Search tips
Search criteria

Results 1-25 (38)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
1.  Echinococcus multilocularis Detection in Live Eurasian Beavers (Castor fiber) Using a Combination of Laparoscopy and Abdominal Ultrasound under Field Conditions 
PLoS ONE  2015;10(7):e0130842.
Echinococcus multilocularis is an important pathogenic zoonotic parasite of health concern, though absent in the United Kingdom. Eurasian beavers (Castor fiber) may act as a rare intermediate host, and so unscreened wild caught individuals may pose a potential risk of introducing this parasite to disease-free countries through translocation programs. There is currently no single definitive ante-mortem diagnostic test in intermediate hosts. An effective non-lethal diagnostic, feasible under field condition would be helpful to minimise parasite establishment risk, where indiscriminate culling is to be avoided. This study screened live beavers (captive, n = 18 or wild-trapped in Scotland, n = 12) and beaver cadavers (wild Scotland, n = 4 or Bavaria, n = 11), for the presence of E. multilocularis. Ultrasonography in combination with minimally invasive surgical examination of the abdomen by laparoscopy was viable under field conditions for real-time evaluation in beavers. Laparoscopy alone does not allow the operator to visualize the parenchyma of organs such as the liver, or inside the lumen of the gastrointestinal tract, hence the advantage of its combination with abdominal ultrasonography. All live beavers and Scottish cadavers were largely unremarkable in their haematology and serum biochemistry with no values suspicious for liver pathology or potentially indicative of E. multilocularis infection. This correlated well with ultrasound, laparoscopy, and immunoblotting, which were unremarkable in these individuals. Two wild Bavarian individuals were suspected E. multilocularis positive at post-mortem, through the presence of hepatic cysts. Sensitivity and specificity of a combination of laparoscopy and abdominal ultrasonography in the detection of parasitic liver cyst lesions was 100% in the subset of cadavers (95%Confidence Intervals 34.24–100%, and 86.7–100% respectively). For abdominal ultrasonography alone sensitivity was only 50% (95%CI 9.5–90.6%), with specificity being 100% (95%CI 79.2–100%). For laparoscopy alone sensitivity was 100% (95% CI 34.2–100%), with specificity also being 100% (95% CI 77.2–100%). Further immunoblotting, PCR and histopathological examination revealed one individual positive for E. multilocularis, whilst the other individual was positive for Taenia martis.
PMCID: PMC4500463  PMID: 26167927
2.  Prevention and Immunotherapy of Secondary Murine Alveolar Echinococcosis Employing Recombinant EmP29 Antigen 
PLoS Neglected Tropical Diseases  2015;9(6):e0003795.
Alveolar echinococcosis (AE) is caused by infection with the larval stage of the tapeworm Echinococcus multilocularis. An increasing understanding of immunological events that account for the metacestode survival in human and murine AE infection prompted us to undertake explorative experiments tackling the potential of novel preventive and/or immunotherapeutic measures. In this study, the immunoprotective and immunotherapeutic ability of recombinant EmP29 antigen (rEmP29) was assessed in mice that were intraperitoneally infected with E. multilocularis metacestodes. For vaccination, three intraperitoneal injections with 20μg rEmP29 emulsified in saponin adjuvants were applied over 6 weeks. 2 weeks after the last boost, mice were infected, and at 90 days post-infection, rEmP29-vaccinated mice exhibited a median parasite weight that was reduced by 75% and 59% when compared to NaCl- or saponin–treated control mice, respectively. For immunotherapeutical application, the rEmP29 (20μg) vaccine was administered to experimentally infected mice, starting at 1 month post-infection, three times with 2 weeks intervals. Mice undergoing rEmP29 immunotherapy exhibited a median parasite load that was reduced by 53% and 49% when compared to NaCl- and saponin–treated control mice, respectively. Upon analysis of spleen cells, both, vaccination and treatment with rEmP29, resulted in low ratios of Th2/Th1 (IL-4/IFN-γ) cytokine mRNA and low levels of mRNA coding for IL-10 and IL-2. These results suggest that reduction of the immunosuppressive environment takes place in vaccinated as well as immunotreated mice, and a shift towards a Th1 type of immune response may be responsible for the observed increased restriction of parasite growth. The present study provides the first evidence that active immunotherapy may present a sustainable route for the control of AE.
Author Summary
Current medical management of AE that relies on surgery and continuous benzimidazole administration is of limited effectiveness. Therefore, alternative preventive and therapeutic tools need to be explored. Here, we demonstrate that vaccination with recombinant antigen EmP29 (rEmP29), prior or after secondary infection of BALB/c mice, resulted in a significant reduction of the median parasite weight when compared to different control groups. We then characterized the transcription level of splenic IL-4 and IFN-γ cytokines as hallmarks for AE-anti-protective humoral immune reaction (Th2) and for AE-effective (restrictive) cellular response (Th1), respectively. Results revealed that vaccinated mice in pre- or post-infection situation exhibited the lowest IL-4/IFN-γ mRNA ratios. In addition, those groups showed also significantly low levels of IL-10-encoding mRNA coding (immunosuppressive cytokine), as well as IL-2. These findings suggest that reduction of parasite load in rEmP29-vaccinated mice (in pre- or post-infection status) might be triggered by a decline of the immunosuppressive environment and a change of the host immune reaction towards a Th1-re-oriented cell-mediated immune defense. A similar non-specific effect appears also to be yielded by the immunostimulating adjuvants. This study provides the first insight into the potential benefits of antigen-specific immunotherapy as new treatment option of AE.
PMCID: PMC4460070  PMID: 26053794
3.  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 
PLoS Neglected Tropical Diseases  2015;9(5):e0003755.
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.
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.
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.
Author Summary
In larval E. multilocularis infection causing alveolar echinococcosis (AE) in humans as well as mice, immune tolerance and/or down-regulation of protective immunity is a marked characteristic of this chronic disease. Our study provides a comprehensive evidence for a major involvement of the recently identified CD4+ CD25+ Regulatory T Cell Effector Molecule FGL2 to the outcome of AE. Our major findings are as follows: 1) FGL2 is mostly secreted by Tregs and partly contributes to their functions; 2) FGL2 can down-regulate the maturation of DCs, suppress Th1 and Th17 immune responses, and support Th2 and Treg immune responses, and finally 3) IL-17A contributes to FGL2 secretion. Based on the present findings in mice, we will investigate FGL2 as a potential marker of progression of AE in human patients, or as a potential immunotherapeutical target. Early prediction of parasite regression (currently not yet possible) would allow clinicians to plan for withdrawing benzimidazole treatment, which is currently administered for life. Then, FGL2 should be investigated as a target for an anticipated immunomodulatory treatment of patients with progressive AE, especially of those who are non- or low-responders to benzimidazole treatment, or who suffer from side-effects due to chemotherapy.
PMCID: PMC4425495  PMID: 25955764
4.  Hepatic alveolar hydatid disease (Echinococcus multilocularis) in a boxer dog from southern Ontario 
The Canadian Veterinary Journal  2014;55(6):551-553.
A 2-year-old boxer dog from southern Ontario was evaluated because of acute onset lethargy. Exploratory laparotomy revealed a hemorrhagic, destructive, liver mass. Histology, immunohistochemistry, and polymerase chain reaction confirmed Echinococcus multilocularis as the cause of the hepatic mass. This constitutes the first description of endemic E. multilocularis in Ontario.
PMCID: PMC4022022  PMID: 24891637
5.  Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri 
BMC Genomics  2014;15(1):496.
The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media.
As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles.
This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-496) contains supplementary material, which is available to authorized users.
PMCID: PMC4082629  PMID: 24950717
Naegleria fowleri; Primary amoebic meningoencephalitis; Whole genome sequencing; RNA sequencing; Comparative proteomics; Pathogenicity factors
6.  Comparative Assessment of ELISAs Using Recombinant Saposin-Like Protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of Human Fasciolosis 
Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.
Author Summary
To improve the serodiagnosis of human fasciolosis caused by Fasciola hepatica, we comparatively evaluated the accuracy of two different enzyme-linked immunosorbent assays (ELISAs) based on the use of two published recombinant antigens. The best performance was achieved with the recombinant F. hepatica saposin-like protein-2 antigen (recSAP2). Although the F. hepatica E/S antigen exhibited a slightly higher diagnostic sensitivity, the higher specificity performance of recSAP2 renders this antigen very suitable for application in low endemic areas, especially when coupled to an easy and standardized production facility as compared to the relatively complex production procedure for an E/S antigen. Conclusively, the recSAP2-ELISA can be used as a routine individual serodiagnostic test for human fasciolosis, especially when backed up by a compatible clinical history together with other serodiagnostic technique for other helminth infections of the liver, e.g. alveolar or cystic echinococcosis.
PMCID: PMC4055579  PMID: 24922050
7.  Echinococcus P29 Antigen: Molecular Characterization and Implication on Post-Surgery Follow-Up of CE Patients Infected with Different Species of the Echinococcus granulosus Complex 
PLoS ONE  2014;9(5):e98357.
The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.
PMCID: PMC4031130  PMID: 24851904
8.  Transcriptional Profiles of Cytokine/Chemokine Factors of Immune Cell-Homing to the Parasitic Lesions: A Comprehensive One-Year Course Study in the Liver of E. multilocularis-Infected Mice 
PLoS ONE  2014;9(3):e91638.
Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.
PMCID: PMC3956718  PMID: 24637903
10.  Echinococcus metacestode: in search of viability markers 
Parasite  2014;21:63.
Epidemiological studies have demonstrated that most humans infected with Echinococcus spp. exhibit resistance to disease. When infection leads to disease, the parasite is partially controlled by host immunity: in case of immunocompetence, the normal alveolar echinococcosis (AE) or cystic echinococcosis (CE) situation, the metacestode grows slowly, and first clinical signs appear years after infection; in case of impaired immunity (AIDS; other immunodeficiencies), uncontrolled proliferation of the metacestode leads to rapidly progressing disease. Assessing Echinococcus multilocularis viability in vivo following therapeutic interventions in AE patients may be of tremendous benefit when compared with the invasive procedures used to perform biopsies. Current options are F18-fluorodeoxyglucose-positron emission tomography (FDG-PET), which visualizes periparasitic inflammation due to the metabolic activity of the metacestode, and measurement of antibodies against recEm18, a viability-associated protein, that rapidly regresses upon metacestode inactivation. For Echinococcus granulosus, similar prognosis-associated follow-up parameters are still lacking but a few candidates may be listed. Other possible markers include functional and diffusion-weighted Magnetic Resonance Imaging (MRI), and measurement of products from the parasite (circulating antigens or DNA), and from the host (inflammation markers, cytokines, or chemokines). Even though some of them have been promising in pilot studies, none has been properly validated in an appropriate number of patients until now to be recommended for further use in clinical settings. There is therefore still a need to develop reliable tools for improved viability assessment to provide the sufficient information needed to reliably withdraw anti-parasite benzimidazole chemotherapy, and a basis for the development of new alternative therapeutic tools.
PMCID: PMC4245873  PMID: 25429386
Echinococcus multilocularis; Alveolar echinococcosis; Surgery; Medication; Follow-up; Em18; Em2; FDG-PET
11.  Treatment of echinococcosis: albendazole and mebendazole – what else? 
Parasite  2014;21:70.
The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival.
PMCID: PMC4271654  PMID: 25526545
Alveolar echinococcosis (AE); Echinococcus multilocularis chemotherapy; In vitro culture; Drugs; Host-parasite interaction
12.  Fatal Alveolar Echinococcosis of the Lumbar Spine 
Journal of Clinical Microbiology  2013;51(2):688-691.
For the last 10 years, the southern part of Belgium has been recognized as a low-risk area of endemicity for alveolar echinococcosis. This infection, caused by Echinococcus multilocularis, usually induces a severe liver condition and can sometimes spread to other organs. However, alveolar echinococcosis involving bones has been described only very rarely. Here, a fatal case of spondylodiscitis due to E. multilocularis contracted in southern Belgium is reported.
PMCID: PMC3553908  PMID: 23175265
13.  Subcutaneous Infection Model Facilitates Treatment Assessment of Secondary Alveolar Echinococcosis in Mice 
Alveolar echinococcosis (AE) in humans is a parasitic disease characterized by severe damage to the liver and occasionally other organs. AE is caused by infection with the metacestode (larval) stage of the fox tapeworm Echinococcus multilocularis, usually infecting small rodents as natural intermediate hosts. Conventionally, human AE is chemotherapeutically treated with mebendazole or albendazole. There is, however still the need for improved chemotherapeutical options. Primary in vivo studies on drugs of interest are commonly performed in small laboratory animals such as mice and Mongolian jirds, and in most cases, a secondary infection model is used, whereby E. multilocularis metacestodes are directly injected into the peritoneal cavity or into the liver. Disadvantages of this methodological approach include risk of injury to organs during the inoculation and, most notably, a limitation in the macroscopic (visible) assessment of treatment efficacy. Thus, in order to monitor the efficacy of chemotherapeutical treatment, animals have to be euthanized and the parasite tissue dissected. In the present study, mice were infected with E. multilocularis metacestodes through the subcutaneous route and were then subjected to chemotherapy employing albendazole. Serological responses to infection were comparatively assessed in mice infected by the conventional intraperitoneal route. We demonstrate that the subcutaneous infection model for secondary AE facilitates the assessment of the progress of infection and drug treatment in the live animal.
Author Summary
Alveolar echinococcosis is a disease which affects humans and inflicts severe damage to the liver and other organs. It is caused by a parasite whose definitive host is the fox. Despite being a relatively rare disease, an increasing number of new cases has been reported in central and eastern European countries more recently. The current therapy in human AE patients consists of benzimidazoles. The treatment has to be taken on a daily basis for very long periods of time, or even lifelong. New options are currently being searched for, mainly based on compounds that show efficacy in experimental animal infection models. The infection is commonly done by injecting parasites directly into the peritoneal cavity of the animals, with risk of damage to the surrounding organs. The efficacy of applied treatments can only be evaluated at the end of the studies by dissection of the animals. In this study we show that the subcutaneous infection model can be applied for drug treatment trials and enables the direct monitoring of treatment effects during the entire study period.
PMCID: PMC3662659  PMID: 23717701
14.  Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region 
The Canadian Veterinary Journal  2012;53(8):870-874.
An adult dog that lived in central British Columbia was examined because of a history of lethargy and vomiting. Histology, immunohistochemistry, and polymerase chain reaction (PCR) examination of a hepatic mass confirmed the presence of an alveolar hydatid cyst, the first description of Echinococcus multilocularis in British Columbia. We provide recommendations for case management and remind practitioners in endemic areas of western Canada that dogs can serve as definitive and, rarely, intermediate hosts for E. multilocularis.
PMCID: PMC3398525  PMID: 23372195
15.  A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex 
Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.
Author Summary
The dog tapeworm Echinococcus granulosus (E. granulosus) is a cosmopolitan parasite. The adult worms reside in the small intestine of their definitive hosts (dogs). Infective eggs are shed with the feces into the environment and are orally ingested by intermediate hosts where they develop into the metacestode (larval) stage, causing cystic echinococcosis (CE) in humans and livestock. Ten intraspecific genotypes of E. granulosus (G1 to G10) have been reported from different intermediate host species. Based on the recently established molecular phylogeny, E. granulosus is now considered a complex consisting of four species: E. granulosus sensu stricto (G1/G2/G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–G10). Simple and highly discriminative molecular epidemiological approaches are needed to explore dynamics, life cycle patterns, and the pathogenicity of the members of this complex. We here introduce a one-step multiplex PCR (mPCR) protocol for the genotyping and discrimination of the different members of the E. granulosus complex, allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex, and (iii) genetic variants within the E. granulosus complex. The relatively complicated task of E. granulosus complex speciation and genotyping is clearly simplified by mPCR, and this technique therefore represents a useful tool for routine practice.
PMCID: PMC3547860  PMID: 23350011
16.  Detection of European Strain of Echinococcus multilocularis in North America 
Emerging Infectious Diseases  2012;18(6):1010-1012.
PMCID: PMC3358155  PMID: 22608114
Echinococcus; Echinococcus multilocularis; alveolar hydatid cyst; zoonoses; European strain; North America; Canada; parasites; cestodes; dogs; canine; canid
17.  Echinococcus spp. and echinococcosis 
Acta Veterinaria Scandinavica  2010;52(Suppl 1):S5.
PMCID: PMC2994311
18.  Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray 
Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.
Methodology/Principal Findings
Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.
This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.
Author Summary
Crude or purified, somatic or metabolic extracts of native antigens are routinely used for the serodiagnosis of human helminthic infections. These antigens are often cross-reactive, i.e., recognized by sera from patients infected with heterologous helminth species. To overcome limitations in antigen production, test sensitivity and specificity, chemically synthesized peptides offer a pure and standardized alternative, provided they yield acceptable operative characteristics. Ongoing genome and proteome work create new resources for the identification of antigens. Making use of the growing amount of genomic and proteomic data available in public databases, we tested a bioinformatic procedure for the selection of potentially antigenic peptides from a collection of protein sequences including conceptually translated nucleotide sequence data of Echinococcus multilocularis and E. granulosus (Plathyhelminthes, Cestoda). The in silico selection was combined with high-throughput screening of peptides on microarray and systematic validation of reactive candidates in enzyme-linked immunosorbent assay. Our study proved the applicability of this approach for selection of peptide antigens with good diagnostic characteristics. Our results suggested the pooling of several peptides to reach a high level of sensitivity required for reliable immunodiagnosis.
PMCID: PMC2914747  PMID: 20689813
19.  Hepatic Gene Expression Profile in Mice Perorally Infected with Echinococcus multilocularis Eggs 
PLoS ONE  2010;5(4):e9779.
Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown.
Methodology/Principal Findings
We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p≤0.05), 35 genes were assigned to the functional gene ontology group , while 3 associated with the functional group . Upregulated genes associated with could be clustered into functional subgroups including , , , and . Two downregulated genes related to and , respectively. The genes either associated with an or an pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods.
Based on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.
PMCID: PMC2848562  PMID: 20368974
20.  Echinococcus multilocularis and Its Intermediate Host: A Model of Parasite-Host Interplay 
Host-parasite interactions in the E. multilocularis-intermediate host model depend on a subtle balance between cellular immunity, which is responsible for host's resistance towards the metacestode, the larval stage of the parasite, and tolerance induction and maintenance. The pathological features of alveolar echinococcosis. the disease caused by E. multilocularis, are related both to parasitic growth and to host's immune response, leading to fibrosis and necrosis, The disease spectrum is clearly dependent on the genetic background of the host as well as on acquired disturbances of Th1-related immunity. The laminated layer of the metacestode, and especially its carbohydrate components, plays a major role in tolerance induction. Th2-type and anti-inflammatory cytokines, IL-10 and TGF-β, as well as nitric oxide, are involved in the maintenance of tolerance and partial inhibition of cytotoxic mechanisms. Results of studies in the experimental mouse model and in patients suggest that immune modulation with cytokines, such as interferon-α, or with specific antigens could be used in the future to treat patients with alveolar echinococcosis and/or to prevent this very severe parasitic disease.
PMCID: PMC2842905  PMID: 20339517
21.  Epidemiology, Diagnosis, Treatment, and Control of Trichinellosis 
Clinical Microbiology Reviews  2009;22(1):127-145.
Summary: Throughout much of the world, Trichinella spp. are found to be the causative agents of human trichinellosis, a disease that not only is a public health hazard by affecting human patients but also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts in many countries have focused on the control of Trichinella or the elimination of Trichinella from the food chain. The most important source of human infection worldwide is the domestic pig, but, e.g., in Europe, meats of horses and wild boars have played a significant role during outbreaks within the past 3 decades. Infection of humans occurs with the ingestion of Trichinella larvae that are encysted in muscle tissue of domestic or wild animal meat. Early clinical diagnosis of trichinellosis is rather difficult because pathognomonic signs or symptoms are lacking. Subsequent chronic forms of the disease are not easy to diagnose, irrespective of parameters including clinical findings, laboratory findings (nonspecific laboratory parameters such as eosinophilia, muscle enzymes, and serology), and epidemiological investigations. New regulations laying down rules for official controls for Trichinella in meat in order to improve food safety for consumers have recently been released in Europe. The evidence that the disease can be monitored and to some extent controlled with a rigorous reporting and testing system in place should be motivation to expand appropriate programs worldwide.
PMCID: PMC2620635  PMID: 19136437
22.  Genetic Diversity of the Cestode Echinococcus multilocularis in Red Foxes at a Continental Scale in Europe 
Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.
Methodology/Principal Findings
The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas.
The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.
Author Summary
Echinococcus multilocularis is a tapeworm of the red fox, which represents a considerable health threat to respectively infected humans. Main endemic areas are located in China, Siberia, and central Europe. Alarmed by an emerging or reemerging situation in Europe, the question of how the parasite gets spatially and temporally spread and transmitted becomes essential to prepare appropriate control programs. The question was tackled by using genetic data on a large sample size of E. multilocularis adult stage tapeworms, combined with geographical site location data input. The historically documented endemic area, represented by the northern Alpine arch, was shown to harbour the highest genetic richness and diversity, as compared to surrounding areas in northern and eastern Europe. The spatial and temporal spread of different E. multilocularis genotypes in Europe seems to be ruled by a founder event, linked to exportation of parasites from the central core to newly identified (western and eastern) areas or subregions, where these parasites could subsequently disseminate under geographical separation from the original foci.
PMCID: PMC2685985  PMID: 19513103
23.  The EG95 Antigen of Echinococcus spp. Contains Positively Selected Amino Acids, which May Influence Host Specificity and Vaccine Efficacy 
PLoS ONE  2009;4(4):e5362.
Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.
PMCID: PMC2671473  PMID: 19401778
24.  In Vitro and In Vivo Treatments of Echinococcus Protoscoleces and Metacestodes with Artemisinin and Artemisinin Derivatives▿  
In vitro treatment of Echinococcus multilocularis and Echinococcus granulosus larval stages with the antimalarials dihydroartemisinin and artesunate (10 to 40 μM) exhibited promising results, while 6 weeks of in vivo treatment of mice infected with E. multilocularis metacestodes (200 mg/kg of body weight/day) had no effect. However, combination treatments of both drugs with albendazole led to a substantial but statistically not significant reduction in parasite weight compared to results with albendazole alone.
PMCID: PMC2533465  PMID: 18625777
25.  Assessment of Echinococcus granulosus Somatic Protoscolex Antigens for Serological Follow-Up of Young Patients Surgically Treated for Cystic Echinococcosis▿  
Journal of Clinical Microbiology  2008;46(5):1631-1640.
Echinococcus granulosus protoscolex soluble somatic antigens (PSSAs) were assessed for their prognostic value in the serological follow-up of young patients treated for cystic echinococcosis (CE), compared to conventional hydatid fluid (HF) antigen. Based on different clinical courses and outcome of infection, as well as imaging findings, patients were retrospectively classified into two different groups including either cured CE (CCE; i.e., absence of active cysts or presence of inactive cysts, respectively) and noncured CE (NCCE) patients still presenting active cysts at the end of an up to 5-year follow-up period. An immunoglobulin G (IgG)-PSSA enzyme-linked immunosorbent assay (ELISA) showed a gradual decrease in antibody levels in CCE cases, reaching seronegativity in 20% of the cases at least within 5 years postsurgery. In comparison, the conventional IgG-HF ELISA showed a significantly lower progressive decrease in antibody levels, serology becoming negative in only 15% of CCE patients at the endpoint of the follow-up period. Serological analysis of PSSA by immunoblotting yielded an interesting immunoreactive double band of 27 and 28 kDa that, in 15 (75%) of 20 CCE cases, exhibited a rapid decrease and subsequent disappearance of respective antibody reactivities within 3 years postsurgery. Conversely, anti-27- and -28-kDa antibody reactivity strongly persisted until the endpoint of the follow-up period in all of the five NCCE patients. Further analysis of the 27- and 28-kDa doublet by using affinity-purified antibodies showed that the double band was not detectable in HF. Furthermore, a predominantly IgG4 subclass-restricted humoral immune response against the 27- and 28-kDa antigens was demonstrated in seroreactive CE patients. Overall, an anti-27- and -28-kDa response appeared to correlate with cyst activity. In conclusion, PSSA represents a useful candidate to carry out a serologic follow-up of CE subsequent to treatment and deserves further respective evaluation for other age groups of CE patients.
PMCID: PMC2395101  PMID: 18367566

Results 1-25 (38)