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PubMed2005

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in gerbils: modified lymphocyte responses due to the parasite antigen.

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in Mongolian gerbils were investigated. Gerbils not treated with prednisolone expelled most of the parasites within 3 days post-infection and induced parasite-specific intestinal IgA secretion after the oral inoculation with protoscoleces. In contrast, prednisolone-treated gerbils harbored notable numbers of parasites, and the parasite-specific intestinal IgA secretion was lower. In gerbils not treated and orally inoculated with protoscoleces (infected group), parasite-specific antibody levels in sera and intestinal washings were elevated, but blastogenesis against protoscolex antigens was observed only in cells from Peyer's patches at 14 days post-infection. Concanavalin A-induced proliferative lymphocytes from both infected and naive gerbils were suppressed by adding protoscolex somatic antigen. These data indicate that while parasite-specific antibody responses were observed, lymphocyte proliferative responses were suppressed by parasite infection/antigens in the Mongolian gerbil, prednisolone-untreated rodent definitive host model.

Administration, Oral↗
PubMed1999

Cytokine and chemokine secretion by human peripheral blood cells in response to viable Echinococcus multilocularis metacestode vesicles.

In human alveolar echinococcosis, asexually proliferating metacestodes of Echinococcus multilocularis progressively infiltrate host tissues and cause serious pathology to the affected organs. This study employed an in vitro culture of E. multilocularis and examined the production of cytokines and chemokines by peripheral blood cells from echinococcosis patients in response to viable proliferating E. multilocularis metacestode vesicles (Em-vesicles). A significant interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production was elicited in echinococcosis patients when their cells were cocultured with viable Em-vesicles and autologous immune sera. Furthermore, in echinococcosis patients, substantial amounts of cytokines were detected; and the levels of IL-12 and IL-13 found in patients correlated with the actual state of clinical disease. These observations suggest that viable E. multilocularis vesicles will induce significant cellular production of cytokines and chemokines in patients, and that such immune mediators may activate and enhance antibody-dependent cellular effector mechanism against proliferating metacestodes of E. multilocularis.

Adult↗
PubMed1991

Structure of the Echinococcus multilocularis U1 snRNA gene repeat.

The gene encoding U1 snRNA in Echinococcus multilocularis has been cloned and sequenced. This gene is contained within a 1300-bp sequence which is tandemly repeated in the E. multilocularis genome. E. multilocularis U1 snRNA is 50-70% homologous to U1 snRNAs of other species. E. multilocularis U1 snRNA could assume a predicted secondary structure similar to that proposed for other U1 snRNAs, and appears shorter (157 bases) than the U1 snRNAs of higher eukaryotes (163-166 bases).

Amino Acid Sequence↗
PubMed2001

Immunisation with Salmonella typhimurium-delivered glyceraldehyde-3-phosphate dehydrogenase protects mice against challenge infection with Echinococcus multilocularis eggs.

Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.

Amino Acid Sequence↗
PubMed1993

Echinococcus multilocularis: molecular and immunochemical characterization of diagnostic antigen II/3-10.

A recombinant Echinococcus multilocularis antigen (II/3-10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3-10 antibodies identified the corresponding epitopes on bands with identical M(r) in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3-10 antibodies reacted with antigens of different M(r) from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

Amino Acid Sequence↗
PubMed2001

A major Echinococcus multilocularis antigen is a mucin-type glycoprotein.

The metacestode of Echinococcus multilocularis is surrounded by a carbohydrate-rich laminated layer, which plays a key role in the establishment of the infection in the mammalian host. A major component of the laminated layer is an antigen referred to as Em2(G11). This highly species-specific antigen has been used for serodiagnoses of alveolar echinococcosis and is suggested to contain carbohydrates as major constituents. The results of this work have shown that immunoaffinity-purified Em2(G11) subjected to size-exclusion chromatography eluted mainly in the void volume, indicating a high molecular weight structure of this antigen. Amino acid analysis revealed a large proportion of threonine and proline residues in Em2(G11). The carbohydrate moiety of the antigen was found to be composed of galactose, N-acetylgalactosamine, and N-acetylglucosamine with a ratio of 2.4:1.0:0.5 as determined by gas-chromatography/mass spectrometry. An isotope tag was introduced to the beta-eliminated glycans, and an integrated mass spectrometric O-glycan profiling and sequencing approach was employed to obtain detailed sequence and linkage information of the unseparated glycoform pool. Novel glycoforms containing mucin-type core Gal1-3GalNAc and branched core structures attached to both serine and threonine residues are described. The data presented reveal that the Em2(G11) antigen is a mucin-type glycosylated protein.

Animals↗
PubMed1983

Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (cestoda).

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.

Animals↗
PubMed1995

A model for the control of Echinococcus multilocularis in France.

In some areas of France the prevalence of Echinococcus multilocularis in foxes is as high as 50%, whereas less than one in a thousand voles (principally Microtus arvalis) are infected. In these regions the control of rabies in foxes is achieved by using helicopters to spread bait containing oral vaccine in capsules. A mathematical model has been constructed in an attempt to determine if the addition of praziquantel to bait would be effective in eradicating E. multilocularis, or at least achieve a useful measure of control. It has been shown that the qualitative population dynamics of E. multilocularis are not affected by the detail of its epidemiology in the intermediate host population. The model is, however, sensitive to assumptions about the distribution and longevity of the adult worm in the definitive host. Given these assumptions, a method is provided that determines the feasibility of eradication conditional on the pre-control prevalence in foxes, or predicts the post-control prevalence if eradication is not feasible. If experiments could be designed to provide better information about the biological factors that determine the epidemiology of this parasite in the definitive host, a more reliable assessment of the feasibility of control would be achieved.

Animals↗
PubMed1991

Immune suppression induced by protoscoleces of Echinococcus multilocularis in mice. Evidence for the presence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis.

Immunoregulatory states induced by i.p. inoculation with the metazoan parasite Echinococcus multilocularis in the murine system were investigated. Proliferative responses and IL-2 production induced by Con A in spleen cells from BALB/c mice were significantly depressed at an early stage after infection with E. multilocularis protoscoleces (PSC). Addition of plastic-adherent cells from normal syngeneic mice to the nonadherent spleen cells from infected mice did not restore the depressed Con A responsiveness. On the other hand, exogenous IL-2 reconstituted completely the proliferative responses to Con A. Flow cytometry analysis revealed that CD4- CD8+ cells with a low density of CD8 Ag (CD8dull cells) increased in spleens from infected mice 2 weeks after inoculation. Addition of the spleen cell subpopulation containing the CD8dull cells, but not that depleted of the CD8dull cells, to normal spleen cells resulted in marked suppression of the Con A responses. These findings suggest that the CD8dull cells detected in spleens of mice inoculated with E. multilocularis PSC may play a key role in the suppressive regulation of immune responses. The relevance of the immune suppression seen in the early stages of experimental infection with E. multilocularis PSC to the eventual establishment of a host-parasite relationship is discussed.

Animals↗
PubMed2005

Detection of Echinococcus multilocularis in wild boars in France using PCR techniques against larval form.

Recently, new data have been collected on the distribution and ecology of Echinococcus multilocularis in European countries. Different ungulates species such as pig, goat, sheep, cattle and horse are known to host incomplete development of larval E. multilocularis. We report a case of E. multilocularis portage in two wild boars from a high endemic area in France (Department of Jura). Histological examination was performed and the DNA was isolated from hepatic lesions then amplified by using three PCR methods in two distinct institutes. Molecular characterisation of PCR products revealed 99% nucleotide sequence homology with the specific sequence of the U1 sn RNA gene of E. multilocularis, 99 and 99.9% nucleotide sequence homology with the specific sequence of the cytochrome oxydase gene of Echinococcus genus and 99.9% nucleotide sequence homology with a genomic DNA sequence of Echinococcus genus for the first and the second wild boar, respectively.

Animals↗
PubMed2004

Processes leading to a spatial aggregation of Echinococcus multilocularis in its natural intermediate host Microtus arvalis.

The small fox tapeworm (Echinococcus multilocularis) shows a heterogeneous spatial distribution in the intermediate host (Microtus arvalis). To identify the ecological processes responsible for this heterogeneity, we developed a spatially explicit simulation model. The model combines individual-based (foxes, Vulpes vulpes) and grid-based (voles) techniques to simulate the infections in both intermediate and definite host. If host populations are homogeneously mixed, the model reproduces field data for parasite prevalence only for a limited number of parameter combinations. As ecological parameters inevitably vary to a certain degree, we discarded the homogeneous mixing model as insufficient to gain insight into the ecology of the fox tapeworm cycle. We analysed five different model scenarios, each focussing on an ecological process that might be responsible for the heterogeneous spatial distribution of E. mulitlocularis in the intermediate host. Field studies revealed that the prevalence ratio between intermediate and definite host remains stable over a wide range of ecological conditions. Thus, by varying the parameters in simulation experiments, we used the robustness of the agreement between field data and model output as quality criterion for the five scenarios. Only one of the five scenarios was found to reproduce the prevalence ratio over a sufficient range of parameter combinations. In the accentuated scenario most tapeworm eggs die due to bad environmental conditions before they cause infections in the intermediate host. This scenario is supported by the known sensitivity of tapeworm eggs to high temperatures and dry conditions. The identified process is likely to lead to a heterogeneous availability of infective eggs and thus to a clumped distribution of infected intermediate hosts. In conclusion, areas with humid conditions and low temperatures must be pointed out as high risk areas for human exposure to E. multilocularis eggs as well.

Animals↗
PubMed1985

[Increased risk of infection by Echinococcus multilocularis for people in the endemic "Schwaebische Alb" region?].

The infestation by the larva of Echinococcus multilocularis, also known as alveolar echinococcosis, is the most dangerous parasitic disease of man in Middle Europe. This is due to the location in the liver but still more so because of the proliferative and infiltrating growth of the larval tissue. The basic infective cycle of the parasite is a zoonosis between foxes as final hosts and small rodents such as common voles as intermediate hosts. Man can be a false intermediate host and thus a carrier of the larval stage. The infection arises from oral ingestion of the tapeworm eggs either on wild berries or in dust. Dogs and cats are a further important source of infection. They can be facultative carriers of adult E. multilocularis and are thus able to excrete eggs or proglottids of the tapeworm. In west Germany, in particular the "Schwäbische Alb" must be regarded as an endemic region for E. multilocularis. According to the examinations of Zeyhle, the infection rate of foxes here is more than 15% on an average, in some local areas as for example in the district of Reutlingen it is over 25%. In order to estimate the danger of infection for man in hyperendemic areas the population of two villages of this district and also specially endangered occupational groups (hunters, foresters) of the whole "Schwäbische Alb" have been examined for echinococcosis by serological means. A stepless ELISA was used as screening test. Thus circa 2200 persons liable to E. multilocularis could be examined. Among the population of the district of Reutlingen nine highly suspective sera could be detected. Computer tomography of these nine persons showed only one case of Echinococcus which could be identified as E. multilocularis after operation. Presumably serological positive cases come up as a consequence of a real contact with E. multilocularis. But in most cases the larval cestode tissue might soon degenerate because man is a relatively poor intermediate host. It cannot be excluded, however, that the Echinococcus-larva could not yet be identified clinically because of its early stage. According to most cautious estimation the prevalence of E. multilocularis in this endemic area might be one per thousand, the contact frequency, however, up to one per cent.

Animals↗
PubMed1985

Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones).

Four isolates of larval Echinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2-4 degrees C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (-196 degrees C). After storage for one week the samples were rapidly thawed at +37 degrees C for approximately 3 min, washed in fresh EMEM/A (37 degrees C) and transferred into the peritoneal cavity of Meriones for viability testing. As judged by histological examinations and metacestode weights of each 24 Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at -28 degrees C, 30 min at -80 degrees C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7 degrees C min-1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65 degrees C min-1 for step 3. These results demonstrate that metacestodes of E. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.

Animals↗
PubMed2006

Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients.

Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.

Adolescent↗
PubMed2006

Echinococcus multilocularis in the red fox Vulpes vulpes from the East Carpathian region of Poland and the Slovak Republic.

The occurrence of Echinococcus multilocularis in the Poland-Slovak frontier zone of the East Carpathian region was assessed, for comparison with that in adjacent regions in both countries. A total of 392 red foxes from Poland and 427 red foxes from the Slovak Republic were examined from 2001 to 2004. Significant differences in prevalences were observed in foxes captured from the borderland and adjacent zones in both countries. The mean prevalence of E. multilocularis in the Polish borderland reached 45.7+/-18.6% and in the Slovak border 35.0+/-10.7%. In both countries, the prevalence of E. multilocularis in red foxes from adjacent districts, outside the frontier Carpathian region, was considerably lower (18.9+/-9.2% in Poland and 20.8+/-9.0% in Slovakia). These differences are probably due to geomorphological and ecological factors, which contribute to the survival of the tapeworm eggs and the subsequent spread of infection. The Carpathian regions of northeast Slovakia and southeast Poland are characterized by specific climatic conditions such as low mean annual air temperatures, low temperatures in active soil surfaces, high soil humidities and a high mean annual rainfall.

Animals↗
PubMed1993

Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification.

In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol-chloroform extraction and a purification step on a matrix (Prep-A-Gene). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.

Animals↗
PubMed1993

Molecular cloning of an echinococcal microtrichal antigen immunoreactive in Echinococcus multilocularis disease.

A cDNA expression library of the larval stage of the cestode worm Echinococcus multilocularis has been established in the phage lambda ZAPII system. By immunoscreening with pooled sera from patients with alveolar echinococcosis an immunoreactive clone, termed pEM13, was isolated. EM13 was expressed using the expression vector pGEX-3X, resulting in the synthesis of a glutathione S-transferase fusion protein. 82% of the sera from 28 patients suffering from E. multilocularis disease had antibodies against EM13, whereas none of the 55 sera obtained from Echinococcus granulosus-infected patients and none of the 15 sera from patients with other helminthic infections reacted with recombinant EM13. By use of a polyclonal rabbit anti-EM13 hyperimmune serum native EM13 protein could be detected only in the protoscolices of E. multilocularis, but not in E. granulosus larvae or hydatid fluid. Immunoelectron microscopy suggested that EM13 is located in the microtriches on the surface of the larvae. In contrast, EM13 mRNA could be detected by Northern blot analysis in both E. multilocularis and E. granulosus larval RNA preparations. Nucleotide and amino acid sequence analysis of a cDNA clone coding for the corresponding antigen of E. granulosus larvae, termed EG13, revealed a 21-bp insertion, a 51-bp deletion and additional 22 nucleotide exchanges resulting in a 96.3% identity at the nucleotide sequence level and a 96.6% identity at the amino acid sequence level to the coding region of the cDNA pEM13. Cross-reactivity of the polyclonal anti-EM13 serum with the recombinant EG13 indicates a posttranscriptional regulation mechanism, resulting in an EG13 negative phenotype in E. granulosus.

Amino Acid Sequence↗