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.

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice.

BACKGROUND The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

[Lung and pleural lesions before and after implementation of a national eradication program against enzootic pneumonia and actinobacillosis as well as changes of slaughter carcass organs in slaughter pigs in Switzerland].

In a representative cross-sectional study during 12 months of the years 2008/2009 in four abattoirs in Switzerland, lung and pleura lesions as well as lesions of slaughter carcasses and organs of 34 706 pigs were studied for frequency and type of macroscopic lesions. Of the 24276 examined pigs, 91.2% of the lungs, 94.4% of the heart and 95.5% of the livers showed no macroscopically visible lesions. Pigs that were produced for a label program had significantly less bronchopneumonia and pneumonia residuals, pleuritis and liver lesions due to echinococcosis. Pigs supervised by the Swiss Pig Health Service (SGD), showed significantly less bronchopneumonia and pneumonia residuals, diffuse pleuritis, pleuritis/pericarditis and milkspots compared to the non-SGD supervised farms. Thanks to the national eradication program for enzootic pneumonia (EP) and actinobacillosis, the health-status of lungs has been considerably improved and the prevalence of pleurisy decreased considerably. The results of this study indicate a good herd health in Swiss pig production.

Taxonomy, phylogeny and molecular epidemiology of Echinococcus multilocularis: From fundamental knowledge to health ecology.

Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the most severe parasitic diseases in humans and represents one of the 17 neglected diseases prioritised by the World Health Organisation (WHO) in 2012. Considering the major medical and veterinary importance of this parasite, the phylogeny of the genus Echinococcus is of considerable importance; yet, despite numerous efforts with both mitochondrial and nuclear data, it has remained unresolved. The genus is clearly complex, and this is one of the reasons for the incomplete understanding of its taxonomy. Although taxonomic studies have recognised E. multilocularis as a separate entity from the Echinococcus granulosus complex and other members of the genus, it would be premature to draw firm conclusions about the taxonomy of the genus before the phylogeny of the whole genus is fully resolved. The recent sequencing of E. multilocularis and E. granulosus genomes opens new possibilities for performing in-depth phylogenetic analyses. In addition, whole genome data provide the possibility of inferring phylogenies based on a large number of functional genes, i.e. genes that trace the evolutionary history of adaptation in E. multilocularis and other members of the genus. Moreover, genomic data open new avenues for studying the molecular epidemiology of E. multilocularis: genotyping studies with larger panels of genetic markers allow the genetic diversity and spatial dynamics of parasites to be evaluated with greater precision. There is an urgent need for international coordination of genotyping of E. multilocularis isolates from animals and human patients. This could be fundamental for a better understanding of the transmission of alveolar echinococcosis and for designing efficient healthcare strategies.

Immunoregulation in larval Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a clinically very severe zoonotic helminthic disease, characterized by a chronic progressive hepatic damage caused by the continuous proliferation of the larval stage (metacestode) of Echinococcus multilocularis. The proliferative potential of the parasite metacestode tissue is dependent on the nature/function of the periparasitic immune-mediated processes of the host. Immune tolerance and/or down-regulation of immunity are a marked characteristic increasingly observed when disease develops towards its chronic (late) stage of infection. In this context, explorative studies have clearly shown that T regulatory (Treg) cells play an important role in modulating and orchestrating inflammatory/immune reactions in AE, yielding a largely Th2-biased response, and finally allowing thus long-term parasite survival, proliferation and maturation. AE is fatal if not treated appropriately, but the current benzimidazole chemotherapy is far from optimal, and novel options for control are needed. Future research should focus on the elucidation of the crucial immunological events that lead to anergy in AE, and focus on providing a scientific basis for the development of novel and more effective immunotherapeutical options to support cure AE by abrogating anergy, anticipating also that a combination of immuno- and chemotherapy could provide a synergistic therapeutical effect.

Activities of fenbendazole in comparison with albendazole against Echinococcus multilocularis metacestodes in vitro and in a murine infection model.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE.

Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development.

BACKGROUND The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.