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PubMed2000

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes.

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

Animals↗
PubMed1995

Echinococcus multilocularis in germany: increased awareness or spreading of a parasite?

The 'small fox tapeworm' Echinococcus multilocularis has recently become a matter of intense interest in Germany. A long-term increase of its prevalence in foxes has been noted in the well-known endemic areas in Southern Germany, and reports on the occurrence of the parasite in other parts of the country suggest that the parasite is actually much more widespread than previously thought. As nearly all of the relevant studies are published in the German language in a veterinary journal and in the hunting press, accessibility to the information is limited. Richard Lucius and Brigit Bilger here describe the situation, and discuss the possible reasons and consequences.

Journal Article↗
PubMed2005

The first report of Echinococcus multilocularis strain isolation from human in Turkey.

The adult form of the small cestode Echinococcus multilocularis (E. multilocularis) is found in carnivorous animals, especially in the fox. This cestode, which is observed in the northern hemisphere of the world, is the cause of a generally fatal, progressing disease in humans, known as "alveolar echinococcosis" (AE). The metacestodes of E. multilocularis can be experimentally developed in the Meriones unguiculatus species of rodents, and it is possible to use these metacestodes for numerous purposes, primarily for antigens required for diagnosis. It is with this aim that in this study for the first time in Turkey, E. multilocularis metacestodes were developed in Meriones unguiculatus and an indigenous strain was isolated using the surgical material from a patient diagnosed with alveolar echinococcosis.

Journal Article↗
PubMed2006

Expression and diversity of Echinococcus multilocularis AgB genes in secondarily infected mice: evaluating the influence of T-cell immune selection on antigenic variation.

The T-cell-mediated immune response exhibits a crucial function in the control of the intrahepatic proliferation of Echinococcus multilocularis larvae in mice and humans, both being natural intermediate hosts of the parasite. Antigen B (AgB), a metabolized Echinococcus spp. lipoprotein, contributes to the modulation of the T-cell immune response, and distinct sites of the corresponding AgB1, AgB3 and AgB4 genes were shown to be under positive selection pressure. Since several AgB gene variants are present in a single Echinococcus metacestode, we used secondary E. multilocularis infections in BALB/c and in athymic nude mice (devoid of T-cell responses) to analyze the effect of the cellular immune response on the expression and diversity of EmAgB1-EmAgB4 genes. We demonstrated hereby that EmAgB transcripts were less abundant in nude mice during the early phase of infection (at one month post-infection), and that EmAgB2 is simultaneously down-regulated when compared to the other three genes. A negative relationship exists between the level of transcription and diversity of EmAgB genes. Moreover, no excess of non-synonymous substitutions was found among the distinct EmAgB alleles from a single host. Together, these results pointed to the effect of purifying selection, which seemed to eliminate the detrimental AgB variants generated during the development of the metacestode within the peritoneal cavity of its intermediate host.

Animals↗
PubMed1998

Morphological changes of larval Echinococcus multilocularis in mice treated with albendazole or mebendazole.

Using DBA/2J mice, tissue homogenates of larval Echinococcus multilocularis were injected into the mesenteric veins to generate the liver infection. Mice were treated with either albendazole or mebendazole for prolonged periods to examine the morphological changes of the metacestode. Albendazole induced disorganization of both laminated and germinal layers and suppressed the maturation of vesicles. Amorphous but loosely laminated PAS-positive material was observed inside the damaged vesicles, although new vesicles slightly developed inside or outside of the damaged ones. Active proliferation of vesicles occurred after treatment with albendazole was terminated. Hydatid cysts were more severely damaged in mice treated with mebendazole and new vesicles did not develop around the damaged ones. Also, hydatid cysts reappeared after treatment with mebendazole was terminated. These results indicate that these drugs do not eliminate larval E. multilocularis in the long-term, but mebendazole has a higher suppressive effect on multivesiculation than albendazole.

Albendazole↗
PubMed2005

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats.

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.

Animal Diseases↗
PubMed2005

Echinococcus multilocularis: cloning and characterization of a member of the SNW/SKIP family of transcriptional coregulators.

We have isolated a cDNA from the fox-tapeworm Echinococcus multilocularis that encodes EmSkip, a novel member of the SNW/SKIP family of transcriptional coregulators. EmSkip displays significant amino acid sequence homologies to already known members of the protein family and contains all the characteristic amino acid residues at their corresponding positions. RT-PCR experiments showed that the EmSkip encoding gene, emskip, is expressed in the Echinococcus larval stages metacestode and protoscolex during an infection of the intermediate host. By yeast two-hybrid analyses, EmSkip was found to be capable of forming homodimers in vivo. Furthermore, EmSkip was found to interact with EmSmadA and EmSmadB, two previously identified TGF-beta/BMP signal transducers of E. multilocularis, indicating a role of this protein in TGF-beta signaling processes in the parasite. In view of the role played by SNW/SKIP proteins in splicing mechanisms and intracellular signaling, the data presented herein should facilitate the identification of Echinococcus factors involved in such processes.

Amino Acid Sequence↗
PubMed1995

Effects of Isoprinosine on Echinococcus multilocularis and E. granulosus metacestodes.

An in vivo study of the effect of Isoprinosine on Echinococcus multilocularis and E. granulosus metacestodes (Cestoda) was performed. Short- and long-term treatments with different doses were tested in experimental hosts: jirds for E. multilocularis and mice for E. granulosus. Modifications in the weight of animals as well as macroscopical and ultrastructural aspects were registered for each animal group. The results obtained showed a considerable ultrastructural alteration in E. multilocularis metacestodes after short-term treatment with the highest dose, and the tissue transplants were negative. In E. granulosus cysts the ultrastructural damage was also very important and its severity increased as the dose was raised. For the two parasites, the macroscopical aspect and weight of the lesions were profoundly changed.

Abdominal Muscles↗
PubMed2003

Statistics and sample design in epidemiological studies of Echinococcus multilocularis in fox populations.

In this paper possible sampling strategies for estimating the prevalence of Echinococcus multilocularis infections in foxes are discussed. To draw valid conclusions from the analysis of fractions of a total fox population, each member of the total population must have the same chance of being selected for the investigation (random sampling), the sample must be representative with respect to all epidemiologically relevant conditions in the population (e.g. age, endemic status, seasonal effects, population density), and it must be large enough to obtain results with the required precision. For detection/exclusion of infections at a pre-specified prevalence threshold and confidence level (e.g. 99%), the required sample is rather small, but the information obtained from the data is limited. For prevalence estimates, the required sample sizes depend on the expected prevalence, the desired precision of the estimate and the chosen confidence level (e.g. 90, 95, or 99%). The samples need to be taken in spatial units where the variation of the conditions potentially influencing the infection can be neglected. A first impression of the spatial distribution of E. multilocularis infections in foxes can also be obtained by mapping the investigated sample (infected and uninfected animals) using the municipalities where they were shot or found as a spatial grid. To analyse the local influence of environmental factors, data on the geographical positions where the animals were sampled need to be collected and analysed in the context of a Geographic Information System (GIS).

Age Factors↗
PubMed1992

Synthesis and in vitro activity of alkylaminoalkyloxy chromones on protoscoleces of Echinococcus multilocularis (Cestoda).

Twelve derivatives with alkylaminoalkyloxy chromone structures were synthesized and tested upon protoscoleces of Echinococcus multilocularis metacestode kept alive in vitro. Assays were performed with protoscoleces attached to the germinal layer in open and in closed vesicles. Compounds IVb and IIIe at the concentration of 0.1 mmol.L-1 killed 50% of the protoscoleces in open vesicles in 48 hours and compound IVb killed 100% of the protoscoleces in open vesicles within 96 hours at the same concentration. In closed vesicles after four days compound IVb killed all protoscoleces, compounds IIIe and IVd half of them, whereas in the controls all protoscoleces were alive. Trifluoperazine (TFP) was the reference compound; none of the new compounds showed better activity than TFP.

Animals↗
PubMed1994

Seroepidemiologic screening of Echinococcus multilocularis infection in a European area endemic for alveolar echinococcosis.

In a serologic survey for Echinococcus multilocularis infection, we screened sera from 7,884 subjects from the Doubs Departement in France, an area endemic for alveolar echinococcosis (AE) of the liver. An enzyme-linked immunosorbent assay (ELISA) with a highly species-specific antigen (Em2) and an E. multilocularis crude antigen (Emc) was used for screening. An evaluation of the cost/benefit relationship of this screening, followed by therapeutic management of patients, was made and compared with the actual cost of the follow-up and treatment of the disease in symptomatic cases in this endemic area. Antibody reactions to Em2 and/or Emc made possible the detection of eight asymptomatic clinical cases (seroprevalence averaging 1/1,000), with typical lesions of active AE revealed by abdominal ultrasonography and computed tomography. All were seropositive using the Emc ELISA but two were seronegative using the Em2 ELISA. In five additional seropositive cases, the radiologic investigations revealed small calcified lesions similar to the lesions of abortive AE previously found in Alaska. The cost of this serologic screening program per screened subject and per diagnosed case averaged 50.00 French Francs (FF) (U.S. $8.60) and 60,000.00 FF (U.S. $10,909.00), respectively. The cost of diagnosis, follow-up and treatment of the patients was 5,086.00 FF (U.S. $929.00) per patient per month in the case of diseases diagnosed by the screening program and 7,086.00 FF (U.S. $1,288.00) per patient per month for patients with symptomatic AE. This survey indicates a high prevalence of AE in the target area; it confirms the long latency period of the larval growth in human AE and shows that abortive AE is present in Europe. The use of both the Emc and Em2 ELISAs seems to be better than using the Em2 ELISA alone. The cost of the hospitalization and treatment of the eight screened patients would appear to be relatively high. Even though two of them were asymptomatic, they had very severe forms of the disease. In fact, the total cost was much lower than the actual cost of the disease when diagnosed from clinical symptoms.

Adolescent↗
PubMed2006

Implications of increased susceptibility to predation for managing the sylvatic cycle of Echinococcus multilocularis.

The ability to increase the chances that infectious prey are taken by predators is an observed feature of many parasites that rely on one or more predator-prey relationships to complete their life-cycle. In the sylvatic life-cycle of Echinococcus multilocularis - the causative agent of human alveolar echinococcosis-- foxes are the final host, with voles acting as intermediate hosts. Here we review the evidence that E. multilocularis causes increased susceptibility to predation and present a general mathematical model for the sylvatic life-cycle. The ability to increase susceptibility to predation in the intermediate host reduces the sensitivity of the parasite population to adverse conditions. For example, there is no critical density of foxes below which the parasite is expected to die out, even if the effect of the parasite on infected prey is very small. We suggest that increased susceptibility to predation is a plausible explanation for the observed resilience of E. multilocularis during and following field trials of praziquantel baiting.

Animals↗
PubMed2006

In vitro activities of itraconazole, methiazole, and nitazoxanide versus Echinococcus multilocularis larvae.

Albendazole (ABZ) and mebendazole are the only drugs licensed for treatment of human alveolar echinococcosis. In order to augment the armamentarium against this deadly disease, we tested a series of drugs for their efficacy against Echinococcus multilocularis larvae. E. multilocularis larvae grown intraperitoneally in Mongolian gerbils were transferred into tissue culture. Vesicles budded from the tissue blocks and after 6 weeks, drugs were added, and the effect on the vesicles was observed. We tested the following drugs at various concentrations: ABZ, artemether, caspofungin, itraconazole (ITZ), ivermectin, methiazole (MTZ), miltefosine, nitazoxanide (NTZ), rifampin, and trimethoprim-sulfamethoxazole. ABZ, ITZ, MTZ, and NTZ effectively destroyed parasite vesicles in this in vitro culture system. At high NTZ doses of 10 microg/ml, disintegration of all vesicles was observed after 7 days and was significantly more rapid than with ABZ at equal concentrations (21 days). After drug discontinuation, regrowth of vesicles occurred between 7 and 14 days for all four drugs, indicating a parasitostatic effect. Combination treatment with NTZ-ABZ at concentrations between 1 and 10 microg/ml for either 3 weeks, 3 months, or 6 months yielded no vesicle regrowth during 8 months after drug discontinuation. The treated larval tissue was injected intraperitoneally into gerbils, and no regrowth of larval tissue was observed, suggesting a parasitocidal effect after combined treatment. ITZ, MTZ, and NTZ are potent inhibitors of larval growth, although they proved to be parasitostatic only. The combination of NTZ plus ABZ was parasitocidal in vitro. Animal experiments are warranted for studies of dose, toxicity, and drug interactions.

Animals↗
PubMed1998

[Comparative observation on experimental infection with Echinococcus multilocularis in Cricetulus Migratorius and Meriones meridianus].

AIM: To search for an animal model with higher susceptibility to Echinococcus multilocularis and shorter experimental period. METHODS: Cricetulus migratorius, Meriones meridianus and NIH mice were experimentally infected with protoscolices of E. multilocularis and were autopsed at different times after their infection. The development of E. multilocularis in these three species of rodents was observed and compared. RESULTS: The average wet cyst weight and the ratio of cyst weight to body weight on the 91st day after infection were 15.0 +/- 2.1 g and 32.3% for C. migratorius, 3.3 +/- 1.1 g and 6.9% for M. meridianus, 0.6 +/- 0.6 g and 2.4% for NIH mice; and the corresponding figures on the 188th day after infection were 11.8 +/- 2.7 g and 21.7% for C. migratorius, 8.1 +/- 5.1 g and 15.4% for M. meridianus and 8.4 +/- 8.3 g and 26.7% for NIH mice, respectively. On the 91st day, matured protoscolices were found in C. migratorius and M. meridianus, whereas NIH mice had matured protoscolices on the 188th day after infection. CONCLUSION: C. migratorius has higher sensitivity to E. multilocularis infection than the other two species of animals as manifested by high infection rate, well-developed vesicles and rapid growth speed. C. migratorius proved to be an ideal experimental animal for E. multilocularis.

Animals↗
PubMed1990

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.

Animals↗
PubMed2003

Predictive values and quality control of techniques for the diagnosis of Echinococcus multilocularis in definitive hosts.

By the end of the 1980s the only reliable technique for diagnosing the intestinal Echinococcus multilocularis infection of definitive hosts was parasite detection at necropsy. Currently, several techniques for the post mortem and in vivo diagnosis are available, including classical and modern methods. The sedimentation and counting technique (SCT) is used for the exact determination of the worm burden in the intestine after necropsy. The SCT has high sensitivity and specificity values (both close to 100%) and can be regarded as 'gold standard'. The principle of the intestinal scraping technique (IST) is the stereomicroscopic examination of at least 15 intestinal thick smears. This technique has a sensitivity of 78% and a specificity approximating 100%. In recent years, the IST has been successfully used in large post mortem surveys of foxes for E. multilocularis. The newer techniques for detecting coproantigens by ELISA (CA-ELISA) exhibit rather high sensitivities between 84 and 95%, combined with very high specificities of >96%, the latter regarding non-Echinococcus cestodes and other parasites. However, cross-reactivity may occur with E. granulosus. Copro-DNA detection by PCR is also highly sensitive (89-94%) and specific (100%). With the SCT, IST and Copro-PCR one person can only examine about 10-20 animals per day, whereas the CA-ELISA allows the examination of 200 samples. Therefore, the latter test is suited for mass-screening of definitive host populations. Both the CA-ELISA and the Copro-PCR allow the examination of materials from dead and living animals, including faecal samples collected in the field. Quite often diagnostic techniques have been used without adequate quality control and proper definition of their performance characteristics, including diagnostic sensitivity, specificity, predictive values and some other parameters. Examples are presented with the aim to demonstrate the need and the value of calculating the predictive values for assays used to diagnose the E. multilocularis infection in individual animals and in definitive host populations.

Animals↗
PubMed1988

Production of a recombinant antigen of Echinococcus multilocularis with high immunodiagnostic sensitivity and specificity.

A cDNA library derived from mRNA of metacestodes from Echinococcus multilocularis was constructed in the Escherichia coli expression vector lambda gt11 and screened with human patients' sera. The recombinant proteins of 11 positive phages were further evaluated for their potential as immunodiagnostic reagent. One isolated clone (from lysogen II/3) synthesized a fusion protein which was rapidly degraded. This intracellular degradation process provided two distinct polypeptides with Mr of about 31,000 and 33,000, both showing strong binding activity with specific patients' antibodies. The diagnostic value of the II/3-antigen was evaluated by mini-Western-blot with sera from 41 patients infected with E. multilocularis and sera from 77 patients with other helminthic infections, resulting in a diagnostic sensitivity of 98% and an over-all specificity of 96%. These results suggest the usefulness of the recombinant II/3-antigen for immunodiagnosis of human alveolar echinococcosis.

Animals↗