Dot-ELISA与IHA方法诊断肝片形吸虫病的比较

用斑点酶联免疫吸附试验(Dot-ELISA)对200份经粪检和间接血凝试验(IHA)血检的血清样品进行肝片形吸虫感染的检测,同时在屠宰场现场对50份样品进行检测,并与剖检相比较。结果显示Dot-ELISA与IHA法相比,阳性符合率较低(84.21%),阴性符合率高(95.58%),总符合率94.50%;Dot-ELISA与粪检结果相比,阳性符合率(94.74%)明显高于IHA法;与剖检法比较,Dot-ELISA对屠宰场现场样品的检出率和阳性符合率均为100%。结论:Dot-ELISA较IHA法敏感性高、特异性强,适宜用作牛、羊肝片形吸虫感染的现场检测。     

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.     

豚鼠感染肝片形吸虫后体内抗体水平变化

ELISA分析结果显示，豚鼠感染肝片形吸虫后，血清中抗体水平因感染数量的不同而有明显变化。感染囊蚴(metacercaria)20个时，P／N值出现两个波峰，第二峰值较高并维持至豚鼠死亡。感染囊蚴10个时，P／N值也出现两个波峰，在感染后85～95天开始下降。感染量为5个时，P／N值只出现一个波峰，出现的时间各只豚鼠略有不同。     

基于GAPDH重组蛋白建立羊肝片吸虫病间接ELISA检测方法

为了建立早期羊肝片吸虫病的血清学快速检测方法,本试验成功克隆并表达肝片吸虫GAPDH重组蛋白,Western blot结果显示,该重组蛋白具有良好的抗原活性。用肝片吸虫GAPDH重组抗原包被,建立肝片吸虫病血清抗体的间接ELISA方法;随后优化反应的最佳血清稀释度和抗原包被量,同时筛选其他反应条件。结果显示,间接ELISA方法批内和批间重复试验的最大变异系数均小于10%,GAPDH重组抗原与华枝睾吸虫、日本血吸虫和捻转血矛线虫阳性血清均无交叉反应。对来自黑龙江省各地区的96份羊血清进行检测,阳性率为16.67%(16/96)。结果表明本试验建立的方法具有良好的敏感性和特异性,能够为早期诊断羊肝片吸虫病提供参考。     

应用肝片吸虫ES抗原检测实验感染山羊IgG的动态水平

用肝片吸虫排泄分泌抗原 ( ESA g)的酶联免疫吸附试验 ( ELISA)检测人工感染肝片吸虫的山羊血清中特异性 Ig G抗体动态变化。ESAg用量为 13.8μg/孔 ,抗体稀释 10 0 0倍 ,二抗 1∶ 2 0 0 0稀释 ( 3.5μg/孔 ) ,HRP标记的葡萄球菌 A蛋白 ( SPA* )工作浓度为 1∶ 4 0。检测结果表明 ,2组实验山羊 (第 1组每只羊口服2 0 0个囊蚴 ,第 2组每只羊口服 50 0个囊蚴 )在感染后第 3周血清中的特异 Ig G即开始升高 ,呈现动态变化趋势 ;第 2组于第 6周 ( 42 d) Ig G水平升到高峰 ,随后稍有下降 ,第 1组于第 9周 ( 6 3d) Ig G水平升到最高峰 ,随后又稍有下降 ,一直呈波动趋势 ;在试验的 3～ 17周期间 ,总体上虽第 2组抗体水平比第 1组高 ,统计分析无显著差异 ( P >0 .0 5) ,但均比对照组保持较高的水平 ,有显著差异 ( P <0 .0 5或 P <0 .0 1) ,表明山羊在肝片吸虫入侵后 ,很快产生了高水平的体液免疫 ,而 Ig G的波动可能与虫体的移行有关 ,与虫卵数量则无关。     

不同方法及材料检测大片吸虫感染的比较试验

为片形吸虫病诊断提供高效、简便的新技术,以大片吸虫分泌排泄抗原（ES抗原）,用于斑点酶联免疫吸附试验（Dot－ELISA）、琼脂扩散酶联免疫吸附试验（Dig－ELISA）、免疫胶体金滴渗法（DIGFA）检测动物体内的大片吸虫（Fasciolagigantica）感染。通过检测血清、血纸、牛奶等几种材料,多重试验比较,结果三种方法均具有敏感性高、特异性强、准确性可靠等优点,并各有特长,如操作简便快速、费用低廉,易于在基层推广应用等,同时证明用血纸和牛奶检测抗体效果同样可靠。本试验首次报道用Dot－ELISA、DIGFA在奶中检测片形吸虫抗体,为该寄生虫病普查提供一种新思路。     

牛病毒性腹泻-黏膜病病毒双抗体夹心Dot-ELISA检测方法的建立

采用辛酸-硫酸铵法提取兔抗牛病毒性腹泻-黏膜病毒(BVDV)高免血清中的免疫球蛋白IgG,辣根过氧化物酶标记提纯后IgG,建立了检测BVDV抗原的双抗体夹心斑点酶联免疫吸附试验(Dot-ELISA)法。结果显示,抗体最佳包被量为300μg/mL,酶标记抗体的最适浓度为1∶50倍稀释,以5%牛血清作为封闭液、封闭45min效果最佳,抗原最小检出量是1.34μg/mL。应用建立的检测方法对河北省内78份腹泻奶牛血样进行了检测,阳性检出率为57.69%;经卡方检验分析,建立的Dot-ELISA与琼脂扩散(AGP)法相比较,阳性检出率差异显著。试验证明,该方法具有简便快速、特异性强、重复性高的优点,适合于基层兽医的检测诊断。     

基于N蛋白的牛冠状病毒抗体间接ELISA检测方法的建立与应用

为建立牛冠状病毒(Bovine coronavirus,BCoV)抗体间接ELISA检测方法,对BCoV的N基因进行克隆,利用原核表达制备重组N蛋白,以纯化的重组N蛋白作为包被抗原,建立ELISA检测方法,并对随机收集的奶牛血清样本进行检测.结果显示,重组N蛋白大小为50 ku,经Western blot鉴定重组N蛋白...     

间接法Dot-ELISA检测猪流行性腹泻抗体的研究

使用非洲绿猴肾（Ｖｅｒｏ）细胞增殖适应传代细胞培养的猪流行性腹泻病毒（ＰＥＤＶ），并经聚乙二醇（ＰＥＧ）沉淀法分离纯化ＰＥＤＶ抗原，建立斑点酶联免疫吸附试验（Ｄｏｔ－ＥＬＩＳＡ）检测猪流行性腹泻（ＰＥＤ）抗体。在最适工作条件下，进行了敏感性和特异性试验，结果表明，该法检测ＰＥＤＶ抗体，敏感、特异、重复性好，且方便、快捷，适用于大批量试样的检测，可作为一种诊断ＰＥＤ的比较理想的方法。采用此方法分别对来自加拿大、台湾省进口的种猪和海南省和广东省内种猪群的血清样共８３４份进行了检测，检测得ＰＥＤＶ抗体阳性率达２１％。     

水牛肝片吸虫病间接ELISA诊断方法的建立及应用

以肝片吸虫的分泌－排泄物作为抗原（ES抗原），建立水牛肝片吸虫病间接ELISA诊断方法，并用此方法与粪便检查法检测江苏和安微2省10县（市）302户的307头水牛的血清和粪便；间接ELISA诊断法的最佳工作条件，每乳包被0．2μg抗原，血清稀释度为1：960，二抗稀释度为1：600。结果粪便检查出虫卵的阳性水牛43头，阳性率为14．01％，其中安徽省为11．61％（18／155），江苏省为16．45％（25／152）；用间接ELISA法检测299头水牛血清，阳性87头，阳性率为29．19％，其中安徽省为23．53％，江苏省为34．92％，两种方法的符合率为86．05％。     

Immunodiagnosis of current fasciolosis in sheep naturally exposed to Fasciola hepatica by using a 2.9 kDa recombinant protein

A 2.9 kDa recombinant-Fasciola hepatica protein (FhrAPS) was employed to estimate the prevalence of fasciolosis in sheep maintained under field conditions. For this purpose, 340 samples with known status in relation to fasciolosis by using a direct-ELISA and the coprological sedimentation were used. These samples were analysed by using an indirect-ELISA (iELISA) and the FhrAPS recombinant protein and excretory/secretory antigens (FhES) of this trematode. Current fasciolosis (CF) was named when results were positive to antigenemia and/or coprology. Out of 198 sheep with current fasciolosis, 68% were positive to the FhrAPS-ELISA test and 53% to the FhES. We observed 14% of the CF-neg sheep were positive to the FhrAPS, whereas this percentage was 52% with the FhES. A significant correlation between FhrAPS and current fasciolosis was obtained (r2=0.513, p=0.001). We concluded that the FhrAPS provides a more suitable antigen than FhES for developing field trials to know the prevalence of early and current fasciolosis.     

Response of sheep to challenge infection with Fasciola hepatica

Sheep were infected with 100 metacercariae of Fasciola hepatica and reinfected 16 weeks later with a further 100 metacercariae. Serum samples were taken weekly for 36 weeks after primary infection. Serum was assayed for the presence of the enzymes glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gamma-GT), as indicators of liver and bile duct damage respectively, and for levels of precipitating antibody. Antibody and GLDH levels rose following the primary infection but fell after patency had been reached . A peak in gamma-GT activity was associated with the onset of patency. After the challenge infection levels of both enzymes rose substantially and there were persistent fluctuations in activity. Antibody levels did not rise markedly following challenge but fluctuated at low levels until autopsy, 20 weeks after challenge. There was no resistance to challenge judged by worm recoveries at autopsy. It is suggested that the presence of adult flukes in the bile ducts suppresses the antibody response to challenge infection. Tissue damage, which is shown by fluctuations in GLDH and gamma-GT levels after adult flukes have become established in the bile ducts, is considered to be due to the feeding activity of adult flukes and the deposition of immune complexes in the liver parenchyma.     

An experimental study on triclabendazole resistance of Fasciola hepatica in sheep

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted.The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.     

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.