实时荧光定量PCR检测猪病料中的PRRSV与CSFV

根据GenBank发表的PRRSV、CSFV基因核苷酸序列,应用Primer 5.0软件分别设计了2对特异性引物.以重组质粒作为阳性标准品,应用SYBR Green I real-time PCR检测两种病毒的核酸含量.在样品稀释到10.1～10<'-11>拷贝时,能得出良好的线性关系,相关系数为R<'2>=0.999...     

Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.     

三种鉴别诊断PRRSV美洲型经典毒株和变异株的方法比较

我国流行的猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）包括美洲型经典毒株和变异株。本研究应用RT—PCR、直接免疫荧光（ direct immunofluorescence asay, DFA ）及病毒分离3种检测方法对99份不同类型的猪繁殖与呼吸综合征临床疑似病料和人工感染病料进行检测。结果显示,RT—PCR检测试剂盒敏感性最高,DFA鉴别诊断试剂盒次之,病毒分离最差。DFA与病毒分离、RT—PCR的总符合率分别为92％和85．5％。RT—PCR灵敏性高,可作为PRRSV鉴别诊断的首选方法;DFA鉴别诊断法所需时间较短、特异性好,但需要一定的操作经验;病毒分离敏感性低、操作繁琐,阳性分离需要其它方法验证,不适合于PRRSV的鉴别诊断。     

2007-2010年江苏地区猪繁殖与呼吸综合征病毒RT-PCR检测及部分Nsp2基因序列分析

本研究参考GenBank已发表的猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）Nsp2基因序列,在高致病性病毒Nsp2基因缺失区的两端保守区设计并合成了一对引物,建立并优化了能够区分经典PRRSV和高致病性PRRSV的RT-PCR诊断方法,并利用该方法对2007～2010年间江苏地区的45份可疑临床病料进行了检测。结果表明临床病料阳性率为40％,所有毒株均属于高致病性毒株。对PRRSV阳性病毒的Nsp2基因序列分析表明,所有18株PRRSV均属于美洲型毒株,与中国高致病性PRRSV代表毒株JXA1、WUH1的氨基酸同源性分别在82．2％～97．6％、80．4％～95.3％。此外,18株病毒的Nsp2共同存在不连续的30个氨基酸缺失,缺失位置与同期中国高致病性PRRSV具有相同的特征。通过本研究掌握了江苏地区2007～2010年间PRRSV流行情况及Nsp2基因变异特征,为地方猪繁殖与呼吸综合征的临床诊断和防治提供参考依据。     

CSFV免疫状态下外周血T淋巴细胞亚群变化及对PRRS的免疫保护作用

建立CSFV疫苗免疫状态下猪繁殖与呼吸综合征病毒（PRRSV）感染动物模型。测定CSFV抗体阻断率和PRRSV抗体S/P值,根据CSFV抗体阻断率和PRRSV感染情况分析猪外周血中T淋巴细胞亚群变化和对PRRS的保护促进作用。采用流式细胞术（FAC）检测猪外周血T淋巴细胞CD3、CD4、CD8亚群的变化。结果,CSFV...     

靶向GP5基因脱氧核酶抑制PRRSV在MARC-145细胞中的复制

针对猪繁殖与呼吸综合征病毒（PRRSV）JL/07/SW株GP5基因设计了4个脱氧核酶,经体外切割试验筛选得到具有体外切割活性的脱氧核酶,并通过RT-PCR、TCID50、CPE、间接免疫荧光（IFA）检测具有切割活性的脱氧核酶抑制PRRSV复制效果进行评价。结果针对GP5基因的Dz-GP5-10抑制效率最高可达到82.4%,Dz-GP5-12也表现良好的抑制效果,表明GP5基因的这2个位点可能是PRRSV复制所必需的。本研究为PRRSV复制及基因组功能研究、抗病毒药物开发和转基因动物研究奠定了基础。     

Cytokine expression, glucocorticoid and growth hormone changes after porcine reproductive and respiratory syndrome virus (PRRSV-1) infection in vaccinated and unvaccinated naturally exposed pigs

The objective of this paper was to study the changes of some cytokines and neuroendocrine hormones in vaccinated and unvaccinated pigs that were naturally infected by a PRRSV-1 (porcine reproductive and respiratory syndrome virus) heterologous field strain. We analyzed gene expression of pro-inflammatory (TNF-α, IL-1β, MCP-1, IL-6), pro-immune (IFN-γ) and anti-inflammatory cytokines (IL-10) in PBMC, as well as hormonal (GH and cortisol) levels in blood samples of pigs obtained in a field trial previously reported [Martelli P, Gozio S, Ferrari L, Rosina S, De Angelis E, Quintavalla C, et al. Efficacy of a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: clinical protection and cell-mediated immunity. Vaccine 2009;27:3788-99]. All vaccinated pigs showed an increase in pro-inflammatory and pro-immune cytokine gene expression with respect to controls and a prompt increase in GH that could be consistently associated with pro-inflammatory cytokines in sustaining innate immunity; moreover, the higher levels of cortisol indicates the activation of the hypothalamus-pituitary-adrenal (HPA) axis response. In contrast, unvaccinated pigs showed down-regulation of the cortisol and GH responses, and the pro-inflammatory and pro-immune cytokines remained at a basal or low level, with an increase of TNF-α and IL-6 in association with a higher level of IL-10 in the late phase of natural infection. The associated trends of pro-inflammatory and anti-inflammatory cytokines together with the cortisol level demonstrate that a previous vaccination promotes an early immune responsiveness in pigs and a more efficient control of inflammation in the late phase of infection with a heterologous PRRSV isolate; both events could sustain clinical protection.     

降雨侵蚀力新算法在豫西山区应用的初步研究

降雨侵蚀力是定量监测评价一个地区土壤侵蚀状况的重要因子之一,找到适宜简便的计算方法十分重要.卜兆宏提出的降雨侵蚀力新算法具有简便易用的优点.本文利用位于豫西山区鲁山县的3个水文雨量站10年185次的自记降雨过程资料,以经典算法年R值为基准,对新算法在该区的适用性进行了分析评价.结果表明:新算法结果与经典值存在高度的一致性,一致性高达90.2%,模型有效系数为96.4%,相对误差为7.7%,说明新算法能够在该区应用,可以采取此法对该区的降雨侵蚀力进行深入的分析研究;并同时对资料摘读时应该注意的问题进行了讨论.     

国内外降雨侵蚀力简易计算方法的比较

降雨是引起土壤侵蚀的主要动力 ,用 EI30 指标度量。计算 EI30 时 ,需要降雨过程资料 ,而且计算繁琐和费时 ,为此 ,很多学者采用常规降雨资料计算降雨侵蚀力的简易算法。它们往往利用区域资料经验拟合求得 ,如要推广使用 ,需要进行验证。利用我国 10个气象站降雨过程和常规降雨资料 ,通过对国内外 9种比较有代表性的多年平均年降雨侵蚀力简易计算方法的比较 ,建议采用年雨量的指数函数形式作为估算我国多年平均年降雨侵蚀力的简易计算方法。为保证计算结果的区域稳定性 ,应分不同区域进行拟合。     

PRV、PCV-2、PRRSV与HPs混合感染的核酸检测

为确定青海省某规模化发病猪场的发病原因,在临床诊断的基础上对采集的临床组织病料进行了13种常见病原体的核酸检测,并对检测结果进行了序列鉴定.结果表明,该发病猪场存在猪伪狂犬病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒与副猪嗜血杆菌的混合感染.研究结果提示,利用分子生物学技术对发病猪场的病原进行快速鉴定,是病源快速诊断和防控的有效途径.