Genomic and Pathogenic Studies on a Glycoprotein E Variant Field Isolate of Bovine Herpesvirus 1

Glycoprotein E-negative (gE–) laboratory strains of bovine herpesvirus 1 (BHV-1) were recently introduced as novel marker vaccines, allowing serological discrimination between vaccinated and naturally infected animals on the basis of lack or presence of antibodies against gE epitopes. The applicability of this approach is based on the genetic stability of the gE. However, mutant field variants of BHV-1 with a variable response in anti-gE ELISA have been isolated. The molecular characterization of a gE variant field isolate (Salwa strain) is presented here. By comparing the gE nucleotide and amino acid sequences of the Salwa strain with those of the wild strain Jura, ten mutated bases were found in the gE strain of Salwa, six of which alter the amino acid sequence, leading to changes in five amino acids. Both strains caused respiratory disease in experimentally infected calves, but Salwa generated slightly milder signs. Both viruses were excreted in nasal and ocular discharges, and were reactivated by dexamethasone treatment. In conclusion, the rather close similarities observed in the gE gene structure and pathogenicity features of the gE mutant and of the wild strain of BHV-1 confirm the genetic stability of gE. The findings indicate that the Salwa isolate is virulent, but less virulent than wild strains. Our data support the use of gE-negative marker vaccines in eradication programmes.     

猪瘟病毒广西流行毒株E0基因的克隆与分析

应用RT-PCR方法对猪瘟病毒E0基因进行扩增、克隆及测序,用DNA Star分析软件对6株毒株与猪瘟兔化弱毒苗(HCLV)、石门(Shimen)株、Paderborn株、GXWZ02株的相应片段进行比较分析,构建了CSFV遗传进化树。序列分析表明,广西流行株与HCLV株、Shimen株、Paderborn株、GXWZ02株之间核苷酸的同源性分别为80.8%~81.5%、82.8%~83.3%、93.1%~94.2%、93.7%~94.2%,推导的氨基酸同源性分别为88.3%~89.7%、89.7%~91.1%、96.2%~97.7%、97.7%~99.1%;6株广西CSFV流行毒株与国内外已发表的14株病毒相应序列进行比较,构建遗传进化树,结果表明所比较的20株毒株分为2个基因群,广西流行毒株均属于基因群Ⅱ。本研究成功地对广西流行猪瘟病毒株的E0基因进行了测序分析,表明近年来广西流行毒株未发生较大的变异,但病毒株有远离疫苗株发展的趋势。     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

E．stiedai感染兔免疫器官内球虫抗原的免疫组化定位

用免疫组化SABC法,对斯氏艾关耳球虫感染后兔肝脏、脾脏和肝门淋巴结中球虫抗原的分布进行了观察。结果表明,在肝脏窦状隙、胆管上皮细胞、肝门淋巴结和脾脏内可见到明显的呈棕黄色阳性反应区,表明在这些区域和部位有相应的E．stiedai孢子囊、裂殖子或子孢子抗原存在,且抗原数量以肝脏内最多,其次为脾脏,最少者为肝门淋巴结;显微镜下还可见到大量坏死的肝细胞、淋巴细胞以及朗罕氏巨细胞,说明多核巨细胞也参与了对球虫的吞噬和处理过程。     

微生物法测定婴幼儿配方乳粉中的生物素、叶酸和VB12

目的:采用微生物法测定婴幼儿配方乳粉中生物素、叶酸和VB12.方法:利用不同乳酸菌对特定维生素具有敏感性的特点,通过测定菌株的生长变化来反映样品中相应微生物的维生素含量,对市售的13种婴幼儿配方乳粉维生素进行测定.结果:微生物法对于生物素和VB12测定数据更稳定,重复性好,生物素相对标准偏差为0.056％～4.158％,VB12的相对标准偏差范围为0.112％～3.848％,而叶酸的检测数据之间波动稍大,相对标准偏差为1.236％～8.332％.结论:本法适合婴幼儿配方乳粉中生物素、叶酸和VB12这3种维生素的分析定量研究.     

锌、铁和维生素A互作对肉仔鸡血清和肝脏抗氧化效应的影响

试验选用360只健康1日龄艾维茵肉仔鸡(公、母混养),采用2×2×3三因子析因化设计,将360只试验雏鸡随机分成12组,每组设3个重复,每个重复10只鸡。锌的添加浓度为50、100mg/kg,铁的添加浓度为30、60mg/kg,维生素A的添加浓度为1500、3000、6000IU/kg。测定了肉仔鸡血清、肝脏谷胱甘肽过氧化物酶(GSH-Px)活性,过氧化氢酶(CAT)活性、铜锌超氧化物歧化(酶CuZn-SOD)活性,丙二醛(MDA)含量,以此来研究不同水平的锌、铁、维生素A同时添加,三者在肉仔鸡血清、肝脏抗氧化功能方面的互作效应。结果表明:锌、铁与维生素A三者互作对提高肉仔鸡血清CuZn-SOD活性影响极显(著P<0.01),对提高肉仔鸡肝脏GSH-Px活性和降低MDA含量影响极显(著P<0.01)。这说明锌、铁、维生素A三者之间在肉仔鸡抗氧化方面存在协同效应。     

热研15号刚果臂形草的选育与利用

1991年从哥伦比亚国际热带农业研究中心(CIAT)引入6095刚果臂形草(Brachiaria ruziziensis G.et E.CIAT6095),1992-1993年在网室和田间进行生态适应性观测,1994-1996年进行品种比较试验,1999-2004年进行区域试验,1999-2005年在海南、云南和福建进行生产试验。结果表明:热研15号刚果臂形草产草量达209865.83 kg/hm²·a,极显著(P<0.01)高于热研6号珊状臂形草(B.brizantha cv.Reyan 6)54.43%,高于热研3号俯仰臂形草(B.decumbens cv.Reyan 3)43.76%;热研15号刚果臂形草喜湿润热带气候,适宜在海拔1000~2000 m,年降水1000 mm以上的热带、亚热带地区生长,具有良好的耐旱能力,在pH4.5~5.0和极端贫瘠的土壤具有良好的持久性和丰产性;适于建植高产、优质、耐久的放牧型草地;固土护坡能力强,适于作水土保持的先锋草种。     

大肠杆菌Nissle 1917鞭毛蛋白部分高变区功能初步研究

初步研究大肠杆菌Nissle 1917（E. coli Nissle 1917,EcN）鞭毛蛋白（Flagellin, FliC）高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆（EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in EcNc）为实验菌株,分别构建2个鞭毛蛋白基因（fliC）高变区859-888位、829-858位的缺失突变株和回补菌株。分析突变区域对EcNc的生长性能、生化特性、运动性及抗原性等方面的影响。结果显示：EcNc、两个缺失株和回补株的生长特性无明显差异,且三者生化试验结果一致。缺失突变株在半固体培养基上不能运动,而回补株均恢复了原运动性;缺失突变株均不与H1单因子血清发生凝集,而互补菌株均能产生与EcNc一致的凝集反应。表明高变区的这两个区域尽管不影响菌株的生长性能和生化特性,但与EcN的运动性及抗原性密切相关。这为进一步探索EcN鞭毛展示技术的研究提供了一定的数据。     

猪附红细胞体PCR-微孔板杂交-酶免疫检测方法的研究

以猪附红细胞体和多种血营养菌16S rRNA基因的保守区设计合成引物HM-f/HM-r,在反向引物的5′端用生物素标记,以猪附红细胞体广东株16S rRNA基因的可变区序列设计种特异性寡核苷酸探针,探针的5′端用地高辛标记,成功地建立了猪附红细胞体PCR-微孔板杂交-酶免疫检测方法。通过测定不同浓度PCR产物对应的微孔板杂交-酶反应显色OD值,用SPSS统计软件进行回归分析,得到回归方程为y=2.1012-0.4492×logx,其相关系数R为0.977,显示对未知样品可进行定量检测。该方法与猪的多种细菌性病原、猫血巴尔通氏体CA株和E.wenyonii无交叉反应,其敏感性比常规PCR琼脂糖电泳检测提高了近100倍。用建立的方法对38份临床样品进行检测,结果有12份检测结果为阳性。     

Protecting effect of vitamin E supplementation on submaximal exercise-induced oxidative stress in sedentary dogs as assessed by erythrocyte membrane fluidity and paraoxonase-1 activity

The aim of this placebo-controlled study was to investigate the effects of oral vitamin E supplementation for 10 weeks on exercise-induced oxidative damage in untrained dogs. Eight dogs were randomly assigned to a supplementation (n = 4) or control (n = 4) group and underwent two isolated submaximal exercise sessions, 10 weeks apart. Blood was collected during each session to measure erythrocyte membrane fluidity (EMF), paraoxonase-1 (PON1) activity, plasma malondialdehyde (MDA) and vitamin E concentrations. These biomarkers were measured in venous blood samples collected before (t₀), just after (t, EMF only) and 1 d (t + 1 d) and 7 d (t + 7 d) after the dogs ran on a treadmill.Prior to vitamin E supplementation, exercise induced a significant decrease in PON1 activity, EMF, vitamin E concentration and a significant increase in MDA concentration at t + 1 d. After a 10 week vitamin E supplementation period, these exercise-induced changes in PON1 activity, EMF and MDA concentration were still significant in the control group, but not in the supplemented group. These results suggested that vitamin E supplementation had a protective effect on submaximal exercise-induced oxidative damage in sedentary dogs.