Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd

In more than 10 Spanish dairy cows, a bovine herpesvirus 4 (BHV4) associated postpartum metritis was confirmed by virus isolation, BHV4-glycoprotein B (gB) PCR and/or serology. In this study, 12 cows with, and, at the time of sampling, 3 cows without clinical signs of acute postpartum metritis from one large dairy herd in Spain were examined for bacterial and viral infections. Blood, placenta/caruncles and uterine contents were collected between day 1 and day 20 post-calving, and examined for the presence of bacteria and for viruses by virus isolation, BHV4 DNA by BHV4-gB PCR and/or BHV4 antibody titres. Bovine herpesvirus 4 was detected in 83% of the cases with clinical signs of acute postpartum metritis by virus isolation and/or BHV4-gB PCR. An increase of BHV4 antibodies was detected in all examined postpartum metritis cows and in the 3 cows without clinical metritis. Two of these 3 cows developed severe metritis a few dayss after collecting the first blood sample. A concurrent infections of BHV4 and bacteria, mainly Arcanobacterium pyogenes and Streptococcus sp., were detected in 73% of the examined uterine contents collected from postpartum metritis affected cows. This case-report study showed a clear association between BHV4 infections and acute postpartum metritis in dairy cows. In addition, the BHV4-associated postpartum metritis appeared to be an emerging syndrome in this Spanish herd.     

Development of an indirect enzyme-linked immunosorbent assay for detecting equine serum antibodies to the lipopolysaccharide of Salmonella abortusequi

An indirect enzyme-linked immunosorbent assay (IELISA) was developed for the detection of equine serum antibodies to lipopolysaccharide of Salmonella enterica subsp. enterica serovar Abortusequi (LPS), a causative organism of Equine Paratyphoid. The data presented demonstrates that horses immunized with S. abortusequi LPS developed antibodies detectable by the IELISA. By comparison, the tube agglutination test (TAT) did not detect antibody to S. abortusequi LPS as consistently as the IELISA. The data suggests that the IELISA may be a more suitable test for the detection of serum antibodies to S. abortusequi than the TAT.     

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2)

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10⁹, 10⁶ and 10³ EID₅₀/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10⁹, 10⁶ and 10³ EID₅₀, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 10⁶ EID₅₀/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.     

农产品中基质对酶联免疫分析法检测三唑磷残留的干扰及消除研究

基质干扰是免疫法检测食品中农药残留时经常碰到的问题。以胡萝卜、菠菜、香蕉、梨、稻米和花生等为试材,研究了不同农产品基质对酶联免疫分析法(ELISA)检测三唑磷的影响。结果表明:不同农产品基质对ELISA检测的干扰机制不同,香蕉主要是抑制了辣根过氧化物酶的活性,菠菜、梨和稻米则主要是干扰了抗原和抗体的结合反应,胡萝卜和花生不仅抑制酶的活性,同时还干扰了抗原和抗体的结合反应。将这些农产品的磷酸盐缓冲液(PBS)(含2.5%甲醇)提取液用含有质量分数为0.3%的脱脂奶粉、0.1%的乙二胺四乙酸(EDTA)和含2.5%甲醇的PBS稀释10倍,在12000r/min下离心10min,即可消除其对ELISA检测的干扰。研究结果表明, ELISA法是可用于农产品中三唑磷残留检测的一种简单、快速、有效的方法。     

发根农杆菌介导不同基因型大豆转化效率的筛选

基因型是影响发根农杆菌转化大豆的主要因素之一,为筛选转化效率高的大豆基因型,利用不同基因型大豆的下胚轴接种发根农杆菌K599(含GUS基因),待长出毛状根后,根据不同基因型大豆发根诱导率和发根数的差异,选择适宜大豆基因型进行GUS基因的组织化学染色和RT-PCR分析。结果表明,34个大豆基因型发根诱导率和发根数有很大差异,选择发根诱导率大于60%以及发根数大于1.6条的20个大豆基因型毛状根进行GUS基因组织化学染色,其中Y091、Z184、P108、L010等4个大豆基因型的GUS基因转化效率较高,分别为96.2%、91.7%、87.5%、86.4%;进一步对以上4个大豆基因型毛状根进行RT-PCR分析,结果表明,GUS基因均得到了正确转录。表明大豆基因型Y091、Z184、P108、L010是发根农杆菌转化大豆较为理想的受体材料。     

小反刍兽疫病毒细胞膜受体的鉴定

【目的】对小反刍兽疫病毒（Peste des petits ruminants virus,PPRV）H蛋白胞外区（tH）细胞膜受体进行鉴定,为PPRV致病机制的研究奠定基础。【方法】克隆PPRV H基因胞外区（tH）,在毕赤酵母(Pichia pastoris)中进行真核表达,纯化后免疫家兔,获得兔抗PPRVtH蛋白特异性抗体;提取山羊外周血淋巴细胞膜蛋白,经SDS-PAGE检测后,湿转印法转印至NC膜,分别利用纯化的重组tH蛋白和PPRV进行病毒铺覆蛋白结合试验（VOPBA）,对受体进行鉴定。【结果】成功克隆了1 653 bp的PPRV tH基因,构建其重组酵母表达质粒pPIC9K-tH,诱导表达后获得了60 ku目的蛋白。重组tH蛋白免疫家兔后获得了效价为1∶200的抗血清。Westernblotting分析发现,该重组蛋白可与PPRV多克隆抗体发生特异性反应,山羊外周血淋巴细胞膜蛋白上有2个与PPRV和重组tH蛋白结合的蛋白带,分子质量约为38和100 ku。【结论】从山羊外周血淋巴细胞膜蛋白上鉴定到了2种与PPRV结合的蛋白组分,其特性有待于进一步研究。     

5′UTR序列对DR5GUS基因瞬时表达的影响

利用生长素响应原件(DR5)构建了DR5::GUS报告基因表达载体,并在载体构建时,对GUS的5′UTR序列进行2种设计:一种是利用载体GUS基因原有的5′UTR构建成pBI121-DR5::GUS;另一种采用植物增强表达的常用原件烟草花叶病毒(TMV)的5′端序列(Ω′)替换GUS基因原有的5′UTR序列,构建成pBI121-DR5(Ω′)::GUS。将2种载体转化根癌农杆菌后,采用烟草叶片注射浸染的瞬时表达检测法对2个载体的植物表达效果进行检测,2种载体分别转化烟草叶片2 d后,对浸染的叶盘进行GUS染色分析,pBI121-DR5::GUS的转化叶片有明显的GUS反应,而pBI121-DR5(Ω′)::GUS的转化叶片则无GUS反应。分离注射浸染部位叶盘RNA后,对GUS特异引物进行RT-PCR分析,2种转化的基因都已有效转录出RNA,但前者能翻译出相应的Ω-葡萄糖苷酸酶,后者则不能完成翻译过程,说明Ω′序列的引入并未有效提高GUS基因的表达,反而抑制了mRNA的翻译。     

胶体金免疫层析法检测猪瘟(强弱毒区分)抗体研究

以原核表达的重组猪瘟病毒E2蛋白为弱毒抗原,以猪瘟病毒(HCV)强毒株细胞培养物为强毒抗原,利用胶体金标记技术国内首次建立了HCV抗体检测(强弱毒区分)的免疫层析试纸条(双抗原夹心法)。研究结果表明,检测时只需将80μL待检测猪血清加于加样端20 min后观察结果即可。若观察区上端仅1条红色条带,即为HCV抗体阴性;2~3条条带即为HCV抗体阳性;若出现2条条带的位置为观察区上端和中间部位,即为HCV弱毒抗体阳性;若出现2条条带的位置位于观察区上端和下端部位,则为HCV强毒抗体阳性;若出现3条条带则HCV强弱毒抗体均为阳性。本试纸可很好地避免因试验操作造成的假阳性、假阴性,且操作简单、快速、简便、灵敏度高、特异好、不需特殊设备、价格低廉,20 min即可判断结果,且可区分强弱毒感染,适于基层实验室和现场检测。     

贵州矮杨梅营养器官总黄酮测定方法及含量积累规律

矮杨梅为我国特有,其植物体中含有大量的天然黄酮类化合物,对其测定方法和积累规律进行考察和探讨。结果表明,对总黄酮测定方法进行考察得出,线性回归方程为Y=8.5257X-0.0184,相关系数r=0.9996;在0~60 min范围内于波长510 nm条件下测定,结果准确、重现性好(RSD=0.34),回收率高(RSD=2.13%)。相同生育期的总黄酮含量变化规律为:雌株除营养期外均为根>茎>叶;雄株则为营养期和孕花期茎>叶>根,盛花期和果期根>茎>叶。不同生育期矮杨梅总黄酮含量动态变化规律为:雌株根为果期最高,营养期次之,孕花期最小;茎为盛花期>果期>营养期>孕花期;叶为营养期>果期>孕花期>盛花期。雄株根为果期>盛花期>孕花期>营养期;茎和叶为孕花期>果期>营养期>盛花期。     

大型溞胆碱酯酶间接非竞争酶联免疫吸附定量分析法的建立

用达到电泳纯的大型溞(Daphniamagna)胆碱酯酶(cholinesterase,ChE)免疫BALB/c小鼠,得到效价达1∶8000的鼠抗大型溞ChE多克隆抗血清;通过优化抗原抗体反应条件,建立定量分析大型溞ChE含量的间接非竞争酶联免疫吸附法(indirectandnon‐competitiveenzyme‐linkedimmunosorbentassay).结果表明:此方法检测灵敏度为24ng爛mL-1;经免疫交叉反应鉴定,所获抗血清与5、10μg爛mL-1标准蛋白质和其他生物如花翅摇蚊、尖额溞、斑马鱼(脑、脑除外的整体)、家蚕、意大利蜜蜂、赤子爱胜蚯蚓、非洲爪蟾(蝌蚪)等来源的ChE交叉反应率分别为:<0畅25%、<0畅25%、 2畅13%、 10畅19%、3畅88%、 3畅56%、 2畅81%、 3畅93%、 5畅00%和2畅75%,具较高的特异性.说明用本试验得到的抗血清建立的间接非竞争酶联免疫吸附法可用于大型溞体内ChE含量的测定,以利于在农药暴露环境下大型溞体内ChE活性的精确检测.