现宿主在感染CIV早期炎症相关基因转录水平显著降低,而在感染H3N2 CIV后期的炎症相关基因转录水平比早期显著升高.进一步克隆犬NLRP3、ASC和Caspase-1基因并进行原核表达,制备多克隆抗体,通过免疫组织化学方法对未感染以及感染 H3N2 CIV 早期和后期的比格犬组织中的 NLRP3、ASC(apoptosis-associated speck-like protein containing CARD)和Caspase-1蛋白的表达水平进行检测.结果发现在感染H3N2 CIV早期的犬组织中NLRP3、ASC和Caspase-1蛋白的阳性反应与对照组相比总体上没有明显的变化,而在感染后期犬组织中NLRP3、ASC和Caspase-1蛋白阳性反应明显增强.本研究为进一步研究流感病毒与宿主炎症反应的关系提供了思路,为后期更好地预防和治疗犬流感提供了理论依据.

Inflammation is an innate immune response of the body against pathogens and other irritants. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is a major player in the inflammatory response against pathogenic microorganisms. In this study, we analyzed the relationship between the NLRP3 inflammasome and the influenza virus NS1 protein, which is involved in host immune escape. The canine influenza virus NS1 protein transcriptionally attenuated proinflammatory cytokines by inhibiting the nuclear factor-κB (NF-κB) activator. NS1 also directly interacted with NLRP3 and blocked ASC (Apoptosis-associated speck-like protein containing CARD) oligomerization, which deactivated the NLRP3 inflammasome. In addition, NS1 inhibited pro-caspase 1 cleavage into caspase-1, which prevents maturation of IL-1β and IL-18 from their respective precursors, eventually reducing the inflammatory response. Taken together, the influenza NS1 protein evades host immunity, and our findings provide a theoretical basis for the prevention and treatment of canine influenza.

猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是目前引起猪群病毒性腹泻的主要病原之一,给我国乃至世界养猪业造成巨大经济损失.临床上7日龄以内的仔猪发病率最高,病死率可达100％,目前,疫苗仍是预防PEDV的重要手段.本文从流行病学、临床症状与疫苗研究现状等方面分析传统疫苗免疫效果差的原因,阐述PEDV的基本情况及其免疫防控,为新型疫苗研制提供理论参考.

轮状病毒感染可使感染者(人或动物)出现严重的腹泻,年龄较小、较大或免疫系统功能障碍的感染者都可能出现严重脱水等症状,严重者可导致死亡.全球每年约20万婴幼儿因轮状病毒感染而死亡,给世界公共卫生事业带来巨大挑战.同时,在农业生产方面,猪、牛、羊等关乎民生的经济动物的幼仔,也会因感染轮状病毒而死亡,对养殖业造成一定的经济损失.本文从轮状病毒的基因组特征、感染与增殖特点、诱导机体的免疫应答以及疫苗现状进行了总结,为开发轮状病毒相关疫苗或治疗药物提供借鉴.

Background: Porcine epidemic diarrhea virus belongs to family of coronaviruses which are notorious for rapid spread of severe diarrhea among suckling piglets. The virus mainly replicates in the epithelial cells of duodenum, jejunum, ileum and colon and is a life threatening condition in pigs. A highly virulent strain “CHYJ130330” having high mortality rate was isolated from a field outbreak, identified as a new virulent genotype II/G2-b strain and adapted successfully to vero cells was used to prepare inactivated vaccine against PEDV. This newly prepared vaccine was given through intranasal route and is compared with the commercially available bi-combined (PEDV and TGEV) vaccine given by intramuscular injection. In this study milk or mucosal IgA and IgG antibody levels have been used to predict vaccine efficacy and the level of protective immunity against PED virus. Antibody titers in the milk of sows and intestines of suckling piglets were compared by enzyme-linked immunosorbent assay (ELISA). Results: It was shown that CHYJ vaccine induced significantly higher levels of PEDV IgA antibody in milk of sows and intestines of piglets as compared to commercial bi-combined vaccine. Both CHYJ and commercial vaccines were not able to induce detectable IgG levels in the intestines of piglets; the later however induced higher IgG levels when detected in the sow’s milk. Protective efficacy of vaccines was determined against a highly virulent PEDV strain. CHYJ intranasal vaccine gives a better protection 80% (4/5) rate as compared to commercial i.m. vaccine conferring 60% (3/5) immunity in suckling piglets. Conclusions: It is therefore concluded that PEDV inactivated CHYJ vaccine confer better lactogenic immunity and gives more protection to suckling piglets than available bi-combined TGEV and PEDV vaccine through passive immunization.

为调查广东省鸭圆环病毒(DuCV)流行情况,本研究根据已报道的PCR方法,对2018年12月-2020年7月采自广东省9个不同地区的910份临床鸭组织病料进行鸭圆环病毒(DuCV)、大肠杆菌(E.coli)、腺病毒(FAV)、细小病毒(DPV)、禽流感病毒(AIV)和鸭坦布苏病毒(DTUMV)检测.结果表明:DuCV总检出率为23.18％(211/910);2019年第三季度较高,为34.61％;DuCV单重感染为16.24％(32/197),二重感染和三重感染分别为62.43％(132/197)和21.31％(42/197).研究结果表明,DuCV在广东省鸭养殖场中存在不同程度的流行,夏季和冬季为多发,DuCV的感染会造成免疫抑制进而导致其他病原的感染,加重疾病的严重程度和死亡率.本研究调查结果为广东省养殖户了解广东地区DuCV的流行态势提供可支撑的数据.

目的 观察2种化学消毒剂对猪流行性腹泻病毒(PEDV)的灭活效果.方法 采用细胞培养法和悬液定量病毒灭活试验方法,对过硫酸氢钾复合盐和二氧化氯泡腾片2种化学消毒剂灭活PEDV的效果进行观察.结果 在常温下,用浓度为115 mg/L的过硫酸氢钾复合盐或300 mg/L二氧化氯的消毒泡腾片水溶液对悬液内PEDV作用5 min,对悬液内浓度为105.25 TCID50/0.1 mL的PEDV灭活对数值>4.0.结论 过硫酸氢钾复合盐和二氧化氯泡腾片对PEDV均有较好的杀灭作用.

试验旨在建立快捷、高效而准确的鉴别诊断猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)与传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)的双重实时荧光定量PCR方法.通过绘制双重实时荧光定量PCR的标准曲线,检验该方法的特异性、敏感性和重复性,并对临床样品进行检测.结果显示,双重实时荧光定量PCR方法的循环阈值与PDCoV和TGEV质粒拷贝数的对数之间存在良好的线性关系,且对应的相关系数分别为 R(P)=0.9994和 R2(T)=0.996;能特异性地检测 PDCoV 和 TGEV,而与 PEDV、PRV、PRRSV、CSFV 和 RV无交叉反应,具有较强的特异性;检验PDCoV与TGEV质粒标准品的最低检测限度分别达到2和20拷贝/μ L,且分别比常规RT-PCR高1 000和100倍,具有较高的敏感度;PDCoV与TGEV的批内和批间重复性检测的Ct均值基本相同,且变异系数(CV)均＜2％,具有较好的重复性.用该方法对114份仔猪腹泻样品检测结果显示,PDCoV和TGEV的阳性率分别为5.6％(6/114)和8.8％(10/114),混合感染检出率为4.6％(5/114),比常规RT-PCR具有更高的检出率和敏感性.结果表明,本试验建立的双重实时荧光定量PCR方法具有特异性强、灵敏度高、重复性和稳定性好等优点,适用于病毒早期诊断和批量临床样品检测,为疾病防控、流行病学调查及相关性研究提供了技术支持及数据参考.

猪传染性胃肠炎病毒(TGEV)是全球范围内造成仔猪腹泻的原因之一,且常造成混合感染和继发感染,仔猪死亡率可达100％.猪氨肽酶N是TGEV的特异性受体,唾液酸对TGEV有保护作用,如保护TGEV通过胃酸时不会失活.TGEV可以诱导I型干扰素在肠道产生,结构蛋白M、E和非结构蛋白nsp14在其中发挥着重要作用.TGEV已经发展出一系列策略来对抗宿主天然免疫反应,包括基因组中的GAIT样结构、ORF7、非结构蛋白nsp1等自身因素,以及操纵宿主细胞途径,如非折叠蛋白反应.

目的 观察过硫酸氢钾复合盐和二氧化氯泡腾片对猪链球菌和副猪嗜血杆菌的杀灭效果,为畜牧养殖场消毒提供依据.方法 采用悬液定量杀菌试验法,对过硫酸氢钾复合盐和二氧化氯泡腾片对猪链球菌和副猪嗜血杆菌的杀灭效果进行观察.结果 室温下浓度为57.5 mg/L过硫酸氢钾复合盐对悬液内猪链球菌和副猪嗜血杆菌作用5 min,杀灭对数值>5.00.用浓度为0.63 mg/L二氧化氯对悬液内猪链球菌和副猪嗜血杆菌作用5 min,杀灭对数值>5.00.2种消毒剂在试验温度10～30℃范围内,杀菌效果随温度升高而提高.结论 过硫酸氢钾复合盐和二氧化氯泡腾片对猪链球菌和副猪嗜血杆菌均有较好的杀灭效果,升高温度有助于提高杀灭效果.

为了解猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV) CH7328株的致病性,并分析其基因变异和遗传演变规律,试验通过病毒培养、间接免疫荧光试验(IFA)、病毒生长曲线及仔猪致病性试验鉴定病毒的生长特性和致病性,并分析该毒株的全基因组序列.结果 显示,感染PDCoV CH7328株的ST细胞出现肿大变圆、细胞呈碎玻璃状且拉丝、细胞集聚成簇和脱落等病变.IFA结果显示,PDCoV CH7328株主要分布在细胞质中.生长曲线结果显示,PDCoV CH7328株在6h就已开始增殖,在36 h时达到峰值,对应的TCID50为10-8.0/mL,随后逐渐下降.仔猪致病性结果显示,攻毒组仔猪出现腹泻、脱水和厌食等临床症状,仔猪小肠明显变薄、透明而充盈,小肠绒毛严重脱落、萎缩或减少、肠绒毛细胞坏死等.粪便病原检测结果显示,在攻毒第2天便能检测到病毒,之后排毒持续增加,而正常对照仔猪无任何临床现象.全基因组测序及序列分析显示,PDCoV CH7328株的基因组全长为25420 bp,与国内GD株的相似性最高,达到99.9％;遗传演化结果显示,PDCoV CH7328株与国内HKU15-155、CHN-HN-2014和GD株处在同一进化分支,亲缘关系最近.本研究结果为中国PDCoV疫苗候选株的筛选及猪场疫病防控提供了参考数据和理论依据.

对3株(GD1406、FS2015、FJFZ株)变异伪狂犬病病毒(PRV)的gB和gC全基因进行扩增、测序及生物信息学分析,应用微量交叉中和试验分析抗原差异性.基因序列比对结果显示,3株PRV变异株之间gB、gC基因核苷酸和氨基酸同源性分别为99.7％～100.0％和99.6％～100.0％,100.0％和99.0％～100.0％;gB和gC基因系统进化树遗传进化分析显示:系统进化树分为2大支,其中一个大支为国内经典毒株群、2011年后分离的变异毒株群2个小分支所占据的中国毒株群;另一个大支为欧洲分离株和美洲分离株组成的欧美毒株群.微量交叉中和试验结果显示,PRV-Bartha K61高免血清中和PRV-GD1406和PRV-Bartha K61的效价分别为22和53,PRV-GD1406高免血清中和PRV-GD1406和PRV-Bartha K61的效价分别为37和44;PRV-GD1406毒株异源血清中和效价/同源血清中和效价约为0.83,PRV-Bartha K61毒株异源血清中和效价/同源血清中和效价约为0.59;2个毒株(PRV-GD1406、PRV-Bartha K61)间交叉反应程度相关系数R=0.72.

为了探索伪狂犬病病毒(PRV)流行株的基因变异及遗传演变规律,本试验对福建福州疑似发生猪伪狂犬病的猪场病料,首先选用PCR技术、小鼠接种试验、细胞接种试验及间接免疫荧光鉴定等方法分离鉴定病原,再对病原进行毒价测定、TK和gE基因克隆及序列比对分析.结果 显示,病原为PRV野毒,命名为PRV-FJFZ株;PRV-FJFZ株TCID50为10-7.2/0.1 mL,LD50为10-3.56/0.1 mL,对比分析PRV-FJFZ与国外PRV参考毒株的TK和gE基因序列同源性发现,核苷酸的同源性分别为99.5％～100.0％和99.8％～99.9％.TK和gE基因遗传进化树表明,PRV-FJFZ株与当前国内流行的变异PRV毒株在同一分支上,PRV-FJFZ株与国外PRV参考毒株在不同的分支上.本研究结果可为我国PRV的防控净化工作和疫苗株的筛选提供理论依据.

SIgA是评价黏膜免疫的重要指标,分泌片(SC)是SIgA的特有成分,为了更方便的获得大量的SIgA的分泌片,本研究应用RT-PCR方法扩增出SC基因片段,将SC基因片段克隆到pGEX-4T-1原核表达载体上,经酶切和测序验证获得pGEX-4T-1-SC重组质粒;将获得的pGEX-4T-1-SC转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达重组蛋白rSC.重组蛋白rSC经鉴定和纯化后,按常规免疫程序制备多克隆抗体;利用Western blot对抗重组蛋白rSC的多克隆抗体进行鉴定.结果显示,该抗体能特异性识别猪初乳中的SIgA免疫球蛋白,而与猪初乳中的IgG不发生反应.因此,抗SC重组蛋白的多克隆抗体可以作为抗SIgA免疫球蛋白的特异性抗体,为进一步建立SIgA的检测方法及黏膜免疫的研究奠定基础.