三种猪瘟抗体检测技术的应用比较

应用猪瘟正向间接血凝法 (IHA)、斑点酶联免疫吸附试验 (Dot ELISA)以及猪瘟抗体免疫金标试纸条对规模化养猪场 1 38份不同阶段猪血清进行猪瘟免疫抗体水平的同步监测 ,检测结果 :IHA ,Dot ELISA ,金标试纸条测出经产母猪的猪瘟抗体阳性率分别为 83 3 %、80 0 %、80 0 % ,其平均值为 81 1 % ;三种方法测出后备母猪的猪瘟抗体阳性率分别为 53 3 %、53 3 %、50 0 % ,其平均值为 52 2 % ;三种方法测出 2 0日龄仔猪的猪瘟抗体阳性率分别为 69 2 %、76 9%、76 9% ,其平均值为 74 3 % ;测出 40日龄猪的猪瘟抗体阳性率分别为 76 9%、80 8%、80 8% ,其平均值为 79 5 % ;测出育肥猪的猪瘟抗体阳性率分别为 30 8%、30 8%、34 6 % ,其平均值为 32 1 %。经过统计学x2 检验 ,对于不同阶段猪三种方法两两之间检测结果均差异不显著 (P >0 0 5) ,表明上述三种方法均可用于猪瘟抗体的检测。     

应用单克隆抗体夹心酶联免疫吸附试验检测兔出血症病毒

应用建立的单克隆抗体夹心酶联兔疫吸附试验（ＭｃＡｂ－ＥＬＩＳＡ）,检测了人工感染兔出血症病毒（ＲＨＤＶ）ＤＪＲＫ细胞毒、肝毒的兔以及自然感染ＲＨＤＶ的兔的组织样品。结果表明,感染死亡兔的肝、脾、肾、骨髓样品病毒抗原的检出率为１００％,淋巴结和肌肉的检出率分别为９７．５％和７９．５％。ＭｃＡｂ－ＥＬＩＳＡ能检出肌肉中血凝试验不能检出的ＲＨＤＶ抗原。此外,还用ＭｃＡｂ－ＥＬＩＳＡ检测了肝毒人工感染兔血中ＲＨＤＶ的动态,并对１０份兔出血症脏器灭活苗的效价作了滴定。     

Dot-ELISA检测兔疥螨抗体方法的建立和应用

将从病兔痂皮内收集的疥螨经研磨、冻融、离心后,制成可溶性抗原,作为诊断抗原,建立Dot-ELISA方法检测兔疥螨血清抗体.研究确定了该方法的最佳工作条件.制备的诊断膜片特异性强,不与兔瘟病毒、兔大肠埃希菌、兔附红细胞体等阳性血清反应.膜片具有良好的灵敏性,高免血清作1∶210稀释亦能检出;重复性试验表明该法重复性良好.诊断膜片在4 ℃保存5个月其检测活性不变.结果表明,建立的Dot-ELISA可用于免疥螨抗体的检测.     

应用间接血凝试验检测兔出血症病毒抗体的研究

应用纯化兔出血症病毒抗原,以戊二醛醛化绵羊红细胞为载体,成功地建立了间接血凝试验用以检测兔出血症病毒抗体.与血凝抑制试验和琼脂双扩散试验比较,其敏感性比血凝抑制试验高32倍,比琼脂双扩散试验高1024倍.致敏红细胞于4°C保存四个月仍有效.     

兔出血症巴氏杆菌波氏杆菌魏氏梭菌四联灭活苗疫苗试验研究

应用兔出血症马氏杆菌波氏杆菌魏氏梭菌四联含有免疫佐剂灭活疫苗（简称：兔四联苗）对８组３２只健康敏感家兔人工接种,进行免疫试验,其免疫保护率结果依次为１００％,７５％,７５％,７５％。     

实验性兔出血症病兔肝脏酶组织化学变化研究

用组织化学染色法,对实验性感染兔出血症(RHD)病毒后不同病期(潜伏期、高热期、濒死期和死亡时)病兔的肝脏碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、三磷酸腺苷酶(ATPase)、琥珀酸脱氢酶(SDH)和乳酸脱氢酶(LDH)的活性变化进行了动态观察。结果表明,肝脏AKP、ACP、和LDH活性在潜伏期至高热期增强,以后各期均减弱;ATPase和SDH在病程各期均明显减弱。从而推论,肝细胞的坏死与ACP活性增强、溶酶体活跃有关;黄疸的发生与ATPase活性减弱及肝细胞线粒体受损有关;病兔的肝脏代谢以无氧酵解为主。     

兔出血症病毒VP60蛋白真核表达及其免疫原性研究

将兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)衣壳蛋白VP60基因,克隆到真核表达载体pcDNA3.1(+)中,构建pcDNA-VP60,并将其转染Vero细胞.间接免疫荧光(indirect immunofluorescence assay,IFA)检测pcDNA-VP60在细胞中的表达情况,同时将pcDNA-VP60免疫实验兔,观察血清中特异性抗体变化情况.试验结果表明,本文构建的pcDNA-VP60不仅能在Vero细胞中表达VP60蛋白,而且能够诱导机体产生免疫应答.     

兔IL-6基因真核表达载体的构建及其对pcDNA-VP60DNA疫苗佐剂效应研究

为研究兔白介素6（interleukin6,IL-6）真核表达质粒（pcDNA-IL-6）对核酸疫苗免疫效果的影响,本文构建了真核表达质粒pcDNA-IL-6,并将其与核酸疫苗pcDNA-VP60联合免疫家兔,以pcDNA-VP60和质粒载体pcDNA3．1（＋）作对照,用血凝抑制试验检测试验兔体内特异性抗体水平。结果表明：真核重组质粒pcDNA-IL-6对重组质粒pcDNA-VP60均具有免疫增强作用,从免疫后7d到70d,pcDNA-VP60／pcDNA-IL6联合免疫组抗体水平均高于pcDNA-VP60免疫组,差异具有显著统计学意义。     

间接法DOT—ELISA检测新城疫病毒的研究

本文首次成功地建立了间接法Dot-ELISA检测新城疫病毒,对新城疫发病鸡群口咽、泄殖腔拭子的NDV抗原阳性检出率分别为58.33％(91/156)、62.50％(90/144),两者没有显著差异(P>0.05),结果表明,间接法Dot-ELISA对新城疫的早期诊断具有简单、快速、经济、敏感性高、特异性强和重复性好等优点,是疫病早期诊断与流行病学调查的有效手段。     

兔出血症病毒遗传与变异的分子基础

兔病毒性出血症是由兔出血症病毒（Rabbit hemorrhagic disease virus，RHDV）引起的一种急性、高度致死性传染病，病死率高达100％。近年来，由于该病毒血凝性、抗原性的变异，病毒感染宿主增多，不仅对该病的防控造成了一定的威胁，更造成了严重的经济损失。国内外研究学者对该病毒的基因组结构、基因组编码的产物和功能，病毒的宿主和受体以及病毒变异等相关进行了大量深入的研究和分析，为该病毒的防控和治疗提供了技术基础。本文就RHDV遗传与变异的分子基础作一综述，希望能为深入了解该病毒，以及日后的防控提供一定的理论支持。     

兔出血症病毒缺失突变体的构建及鉴定

为研究兔病毒性出血症病毒（Rabbit hemorrhagic disease virus,RHDV）衣壳蛋白与病毒侵染性的关系,在RHDV侵染性克隆的基础上,构建了缺失衣壳蛋白（VP60）部分编码基因（5 325-6931 nt）的全长cDNA分子克隆。然后,在体外转录合成RNA,转染RK-13细胞,观察RHDV缺失5 325-6 931 nt区域以后,病毒的侵染性以及病毒复制能力的变化情况。结果表明,5 325-6 931 nt区域缺失以后,病毒的侵染性没有明显改变,但是病毒的复制能力有所下降。可见,该区域含有与病毒复制有关的调控序列或元件。     

COMPARISON OF THREE METHODS FOR CALCULATING PORTAL BLOOD FLOW VELOCITY IN DOGS USING DUPLEX-DOPPLER ULTRASONOGRAPHY

Measurement of portal blood flow in dogs using duplex-Doppler ultrasonography has potential clinical utility for diagnosis of cirrhosis and portosystemic shunts. Three methods for calculating portal blood flow were compared to identify a consistent method with minimal potential for intraoperator variation. Sixteen unsedated, normal beagles had their mean portal blood flow velocity determined using three methods: method 1 used a small Doppler sample volume positioned over the center of the portal vein and manual selection of data points on the resulting velocity spectra, followed by arithmetic correction of the calculated maximum velocity to mean velocity using the factor 0.57; method 2 used a small centrally-located Doppler sample volume and computed maximum flow determination, again corrected using the factor 0.57; method 3 used a large Doppler sample volume that overlapped the walls of the portal vein followed by computed mean blood flow velocity determination. The calculated mean (SD) portal blood flow velocity was 15.8 (1.8), 15.1 (1.4), and 14.7 (2.5) cm/s using methods 1–3, respectively. There was no significant difference between the results of the three methods; however, method 3 (uniform insonation) was easier to use and produced a higher amplitude Doppler signal and therefore represents a useful alternative to the peak flow methods.     

方差组分估计方法的比较

本文通过蒙特卡罗模拟产生的八个模拟资料比较了我国常用的方差分析法(不考虑场年季效应)、Henderson方法1、Henderson方法3、最大似然法、改进最大似然法与约束最大似然法。结果表明:方差分析法估计值偏差最大,而约束最大似然法的估计值最准确。     

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。     

兔出血热病毒的血凝性观察

兔出血热病毒k—8602株在pH6.4～8.4的0.01M PBS和生理盐水中,对人的A、O、B、AB型红细胞有明显的凝集作用,并可被相应的兔免疫血清所抑制;而对鸡、猪、兔等的红细胞不凝集。该病毒在室温和4℃条件下,在pH7.0～8.4 PBS和生理盐水中对A型、O型红细胞凝集之后,在37℃下又释放出来,在4℃下又重新凝集,而对B、AB型红细胞则没有这种现象。根据吸附—释放原理,利用A型红细胞吸附提纯了病毒。     

A RADIOGRAPHIC TECHNIQUE FOR PELVIMETRY OF UNANAESTHESTISED EWES AND A COMPARISON OF THREE METHODS OF ESTIMATING THE AREA OF THE PELVIC INLET

SUMMARY A radiographic procedure is described which enables reliable estimation of the area of the pelvic inlet of up to 150 ewes per seven-hour day by combining use of a specialized radiographic table, immobilisation of the subject with a blindfold and 2 restraining belts, and a mobile condenser-discharge x-ray generator powered by a domestic electrical supply or a portable generator. Six pelvic dimensions, the distances between the medial ischiatic tuberosities, the lateral ischiatic tuberosities, the length of the pubic symphysis, the acetabular, transverse and conjugate diameters and the area of the pelvic inlet computed as the product of the transverse and conjugate diameters (pelvic axes), were estimated by three methods which allowed for magnification resulting from the dimensions of interest lying at varying heights above the film. The estimates were compared by linear regression analysis with their respective values obtained directly from the pelves dissected after slaughter, method A. Method B was isometric, using a perspex vaginal isometer with radiopaque calibrations. Methods C and D applied the theorem of similar triangles. Method C assumed that the dimensions of interest lay in a horizontal plane halfway through the dorso-ventral or lateral thickness of the subject when thickness was measured on a level with the femoral greater trochanters. Method D assumed that the dimensions of interest lay in the same horizontal plane as the height of the subject's anus above the table top when measured in sternal or lateral recumbency. The test subjects were groups of aged, non-pregnant Dorset Horn or Merino ewes. Method B estimated the pelvic axes unreliably in both breeds. Method D, although almost as reliable as method C in Dorset Horn ewes, was not used in merinos because it was cumbersome. Method C was preferred because the estimates were highly correlated with the exact values for both breeds and it was the easiest to apply since measuring body thickness was essential for calculating exposures. Using method C, R values in Dorset Horns for the transverse and conjugate diameters, and the area of the pelvic inlet, were 0.96, 0.78 and 0.81 respectively. For Merinos the corresponding values were 0.88, 0.80 and 0.84 respectively. As method C over-estimated the area of the pelvic inlet, a prediction equation was derived to minimise this tendency, resulting in revised estimates, accurate within 2.7% and 3.9% respectively, of the values obtained at the autopsy of the merino and Dorset Horn test subjects. All methods were unsatisfactory for estimating dimensions other than the transverse and conjugate diameters, and the area of the pelvic inlet. The procedure recommended has been used in several breeding flocks to determine the role of mature maternal pelvic size in feto-pelvic disproportion, birth injury to the fetal central nervous system and repeated maternal rearing failure.