小反刍兽疫疫苗毒与野毒实时荧光RT—PCR鉴别检测方法的建立

为了建立鉴别检测小反刍兽疫疫苗毒与野毒的快速分子生物学检测方法,通过对自行测序的疫苗毒基因组序列及GenBank中登录的野毒基因组序列进行比对分析,设计了2套引物和TaqMan荧光探针,对实时荧光RT-PCR反应条件进行优化,建立了小反刍兽疫疫苗毒与野毒实时荧光RT-PCR鉴别检测方法。特异性试验证实,该检测方法只能检测到目的病毒核酸,表明其具有良好的特异性。灵敏性试验发现,检测疫苗株的最低检测限可达1．38mg／L的总RNA,检测野毒株的最低检测限为0．16mg／L的核酸。对同一样品进行重复性检测,检测的荧光扩增曲线阈值完全重舍,证明其重复性极好。从180份临床样品中,检测出2份疫苗病毒株阳性样品,其余均为阴性。结果表明,本研究所建立的实时荧光RT-PCR方法能对小反刍兽疫疫苗毒及野毒进行鉴别检测,具有特异性好、灵敏度高、重复性极好的优点,是开展小反刍兽疫疫情监测的有力工具。     

小反刍兽疫病毒M基因截短表达及鉴定

目的将小反刍兽疫病毒M蛋白基因截短表达后用于特异性单抗制备及临床抗体水平检测。方法:用在线分析软件BepiPred分析小反刍兽疫病毒M蛋白潜在的B细胞线性表位,以本实验室构建的pCR2.1T-PPRV M质粒为模板,扩增三段截短的M基因,纯化后的PCR产物分别与克隆载体pCR2.1T连接,筛选出的阳性重组质粒经双酶切后,分别与表达载体pET-32a(+)及pGEX-6p-1连接,再将鉴定为阳性的重组质粒转化入E.coli BL21(DE3)菌株诱导表达,并用SDS-PAGE及Western blot验证。结果 PCR产物电泳,得到与预期大小相符的特异性片段。对连接克隆载体及表达载体的重组质粒双酶切后,均出现与预期一致的片段,DNA测序表明,插入片段序列与小反刍兽疫Nigeria75/1株M蛋白基因完全一致。重组蛋白经SDS-PAGE及Western blot鉴定,证明所构建的重组蛋白均获得高效表达,并均具有良好的反应原性。结论成功表达了小反刍兽疫截短M基因的蛋白,为制备特异性单抗及小反刍兽疫抗体检测奠定了一定基础。     

小反刍兽疫的流行、诊断与防控

小反刍兽疫是一种传染性极强的病毒性疾病,主要影响家养以及野生的小反刍动物。该病的主要特征是发热、口腔炎、结膜炎、肠胃炎和肺炎。小反刍兽疫也是世界动物卫生组织（0IE）规定必须上报的法定疾病之一。文章对该病的流行、传播方式、诊断方法以及防控措施进行了综述。     

我国首例小反刍兽疫诊断报告

2007年7月我国西藏发生不明山羊疫情,国家外来动物病诊断中心对当地动物疫病控制中心送检的14只病死山羊病料和一批血清样品分别进行病原学和血清学检测。利用较敏感的小反刍兽疫病毒(PPRV)特异性荧光定量RT-PCR方法,在11只病羊组织中检测到小反刍兽疫病毒核酸。利用次敏感的PPRV普通RT-PCR方法,从8只病羊组织中检测到PPRV核酸。针对1号样本病原核酸N基因和F基因片段进行遗传发生分析,该病原属于4系。利用竞争ELISA试剂盒对送检的13份血清样本进行抗体检测,结果全部阳性。将1号组织样本接种Vero细胞分离病毒,透射电镜观察下,发现了500纳米左右的病毒粒子。对分离毒株进行PCR检测同样证实其为PPRV。     

Regional Conservation in the Virunga-Bwindi Region

SUMMARY

Bwindi Impenetrable National Park and the Virunga Volcano region are the last remaining habitats of the highly endangered mountain gorilla (Gorilla beringei beringei). This conservation area, shared by Uganda, Rwanda and the Democratic Republic of Congo, also acts as a refuge for biodiversity and high endemism in the Albertine Rift of Central East Africa. This particular region has suffered from a number of years of civil strife, which have severely affected the management of these forests. This paper will explore the experiences of the International Gorilla Conservation Programme (IGCP) obtained throughout the development of regional processes that are working towards establishing a transboundary-protected area. The establishment of a regional framework and tools utilized are described. The costs and constraints incurred by the IGCP and the resulting positive impacts on conservation are also highlighted.     

小反刍兽疫病毒及山羊痘病毒N-ITR基因二联实时荧光定量RT-PCR检测方法的建立

Peste des petits ruminants virus (PPRV) and goat pox virus (GTPV) are the causative agents of two kinds of goats’ diseases-peste des petits ruminants and goat pox which can cause disaster economic losses. In order to detect the two viruses simultaneously and quickly, two sets of primers and relative probes were designed based on the nucleoprotein (N) gene of PPRV and the inverted terminal repeat (ITR) segment of GTPV， respectively. In order to work together in the same reaction, the probes were labeled with different fluorescent materials 5′FAM-TAMRA3′and 5′JOE-Eclipse3′，respectively. Results showed that the duplex Real-time RT-PCR assay was identified to be specific for PPRV and GTPV only and specific fluorescent signal could be detected, but the related viruses including fowl pox virus（FPV）and canine distemper virus (CDV) had no specific fluorescent signal. Positive recombinant plasmids (PPRV pMD18-T-N and GTPV pMD18-T-ITR) were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe N-ITR was found to be highly specific and sensitive with a detection limit of 10² copies/μL cDNA and 10³ copies/μL DNA for PPRV and GTPV， respectively. Finally, the duplex Real-time RT-PCR assay for simultaneous detection of PPRV and GTPV was established preliminarily in the study.     

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.     

反刍动物日粮组合效应的研究进展

从传统饲养体系的局限性到组合效应的概念、衡量指标类型、试验研究、发生机制、等方面综述了反刍动物日粮组合效应的研究方法与应用。     

小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内代谢消长规律的研究

为研究小反刍兽疫病毒N蛋白抗体与H蛋白抗体在羊体内的代谢消长规律,试验通过建立小反刍兽疫N蛋白双抗原夹心ELISA抗体检测方法、H蛋白阻断ELISA抗体检测方法与血清中和抗体试验方法,分别检测了羊免疫疫苗后体内N蛋白抗体与H蛋白抗体在每个免疫阶段的代谢消长变化。结果表明:羊免疫小反刍兽疫疫苗后,血清内的N蛋白抗体与H蛋白抗体在6~8周时血清抗体效价较高,对应的羊体内中和抗体效价为1∶512,且效价至少可以持续到10周以上,2种抗体具有一定消长代谢规律的相关性。该研究结果也为以N抗原与H抗原为基础建立相应的抗原捕获ELISA方法、竞争ELISA检测方法、间接ELISA检测方法以及双抗原夹心ELISA检测方法奠定了一定的理论依据。     

复合营养舔砖对山羊的饲用效果和作用机理研究 Ⅰ.补饲营养舔砖对增重速度、胴体性状和经济效益的影响

为验证复合营养舔砖对山羊的饲用效果,进一步完善其配方,改进产品,促进山羊生产发展,通过饲养试验和屠宰测定等手段,研究了复合营养舔砖对山羊增重速度、胴体性状和经济效益的影响．结果表明：补饲舔砖Ⅰ号和Ⅱ号的试验Ⅰ组和Ⅱ组羊只的平均增重速度分别比对照组提高了４３．６６％和３７．８６％;羊只胴体重、肌肉重、屠宰率和胴体瘦肉率也均显著或极显著地高于对照组,尤以试验Ⅰ组为佳;每只羊平均每天比对照组羊只分别净增纯利０．１７,０．１５元;每吨舔砖产生的直接经济效益分别在２．４１,２．０６万元以上．