犬粪便棘球绦虫抗原的双抗体夹心酶联免疫吸附试验检测法建立与应用  

An Immunological Coproantigen Test Used to Detect Infection of Echinococcus in Dogs

作　　者：黄燕[1] Heath D David Antone J Christine 邱加闽[1] 王谦[1] 

机构地区：[1]四川省疾病预防控制中心 [2]AgResearch,AgResearch,Wallaceville Animal Research CentrePO Box 40063,Upper Hutt,New Zealand

出　　处：《预防医学情报杂志》2008年第6期407-411,共5页Journal of Preventive Medicine Information

基　　金：中国-新西兰合作项目"Ganzi County Hydatid Disease Control and Community Health Education Pilot Program"(227/中华人民共和国102/0103)

摘　　要：目的研究双抗体夹心酶联免疫吸附检测犬粪便棘球绦虫虫体抗原的方法并评价其在实际工作中的应用价值。方法实验犬12只，分实验组和对照组，实验组用活化绵羊源原头蚴（G1型）作人工感染；感染后0～8周内收集粪便，56d宰杀实验犬，收集成虫；制备成虫抗原和虫体代谢／分泌抗原，蛋白酶K处理抗原并分别免疫成年绵羊和新西兰白兔；绵羊抗血清用80％饱和硫酸铵沉淀、透析，兔抗血清过IgG亲和层析柱，分别获得纯化抗体；观察犬粪便在PBS液中-80℃冷冻3d或在含1％Formalin的PBS液中70℃～80℃加热8h的处理方法对检出结果的影响。运用该方法对四川省甘孜县达通玛区4个乡的580只家犬吡喹酮每6月一次驱虫前后的粪抗原进行监测。结果试验系统的敏感性为92．3％，特异性为80％；粪抗原从感染后的第2～3周可检出；两种样品处理方法都可以灭活虫卵但不影响检出效果；检测方法可同时用于对细粒棘球绦虫和多房棘球绦虫感染的诊断；达通玛区家犬粪抗原阳性率从2000年的50％下降到2005年的17％。结论检测方法具有较高的特异性和敏感性；推荐粪便用加热的方法处理可以灭活虫卵以防止生物污染；该方法可以在基层推广应用。Objective To establish an immunological coproantigen test used to detect infections of Echinococcus in dogs and to evaluate its application in field work. Methods A Double-Sandwich ELISA （enzyme-linked iramunosorbent assay ）method was developed by using serial faeces obtained from 6 experimentally infected dogs with protoscoleces （ G1 type） and from 6 control dogs. Dog faeces were collected at the time of infection, and weekly thereafter, and each dog fecal sample was individually collected at weeks 1 -8 after infection. Infective dogs were necropsied 56 days after infection to harvest Echinococcus spp. worms. The antigens were either obtained from culture fluid of worms maintained in vitro, or freeze-thaw of adult worms. Both antigens were treated with Proteinase-K to remove any protein. Antisera were raised in sheep and rabbits, and the IgG fractions were purified. Both PBS extract of faeces and 1% formalin in PBS extract of faeces, heated overnight to kill the eggs, were screened by this technique. This assay was applied to survey the copro-positive changes before and after praziquentel treatments at 6-monthly interval for 580 of owned dogs in Datongma District, Ganzi County, Sichuan Province. Results The test had a sensitivity of 92.3% and a specificity of 80% respectively. Coproantigen in dog faecal extracts could be positive in dog faeces after 2-3 weeks infections. Two treatments could be used to kill eggs in dog faeces and would not affect the coproantigen detection results. This test system can be used to detect both Echinocuccus granulosus infections in dogs and E. multilocularis infections in foxes. The copropositive rate of owned dogs in Datongma was decreased from 50% in 2000 to 17% in 2005. Conclusion This test method has a higher specificity and sensitivity and can be wildly used in field work. Heating faeces is recommended to kill eggs in dog faeces for avoiding biohazard.

关 键 词：棘球属 粪抗原 双抗体夹心酶联免疫吸附试验 犬 诊断 控制 

分 类 号：R383.3[医药卫生—医学寄生虫学]