Specific antibody responses in dogs experimentally infected with Echinococcus granulosus

Six dogs reared helminth-free were divided into 2 groups. Four dogs were infected per os with 200,000 protoscoleces each of Echinococcus granulosus and 2 were kept as uninfected controls. All the dogs were kept together until 32 days after infection, when 1 infected dog was killed, its intestine removed and the contents examined to confirm that the infection with E. granulosus had been successful. The remaining 3 infected dogs were transferred to high security housing and their feces inspected daily to establish the time infections became patent. The infected and control dogs were bled every 5 days for 75 days from the time of infection and the sera were stored at -70 degrees C. Sera were tested by the enzyme-linked immunosorbent assay (ELISA) for antibodies to E. granulosus scolex excretory/secretory (ES) antigen, protoscolex antigen and oncosphere antigen. Antibodies to scolex ES antigen and protoscolex antigen were detected in the sera of infected dogs within 2 weeks of infection. Antibody titers rose rapidly and remained at a high level until the dogs were killed 75 days after infection. Antibodies in these sera did not cross react with antigens prepared from Taenia ovis, T. hydatigena, T. pisiformis, Ancylostoma caninum, Trichuris vulpis and Toxocara canis.     

Serum antibody response following parenteral immunization with hydatid cyst fluid in sheep infected with Echinococcus granulosus

Production of specific serum antibodies following immunization with hydatid cyst fluid antigens was investigated in sheep with Echinococcus granulosus infection and in noninfected controls. Six of 10 infected animals responded to intramuscular injection of antigen by rapid production of antibodies detected in indirect hemagglutination assays. Similar responses did not occur in any of 10 noninfected controls. It is suggested that differences in the rate of response to immunization with cyst fluid antigens between groups of sheep could be exploited in serodiagnosis of E. granulosus infection in sheep. The results also suggest that low levels of antibody found in the serum of sheep infected with E. granulosus are not the result of immunosuppression or immunological tolerance, but are due to sequestration of antigen from the immune system of the host.     

Rapid dot-ELISA for the detection of specific antigens in the cyst fluid from human cases of cystic echinococcosis

A rapid dot-ELISA was developed for the detection of specific antigens in samples of cyst fluid from human cases of Echinococcus granulosus infection, permitting the confirmation of cystic echinococcosis (CE). Peroxidase-conjugated antibodies against antigen B (derived from the fluid in the cysts of sheep infected with E. granulosus) were used to test cyst-fluid samples from 22, surgically confirmed cases of human CE and 21 domestic animals (horses, sheep, buffalo, a cow and a camel) with CE. All of these samples were found to be strongly positive in the dot-ELISA, by direct reading with the naked eye. In contrast, fluid samples from seven, non-parasitic liver cysts of human origin were all negative, and a sample taken from the residual cavity left in a patient after previous CE surgery showed a weakly positive reaction. The antigen-detection assay, which can be completed within 10 min, may be a useful 'bedside' test for the differential diagnosis of cysts during surgery or percutaneous treatment for suspected CE.     

Examination of murine antibody response to secondary hydatidosis using ELISA and immunoelectrophoresis

Summary Antibody responses in mice with up to 64 weeks of secondary Echinococcus granulosus hydatidosis were examined by ELISA using hydatid protoscolex antigen (Px), Antigen 5 (Ag5) and Antigen B (AgB), and by immunoelectrophoresis (IEP) using sheep hydatid cyst fluid (SHCF). Anti-Px IgG antibodies, evident from 3–5 days post infection (p.i.), increased steadily until 16 weeks and maintained a high level afterwards. Anti-Ag5 IgG antibodies were negligible up to two weeks, but they showed a small increase around 2–3 weeks which was followed by a big increase around 16 weeks p.i. The high level of anti-Ag5 IgG antibodies persisted to the end of experiment. The level of anti-AgB IgG antibodies remained relatively low throughout infection. Anti-Px IgM antibodies appeared in the early period of infection, but became insignificant as the infection proceeded. Specific IgM antibodies to Ag5 and AgB showed two waves of increase, one between 3 days to 4 weeks p.i. and the other between 16 weeks to 46 weeks p.i. The level of IgA antibodies to Ag5 and AgB was low and only a moderate amount of anti-Px IgA antibodies was detected. Generally, a higher level of serum antibodies are associated with a larger number of mature cysts. Serum samples from 5 of 8 mice harbouring hydatid cysts formed 1–3 bands with SHCF in IEP, including Arc 5, but a precipitation arc with AgB was not observed. Analysis of hydatid cyst fluid from the infected mice (MHCF) in IEP also failed to demonstrate AgB. Despite the high levels of antihydatid antibodies generated in the infected mice, protoscoleces appeared to be unhindered in their growth to mature cysts.     

Immunoglobulin profiles in a murine intermediate host model of resistance for Echinococcus granulosus infection

We have shown previously that primary infection of Chinese Kunming (CKM) mice with Echinococcus granulosus oncospheres is protective against subsequent challenge. Nine groups of mice were infected with the oncospheres of E. granulosus by different routes (intraperitoneal, subcutaneous or intravenous injection). After infection, serum was collected after different periods of time and serum antibodies were tested by ELISA against oncospheral proteins and hydatid cyst fluid antigens. The results indicated that CKM mice produced low levels of antibodies before a secondary challenge infection given 3 weeks later by a different route. Most mice did not evoke significant antibody responses against oncospheral antigens until 5 weeks after infection. The level of IgG, especially IgG1 against oncospheral antigens increased from week 4 post-infection (p.i.), to a maximum at week 9 p.i. In addition, antibodies against hydatid cyst fluid antigens increased at the same time as the recognition of oncospheral antigens. Immunoblots using hydatid cyst fluid showed that the first antigen that was recognized - an 8-kDa protein, possibly the smallest subunit of Antigen B - appeared 5-6 weeks p.i. and reactivity to this molecule was intensive at week 9 p.i. The results suggest that protection against secondary infection was not principally antibody-mediated during the initial phases of infection, when cellular immune responses may play a pivotal role in the protective mechanism.     

细粒棘球蚴在NIH小鼠体内发育的组织学及组织化学的进一步观察

本文继续观察感染后6～24个月的细粒棘球蚴在NIH小鼠体内发育的组织学及组织化学变化。结果表明,在鼠体内出现育囊的时间为感染后7～8个月,囊液内见到游离原头节及子囊的时间分别为8及10个月,并发现细粒棘蚴体内的糖原、DNA、RNA、碱性蛋白质的含量,AKP、ACP及ATP酶的活力,均以生发层的芽状突起部分及原头节内的较丰富和较强。     

感染细粒棘球蚴绵羊诱发过敏性休克期间IgG、IgG1和IgE水平的探讨

目的　测定感染细粒棘球蚴(E.g)绵羊诱发过敏性休克期间特异性IgG、IgG1和IgE抗体水平。了解抗原B对人工感染E.g绵羊IgG抗体的反应性。　方法　从绵羊E.g囊液中制备抗原B和E.g囊液粗制抗原,ELISA测定感染E.g绵羊诱发休克期间特异性IgG、IgG1和IgE抗体的动态变化。　结果　感染E.g绵羊6个月,特异性IgG、IgG1和IgE抗体水平较正常绵羊显著升高;诱发休克后特异性IgE抗体水平显著下降,尤其因休克致死的绵羊下降更为明显;IgG及IgG1抗体的衰减时间不同,趋势各不相同;抗原B和E.g囊液粗制抗原与血清IgG抗体反应阳性率分别为91%、32%。　结论　特异性IgE是导致棘球蚴病所致过敏性休克的主要抗体,而IgG和IgG1抗体也起着重要作用。抗原B与感染E.g绵羊IgG抗体的血清反应性较好,可作为一种血清免疫学诊断方法监测绵羊感染E.g的状况。     

The identification of eggs of Echinococcus by immunofluorescence using a specific anti-oncospheral monoclonal antibody

A relatively simple and specific test has been developed to distinguish eggs of Echinococcus from those of other morphologically identical taeniid species. A specific anti-Echinococcus oncosphere monoclonal antibody was produced which binds in an indirect immunofluorescence test to egg-derived oncospheres of E. granulosus but not to those of other taeniid species, such as Taenia hydatigena, T. saginata, T. pisiformis, T. ovis, T. multiceps, or T. taeniaeformis. Specific fluorescence was obtained with oncospheres of E. granulosus derived from either hatch/activated viable eggs using artificial intestinal fluid or from hypochlorite/detergent treated eggs. The potential use of this test in the study of the transmission of Echinococcus in Turkana, Kenya, is discussed.     

Lymphocyte subpopulations of sheep in protective immunity to Taenia hydatigena

Lymphocyte subpopulations entering the liver and surrounding the rejection sites during a 9-day period after infection of immune sheep with Taenia hydatigena were identified with the aid of monoclonal antibodies against lymphocyte cell surface markers. Viable lymphocytes were isolated from the liver tissue and stained by indirect immunofluorescence for subsequent flow cytometry analysis. Over the first 6 days after challenge infection a marked increase in the ratio of T-helper to T-suppressor/cytotoxic lymphocytes was observed. SBU-T19+ lymphocytes, a CD5+ T-cell subpopulation uniquely identified in the sheep, were present in small numbers in sheep liver both before and after infection. There was a large, continuous increase of sIg+ B-cells over the 9-day observation period after infection. Eosinophils were the predominant granulocytes in the liver of infected sheep. The exact location of the leucocyte subpopulations in respect to the rejection sites in infected liver was determined by in-situ immunoperoxidase staining of frozen liver sections. The evolution of the parasite-induced leucocyte response was characterized by the appearance of a central core of eosinophils surrounded by increasing numbers of CD4+ helper T-cells. CD8+ (suppressor/cytotoxic) and SBU-T19+ T-lymphocytes were present in much smaller numbers and by day 9 after infection were located predominantly around the periphery of the lesions. Distinct foci of tightly packed B-cells developed within the lesions and increased dramatically in size over the 9-day observation period. At this time, lesions appeared as compact aggregations of leucocytes encircled by a second band of eosinophils. Both T- and B-lymphocytes within the lesions stained positive for class II major histocompatibility antigens.     

Immune responses associated with protection in sheep vaccinated with a recombinant antigen from Taenia ovis

This paper describes the evaluation of the protective antibody response of sheep to vaccination against Taenia ovis infection with a defined recombinant antigen (45W). Sera from 181 vaccinated sheep, collected prior to experimental challenge with T.ovis , were assessed for 45W specific IgA, IgG, IgG₁, IgG₂ and IgM levels and these results correlated with protection data. There were significant relationships (P < 0.001) between IgG, IgG₁ and IgG₂ titres and protection. Serum IgA levels did not correlate with protection and there were no significant levels of 45W specific IgM detected. Killing of several other taeniid cestodes has been shown to be complement mediated and the findings in this study are consistent with the involvement of this immune mechanism in 45W vaccinated sheep. A comparison of the adjuvants used in this study (saponin and oil in water) demonstrated that whereas both adjuvants stimulated the production of similar levels of 45W specific IgG₁, the IgG₂ response was significantly higher in sheep vaccinated with oil adjuvant.     

棘球蚴感染绵羊并诱发休克期间特异性IgG、IgE抗体对特异性抗原的识别

目的 探讨细粒棘球蚴(E.g.)囊液粗制抗原和B抗原(EgB)识别绵羊感染E.g.后及诱发过敏性休克期间特异性IgG和IgE抗体的特异性反应,并对抗原特性及分子量进行描述。 方法 制备E.g.囊液粗制抗原及EgB,应用免疫印迹技术检测经剖杀证实感染E.g.并诱发过敏性休克的20只绵羊血清特异性IgG和IgE抗体对两种抗原的抗体反应性,并对抗原特性和分子量进行描述。 结果 特异性IgE抗体与耐热、低分子量的EgB(8、12和16 kDa)抗原未见明显的反应条带,而与E.g.囊液粗制抗原在43 kDa处可见明显的反应条带。IgG抗体与E.g.囊液粗制抗原未见明显的反应条带,但与EgB抗原反应后在31、43和66.2 kDa处可见较为明显的反应条带。 结论 EgB可能不是特异性IgE抗体的特异性抗原,而是IgG抗体的特异性抗原。E.g.粗制囊液抗原中含有特异性IgE抗体的特异性抗原组分,其分子量大于43 kDa,可能是导致棘球蚴病患者过敏性休克的主要致敏原。     

Purification and N-terminal amino acid sequencing of Echinococcus granulosus antigen 5

Antigen 5, a major parasite-derived lipoprotein of unilocular hydatid ( Echinococcus granulosus ) cyst fluid, comprises subunits of 56–70 kDa which dissociate on disulphide bound reduction to two subunits of approximately 25 and 38 kDa on SDS-PAGE. The 38 kDa component is recognized as a potentially important diagnostic marker for cystic hydatid disease. Single dimensional SDS-PAGE, two dimensional (2-D) gel electrophoresis, modified '2-D' gel electrophoresis, immunoaffinity-purification and HLPC were employed to purify antigen 5. Subsequent N-terminal sequencing suggested that antigen 5 isoforms exist due to the identification of a single amino acid sequence with alternative residues at some positions. An 18 amino acid consensus N-terminal sequence was derived for antigen 5 as a result of comparing the sequences obtained by the different fractionation methods. No homology was revealed to any previously identified protein including putative antigen 5 amino acid sequences. A synthetic peptide, mimicking the N-terminal consensus sequence, did not bind with patient sera (confirmed positive to antigen 5) or an anti-antigen 5 MoAb. Protein sequences (16 residues compared) for the 38 kDa subunit from sheep and horse (representing different strains of E.  granulosus ) cyst fluids were very similar except for differences at three positions. 2-D and modified '2-D' gel electrophoresis, in combination with immunoblot analysis, indicated the presence of more than one protein in the 38 kDa region of SCF capable of binding the anti-antigen 5 MoAb.     

Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, respectively. Sheep received two vaccinations with various combinations of these two delivery systems and/or purified recombinant 45W protein in a conventional vaccine formulation containing Quil A as adjuvant (protein/Quil A vaccine). Sheep receiving two inoculations of either the nucleic acid or the recombinant adenovirus alone, demonstrated only low levels of 45W-specific antibody. However, immunization with either nucleic acid or recombinant adenovirus primed animals to mount an enhanced immune response after a subsequent vaccination with the protein/Quil A vaccine. The most striking result was that sheep initially immunized with the nucleic acid vaccine and boosted with the recombinant adenovirus, mounted IgG₁ responses >65 fold higher than those of sheep receiving either vaccine alone. The level of antibody in these sheep was commensurate with that observed in animals vaccinated twice with the protein/Quil A adjuvanted vaccine. In both cases, host-protection from experimental challenge infection with T. ovis was obtained.     

Analysis of host components in hydatid cyst fluid and immunoblot diagnosis of human Echinococcus granulosus infection.

To improve serodiagnosis of cystic hydatidosis, immunoblotting studies were performed to look for a highly specific parasite antigen(s). First, commercially available hydatid cyst fluid antigen preparations were characterized by SDS-PAGE and by immunoblotting with sera specific for parasite and host animal proteins. One preparation, designed for use in complement fixation tests, did not appear to be suitable for immunoblotting because of the low concentrations of parasite antigens. Several host proteins, including serum albumin and IgG, were detected in the cyst fluid. Sera from patients with Echinococcus granulosus infections and other parasitic diseases were examined by immunoblotting for antibodies against specific cyst fluid parasite antigens. Several parasite antigens were variably recognized. Only one antigen, a 40 kDa protein, was recognized by all E. granulosus-infected patients. Reactivity against this antigen was also observed in all sera from E. multilocularis, cysticercosis, and schistosomiasis patients as well as in some filariasis cases. Two E. granulosus antigens, molecules of 12.5 and approximately 17 kDa, were only recognized by antibodies from some E. granulosus patients.     

绵羊脑多头蚴病45m基因在多头绦虫和细粒棘球绦虫不同发育阶段同源性的比较

目的研究绵羊多头蚴病45M重组蛋白的同源性。方法将多头蚴的45m基因片段亚克隆到pET41b表达质粒,构建45m-pET41b原核表达系统,诱导表达45M重组蛋白,制备小鼠高免血清,进行免疫印迹试验（Western blot）;运用RT-PCR的方法,从多头绦虫的成虫cDNA中分离到45mA。结果45M蛋白的高免血清可被细粒棘球绦虫不同发育阶段的天然抗原所识别,均在63kD处发生交叉反应;从多头绦虫的成虫cDNA中分离到45m基因的同源基因,命名为45mA（Gen-Bank号为EU326106）,45m和45mA的核酸序列和蛋白序列分别有86％和76％的同源性;45M和45MA蛋白与细粒棘球蚴的保护性蛋白Eg95有57％的氨基酸同源性。结论提示45m抗原分子在多头绦虫和细粒棘球绦虫的不同发育阶段均存在,为45M蛋白的免疫原性的分析提供了宝贵的资料。     

Specific detection of circulating surface/secreted glycoproteins of viable cysticerci in Taenia saginata cysticercosis

A mouse monoclonal antibody (MoAb), reactive with a repetitive carbohydrate epitope on lentil-lectin adherent glycoproteins present on the surface and in the secretions of Taenia saginata cysticerci, was used in the construction of a diagnostic ELISA assay to detect these glycoproteins in the serum of T. saginata infected cattle. The MoAb was used as the trapping layer and subsequently bound glycoprotein was revealed using the same MoAb and biotinylated peroxidase-streptavidin complex as the developing system. The assay was suitably specific. Exceptionally low background values were obtained with sera from animals with a range of commonly occurring tropical parasitic infections, including Taenia hydatigena, Echinococcus granulosus and Fasciola gigantica. The minimal detection level was approximately 200 live cysticerci in cattle. Parasite derived glycoprotein could be detected in serum from about 4-5 weeks post-infection onwards and was associated with a current infection, as drug treatment of infected cattle to kill the cysticerci resulted in the disappearance of these components from the circulation, while the titre of anti-parasite antibody remained high. The same assay also detected Taenia solium cysticercosis in humans.     

高强度聚焦超声波辐照细粒棘球蚴包囊的热效应

目的通过检测高强度聚焦超声波(HIFU)照射棘球蚴包囊后囊液、囊壁、包囊周围肝组织温度和原头蚴死亡率,了解HIFU处理后不同部位的升温效应,探索HIFU杀伤棘球蚴的方法和效果。方法采集感染细粒棘球绦虫的新鲜羊肝,选取囊壁较薄,触摸弹性较好,直径介于10mm到45mm之间的包囊42个。采用随机区组设计的方法分组,对照组用普通超声照射,实验组用200W声功率HIFU直线扫描的方法照射,照射时间分别为2min,4min,6min,8min,10min。照射后立即测定囊液、囊壁、距包囊5mm部位肝组织的温度。抽取囊液、涂片,经台盼兰染色后计数原头蚴的死亡率。结果HIFU照射后,棘球蚴囊壁、囊液、包囊周围肝组织的温度均有升高,其中囊壁的温度升高最为明显,且与囊液、包囊周围肝组织之间有显著差异;囊液中的原头蚴死亡率增加。结论HIFU照射使棘球蚴包囊的囊壁产生明显的升温效应,HIFU照射对原头蚴有杀伤作用。     

绵羊细粒棘球蚴囊液抗原诱发小鼠免疫反应及保护性免疫的研究

本文用绵羊细粒棘球蚴囊液抗原制剂,给小鼠3次腹腔注射诱发初次免疫,然后腹腔移植泡球蚴组织作攻击感染,观察保护性免疫。实验提示,注射免疫原制剂后1月能诱发小鼠免疫反应,间接血凝试验(IHA)可测出血清抗体;攻击感染后23周,IHA阳性率达94.4％,而未诱发初次免疫的对照鼠IHA阳性率为90.0％,表明无论是初次免疫或攻击感染,均能诱发小鼠产生抗体,二者无显著差别(P>0.05);但IHA血清滴度1:256百分率分别为94.1％和55.5％,有显著差别(P<0.01),表明攻击感染有强化初次免疫的作用。攻击感染后23周,对比免疫鼠和对照鼠的腹腔泡球蚴湿重均值和病理分级率均无显著差别(P>0.05),提示本文小鼠初次免疫未显保护性免疫之效。     

Identification of a differentially expressed Echinococcus multilocularis protein Em6 potentially related to antigen 5 of Echinococcus granulosus

By a strategy of differential immunological screening of an expression library constructed from adult Echinococcus multilocularis parasites, a partial cDNA sequence encoding a protein termed Em6 was isolated. This molecule displayed high sequence homology to the recombinant antigen 'Eg6' which was previously described as an immunogenic epitope of antigen 5 of E. granulosus. Further Em6 sequences and the corresponding sequences from a cattle isolate of E. granulosus were obtained by a PCR approach. By immunoblot analyses using affinity purified antibodies, expression of Em6 in fertile cysts producing protoscoleces of the E. multilocularis metacestode stage was observed. However, Em6 was absent in non-fertile metacestodes. The demonstration of a protein in E. multilocularis displaying identities to 'antigen 6' of E. granulosus could potentially contribute to the future elucidation of the relationship between antigen 5 and 'antigen 6' in the genus Echinococcus , and shed some lights on the performance of serodiagnostic assays for hydatid disease based on the respective antigens .     

Studies on stage-specific immunity against Taenia Taeniaeformis metacestodes in mice

The possible existence of stage-specific immune responses to Taenia taeniaeformis infection was investigated in C3H/He mice vaccinated with antigens prepared from either the oncosphere or metacestode stages. Mice were immunized twice, 2 weeks apart, with antigen in Freund's complete adjuvant. Two weeks after the second immunization they were challenged with 250 T. taeniaeformis eggs and killed day 0, 5, 10, 15, 20, 25, 30, 45 and 60 after infection. Gross examination of the livers revealed marked differences between oncosphere (TtO) and metacestode (TtM) vaccinated mice. Very few metacestodes were found in the first group but most of those that evaded the initial host attack developed like the cysts found in the control group. In contrast, many degenerating metacestodes were found in the TtM vaccinated group. In a subsequent experiment groups of mice were vaccinated with varying doses of either TtO or TtM to determine whether the qualitative differences observed above were due to antigen dose effects. However, varying antigen doses gave the same results. These data show that vaccination with oncospheres generates an immune response capable of killing invading larvae soon after infection whereas vaccination with TtM results in larvae being killed at a later stage, suggesting that there are stage-specific, host-protective antigens.