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Purification and characterization of a specific antigen from Echinococcus multilocularis.

A polypeptide (Em2a) purified by affinity chromatography from the Echinococcus multilocularis metacestode showed a high degree of purity as assayed by SDS-PAGE and analytical isoelectrical focusing. A minor contamination with host albumin was revealed. Estimation of relative mol. mass gave a value of 54,000. The isoelectric point was found to be 4.8. Antigenic activity of the polypeptide was demonstrated by immunoprecipitation and western blotting. In these assays the protein was recognized only by homologous sera from patients infected with larval E. multilocularis. This antigen (Em2a) did not react in the ELISA with sera from patients infected with heterologous helminths; these sera were highly cross-reacting with antigen from E. granulosus hydatid fluid. Seventy-three (94%) from 78 investigated patients (alveolar echinococcosis) showed a seropositive reaction with the polypeptide Em2a.

Animals↗

Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes.

It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT-PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

14-3-3 Proteins↗

Echinococcus multilocularis: an emerging pathogen in Hungary and Central Eastern Europe?

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is reported for the first time in Red Foxes (Vulpes vulpes) in Hungary. This parasite may be spreading eastward because the population of foxes has increased as a consequence of human interventions, and this spread may result in the emergence of alveolar echinococcosis in Central Eastern Europe.

Animals↗

Comparison of suitability of dogs and cats as hosts of Echinococcus multilocularis.

The suitability of dogs and cats as hosts of Echinococcus multilocularis was studied in animals which had each been infected orally with approximately 15,000 protoscolices. Twenty-one days after injection, the mean number of immature worms recovered from 12 dogs was 874.7 and from 12 cats was 101.7. The mean length of the worms was 1.21 mm in dogs and 1.00 mm in cats. The number and length of worms recovered from dogs were significantly greater (alpha = 0.01).

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Analysis of cDNAs coding for immunologically dominant antigens from an oncosphere-specific cDNA library of Echinococcus multilocularis.

A cDNA library based on mRNA from oncospheres of Echinococcus multilocularis was constructed and screened with an oncosphere-specific rabbit serum. cDNA sequences of three clones that were isolated out of this library are discussed: one codes for a serpin-like proteinase inhibitor, the first isolated from cestodes. Two other clones code for dominant oncosphere antigens and represent homologues of known genes: one is known from several taeniid cestodes as a protective antigen containing fibronectin III domains, the second is related to genes of small heat shock proteins. It contains an internal duplication that might be specific for platyhelminths.

Amino Acid Sequence↗

Interleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in murine lymphocytes.

Mitogenic effects of protoscoleces (PSCs) of Echinococcus multilocularis on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when cocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete interleukin-1 (IL-1). Depletion of plastic- and Sephadex G-10-adherent cells from the spleen cell population significantly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic-adherent cells to these cultures. Furthermore, addition of anti-IL-1 serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on IL-1 secreted by cells of macrophage/monocyte lineage.

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High oral doses of mebendazole interfere with growth of larval Echinococcus multilocularis lesions.

The natural development of the larval stage of Echinococcus multilocularis in man has been studied in 7 patients after presumed radical operation 2 8/12-11 2/12 years prior to detection of a relapse. The volumes of the recurring lesions were assessed by CT-scanning, and assuming linear growth a median increase of 14.8 ml/year (range 3.8-220 ml/year) was calculated. In 6 patients treated for a median duration of 4 5/12 years with high oral doses of mebendazole a median growth rate of -3.0 ml/year (range-470- + 10.2 ml/year) was found, which differed significantly from the natural growth rate (P less than 0.01). Although the patients improved clinically, there was evidence of persistent infection. These data are the first controlled evidence that high oral doses of mebendazole may be parasitostatic in alveolar echinococcosis in man. Although not curative, this pharmacological effect appears to be clinically beneficial.

Adult↗

Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction.

The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic DNA of E. multilocularis, while BG1 and BG3 define a 0.3 kb fragment. A PCR study including 14 independent E. multilocularis isolates in addition to Echinococcus granulosus. Echinococcus vogeli, Taenia spp. and other cestodes revealed that the 2.6-kb fragment was amplified from genomic DNA of all E. multilocularis isolates tested (originating from Switzerland, Alaska, Canada, France, Germany and Japan), but from genomic DNA of none of the other cestode species. PCR with BG1 and BG2 furthermore uniquely resulted in the synthesis of a 0.55-kb fragment specific for Taenia saginata and a 0.6-kb fragment specific for T. taeniaeformis. In contrast to the species specificity of the 2.6-kb BG1/BG2 product, the 0.3 kb (BG1/BG3) product demonstrated genus specificity: the 0.3-kb product was amplified from genomic DNA of all E. multilocularis, E. granulosus and E. vogeli isolates tested, but from genomic DNA of none of the other cestode species. The diagnostic sensitivity of PCR using both primer sets was determined to be 50 pg parasite DNA, suggesting the practical utility of this simple assay in demonstrating parasite DNA in specimens from a variety of sources. At the basic level, the pAL1-derived oligonucleotides may also prove useful in assessing strain variation, RFLPs or other manifestations of genetic variation in E. multilocularis.

Animals↗

Effects of albendazole on Echinococcus multilocularis infection in the Mongolian jird.

Albendazole chemotherapy of larval Echinococcus multilocularis was studied in the Mongolian jird by administration of medicated feed at various concentrations and durations. The effects were evaluated by comparison of treated and control groups in terms of host mortality, larval metastases to the lungs, and final weight and histologic appearance of larval tissue. Viability of larval tissue at necropsy of each animal was tested by inoculation into two noninfected jirds. Albendazole-medicated feed (0.05%-0.10%) significantly inhibited larval growth. Other effects of the drug included larval degeneration and necrosis, inhibition of protoscolex formation, decreased pulmonary metastases, and reduced mortality of hosts. Adverse effects on the parasite correlated significantly with serum albendazole metabolite levels and duration of therapy. However, serum albendazole levels in jirds equal to or exceeding concentrations achieved in humans receiving daily doses of 10 mg/kg of body weight did not kill the parasite.

Albendazole↗

Time-course of antibody response in mice against oral infection with eggs of Echinococcus multilocularis.

The kinetics of serum antibody response against infection with Echinococcus multilocularis eggs was evaluated in AKR mice. The animals were infected by oral inoculation with 300 parasite eggs, and necropsied at 1, 2, 4, 6, 9, 12 and 16 weeks post-infection (p.i.), respectively. The parasite formed the laminated layer at 4 weeks p.i., the brood capsule with a massive proliferation of germinal cells at 9 weeks p.i. and protoscoleces at 16 weeks p.i. Serum antibody responses of the mice to antigen preparations from metacestodes of different stages and protoscoleces were evaluated by ELISA, immunoblotting and immunohistochemistry. In ELISA, the antibody responses began to increase at 4 weeks and became more apparent at 9 weeks p.i. and thereafter. Immunoblots using sera collected at 16 weeks p.i. showed some common bands among the 3 different antigen preparations. In addition to this, the germinal cells and brood capsules of mature metacestodes were stained strongly in an immunohistochemical study. From above, it is suggested that some antigen molecules are expressed in the parasite through these stages and stimulated host antibody responses.

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Protective immune mechanisms against the metacestode of Echinococcus multilocularis.

Infection with the larval stage of the fox tapeworm Echinococcus multilocularis results in a life-threatening hepatic disease concerning humans and intermediate rodent hosts. Immunoepidemiological surveys provided information that a large proportion of infected individuals may demonstrate either constitutional resistance to early post-oncospheral development of the parasite or late resistance to disease by exhibiting an intrahepatic died-out parasite lesion. Similar events have been found in secondary infections of laboratory rodents. Dissection of humoral and cell-mediated immune responses in susceptible versus resistant individuals provides insight into immunological pathways associated with the different outcome of infection. Survival strategy of the metacestode obviously focuses on the crucial role played by the parasite laminated layer. This layer protects the metacestode from host effector mechanisms which can potentially kill the proliferating germinative compartments in case of resistant hosts. Bruno Gottstein and Richard Felleisen here discuss the need to search for more parameters discriminating between the different immune pathways in order to find out (immunogenetic?) predispositions responsible for the respective phenomena.

Journal Article↗

Synthetic studies on glycosphingolipids from Protostomia phyla: total syntheses of glycosphingolipids from the parasite, Echinococcus multilocularis.

Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with benzoyl ceramide in the presence of N-iodosuccinimide (NIS)/TfOH, which gave the desired oligosaccharide derivatives. The fully protected glycosides 13, 20, 22 and 25 were deprotected to give four target glycosphingolipids (1-4).

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Spatial spreading of Echinococcus multilocularis in Red foxes (Vulpes vulpes) across nation borders in Western Europe.

The occurrence of the fox tapeworm Echinococcus multilocularis in Red foxes was studied in Belgium and a neighbouring region in The Netherlands. A total number of 1202 foxes were analysed (1018 in Belgium and 184 in The Netherlands) of which 179 were infected with E. multilocularis (164 in Belgium and 15 in The Netherlands). Further, the spatial distribution of infection among sampled foxes was analysed with an ellipsoidal gradient, demonstrating a decreasing prevalence in northwestern direction. Using this gradient, we showed that the spatial patterns of infection in Belgium and the neighbouring region in The Netherlands correspond, indicating a continuous distribution of E. multilocularis across the nation borders. Part of the Belgian data allowed investigating temporal changes in the spatial distribution of E. multilocularis. This revealed a northwestern spread of E. multilocularis.

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Echinococcus multilocularis in north Italy.

Alveolar echinococcosis is a zoonotic infection caused by the metacestode of the tapeworm Echinococcus multilocularis. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes were detected in Bolzano province (Trentino Alto Adige region) near Austrian border. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples belonging to red fox populations from five Italian regions. A total of 522 faecal samples were analysed from foxes shot in Valle d'Aosta (N = 65), Liguria (N = 44), Lombardy (N = 105), Veneto (N = 67), and Trentino Alto Adige (N = 241) regions. Among these, 24 samples, all from the Trentino Alto Adige region, were found positive. Moreoever, 1406 faecal samples of red foxes were analyzed by CA-ELISAs commercial test kit. This paper provides an update of the epidemiological knowledge of this parasite in north Italy.

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The ability of 67Ga scintigraphy to detect the lesions of Echinococcus multilocularis infection: preliminary results.

AIM: To assess the ability of 67Ga scintigraphy to detect the lesions of Echinococcus multilocularis (EM) infection. MATERIALS AND METHODS: An animal model of EM infection was developed. The infected tissues taken from stock infection were placed into the abdominal cavity of uninfected animals operatively. The success of implantation was controlled 20-25 days after implantation. Five infected and 2 healthy animals were studied. All of the animals were examined by ultrasound before the scintigraphic evaluation. After the injection of 7.4 MBq (200 microCi) 67Ga citrate intravenously, static images from the whole anterior thorax and abdomen were obtained at 24, 48 and 72 hours. Visual and semiquantitative analyses were performed. In semiquantitative analysis, an irregular region of interest was drawn over the thorax as the background, excluding the heart and a second region of interest was drawn over the abdomen, excluding the liver and spleen. Abdomen/ background ratios were calculated using the mean counts. RESULTS: In the visual evaluation, it was noticed that there was considerably increased 67Ga uptake in the abdomens of the infected animals. In infected animals, mean abdomen/background ratios at 48 and 72 hours (3.76 +/- 1.04, 4.13 +/- 0.72, respectively) were increased compared with mean abdomen/background ratios at 24 hours (2.94 +/- 0.77). These increases in abdomen/background ratios were statistically significant at 72 hours (p = 0.04). Between the infected animals and control group, mean abdomen/background ratios were compared, and statistically significant differences were found in the images obtained at 48 and 72 hours. CONCLUSION: Imaging at 72 hours seems to be more suitable imaging time for the diagnosis of alveolar echinococcosis. 67Ga scintigraphy may successfully demonstrate the lesions of EM infection localized intraperitoneally. The method of 67Ga scintigraphy is useful because it is simple, non-invasive and relatively safe.

Animals↗

Spatial modelling and ecology of Echinococcus multilocularis transmission in China.

Recent research in central China has suggested that the most likely transmission mechanism for Echinococcus multilocularis to humans is via domestic dogs which are allowed to roam freely and hunt (infected) small mammals within areas close to villages or in areas of tented pasture. This assertion has led to the hypothesis that there is a landscape control on transmission risk since the proximity of suitable habitat for susceptible small mammals appears to be the key. We have tested this hypothesis in a number of endemic areas in China, notably south Gansu Province and the Tibetan region of western Sichuan Province. The fundamental landscape control is its effect at a regional scale on small mammal species assemblages (susceptible species are not ubiquitous) and, at a local scale, the spatial distributions of small mammal populations. To date the research has examined relationships between landscape composition and patterns of human infection, landscape and small mammal distributions and recently the relationships between landscape and dog infection rates. The key tool to characterize landscape is satellite remote sensing and these data are used as inputs to drive spatial models of transmission risk. This paper reviews the progress that has been made so far in spatial modeling of the ecology of E. multilocularis with particular reference to China, outlines current research issues, and describes a framework for building a spatial-temporal model of transmission ecology.

Animals↗

Survival strategy of Echinococcus multilocularis in the human host.

As exemplified by "aborted" calcified liver lesions commonly found in patients from endemic areas, Echinococcus multilocularis metacestodes develop only in a minority of individuals exposed to infection with the papasite. Clinical research has disclosed some aspects of the survival strategy of E. multilocularis in human hosts. Clinical observations in liver transplantation and AIDS suggest that suppression of cellular/Th1-related immunity increases disease severity. Most of the studies have stressed a role for CD8+ T cells and for Interleukin-10 in the development of tolerance. A spontaneous secretion of IL-10 by the PBMC seems to be the immunological hallmark of patients with progressive forms of alveolar echinococcosis (AE). IL-10-induced inhibition of effector macrophages, but also of antigen-presenting dendritic cells, may be operating and allowing parasite growth and survival. The genetic correlates of susceptibility to infection with E. multilocularis are clearer in humans than in the mouse model. A significant link between MHC polymorphism and clinical presentation of AE has been shown, and the spontaneous secretion of IL-10 in patients with a progressive AE is higher in patients with the HLA DR3+, DQ2+ haplotype. Clustering of cases in certain families, in communities otherwise exposed to similar risk factors, also points to immuno-genetic predisposition factors that may allow the larva to escape host immunity more easily. The first stage of larval development may be crucial in producing "danger signals" stimulating the initial production of cytokines. Therapeutic use of Interferon alpha is an attempt to foil the survival strategy of E. multilocularis.

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[Echinococcus multilocularis in Grisons: distribution in foxes and presence of potential intermediate hosts].

The southern border of the European endemic area of Echinococcus multilocularis runs along the alpine crest. This endemic transition area was analysed in the canton Grisons on a small spatial scale. A total of 543 foxes originating from 10 areas north and 4 areas south of the main alpine divide were investigated. Parasites were isolated using the sedimentation and counting technique on intestinal contents. The mean prevalence of E. multilocularis was 6.4% with significant differences between different areas. In the southern valleys, only foxes from the Val Müstair were infected (14.3%). On the northern side of the main alpine divide, prevalences varied between 0 and 40%. The predation habits of foxes on potential intermediate hosts was investigated by means of stomach content analyses (n=530). Rodents of the genera Microtus/Pitymys were found in 19.6% of fox stomachs, Clethrionomys glareolus in 8.0% and Arvicola terrestris in 0.4%. A small scale analysis based on a 10 x 10 km grid suggested that the predation rate on the genera Microtus/Pitymys significantly correlated with the prevalence of E. multilocularis in foxes (Spearman's r = 0.51). Hence, E. multilocularis appears to occur in the alpine study area on a very small spatial scale. These local sources of infection may persist for decades. This may partly explains why, during the last 34 years, some human cases of alveolar echinococcosis occurred in areas of the Canton Grisons where the parasite is presently endemic in foxes. No such human cases have been recorded in other areas free of E. multilocularis.

Animals↗