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。     

Transport of serum IgA into bile of sheep infected with Fasciola hepatica

Sheep infected with Fasciola hepatica were studied for their ability to transport intravenously injected radiolabelled IgA from serum to bile. The results show that there was an apparent reduction in the selectivity of transport of IgA into bile in infected animals compared with uninfected controls. However, in infected animals the biliary flow rate was approximately twice that of uninfected animals, and the total amount of radiolabelled IgA transported was similar irrespective of infection status. The possible relevance of the elevated biliary flow rate is discussed with respect to the pharmacokinetics of drugs used for the chemotherapy of helminth infections in sheep.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Chemotherapy of infection with Fasciola hepatica in cattle.

Diamphenethide and a mixture of nitroxynil and hexachlorophane, which are effective against Fasciola hepatica in sheep, are effective in cattle if allowance is made for the slower rate of development and the more intense liver reaction which is associated with resistance. Adult parasites are mostly rejected around the time of maturity so that therapy in cattle must be directed against the early developmental stage using an appropriate drug.     

Sero-diagnosis of Fasciola hepatica infections in cattle

An antigen was prepared from metabolic products which were produced by maintaining Fasciola hepatica adults in RPMI tissue culture medium for 24 h. The antigen compared favourably with a soluble extract of adult flukes when used in an enzyme-linked immunosorbent assay (ELISA). Experimentally infected calves were used to evaluate the sensitivity of the test and a survey from areas of both traditionally high and low incidence of the disease was carried out. Evidence is presented which suggests that this metabolic antigen could be used for the sero diagnosis of naturally occurring infections. In addition the use of a microcomputer to read, file and analyse the results enabled a very large number of samples to be processed daily.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

片形吸虫DNA随机扩增多态性分析

为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

The efficacy of triclabendazole (Fasinex) against immature and adult Fasciola hepatica in experimentally infected cattle

Eighteen Chinese cattle were experimentally infected with metacercariae of Fasciola hepatica and randomly assigned to 6 groups. Five groups of cattle were treated with a single oral dose of triclabendazole at a dose rate of 12 mg kg-1. At necropsy, the reduction in fluke burden compared with the untreated group was 85, 99.6, 99.8, 100 and 100% for cattle treated 2, 6, 8, 12 and 16 weeks after infection, respectively. Data are also presented on body weight changes during the experimental period and on serum gamma-GT activity in cattle from selected groups. Triclabendazole is considered to be safer and more efficacious than currently available fasciolicides in China.     

Comparative efficacy of triclabendazole, nitroxynil and rafoxanide against immature and mature Fasciola hepatica in naturally infected cattle

In two trials the fasciolicidal activities of triclabendazole, nitroxynil and rafoxanide were assessed in cattle naturally infected with predominantly immature stages of Fasciola hepatica. Tablets containing 900 mg triclabendazole were administered orally at a dose rate of 12 mg/kg bodyweight. Rafoxanide and nitroxynil were used at a dose rate of 10 mg/kg, rafoxanide being given orally and nitroxynil by subcutaneous injection. Based on faecal egg counts nine weeks after treatment the efficacies were calculated to be 100 per cent for triclabendazole and 95.0 per cent for nitroxynil in the first trial and 98.4 per cent for triclabendazole and 52.9 per cent for rafoxanide 15 weeks after treatment in the second trial. In the first trial five animals from each of the three groups were slaughtered and their fluke burdens counted. Compared with the untreated control group the reductions in the fluke burdens were 96.9 per cent in triclabendazole treated cattle and 76.4 per cent in the nitroxynil treated group.     

Comparative efficacy of clorsulon and albendazole against Fasciola hepatica in cattle

In a dosage-confirmation trial, anthelmintic activities of clorsulon and albendazole against Fasciola hepatica were evaluated and compared. Twenty-eight cattle (8 to 12 months old) with natural F hepatica infections were randomly allotted to 4 groups of 7 cattle each: group 1, no treatment (controls); group 2, clorsulon suspension given orally at 3.5 mg/kg of body weight; group 3, clorsulon suspension given orally at 7 mg/kg; and group 4, albendazole paste given orally at 10 mg/kg. At necropsies, performed 7 and 8 days after treatment, control cattle harbored a geometric mean of 133.2 F hepatica, 16.0 of which were immature. Clorsulon administered at 3.5 mg/kg or 7 mg/kg resulted in greater than 99% removal of F hepatica, including immatures. Albendazole treatment resulted in a 76% overall reduction in F hepatica, including a 91% reduction of immatures. Fascioloides magna also were found in the cattle, but neither clorsulon nor albendazole caused significant reductions of the parasite. Adverse reactions to the 2 drugs were not observed.     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。