Multilocularis Antigen Research Articles

T LYMPHOCYTE CYTOKINE PROFILE AT A SINGLE CELL LEVEL IN ALVEOLAR ECHINOCOCCOSIS

The metacestode Echinococcus multilocularis causes a life-threatening disease in humans, named alveolar echinococcosis (AE). A comparative analysis of the early activation marker CD69 on peripheral blood mononuclear cells (PBMC) of patients with AE and healthy controls after in vitro culture with crude E. multilocularis antigen revealed that specific expression of CD69 was induced in CD4+T lymphocytes as well as in CD8+T lymphocytes. Using a protocol for intracellular staining of cytokines followed by fluorescence activating cell sorting (FACS) analysis, production of interleukin (IL)-2, IL-5 and IL-10 was detected in CD4+as well as in CD8+lymphocytes. Most noteably, there was a definite increase in the expression of IL-10 in CD8+lymphocytes from patients with alveolar echinococcosis. The data support an important role of CD8+lymphocytes in the long persistence of the metacestode.

Management of hydatid disease of the liver—10 years' experience in a non-endemic area

The incidence of hydatid disease of the liver caused byEchinococcus granulosus in Austria, a nonendemic region in central Europe, is documented over a 10-year period. Since 1984, 28 patients (24 women, 4 men) with hydatid disease of the liver have undergone surgery in our department. A definitive diagnosis was established by combining serological tests (an enzyme-linked immunosorbent assay withEchinococcus multilocularis antigen and an indirect hemagglutination assay withEchinococcus granulosus antigen) and abdominal imaging (computed tomography scan and ultrasonography). Twenty-four patients (85.7%) had uncomplicated echinococcosis; in most of them (16) total closed pericystectomy was done. Four patients (14.3%) had complicated echinococcosis, suffering from intrabiliary rupture and spontaneous perforation into the peritoneal cavity, and in two of these patients (7.1%) pyogenic transformation, one with abscess perforation, occurred. Postoperative complications were significantly more common in patients with complicated echinococcosis and included two bile leaks (necessitating prolonged tube drainage), pleural effusion, atelectasis, and wound infection. One recurrence of hydatid infection due to previous spontaneous perforation required reoperation 4 years after the primary event. The low recurrence rate of uncomplicated echinococcosis (0%) over a mean period of 5.6 years and of 3.6% when peritoneal echinococcosis was included may be the result of the radical surgical technique employed.

T-Lymphocyte cytokine mRNA expression in cystic echinococcosis

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

EM18, a New Serodiagnostic Marker for Differentiation of Active and Inactive Cases of Alveolar Hydatid Disease

We determined whether detection of antibody response against a newly detected epitope, designated Em18, among Echinococcus multilocularis antigens could be a reliable marker for differentiation of active cases of alveolar hydatid disease (AHD) from inactive cases. Fifteen Alaskan patients with either active or inactive lesions of AHD previously confirmed clinically, pathologically, and serologically by the Em2-enzyme-linked immunosorbent assay (ELISA) were used for a blind test by Western blotting. Ten and five cases were considered to be active and inactive cases, respectively. One of the 10 cases classified serologically as active was judged to be inactive based on clinical and pathologic criteria; the patient had a recognizable parasitic lesion, and following short-term treatment with albendazole, a biopsy of the liver showed a degenerated lesion that did not grow in rodents. The five cases judged to be inactive included two confirmed inactive cases with cicatrized lesions and three active cases that showed the weakest values in the Em2-ELISA. The most predominant IgG subclass responding to Em18 was IgG4. In general, there were good correlations between 1) the antibody response against Em18 and the presence of active lesions and 2) the antibody response against Em18 and the Em2-ELISA values.

Identification of a cDNA clone from the larval stage ofEchinococcus granulosus with homologies to theE. multilocularis antigen EM10-expressing cDNA clone

Identification of a cDNA clone from the larval stage ofEchinococcus granulosus with homologies to theE. multilocularis antigen EM10-expressing cDNA clone

Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen

Alveolar echinococcosis (AE) in humans is generally a fatal disease when not diagnosed early enough to provide curative treatment such as radical surgery. Immunodiagnosis for early detection of AE was improved by the isolation of an affinity-purified metacestode Em2 antigen and by the synthesis of recombinant Echinococcus multilocularis antigen II/3-10. Both antigens were individually assessed by enzyme-linked immunosorbent assay (ELISA) and demonstrated high specificities and diagnostic sensitivities, although both missed approximately 4 to 11% of diagnostic cases of AE. To provide an optimal serodiagnostic test, we investigated the two purified antigens by using a test employing a mixture of both purified antigens (designated Em2plus antigen) in one assay. For comparative purposes, crude E. multilocularis and Echinococcus granulosus metacestode antigens were investigated as well. The Em2plus ELISA proved to be the optimal diagnostic test with the highest diagnostic sensitivity, 97%, in serum samples from 140 patients with AE and an overall specificity of 99% for infections due to other Echinococcus and non-Echinococcus parasites. The new test combination (Em2plus ELISA) is suggested for the serodiagnosis of AE in patients and for seroepidemiological surveys.

Echinococcus multilocularis Infection: Immunology and Immunodiagnosis

Publisher Summary Echinococcus multilocularis was definitively established as an independent species by Vogel's exact morphological and biological investigations, including the completion of the life cycle of the parasite in experimentally infected hosts. The natural life cycle of E. multilocularis is uniform, although the host assemblages universally differ in accordance with faunal changes southwards from the Arctic. This fact is the principal determinant of the prevalence and distribution of the parasite. Attempts to delineate the three approximate geographical ranges of E. multilocularis have been made but exact distributional data are incomplete. Nevertheless, the geographical distribution of E. multilocularis seems to be uniquely restricted to the northern hemisphere. Alveolar echinococcosis (AE) of humans is biologically comparable to the disease in the natural intermediate hosts. The primary localization of E. multilocularis metacestodes (larvae) in humans (as well as in natural intermediate hosts) is almost exclusively in the liver. Local extension of the lesion and metastatic lesion formation in the lungs, brain, and other organs can follow. This chapter presents the immunological aspect of E. multilocularis and also discusses various diagnostic techniques. New developments in the diagnosis of and vaccination against E. multilocularis are also discussed, but the major and crucial aspect remains concentrated on the identification of E. multilocularis antigens to protect hosts.

The diagnostic value and molecular characterisation of an Echinococcus multilocularis antigen gene clone

A diagnostic Echinococcus multilocularis antigen gene (EM4) has been expressed using the Escherichia coli expression vector pGEX-1 resulting in the high level synthesis of a readily purifiable, soluble, non-denatured peptide fused to the 26-kDa glutathione-S-transferase of Schistosoma japonicum. This recombinant antigen, on testing by enzyme-linked immunosorbent assay (ELISA) with heterologous human antisera, demonstrated 100% E. multilocularis specificity. Specific anti-EM4 antibody immunoprecipitated a single 66-kDa protein from protoscolex total RNA directed in vitro translation products indicating the probable involvement of post-translational modification in the production of the native EM4 antigens. Southern blotting analysis suggests that the EM4 native antigens are coded for by a single-copy gene and that the genomic organisation of the EM4 related genes in other parasites is not conserved. The nucleotide sequence of the cloned EM4 cDNA molecule has been obtained and the derived amino acid sequence shows no significant homology with other existing protein sequences.

Detection of Taenia hydatigena copro-antigens by ELISA in dogs

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 μg 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°, +4° or at −20°C for at least 5 days.

Humoral and cellular immune response in mice and dogs induced by a recombinant Echinococcus multilocularis antigen produced by a Salmonella typhimurium vaccine strain

A gene fragment (II/3-10) from the cestode Echinococcus multilocularis coding for a species-specific antigen was expressed in the live attenuated Salmonella typhimurium vaccine strain LT2 M1C. The recombinant vaccine (S. typhimurium + pVM II/3-10) was assessed for its potential to induce both a humoral and cell-mediated immune response in mice and dogs. Both subcutaneous and peroral administration of the vaccine resulted in antibody synthesis and lymphocyte priming of C57BL/6J mice against S. typhimurium antigens as well as against the recombinant E. multilocularis antigen. Two vaccinated (subcutaneous and peroral) dogs showed a strong humoral immune response to S. typhimurium antigens and to the recombinant E. multilocularis antigen, but proliferation of peripheral blood lymphocytes was not detectable against the bacterial S. typhimurium antigens. Regarding the recombinant E. multilocularis antigen, borderline lymphocyte proliferation was demonstrated following subcutaneous and none following peroral administration of the recombinant vaccine to dogs.

Application of a recombinant Echinococcus multilocularis antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of human alveolar echinococcosis

A highly antigenic polypeptide fragment of the recombinant Echinococcus multilocularis antigen II/3 was produced in Escherichia coli and purified for application in enzyme-linked immunosorbent assay (ELISA). The antigen II/3-encoding 1.0 kb DNA sequence was reduced by sonication into smaller DNA fragments which were subsequently cloned into λgt11. Three clones could be isolated from the sublibrary, all synthesizing a recombinant antigen as a stable β-galactosidase fusion protein. In a further step, the 0.6-kb insert from one positive clone was subcloned into the plasmid pAR 3038, which directed efficient synthesis of the antigen fused to only 11 amino acids from the N-terminus of the phage T7 major capsid protein. The plasmid-encoded antigen (antigen II/3–10) was purified from a bacterial cell extract and then tested in an ELISA. Using sera from 88 patients with an E. multilocularis-infection, a high diagnostic sensitivity of 90% was demonstrated. Investigation of sera from 220 patients with various helminthic infections showed a specificity of 99%, suggesting the suitability of the antigen II/3–10 as an immunodiagnostic tool.

The isolation, by differential antibody screening, of Echinococcus multilocularis antigen gene clones with potential for immunodiagnosis

A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multilocularis protoscolex mRNA. Differential screening of the library with pools of E. multilocularis and Echinococcus granulosus human infection sera has revealed 13 potentially immunodiagnostic clones. On the basis of plaque immunoassays and lysogen characteristics, two of these clones, designated EM2 and EM4, have been further characterised. The recombinant fusion-peptides have been purified and their potential as immunodiagnostic reagents has been assessed by immunoblotting and, in the case of one fusion-peptide (EM4), by enzyme-linked immunosorbent assay (ELISA). Furthermore, the native parasite antigens coded for by these clones have been identified. EM2 corresponds to a 70 kDa protein and epitopes coded for by EM4 have been found on three antigens of 62, 49 and 44 kDa. The native antigens of both clones are present in the protoscolex and those corresponding to EM4 appear to be excreted/secreted products. They are not recognised in ELISA by a variety of human parasitic infection sera other than sera taken from patients infected with E. multilocularis. Nevertheless, the native antigens for both clones are present in E. granulosus protoscoleces and Taenia solium cysticerci. These antigens are not detectable in E. granulosus cyst fluid, and this may, in part, explain the lack of immune response to them in human E. granulosus and T. solium infections.

Combined Application of Enzyme-linked Immunosorbent Assay (ELISA) and Indirect Haemagglutination Test (IHA) as a Useful Tool for the Diagnosis and Post-operative Surveillance of Human Alveolar and Cystic Echinococcosis

A combined application of enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA) for the diagnosis and post-operative surveillance of human alveolar and cystic echinococcosis is described. Since January 1985 each serum sample submitted for the detection of specific antibodies was examined with both an ELISA using Echinococcus multilocularis antigen (EmELISA) and an IHA using E. granulosus antigen (EgIHA). In the course of our study altogether 72 human cases of Echinococcus infections were diagnosed. All 16 cases of alveolar echinococcosis (= 100%) and 48 out of 56 cases of cystic echinococcosis (= 86%) were revealed as Echinococcus infection at least in one of the two tests. Although crude antigens were used in both, EmELISA and EglHA, species-specific diagnosis was achieved in 57 (= 89%) of 64 cases of the infections with E. multilocularis or E. granulosus. The diagnostic value of EmELISA and EglHA for the post-operative surveillance is demonstrated by the follow-up of the immune response of one case of alveolar and three cases of cystic echinococcosis.

Demonstration of a specific echinococcus multilocularis antigen in the supernatant of in vitro maintained protoscolices

Serodiagnosis of echinococcosis is still often met with difficulties resulting from unspecific reactions due to crude antigens which contain numerous host-derived proteins. In order to eliminate host protein contamination we produced Echinococcus multilocularis antigen by methods of in vitro technique: Evaginated protoscolices of Echinococcus multilocularis isolated from experimentally infected Mongolian gerbils ( Meriones unguiculatus) were maintained in RPMI 1640 medium. Although no serum proteins were added, protoscolices could be kept alive for more than 2 weeks. The supernatants harvested from protoscolices cultures were tested for their immunoreactivity against sera of patients with confirmed alveolar or cystic echinococcosis, cysticercosis, schistosomiasis or fascioliasis by means of SDS-PAGE and immunoblotting. A polypeptide band at about 62,000 mol. mass was identified which proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis, whereas sera from patients with cystic echinococcosis or with other helminthic infections as well as sera from healthy blood-donors did not react with this polypeptide band. Thus, the use of supernatant antigen in immunoblotting technique allows a serological differentiation between infections with Echinococcus multilocularis and those with Echinococcus granulosus and provides an accurate diagnosis of alveolar echinococcosis.

Echinococcus multilocularis: Immunoglobulin and antibody response in [formula omitted] mice

Each of 50 male C57BL/6J mice was infected intraperitoneally with 50 cysts of Echinococcus multilocularis. At 2, 4, 6, 8, and 14 weeks after infection, 10 mice were sacrificed, their larval cyst masses weighed, and their sera collected. Each serum sample from uninfected control and infected mice was adsorbed twice with two batches of E. multilocularis antigen conjugated to Sepharose beads. The concentrations of IgG1, IgG2a, IgG2b, IgM, and IgA in unadsorbed and IgG1, IgG2b, and IgM in adsorbed sera were quantified by the radial immunodiffusion technique. Hydatid mice produced increasingly large amounts of IgG1 and IgM; small measurable increases of IgG2b and no significant increases of IgG2a and IgA were observed during the course of infection. During the rapid growth phase of the cysts (6 to 14 weeks) IgG1 antibodies were found to range from 86 to 93% and IgM antibodies from 17 to 33% of the total IgG1 and IgM. However, the actual protein concentrations of IgM antibodies (761 and 1215 mg/dl) were higher than the sum of the protein concentrations of IgG1 and IgG2b antibodies (411 and 779 mg/dl). The significance of the relative concentrations of IgM, IgG1, IgG2a, and IgG2b antibodies is discussed with reference to their effectiveness in antibody-dependent cellular cytotoxicity and complement-mediated lysis in the control of alveolar hydatid disease.

Studies on Echinococcosis: Nonspecific Serologic Reactions of Hydatid-Fluid Antigen with Serum of Patients Ill with Diseases Other Than Echinococcosis

Summary Sera from 129 patients ill with liver cirrhosis, nephrosis, lupus erythematosus, multiple myeloma and diseases other than echinococcosis were tested with the hemagglutination test and flocculation tests. In the entire sample nine sera (7%) were positive by hemagglutination and 11 sera (8.5%) by flocculation; however, 36 of 129 (28%) showed some degree of serologic reactivity. In the cirrhosis sample, 17 of 35 sera (49%) were serologically reactive and in the other diseases from 10–25% of the sera showed reactivity. Analysis of some of the sera by paper electrophoresis showed no correlation between A/G ratios and reactive and negative sera. Analysis of Echinococcus granulosus antigens of porcine origin and E. multilocularis antigens from cotton rats revealed the presence of host protein in these antigens. Studies by agar double diffusion methods revealed that E. granulosus and E. multilocularis antigens have common antigens with host liver. Serologic activity with echinococcus antigens in patients with liver disease is believed to be due to the presence of autoantibodies which cross-react with host protein in the hydatid fluid antigen.