Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome

Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin-biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis studies and diagnostic applications.     

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.     

Single nucleotide polymorphism identification, linkage and radiation hybrid mapping of the porcine pituitary adenylate cyclase-activating polypeptide type I receptor gene to chromosome 18

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a neuropeptide with diverse biological actions. Type I PACAP receptors (PACAPR) are specific for PACAP, whereas type II and III PACAPRs are less restricted. To localize and analyse the variation of this gene, a 559‐bp long intronic fragment of the porcine PACAPR gene was amplified by polymerase chain reaction and sequenced in samples from five different pig breeds. One single nucleotide polymorphism was identified and its allele frequency was determined in all five breeds. Linkage analysis in a Berkshire × Yorkshire reference family placed the PACAPR gene on chromosome 18, between SW787 and S0062 (SW787– 8.1 cM –PACAPR– 3.0 cM –S0062). Radiation hybrid mapping confirmed that the PACAPR gene was linked to SW1682 on chromosome 18 (28.8 cR₃₀₀₀; LOD = 10.4).     

序贯培养法对猪孤雌激活胚胎发育能力的影响

经体外成熟、孤雌激活和培养获得猪胚胎,研究了不同培养体系、共培养体细胞和序贯培养对猪孤雌激活胚胎发育的影响。试验表明:孤雌激活卵母细胞在SOF 10?S培养体系中分裂效果最好,添加胎牛血清的NCSU-23和颗粒细胞对胚胎发育有促进作用,培养6d后发育到桑囊胚的比率增加(P<0.05)。在序贯培养的前3d,SOF培养基(不含葡萄糖)和颗粒细胞对胚胎的发育有促进作用,分裂率(P<0.05)和突破4细胞阻滞的数目显著增加,在培养的后3d,添加胎牛血清的NCSU-23和输卵管上皮细胞能支持较多胚胎发育到桑囊胚,桑囊胚的发育率为(59.5±3.2)%(P<0.05)。结果表明,SOF培养基和颗粒细胞 添加胎牛血清的NCSU-23和输卵管上皮细胞的序贯培养系统能较好的促进胚胎的发育。     

不同方式制备的猪胃消化液对培养猪多杀性巴氏杆菌（EO630株）的影响

在猪多杀性巴氏杆菌(EO630株)的菌液生产中,使用不同方式制备的猪胃消化液马丁汤培养基生产猪多杀性巴氏杆菌菌液,菌液菌数差异明显。结果表明用温室消化的猪胃消化液制备的马丁汤培养基有利于提高培养菌数。     

猪繁殖与呼吸综合征病毒感染对猪肺泡巨噬细胞外源性抗原加工递呈相关分子和共刺激分子的影响

采用定量竞争PCR技术对猪繁殖与呼吸综合征病毒（PRRSV）感染后不同时相猪肺泡巨噬细胞（PAM）中外源性抗原加工递呈相关分子SLA-DR、Ii链和SLA-DM分子及共刺激分子CD40、CD80、CD86的mRNA转录动态进行了定量检测。结果表明,PRRSV感染后PAM的SLA-DR mRNA转录水平在3、7和14 d下调,低于对照组;感染后3 d,Ii链、SLA-DM和CD40的mRNA转录水平极显著下调（P〈0.01）;CD80和CD86mRNA转录水平也在感染后3 d明显下调（P〈0.05）。用流式细胞术检测PAM表面的SLA-DR抗原的结果表明,在感染后3 d短暂上升,7 d之后一直低于对照组。由此说明,PRRSV感染早期对猪肺泡巨噬细胞的外源性抗原加工和递呈功能产生显著影响,导致其外源性抗原递呈功能下降,进而影响机体的体液免疫应答。     

抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定

以新生乳猪增殖猪流行性腹泻病毒（ＰＥＤＶ）,通过蔗糖密度梯度离心将其纯化,获得较为完整的病毒颗粒。病毒ＥＬＩＳＡ效价为１∶３×１０５,蛋白含量为３．９６ｇ／Ｌ。将提纯的ＰＥＤＶ免疫ＢＡＬＢ／ｃ小鼠,取免疫鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合,融合率为９５．６％。用间接ＥＬＩＳＡ进行筛选,共检出３８个阳性孔,阳性率为２８．８％。对其中１５个阳性值较高的孔进行３次再克隆,阳性率达１００％,最后获得１５株稳定分泌特异性抗ＰＥＤＶ单抗（ＭｃＡｂ）的杂交瘤细胞。ＥＬＩＳＡ阻断试验与交叉试验证明,其分泌的抗体具有高度特异性。腹水和细胞培养上清的ＥＬＩＳＡ效价分别为１∶１０３～１∶１０６和１∶１２８～１∶５１２。经鉴定,１５株ＭｃＡｂ的分子类型均为ＩｇＧ,均无免疫沉淀特性。杂交瘤细胞的染色体数目为９０～１１０,平均９５。尤为重要的是,这些杂交瘤细胞所分泌的抗体,有的对ＰＥＤＶ具有中和作用,能够阻断病毒对猪只的侵害作用。     

仔猪暴发猪繁殖与呼吸道综合征

调查了１９９５年广东省３个猪场暴发的疫病,根据流行病学、临床症状、病理变化、人工感染试验,结合间接免疫荧光试验及电镜观察等实验室诊断,确诊为猪繁殖与呼吸道综合征。     

由猪囊胚内细胞团分离胚胎干细胞的研究

以 PMEF为饲养层培养猪 7～ 9日龄囊胚分离 ES细胞 ,ICM初次传代时间为 4～ 7d,ES细胞传代时间为 4～ 5 d,ES细胞传至 1～ 5代的胚胎数分别为 1 2 (1 8.8% ) ,8(1 2 .5 % ) ,5 (7.8% ) ,3(4 .7% ) ,2 (3.1 % ) ,并进行体外分化和 AKP染色鉴定。对影响猪 ES细胞分离与克隆的因素进行比较 ,结果表明 :(1 ) 9和 1 4日龄小鼠胎儿 PMEF饲养层培养 9日龄猪囊胚 ,贴壁率 (83.3% ,88,9% ) ,ICM形成率 (75 .0 % ,77.8% )和 1代 ES细胞克隆率(2 5 .0 % ,2 2 .2 % )无显著性差异 (P>0 .0 5 )。 (2 ) 7,8和 9日龄胚胎随胚龄增加 ,胚胎贴壁率 (5 .9% ,5 7.1 % ,87,9% )和 ICM形成率 (0 .0 % ,2 8.6 % ,81 .8% )升高 ,差异显著 (P<0 .0 1 )。(3) 9日龄囊胚直径 0 .8～ 1 .2 m m培养36～ 4 8h贴壁 ,贴壁率 1 0 0 % ,ICM形成率 1 0 0 % ;直径 >1 .2 mm囊胚贴壁时间为 4 8～ 72 h,贴壁率 5 7.1 % ,ICM形成率 5 7.1 % ,差异显著 (P<0 .0 5 )。去除直径 >1 .2 m m囊胚部分滋养层细胞培养 36～ 4 8h,贴壁率 91 .7% ,ICM形成率 83.3% ;(4 )添加外源 L IF没有提高贴壁率 (86 .4 % ,90 .9% ;P>0 .0 5 )和 ICM形成率 (81 .8% ,81 .8% ;P>0 .0 5 )     

猪流行性腹泻病毒和猪传染性胃肠炎病毒一步法双重RT-PCR检测方法的建立与应用

本研究旨在建立一种用于临床腹泻病例中猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）快速检测的一步法双重RT-PCR方法。根据GenBank中收录的PEDV和TGEV的基因序列,分别设计两对特性引物,经最佳反应条件的优化和选择,结果显示,该一步法双重RT-PCR检测方法能同时特异性扩增PEDV和TGEV,该方法可检测到稀释度1×10^-5的疫苗毒。应用本方法检测2015年度实验室收集的疑似样品,PEDV阳性率为100%。本研究建立的方法快速、敏感性高、特异性强,可用于临床PEDV和TGEV的快速检测及流行病学调查。