斑点免疫金渗滤法检测猪生殖与呼吸综合征抗体的研究

应用提纯的猪生殖与呼吸综合征病毒(PRRSV)抗原包被硝酸纤维素膜，然后用胶体金标记的金黄色葡萄球菌A蛋白(SPA)建立了检测PRRS抗体水平的斑点免疫金渗滤法(DIGFA)。该法通过胶体金标记SPA直接显色，阳性者出现红色斑点，整个试验过程仅需5min即可判断结果；与猪瘟、猪伪狂犬病、猪细小病毒病阳性血清不发生交叉反应；纯化PRRSV抗原的最低检测量为0．0553mg／mL，即55．3ng／点。对200份猪血清用该法与ELISA同时进行PRRS抗体检测，两种方法的符合率达98％。     

应用斑点免疫金渗滤法诊断犬弓首蛔虫病

根据免疫渗滤原理,采用犬弓首蛔虫抗原蛋白和胶体金标记SPA,建立抗犬弓首蛔虫抗体的斑点免疫金渗滤法(D IGFA)检测200份犬血清样本。结果:阳性检出率为8.00%(16/200);血清样本对应犬粪便用饱和盐水漂浮法检查虫卵,阳性检出率为2.0%(4/200)。结论:斑点免疫金渗滤法具有操作简便、灵敏度高、特异性强的优点,可用于犬弓首蛔虫病的早期、快速检测。     

斑点免疫金渗滤法检测猪瘟抗体的研究

以猪瘟抗原包被硝酸纤维素膜，然后用胶体金标记SPA，建立检测猪瘟抗体水平的斑点免疫金渗滤法检测试纸盒。通过胶体金标记金黄色葡萄球菌A蛋白(SPA)直接显色，阳性者出现红色斑点，结果易于判断。整个试验过程仅需5min，操作简单，与猪兰耳病、猪伪狂犬病、猪细小病毒、新城疫和禽流感等阳性血清不发生交叉反应。同时将该法与目前猪瘟的常规检测方法Dot—ELISA法和间接血凝试验同时对200份猪血清进行猪瘟抗体检测比较，符合率达98．4％和98．9％。说明该法微量、特异、敏感可靠，检测时间短，效果直观，非常适用于猪瘟的早期诊断和普查以及疫苗免疫效果后抗体水平的检测。     

斑点金标免疫渗滤法快速检测牛血吸虫病抗体的效果观察

目的观察斑点金标免疫渗滤法检测牛血吸虫病抗体的效果。方法使用斑点金标免疫渗滤诊断试剂盒检测粪检血吸虫病牛血清(血纸)146头份,疫区耕牛血清(血纸)562头份,并用间接血凝法(indirect haem aggluti-nation assay,IHA)法作对比。结果146头份粪检阳性血清(血纸)检测为阳性者140头份,阳性检出率95.9%,IHA法检测为阳性者141头份,阳性检出率96.6%。结论斑点金标免疫渗滤法检测牛血吸虫病具有快速、简便、灵敏度高、特异性强等优点,但仍有一定漏检,有待于进一步研究提高。     

斑点免疫金渗滤法检测赤羽病抗体的研究

以亲和层析原理为基础,将提纯的AKAV抗原包被在硝酸纤维膜上,利用胶体金标记SPA显色,建立了斑点免疫金渗滤法诊断赤羽病抗体的方法。其中最佳点样抗原质量浓度是0.099mg/mL,封闭液选择含1%BSA,0.5%Tween-20的0.01mol/LpH7.2的PBS缓冲液,SPA胶体金最佳稀释度为1∶4。特异性试验表明,该方法特异性好,与牛病毒性腹泻病毒、牛传染性鼻气管炎、牛白血病、口蹄疫、蓝舌病等阳性血清不发生交叉反应。将DIGFA与ELISA进行对比,差异不显著。研究结果表明,该方法操作简单,结果易于判断,适用于赤羽病的临床诊断和流行病学调查。     

斑点金免疫渗滤测定法检测耕牛血吸虫病的效果观察

免疫胶体金技术是以胶体作为示踪标志物应用于抗原抗体的一种新型免疫标记技术，没有潜在致癌物质的酶显色底物。斑点金免疫渗滤测定法是将斑点酶标与免疫胶体金结合起来的一种新型免疫标记技术。该测定方法是将被检抗体（血清或血纸浸出液）直接点样在硝酸纤维素膜上，滴加胶体金标记的血吸虫抗原，特异性抗原与抗体很快结合，在反应处发生金颗粒聚集，1min左右形成肉眼可见的红色斑点，判定检测效果。     

斑点金免疫渗滤测定法检测耕牛血吸虫病

免疫胶体金技术是以胶体金作为示踪标志物应用于抗原抗体的一种新型的免疫标记技术，没有潜在致癌物质的酶显色底物。斑点金免疫渗滤测定法是将斑点酶标与免疫胶体金结合起来的一种新型的免疫标记技术。将被检抗体（血清或血纸浸出液）直接点样在硝酸纤维素膜上，滴加胶体金标记的血吸虫抗原，特异性抗原与抗体很快结合，在反应处发生金颗粒聚集，1min左右形成肉眼可见的红色斑点。     

应用斑点免疫金渗滤法检测羊东毕吸虫病的研究

应用自行研制的胶体金探针标记ＳＰＡ，建立了诊断羊东毕吸现的新方法－斑点免疫金渗滤法（ＤＩＧＦＡ），该法与ＥＬＩＳＡ法具有良好的相符性，将抗原、等检血清滴加于膜上进行反应，数分钟内即可用肉眼进行结果判定。     

猪乙型脑炎血清抗体DIGFA检测方法的建立与应用

本试验建立了一种以固相滤膜为载体，以红色胶体金颗粒标记的免抗猪IgG作为探针，特异性地检测猪流行性乙型脑炎病毒抗体的斑点金免疫渗滤测定法（DIGFA）。与血凝抑制试验（HI）对比检测了54份猪血清临床样本，两法的阳性符合率为71.4%、阴性符合率为65.5%、总符合率为81.4%。本法中抗原抗体通过渗滤在膜上进行反应，数分钟内即可用肉眼观查结果，具有快速，操作简单，敏感性高，特异性强，重复性好，易于判读等优点，特别适合于猪流行性乙型脑炎快速诊断和大规模血清流行病学调查。     

应用斑点免疫金染色法检测猪细小病毒病抗体的研究

本研究初步建立了斑点免疫金染色法 (Dot- IGS)检测 PPV抗体的方法。试验采用经蔗糖密度梯度离心制备的 PPV提纯抗原和 p H7.4、直径 5 0～ 6 3.5 mm的胶体金来检测 PPV抗体 ,取得了较为满意的效果 ;该法对猪细小病毒病抗体最小检测量为1.0 0 8× 10 - 1 0 g/ ml,灵敏性高 ;不同猪布鲁氏菌病、猪伪狂犬病、猪衣原体病、猪口蹄疫、猪瘟、猪弓形体病等的阳性血清出现交叉反应 ,特异性强且重复性好。另该法还具有胶体金制备容易、操作简单快捷等优点 ,是对 PPV抗体检测的重要方法。     

应用猪囊虫匀浆抗原间接血凝试验检测猪囊虫病抗体的研究

从囊虫匀浆浸提液中分离出一种抗原性与特异循环抗原相同的成分,以这种抗原成分通过间接血凝试验检测病、健猪的血清抗体,结果病猪抗体阳性率为93.2％(96/103),健猪阴性(?)合率为100％。     

酶标葡萄球菌A蛋白染色法用于猪囊尾蚴病诊断的初步研究

用自制的猪囊尾蚴头节“颗粒性抗原”,以HRP—SPA作为第二抗体,进行间接免疫酶染色诊断猪囊尾蚴病。检测61份猪囊尾蚴病血清,阳性59份,检出率为96.6％;健康猪血清69份、细颈囊尾蚴病和肺丝虫病猪血清各10份,均为阴性。此法简便易行,快速、准确,适于基层使用。     

Detection of Taenia solium Cysticercosis in Pigs by ELISA with an Excretory-Secretory Antigen

Serodiagnosis of Taenia solium cysticercosis in pigs was conducted by an enzyme-linked immunosorbent assay with an excretory-secretory antigen. The antigen was obtained by in vitro cultivation of the cysticerci in a synthetic medium RPMI 1640. The sensitivity and specificity of the ELISA in detecting infection in pigs reared on free range was 92% and 100%, respectively. In addition, 33.33% of pigs in which infection could not be detected at meat inspection were found positive by ELISA. However, none of the sera from a group of farm-reared pigs were positive. No cross reactions were observed in pigs that contained either the cysticerci of Taenia hydatigena or hydatid cysts.     

猪囊虫病基因工程疫苗的研制及应用

从猪带绦虫六钩蚴cDNA文库中筛选目的基因并进行克隆表达，重组抗原用血清学方法和猪体免疫试验进行鉴定，筛选保护性抗原基因。将具有免疫保护作用的重组抗原纯化，并与免疫刺激复合物佐剂结合，制成猪囊虫病基因工程疫苗。对工程菌的培养发酵、重组抗原的下游纯化及疫苗的配制、疫苗的中间试制进行了研究，确定了一套比较完善的生产工艺。应用昆明小鼠建立了猪囊虫病的实验动物模型，应用该模型进行上述疫苗的免疫预防试验，免疫1次和2次的免疫保护率分别为96．9％和98．4％。本动物的免疫预防试验显示，疫苗安全性好，免疫保护率达92．2％，且免疫组发现的囊虫多数已死亡。在流行区进行了田间和区域试验。免疫猪未有不良反应，囊虫感染率分别由20％降为1．1％和5．4％降为0．21％。用该疫苗对人工感染的囊虫病猪进行免疫治疗，发现在感染早期的治疗效果较好。免疫动物的体液免疫和细胞免疫的检测结果显示，该疫苗刺激机体产生抗体的时间早、持续时间长、效价高；可显著提高淋巴细胞转化率及E－RFC和Ea-RFC细胞、ANAE^ 细胞和粗粒型ANAE^ 细胞、抑制／杀伤性T细胞亚群的数量。以上结果表明，用大肠杆菌表达的重组抗原制备的猪囊虫病基因工程疫苗安全、高效，且成本低廉，可规模化生产，有成为预防猪囊虫病的一种新型生物制剂。     

The prevalence of porcine cysticercosis in Eastern and Southern provinces of Zambia

The objective of this study was to determine the prevalence and importance of porcine cysticercosis in rural areas of Zambia. The study involved an abattoir survey of 1316 pigs at a slaughter slab in Lusaka and two field surveys in villages in Southern and Eastern provinces. Lingual examination of live pigs and visual inspection of their carcass as well as blood sampling for measuring circulating parasite antigen by enzyme-linked immunosorbent assay (Ag-ELISA) were used as parameters to measure infection. In the field surveys, a questionnaire was administered to every household whose pigs were examined to obtain information on pig husbandry practices and to study risk factors for the infection. Out of the 1316 pigs examined at the slaughter slab, 143 (10.9%) and 271 (20.6%) were positive by lingual examination and meat inspection, respectively. Most of the pigs were very heavily infected with predominantly live cysts. The field surveys revealed that eight (8.2%) out of 98 pigs from Southern province and eight (5.2%) out of 151 pigs from Eastern province were positive for cysticercosis by tongue palpation. Using the Ag-ELISA 20 (20.8%) and 14 (9.3%) pigs were positive in Southern and Eastern provinces, respectively. The questionnaire survey revealed poor pig husbandry practices, absence of meat inspection and control, poor knowledge of the disease and poor sanitation in the surveyed villages. The prevalence of pig cysticercosis found in this study ranks among the highest in the southern African region, in Africa and in the world. The current study suggests the presence of human tapeworm carriers and a high risk of human cysticercosis in the surveyed areas as well as in urban centres where pigs from rural areas are increasingly sold, slaughtered and consumed.     

Prevalence of Taenia solium porcine cysticercosis in the Eastern, Southern and Western provinces of Zambia

Tongue examination and detection of circulating antigen (Ag-ELISA) were used to establish the prevalence of Taenia solium porcine cysticercosis in free-range pigs in selected districts of Eastern, Southern and Western provinces of Zambia, and to determine if prevalence of porcine cysticercosis was associated with age, breed and sex. Households with pigs were identified using the snowballing technique. A total of 1691 pigs were examined out of which 183 (10.8%) were positive on tongue examination. Ag-ELISA gave a sero-prevalence of 23.3%. When considering the factors in a logistic regression analysis, only breed type was significantly associated with porcine cysticercosis (OR=0.72; 95%CI=0.63-0.81). The crossbred pigs were 72% more likely to have had cysticercosis than the Nsenga (dwarf local) breed as determined by Ag-ELISA. The result that crossbred pigs had a higher prevalence of T. solium cysticercosis suggests that pig breeds may display different susceptibility to cysticercosis. The limited use of latrines in these areas implies that people use the nearby bush for defecation, resulting in pigs having access to human faeces. Therefore, investigation of taeniosis and cysticercosis in humans is warranted to better comprehend the local epidemiology and transmission risks. This should then be followed by extension programs to communities so that the control plans that could be instituted are more sustainable.     

囊虫病猪血清CA效价与虫负荷的相关性及CA与Ab的动态观察

应用ELISA双抗体夹心法检测囊虫病猪血清中的循环抗原(CA),对CA效价与虫负荷的相关性作了初步观察,发现两者密切相关.轻度感染猪血清CA效价≤000,中度为1000～10000,重度为10000～100000.人工感染病猪血清一般在感染后1周检出CA,于感染后4～5周达高峰;抗体一般于感染后2周检出.这种方法可检出的最小虫负荷约为20个/猪     

猪囊虫病基因工程疫苗用于免疫治疗的研究

应用猪囊虫病基因工程疫苗分别对实验性猪囊虫病和自然感染的猪囊虫病进行了分组治疗,以探讨该疫苗的免疫治疗作用。１０头人工复制的囊虫病猪,随机分成３组。第１组４头猪,于感染于１个月注射该疫苗,连续３次,间隔１５天;第２组４头猪,于感染后２个月注射疫苗,同上连续３次;第３组２头猪,不注苗。第１组注射疫苗后１个月循环抗原滴度开始降低,２．０、２．５及３．５个月各有１头猪ＣＡ转阴,５个月ＯＤ值分别为０．１４、０．０９、０．１７、０．３８。第２组和第３组各猪血清ＯＤ值一直维持在１．０以上。感染后５个月剖检,第１组４头猪均未检出囊虫;第２组４头猪在咬肌、膈肌、腰肌、股内侧肌、肩胛外侧肌等部位均检测到囊虫,检测部位４０ｃｍ＾２面积发现囊虫３～７个,有部分虫体已钙化,胆汁卵化率为１５．４％;第３组各猪也在各检测部位检测到囊虫,４     

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.     

Evidence that active transmission of porcine cysticercosis occurs in Venezuela

There is a paucity of quantitative data on the status of porcine cysticercosis in Venezuela, information which is essential for understanding the level of disease transmission. This study was, therefore, conducted in a typical small rural community in Yaracuy State, Venezuela, where previous cases of human Taenia solium taeniasis/cysticercosis had been reported and where the free-ranging pig management practices and the lack of rudimentary sanitary facilities indicated an obvious risk for transmission of the disease. Serum samples from 52 village pigs were screened by enzyme-linked immunosorbent assays for anti-cysticercal antibodies (Ab-ELISA), using T. solium cyst fluid as the antigen and the HP10, monoclonal antibody-based, antigen trapping ELISA for parasite antigen (HP10 Ag-ELISA). Significantly, a high proportion of the animals (65.4% for the Ab-ELISA and 42.3% for the HP10 Ag-ELISA) were sero-positive. Five of the pigs, which were selected on that basis of positive tongue palpation, were killed for autopsy, and large numbers of viable cysticerci were found in the carcases. This unequivocal documentation of porcine cysticercosis in Venezuelan pigs presents clear evidence that T. solium is actively transmitted in Venezuela. Further detailed studies and implementation of appropriate control measures are therefore indicated.