An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G₁ that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay.

At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation

     

Adherence of Actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin

The swine pathogen Actinobacillus pleuropneumoniae serotype 1 was investigated for its ability to adhere to swine, rat, and human buccal epithelial cells (BEC). The highest number of bacteria adhered was to swine BEC. This binding ability was affected by heating, extreme pH, treatment with sodium dodecyl sulfate, ethylenediamine tetraacetate, or periodate, and proteolysis, suggesting that cell-surface glycoproteins participate in adherence and that adherence is based mostly on ionic interactions. Mannose and swine fibronectin may play a direct role in this interaction. Convalescent-phase serum from naturally infected pigs inhibited the adhesion. There was a correlation between bacterial pathogenicity as well as host specificity and the capacity for adherence to swine BEC. Adhesion to swine BEC provides a convenient method to study in vitro the adherence of A. pleuropneumoniae and other pathogens of the pig respiratory tract.     

A computer program for calculation of doses and prices of injectable medications based on body weight or body surface area

A computer program (CalcAnesth) was developed with Visual Basic for the purpose of calculating the doses and prices of injectable medications on the basis of body weight or body surface area. The drug names, concentrations, and prices are loaded from a drug database. This database is a simple text file, that the user can easily create or modify. The animal names and body weights can be loaded from a similar database. After typing the dose and the units into the user interface, the results will be automatically displayed. The program is able to open and save anesthetic protocols, and export or print the results. This CalcAnesth program can be useful in clinical veterinary anesthesiology and research. The rationale for dosing on the basis of body surface area is also discussed in this article.     

猪胸膜肺炎放线杆菌外膜蛋白(OMP)5′末端保守区基因的克隆、表达及其免疫活性研究

以猪胸膜肺炎放线杆菌血清7型25-4株基因组DNA为模板,PCR方法扩增外膜蛋白(OMP)5′末端保守区基因片段(OMPc),酶切及核苷酸序列分析鉴定后,与原核表达载体质粒pGEX-6P-1进行连接,构建成重组表达载体pGEX-OM-Pc,转入大肠杆菌BL21中,以IPTG进行诱导,SDS-PAGE电泳分析发现,转化了重组质粒的菌株所表达的融合蛋白相对分子量为34 kD,与实际预测相符,命名为GST-OMPc.GST亲和层析柱进行纯化,ELISA方法对纯化蛋白进行检测.结果表明:纯化蛋白GST-OMPc能够与兔抗猪胸膜肺炎放线杆菌血清7型的阳性血清反应.OMPc蛋白的成功表达为其功能的研究打下基础.     

瘦肉型猪(PIC344)精液碱性磷酸酶的分离纯化及部分性质研究

用正丁醇抽提、硫酸铵分级沉淀、DEAE SepharoseFF和SephacrylS 200柱层系,从瘦肉型猪(PIC344)精液中提取出碱性磷酸酶。纯化倍数11 46,比活为81 23U/mg蛋白。提取液经PAGE和SDS PAGE检测,呈现一条带。该酶相对分子质量为90 12KD,亚基分子质量为48 41KD,该酶为两个相同亚基组成。等电点为8 19,最适pH值9 5,最适温度为40℃,以磷酸苯二钠为底物测得Km值为3 98×10-4mol/L。经分析,Cu2 、Cd2 为酶的抑制剂,Ni2 、Ca2 、Mg2 为该酶的激活剂。选用CuSO4、Cd(NO3)2作酶的抑制类型判断,结果表明,CuSO4为非竞争性抑制,抑制常数为1 32×10-3mol/L,Cd(NO3)2为竞争性抑制,抑制常数为3 33×10-4mol/L。     

仙居鸡染色体G带和Ag-NORs的研究

运用改进的鸡外周血淋巴细胞培养——空气干燥法，分析了仙居鸡染色体G带和Ag—NORs。G带研究结果表明：前10对大型染色体可分为30个区，144条带，同时对G带带型特征进行了具体描述，并绘制了G带模式图。Ag—NORs处理发现：仙居鸡的Ag—NORs常分布于1p、3p、4p、Zp、2q上，均数为3．26，众数为3。不同个体间以及同一个体的不同细胞间。Ag—NORs数目存在多态性；此外研究还发现Ag—NORs联合现象。     

捕食线虫性真菌18S rDNA基因序列与种系发生关系研究

对捕食线虫性真菌——少孢节丛孢菌A1分离株(Arthrobotrys oligospora A1)的18S rDNA基因序列进行了研究。结果表明：其18S rDNA全基因序列为1769bp，在进化树上与同种国外分离株A．oligospora var oligospora 1最为接近，同源性为94．7％。这与二者都具有捕食性结构—菌网的特点相一致。证实捕食线虫性真菌的捕食性结构与捕食线虫性真菌种系发生有关。从少孢节丛孢菌3个分离菌株(Arthrobotrys oligospora A1、A．oligospora 1、A．oligospora2)的进化树及同源性来看。不同国家地区的丛孢菌属(Arthrobotrys)分离株确实存在差异。     

猪原始生殖嵴细胞(PGCs)建系因素的研究

从五指山猪(WSZP)近交系第8～13代培育群中，先后选用21头5～10月龄青年母猪，分别于授精后25～30d采集胎儿106个，进行原始生殖嵴(PGCs)细胞分离、培养等建系技术研究。以DMEM F10(1:1)为基础培养液，按添加或不添加生长因子，将培养液分为A、B、C3种，并以STO细胞作饲养层，在38℃、5.0％CO2和湿润的气相中进行培养建系。结果获得胚胎生殖嵴细胞(EG)细胞系6个细胞株，其中1个EG细胞株传至11代、2个传至5代、1个传至4代、2个传至3代冻存。并进行了AKP染色、体外分化、冷冻-解冻复苏和嵌合体制作等鉴定研究。研究发现：不同胚龄对EG细胞建系具有一定影响，不同培养液对EG细胞建系效果不同，STO细胞饲养层的质量是建株、传代、冷冻-解冻复苏的关键因素之一。EG细胞系的初步建立，为今后筛选进入种系的EG细胞系、实施体外基因操作提供了可能。     

禽流感病毒夹心ELISA快速检测方法的研究

以禽流感病毒(AIV)湖北分离株(H9N2亚型)提纯物为免疫原，制备出鸡抗AIV和兔抗AIV抗体，经用琼脂扩散法测得效价分别为1:32和1:16。以纯化的鸡抗AIVIgG为包被抗体，兔抗AIVIgG为第二抗体，建立了检测AIV抗原的夹心ELISA法。结果表明，鸡抗AIVIgG的最佳包被浓度为1μg/mL，兔抗AIV IgG的最适工作浓度为5μg/mL；对已知的阳性样品，用夹心ELISA法测得的病毒滴度比血球凝集滴度高16倍以上，且能检出其它亚型的禽流感病毒；与新城疫病毒、传染性支气管炎病毒、减蛋综合征病毒、传染性喉气管炎病毒、传染性法氏囊病病毒、鸡痘病毒、马立克氏病病毒等无交叉反应，说明本方法有很高的特异性及敏感性。对14个鸡场送检的、患有呼吸道疾病或有腹膜炎、眼炎、产蛋下降、怀疑为禽流感感染的病鸡进行了检测，结果有7个鸡场为阳性。本方法的建立为禽流感病鸡群的临床检验提供了一种方便、敏感、快速的检测方法。     

应用B-超对小尾寒羊早期胚胎和胎盘发育的研究

应用B-超监测10只健康的妊娠小尾寒羊,以研究早期胚胎和胎盘的发育过程。结果表明,早期胚体发育在妊娠第6周完成;妊娠30～46d,羊膜囊的短径与胎龄之间存在线性关系(R=0.9528,P<0.01),回归方程为Y=0.565065X 18.751970(Y:妊娠天数,X:羊膜囊宽)。胎盘发育期集中在妊娠前11周,在此期间胎盘突直径也可用于预测胎龄,二者之间的方程式为:Y=4.961429X-16.655714(Y:胎盘突直径,X:妊娠周),相关系数R=0.9882(P<0.01)。