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细粒棘球绦虫成虫表膜抗原特异性基因的筛选及克隆 总被引：1，自引：0，他引：1
为筛选细粒棘球绦虫(Echinococcus granulosus,Eg)表膜抗原基因或具有诊断性的特异性基因,提取Eg成虫表膜抗原,经ELISA、Western blot对该抗原的免疫学特性进行初步研究.以Eg成虫表膜抗原高免鼠血清为探针,筛选Eg成虫cDNA文库,将阳性噬菌斑的PCR产物和pGEM-T载体连接,转染到DH5α,对得到的重组子进行测序,并进行同源性分析.ELISA检测显示,表膜抗原免疫鼠诱导产生了特异性抗体,Westem blot鉴定该抗体能识别Eg成虫表膜抗原、原头蚴、Eg发育不成熟、Eg发育成熟抗原.筛选出6个阳性克隆,DNA片段大小在1～2kb之间.对阳性克隆进行同源性分析,结果与EgP-29mRNA同源性为99%,与Eg14-3-3蛋白mRNA同源性80%-99%.筛选Eg成虫cDNA文库所获得的基因,证明了Eg虫体表膜抗原的存在,有望成为犬细粒棘球绦虫的诊断抗原以及犬抗细粒棘球绦虫的候选基因. 相似文献
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《畜牧与兽医》2015,(5):101-104
对细粒棘球绦虫感染家/牧犬常用的检测方法开展比对试验,并予以评估。对人工感染细粒棘球绦虫试验犬含卵粪样在不同时段用饱和蔗糖液(1.28 g/m L)漂浮虫卵,镜检;经化学药物驱虫,收集人工感染试验犬驱虫前、后粪样,流行病学调查中收集的家/牧犬粪样,用粪抗原抗体夹心ELISA检测试剂盒检测,同时,对检出的家/牧犬阳性粪样,再用虫卵漂浮法复检;通过氢溴酸槟榔碱下泻法检查家/牧犬感染细粒棘球绦虫虫体,同时,对下泻犬粪样用粪抗原检测和虫卵漂浮法进行复检。饱和蔗糖液漂浮虫卵最佳时段为2 h,可检出虫卵量与其他时段差异显著(P0.05);人工感染犬驱虫前、后粪样经粪抗原检测,OD值差异极显著(P0.01);对12 288份家/牧犬粪样进行粪抗原检测,阳性样品991份,感染率为8.1%(991/12 288),对991份阳性粪样进行虫卵漂浮法复检,检查出54份粪样中含虫卵。经下泻法检查312只家牧犬,其中,262只犬下泻,检查出27只犬感染细粒棘球绦虫成虫;对262份下泻犬粪样进行粪抗原检测,阳性29份;对262份下泻犬粪样用虫卵漂浮法检查,仅14份粪样检出虫卵。犬粪抗原抗体夹心ELISA检测犬感染细粒棘球绦虫可及时区分驱虫前、后感染状况,其更为安全、快捷、准确。 相似文献
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为了解2019年新疆喀什地区犬细粒棘球绦虫的感染情况,于2019年8—10月从喀什地区各县(市)采集犬粪样品共760份,用细粒棘球绦虫犬粪抗原ELISA检测试剂盒进行检测,共检测出27份阳性样品,总感染率为3.55％(27/760);其中,牧区感染率为5.12％(19/371),农区感染率为2.06％(8/389).表明喀什地区犬细粒棘球绦虫的感染程度较为严重,牧区感染率比农区高.该地区应继续加强犬只的管理和驱虫工作,从而提高包虫病的防控效率,降低牛、羊及人的感染风险. 相似文献
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夏玉芬 《青海畜牧兽医杂志》2018,(3)
为了摸清湟中县犬细粒棘球绦虫感染情况,采用ELISA方法,对采自14个乡镇的驱虫前和驱虫后各90只犬粪便进行细粒棘球绦虫粪抗原检测。结果驱虫前阳性率为12.2%,驱虫后阳性率为3.3%。结果表明调查区的犬细粒棘球绦虫粪抗原阳性率保持在一定范围内,驱虫后阳性率明显低于驱虫前。提示对犬进行定期驱虫、控制传染源非常必要。 相似文献
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《动物医学进展》2021,42(8)
建立多房棘球绦虫粪抗原双抗体夹心ELISA检测方法,为犬感染多房棘球绦虫的早期诊断提供技术支撑。以5E10H5杂交瘤细胞株腹腔接种Balb/c鼠制备的腹水作为包被抗体,多房棘球绦虫成虫可溶性抗原免疫新西兰大白兔制备的多克隆抗体血清作为检测抗体,HRP标记的驴抗兔IgG作为二抗,建立双抗体夹心ELISA方法,检测试验犬(感染多房棘球绦虫)和阴性对照犬犬粪。结果显示,多房棘球绦虫成虫可溶性抗原具有良好的抗原性并能产生高效价抗体;对该方法的灵敏度进行检测,阳性粪样稀释至1∶10 000时仍显示为阳性;在感染动态分析中,该方法最早可在犬感染72 h后,最迟6 d后检测到多房棘球绦虫粪抗原。表明建立的方法可用于诊断犬多房棘球绦虫感染,为后期研制犬多房棘球绦虫粪抗原双抗体夹心ELISA检测试剂盒奠定了基础。 相似文献
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目的:分析在犬细粒棘球绦虫感染的检测当中,现场应用粪抗原检测法的具体效果。方法:选择2020年5月90只细粒棘球绦虫感染病犬为研究样本,对所有病犬进行粪抗原检测法诊断,对具体的诊断效果进行分析。结果:在90分病犬犬粪样本中,阴性样本为90例,阳性检出率为0。结论:针对犬细粒棘球绦虫感染的诊断,在现场应用粪抗原检测法进行诊断的效果较好,值得推广。 相似文献
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本文采用剖检法和ELISA法对青海省民和县22个乡镇的6条犬进行了剖检、180份犬粪样进行了包虫粪抗原检测,以此判定犬细粒棘球蚴绦虫病防治效果。试验结果表明,与2016年相比,2017年犬细粒棘球蚴绦虫感染率为0,犬包虫粪抗原阳性率为2.7%,阳性率明显下降,防治效果显著。 相似文献
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《中国兽医学报》2019,(12):2356-2363
通过了解新疆伊犁地区不同县市包虫病的流行现状及重点县家畜感染包虫的基因型,为调整或完善现有的防控措施提供可借鉴的科学依据。在新疆伊犁地区10个县市的定点屠宰场采用随机抽样的方法检查牛、羊肝/肺包虫感染情况;对采集的犬粪便样品采用细粒棘球绦虫粪抗原抗体夹心ELISA方法进行检测分析;在2个重点县(昭苏县和特克斯县)定点屠宰场收集病变明显的牛、羊肝/肺包囊,通过PCR扩增,分析样品基因型。结果显示,2016年和2018年伊犁地区10个县市牛肝脏包虫总感染率分别为18.53%(241/1 155)和15.14%(154/1 017),牛肺脏包虫总感染率分别为13.68%(158/1 155)和11.26%(137/1 217);羊肝脏包虫总感染率分别为20.41%(565/2 768)和20.46%(414/2 023),羊肺脏包虫总感染率分别为16.54%(442/2 672)和13.59%(275/2 023);犬细粒棘球绦虫粪抗原总阳性率分别为26.89%(434/1 614)和17.18%(101/588)。2年中羊的感染率均高于牛,2018年牛羊包虫病总感染率比2016年略有下降,但差异不显著(P0.05),犬细粒棘球绦虫粪抗原总阳性率下降明显,且存在显著性差异(P0.05);不同县市牛/羊和犬细粒棘球绦虫感染率存在显著性差异(P0.05)。从重点县采集的18份包囊样品,NADH1基因和Cox1基因测序结果均显示为细粒棘球绦虫G1型。2016年和2018年伊犁地区家畜包虫和犬细粒棘球绦虫感染情况较为严重,且存在犬与牛/羊之间循环的传播链,提示该地区包虫病防控工作仍需改善和加强。 相似文献
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R B Gasser M W Lightowlers D L Obendorf D J Jenkins M D Rickard 《Australian veterinary journal》1988,65(12):369-373
Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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M. W. LIGHTOWLERS M. D. RICKARD R. D. HONEY D. L. OBENDORF† G. F. MITCHELL‡ 《Australian veterinary journal》1984,61(4):101-108
Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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A sandwich-ELISA was developed for the detection of soluble Taenia hydatigena antigens in fecal samples of dogs. Affinity-purified polyclonal catching antibodies and alkaline phosphatase-conjugated detecting antibodies were employed, which had been obtained from rabbits hyperimmunized with excretory/secretory antigens derived from in vitro maintained adult Taenia hydatigena. The assay allowed the detection of 800 ng T. hydatigena antigen g-1 of feces as a lower limit. Six helminth-free dogs were each infected with 10 T. hydatigena cysticerci isolated from Swiss sheep. After prepatent periods ranging from 57 to 71 days, the dogs started to excrete Taenia eggs and/or proglottids. The ELISA detected Taenia antigens in all six dogs during the prepatent period starting individually between Day 18 and 45 post-infection (p.i.). Anthelmintic treatment of three dogs at Day 95 p.i. resulted in elimination of the cestodes and within the 5 following days in the disappearance of Taenia antigens from feces. The specificity of the assay was evaluated by testing crude antigens derived from helminths or bacteria. Four Taenia species showed cross-reactivity at concentrations of 5 micrograms protein ml-1. Conversely, no cross-reactions occurred with various antigen batches derived from Echinococcus granulosus, E. multilocularis, Dipylidium caninum, Mesocestoides corti, Diphyllobothrium sp., Toxocara canis and bacterial antigens (Salmonella and Escherichia). Moreover, fecal samples from dogs naturally infected with T. canis (n: 13), hookworms (n: 2), Trichuris vulpis (n: 13) and of 10 dogs with mixed infections with these three nematode groups were tested, and results confirmed the high degree of specificity. The Taenia antigens detectable by this ELISA remained immunologically stable in native feces stored at +25 degrees, +4 degrees or at -20 degrees C for at least 5 days. 相似文献
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Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent. 相似文献
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Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs 总被引：1，自引：0，他引：1
Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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2008-2010年,在内蒙古自治区选择了6个边境旗(县),借助细粒棘球绦虫粪抗原抗体夹心酶联免疫吸附试验,调查了家(牧)犬棘球绦虫感染率;经屠宰场调查,检查了羊、牛棘球蚴感染水平;通过走访当地疾病防控中心,了解人感染包虫痛的基本情况.连续3年的调查结果显示,犬粪便抗原平均阳性率为13.16%,感染严重地区的犬粪便抗原阳性率最高为16.15%,以牧区的阳性率为最高,且越靠近边境地区感染越严重;羊、牛棘球蚴感染率平均为13.66%,感染严重地区的感染率最高为17.90%,平均感染强度达到了1.36个.调查表明,内蒙古边境地区棘球蚴病依然存在,且严重威胁着人类和家畜的健康,应引起重视. 相似文献
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Kittelberger R Reichel MP Jenner J Heath DD Lightowlers MW Moro P Ibrahem MM Craig PS O'Keefe JS 《Veterinary parasitology》2002,110(1-2):57-76
The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus. 相似文献
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为了探明新疆塔城地区绵羊细粒棘球蚴流行株基因型,根据GenBank公布的12S rRNA基因和CO1基因分别设计特异性引物,对58个绵羊细粒棘球蚴包囊进行PCR检测,再通过CO1特异性引物对检测阳性病料进行PCR扩增、测序及基因分型。通过12SrRNA基因检测发现58份样品均为阳性;对所测得的58条CO1基因序列分析,发现新疆塔城地区绵羊细粒棘球蚴流行株基因型均为G1型,且CO1基因序列存在多态性,为塔城地区绵羊细粒棘球蚴病的防控提供了科学依据。 相似文献
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DONG Rui-kai GUO Xiao-yu YUAN Wei-feng JIA Hong XIN Ting HOU Shao-hua ZHU Hong-fei 《中国畜牧兽医》2016,43(10):2716-2723
To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D450 nm) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection. 相似文献