''Arc 5'' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis

The 'arc 5' precipitin band, formed when test human serum is reacted against immunoelectophoresed hydatid cyst fluid antigen, has provided a positive diagnosis of Echinococcus granulosus infection. These antibodies to 'arc 5' antigen have now been found in the sera of sheep. They appear 2 weeks after infection with Taenia ovis, after 3 weeks with T. hydatigena and after 16 weeks with E. granulosus. An antigen similar to the 'arc 5' antigen of E. granulosus cyst fluid was also demonstrated in cyst fluid from T. hydatigena, but it would not be positively identified in T. ovis cyst fluid. The presence of 'arc 5' in immunoelectrophoresis tests is not suitable for specific immunodiagnosis of E. granulosus infections in sheep in New Zealand. 'Arc 5' antibodies were only associated with living E. granulosus cysts and were not present if cysts were dead. The location of the cysts in either liver or lungs and the onset of brood capsule production did not influence the presence of 'arc 5' antibodies.     

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。     

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.     

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.     

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.     

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。     

细粒棘球蚴生发细胞特异性抗原的ELIB分析

应用SDS－PAGE和免疫印渍试验（EITB），证明体外培养的细粒棘球蚴生发细胞和囊液含有特异性抗原，分子量为52kDa和38kDa，而囊壁则含有此2种抗原。生发细胞的52kDa抗原可识别感染小鼠和包虫病患者血清的特异性抗体，对囊虫病人和正常人血清则无反应条带。     

Antigenic polypeptides of Echinococcus granulosus oncospheres and definition of protective molecules

Immunoblotting and in vitro oncosphere-killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus , and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non-denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity-purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.     

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.     

Nonspecific reactions with the intradermal test for hydatidosis in persons with other helminth infections.

A partially purified Echinococcus antigen solution, prepared by boiling hydatid cyst fluid, was used in the intradermal test for hydatid disease in a Peruvian population. An unexpectedly high rate of positive reactions and poor agreement with serological tests suggested the presence of some agent(s) which produced cross-reactions with Echinococcus granulosus antigens in the intradermal test. Testing of hospital patients infected with a variety of helminths demonstrated nonspecific reactions in persons with Hymenolepis nana, Taenia spp., and Fasciola hepatica infections, mixed parasitisms, and several non-helminth pathological conditions. The findings contraindicated the use of the intradermal test for epidemiological surveys of hydatid disease. It is pointed out that intradermal test positivity rates cannot be used as synonymous with infection prevalence and regional differences do not necessarily reflect differences in the prevalence of E. granulosus infection. The greater specificity of some serological tests favor their use for epidemiological purposes.     

Immunochemical localization of major hydatid fluid antigens in protoscoleces and cysts of Echinococcus granulosus from human origin

Monospecific rabbit antisera obtained through experimental immunization with previously purified proteins were used in the structural localization of two hydatid fluid antigens, antigen 5 and antigen B, in cyst membranes and protoscoleces of E. granulosus from human origin. The antigen-antibody reaction was revealed by an avidin-biotin-peroxidase technique. Antigen 5 was not evident in the laminated membrane of the cyst wall, but it was associated with the germinal membrane of the cyst wall and brood capsules. The parenchyma of invaginated and evaginated protoscoleces was heavily labelled. The tegument, the calcareous corpuscles, the suckers and the hooks did not contain antigen 5. Degenerated protoscoleces were also labelled. Antigen B localization was essentially identical to antigen 5, but degenerated protoscoleces were not recognized by anti-antigen B antiserum. Technical aspects and differences with previously published work are discussed.     

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.     

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。     

Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar echinococcosis

Extensive experience has documented that Em2(plus)-ELISA, Em10-ELISA and Em18-immunoblot and Em18-ELISA are reliable serologic methods for detection of alveolar echinococcosis (AE) caused by the metacestodes of Echinococcus multilocularis. Among these, tests based on detection of antibodies to the specific Em18 antigen, either immunoblot or ELISA, appears to be the most specific for AE. Between 90 and 97% of AE cases with characteristic hepatic lesions detectable by image analysis have been positive in Em18-serology. In contrast Antigen B (8 kDa)-immunoblot is the most sensitive for all forms of echinococcosis, although it can not differentiate AE from cystic echinococcosis (CE). Primary serologic screening for echinococcosis, especially for CE using hydatid cyst fluid of Echinococcus granulosus appears to be highly sensitive in endemic areas. Glycoproteins (GPs) purified from cyst fluid of Taenia solium are highly specific for diagnosis of T. solium neuorcysticercosis (NCC). Using currently available antigens it is not difficult to differentiate these three larval cestodiases serologically. We recommend that (1) primary screening of CE in endemic areas should be carried out using hydatid cyst fluid of E. granulosus prepared from cysts in either sheep, human or mouse for immunoblot and from sheep or mouse for ELISA, (2) both primary screening and confirmation of AE in endemic areas should be carried out using Em18-ELISA, Em18-immunoblot or Em2(plus)-ELISA. Serodiagnosis in areas where both AE and CE are endemic, such as in China, should be carried out as a combination of (1) and (2), and (3) serology of NCC should be carried out using GP-ELISA or GP-immunoblot. All samples showing antibody to Em18 are exclusively from echinococcosis cases. There have been no false positive test reactions with sera from other diseases. Strongest Em18 responders are all from patients with AE but some weaker responses may be found in sera of persons with advanced complex lesions of CE. These highly reliable serodiagnostic methods using native, recombinant and synthetic antigens are briefly summarized and experiences with these methods in Japan is reviewed. We believe that use of these specific antigens in screening and confirmation programs for AE in Japan will improve specificity and reduce the confusion, anxiety and expense in persons whose sera give false positive reactions with crude echinococcal antigens.     

18kDa抗原诊断泡型包虫病的评价

目的：评价１８ｋＤａ抗原在泡型包虫病（ＡＥ）诊断中的价值。方法：用免疫印渍法检测３３例泡型包虫病、６９例囊型包虫病（ＣＥ）、３０例囊虫病和８２例健康人血清对泡型棘球蚴（Ｅｍ）和囊型棘球蚴（Ｅｇ）原头节１８ｋＤａ抗原的抗体反应。同时用羊包囊液抗原常规ＥＬＩＳＡ法检测上述血清。结果：ＥＬＩＳＡ试验,ＡＥ血清阳性率为９３．９％、ＣＥ为８５．５％、囊虫病为５０％、健康人为６．１％。显示３种患者血清存在较强的交叉反应。免疫印渍试验,两种原头节的１８ｋＤａ抗原对ＡＥ血清阳性反应均为９０．９％、ＣＥ分别为１０．１％和１３％、囊虫病患者为１３．３％和１６．７％。与健康人血清不发生反应。结论：１８ｋＤａ抗原可以有效的鉴别两型包虫病。     

Antibody response of Echinococcus granulosus infected mice: recognition of glucidic and peptidic epitopes and lack of avidity maturation

The antibody response was followed weekly during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with 2000 Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with 2000 dead PSC. Antibodies against hydatid cyst fluid (HCFA) and its peptidic (periodate-resistant) and carbohydrate (periodate-sensitive) epitopes were titrated by ELISA. Avidity and the antigen recognition pattern of antibodies were also analysed during infection and immunization by ELISA and immunoblot, respectively.
The antibody response of infected mice showed quantitative and qualitative variations during infection, since both titre as well as recognition of peptide and carbohydrate epitopes in HCFA depended on time post infection. No avidity maturation was evident during the course of infection. Sera from infected mice recognized the 38 kDa subunit of Ag5 but did not react with the 8 kDa subunit of AgB. On the contrary, the antibody response of immunized mice showed only one peak of antibodies that recognized both peptidic and carbohydrate epitopes of HCFA. In addition, sera from these mice recognized mainly 60 and 110 kDa bands. Our results suggest that: a) avidity and antigen recognition patterns of antibodies in mice treated with live PSC are different from those treated with dead PSC; b) antibodies against HCFA glucidic or peptidic epitopes appear at different times post infection.     

Serological reactivity to heat shock protein 70 in patients with hydatid disease

Heat shock protein 70 (hsp70) was chosen as a model antigen with which to investigate autoantibody production in humans with cystic hydatid disease. Levels of specific serum antibody were assessed in the sera of patients with surgically confirmed infection with E. granulosus and sera from non-infected controls. Antigens used were human hsp70, obtained from K562 cells grown in culture, and E. granulosus hsp70 obtained by expression of the full length protein in Escherichia coli following cloning of the associated mRNA. Antibody reactivity to human hsp70 was detected in the sera of only a small proportion of hydatid patients (10%) as well as a similar proportion of sera from age matched controls. Specific antibodies reactive with E. granulosus hsp70 were detected in 60% of hydatid patients, although some samples (21%) from healthy controls also reacted with E. granulosus hsp70, the level of reactivity was significantly higher in hydatid patients. This report identifies E. granulosus hsp70 as an immunogen during human hydatid infection but, despite its having a predicted 81% protein sequence homology with human hsp70, it does not appear to induce autoimmune reactivity against the homologous human protein.     

绵羊源细粒棘球蚴原头节感染子午砂土鼠和小鼠的比较观察

子午砂土鼠，ＮＩＨ小鼠和昆明株小鼠在实验感染绵羊源细粒棘球蚴原头节后，在不同时期剖检，观察了细粒棘球蚴在其体内的发育状况，三种鼠平均湿囊重及其占体重百分比，子午砂土鼠均高于其他两种小鼠。子午砂土鼠在感激后２７４天时，部分原头节已发育成熟，在３５７天时，全部成熟，结果表明，子午砂土鼠对细粒棘球蚴的敏感性高于其他小鼠，表现为包囊发育好，生长快，是细粒棘球蚴的一种比较理想的实验动物。     

Further characterization of the 38 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid: evidence for antigenic heterogeneity and reactivity with anti-P1 antibodies

A panel of 5 IgM and 6 IgG1 monoclonal antibodies (MoAbs) was produced against a band, eluted from a reducing SDS-PAGEgel, containing the 38 kDa subunit of antigen 5 (Ag5) from Echinococcus granulosus cyst fluid; seven of the MoAbs were shown subsequently to bind epitopes on Ag5 but none recognizedphosphorylcho-line or periodate-sensitive carbohydrate epitopes. Differences in the fine specificity of the MoAbs were apparent and, upon reduction, heterogeneity in 38 kDa components from hydatid cyst fluids of different intermediate host origin was revealed by peptide fingerprinting and immuno-blotting using the MoAbs. One of the IgGl MoAbs (ED9) was able to distinguish a reduced 38 kDa molecule in cyst fluids from two distinct genotypes–the horse/dog and sheep/dog strains–of E. granulosus and this may have implications for hydatid serology, immunoepidemiology and strain typing. Furthermore, epitopes on this 38 kDa component or on a different molecule with the same or similar M_r are reactive with anti-P1 blood group antigen antibodies and this could result in false-positive reactions where sera from P2 patients with suspected hydatid disease are tested by immunoblot or immunoprecipitation analysis.,     

Standardization and evaluation of an enzyme immunoassay as a screening test for the seroepidemiology of human hydatidosis

The immunodiagnosis of human hydatidosis caused by Echinococcus granulosus is based on the detection of antibodies against arc 5 antigens by the double diffusion (DD5) or counterimmunoelectrophoresis (CIE5) tests. However, neither of those tests is practical for seroepidemiological surveys in which a great number of samples have to be processed. The present study was carried out to evaluate if a solid phase enzyme immunoassay (EIA) standardized to detect total antibodies against E. granulosus cyst fluid antigens would permit the selection of sera with antibody activity against arc 5 antigens detectable by DD5. Groups of sera representing 5 possible results with DD5 were studied by the EIA proposed as a screening test. Using the EIA all the sera with antibody activity against arc 5 antigens were differentiated from all those which did not produce any precipitation band in DD5. It was also possible to differentiate those with no antibodies from all those with antibodies against 3 non-arc 5 antigens, most with antibodies against 2 non-arc 5 antigens and about a fifth with antibodies against 1 non-arc 5 antigen. It is concluded that the EIA, standardized as described, is capable of detecting with a high degree of efficiency all the sera that should be tested by DD5 and excludes most of those in which results with DD5 have no diagnostic significance.