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Transmission dynamics of Echinococcus multilocularis; its reproduction number, persistence in an area of low rodent prevalence, and effectiveness of control.

On the basis of high prevalences of Echinococcus multilocularis in the growing fox populations in Central Europe, its total biomass may have increased significantly in the past 20 years. E. multilocularis is now also found in areas outside the known endemic area in Central Europe. Therefore, E. multilocularis, the causative agent of a serious parasitic zoonosis, might be of major concern for public health and a challenge to control. Some experimental field trials to control E. multilocularis using an anti-worm drug reduced parasite burden in a contaminated region during the control campaign, but failed to eradicate the parasite completely. It was our aim to develop a mathematical model describing the biomass of egg, larval, and adult worm stages of the E. multilocularis life-cycle, and simulate a hypothetical control campaign. Additionally, we derived the reproduction number of this parasite and explored conditions for the persistence of the parasite's life-cycle. Our model shows that while control campaigns rapidly reduce the worm burden in the definitive host, and consequently eggs in the environment, the pool of larvae in the intermediate host remains large. The parasite's life-cycle persists in a region where prevalence in the intermediate host is low (approximately 1%). Therefore, we conclude that the parasite is likely to re-emerge if control is discontinued on the basis of reduced worm population. Continued treatment of the definitive host is required to eradicate the larval stage of the parasite from the intermediate host population.

Animals↗

The immunogenicity of Echinococcus granulosus antigen 5 is determined by its post-translational modifications.

Since its early introduction as a marker for the immunodiagnosis of hydatid disease, antigen 5 (Ag5) has been regarded as one of the more relevant antigens of Echinococcus granulosus, and it is still widely used in different confirmation techniques. In this work we prepared 2 recombinant forms of the antigen, namely, rAg5 (corresponding to the unprocessed polypeptide chain of the antigen) and rAg5-38s (corresponding to its 38 kDa subunit). Their antigenicities were compared to that of the native antigen using a human serum collection. There was a major drop in the reactivity of the sera, particularly against rAg5-38s, which was confirmed by analysis of the cross-reactivity of 2 panels of monoclonal antibodies specific for rAg5-38s and the native antigen. Using the chemically deglycosylated native antigen, we demonstrated that the reduced antigenicity of the recombinants is due to the loss of the sugar determinants, and not to their misfolding. Inhibition experiments using phosphorylcholine confirmed that this moiety also contributes to the reactivity of the antigen, but to a much lesser extent. The presence of immunodominant highly cross-reactive glycan moieties in the Ag5 molecule may involve a parasite evasion mechanism.

Amino Acid Sequence↗

High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

Amino Acid Sequence↗

14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes.

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

14-3-3 Proteins↗

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↗

Molecular genetic analysis of human cystic hydatid cases from Poland: identification of a new genotypic group (G9) of Echinococcus granulosus.

We have used nuclear (ribosomal ITS1) and mitochondrial (ND1) sequences to characterize human and pig isolates of Echinococcus granulosus collected by fine needle aspiration biopsy (FNAB) in Poland. The data indicate clearly that the Polish patients were not infected with the common sheep strain (G1 genotype) of E. granulosus, normally associated with human cystic hydatid infection. Instead, the hydatid parasite infecting the Polish patients shares very similar ND1 sequence with the previously characterized pig (G7) genotype but it also exhibits some clear differences. In particular, E. granulosus DNA from the Polish patients amplified a single ITS1 fragment in PCR and distinct ITS1-RFLP patterns were obtained after restriction digestion. The form of hydatid isolated from the Polish patients appears, therefore, to represent a distinct, previously undescribed genotype (designated G9) of E. granulosus.

Adult↗

Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C.

Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (alpha, beta, gamma and delta), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoforms patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.

Animals↗

Reduced genetic variability within coding and non-coding regions of the Echinococcus multilocularis genome.

Echinococcus multilocularis, a vulpine intestinal tapeworm, is the causative agent of alveolar echinococosis in humans, one of the most severe and lethal parasitic infections in man. To date, there is very little knowledge about the genetical polymorphism of this parasite. To assess sequence polymorphism, we analysed a sample of 33 E. multilocularis isolates from Europe, North America and Asia by PCR-SSCP followed by nucleotide sequencing. This assessment was performed comparatively to sheep, cattle and pig E. granulosus strains. Coding (nuclear antigen B and mitochondrial NADH dehydrogenase genes) and non-coding (introns of actin and homeobox-containing genes) regions of the parasite genome were chosen as targets. Since the estimated nucleotide diversity among genotypes of E. multilocularis were, in general, 10 times lower than among the recognized different strains of E. granulosus, we suggest that the conventional classification of the former species in 2 separated strains (European and North American) should be reviewed.

Animals↗

Identification of a laminated layer-associated protein in Echinococcus multilocularis metacestodes.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction lead to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS-PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

Animals↗

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

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

Animals↗

Monitoring of Echinococcus multilocularis infection in red foxes in Shiretoko, Japan, by coproantigen detection.

The study was conducted to evaluate a mAb-based coproantigen detection assay for monitoring fox prevalence of Echinococcus multilocularis infections in the Shiretoko National Park in Hokkaido, Japan. Eight fox families, each consisting of 2-4 adults and their pups, were selected because their territories have been determined. Faeces of each family (total n = 537) were collected monthly from April to October, 1994. Detection of coproantigen and taeniid eggs was recognized in 58 and 27 faeces, respectively, but mostly restricted in 1 family, in which coproantigen ELISA OD values had dual peaks, one in June and the other in October, whereas taeniid eggs were detected only from April to July. Fox pup faeces (n = 51) collected around the dens used by the positive family were all coproantigen and taeniid egg negative except for 1 faeces. These results suggest that the prevalence and infection pressure in the study area were low and the infected family terminated the infection in the summer and acquired a new one in the early autumn, 1994. This monitoring method for fox infection will be a practical management tool for E. multilocularis infection, especially at the tourist spots in Hokkaido where close contact of fox with humans is frequently observed.

Animals↗

Breeding systems in Echinococcus granulosus (Cestoda; Taeniidae): selfing or outcrossing?

We used the PCR-SSCP method followed by sequencing in order to assess the genetic variability of coding and noncoding parts of the genome of Echinococcus granulosus (Cestoda; Taeniidae) and to test whether or not the parasite populations are mainly self-fertilizing. For this, we analysed a sample of 110 E. granulosus metacestode isolates collected from different geographical regions (Southern Brazil, Europe and Australia) and from different intermediate hosts (ovine, bovine, human, macropod, swine and equine). Using appropriate controls, we were able to identify 4 strains in that sample (sheep, cattle, pig and horse strains). The high degree of genetic differentiation between strains, but not within, and the monomorphism found in most loci (EgAg4, EgActII, EgHbx2 and EgAg6-non-coding-EgAgB/1 and EgND1-coding) indicated that they are largely selfed. On the other hand, outcrossing was also shown to occur, since 5 potential hybrid genotypes between cattle and sheep strains were found in populations of Southern Brazil, but absent in other geographical areas. We suggest that both processes are adaptive. The article also reports, for the first time, the occurrence of the E. granulosus cattle strain in South America.

Animals↗

Ultrastructural localization of an Echinococcus granulosus laminin-binding protein.

Murine antibodies, raised against a purified recombinant 30 kDa laminin-binding protein from Echinococcus granulosus, were used to investigate the tissue distribution of the native protein in protoscoleces and brood capsules. Immunofluorescence, in combination with confocal microscopy, revealed that the protein was distributed in small annular foci near the peripheral regions of the protoscoleces. Immunoelectron microscopy of thawed cryosections demonstrated that the laminin-binding protein was present in the cytoplasm of tegumentary cytons and myocytons, but not in cells of the excretory system. The protein was associated with amorphous regions of the cytoplasm, and was not expressed at the surfaces of cells. This distribution resembles those of other invertebrate laminin-binding proteins, which are thought to act in the cell cycle and cell proliferation events. A low degree of label was consistently detected in extracellular matrices of the protoscolex.

Animals↗

Genetic variation and epidemiology of Echinococcus granulosus in Argentina.

Polymerase chain reaction-ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the carnel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.

Animals↗

Modelling a 3D structure for EgDf1 from Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand.

EgDf1 is a developmentally regulated protein from the parasite Echinococcus granulosus related to a family of hydrophobic ligand binding proteins. This protein could play a crucial role during the parasite life cycle development since this organism is unable to synthetize most of their own lipids de novo. Furthermore, it has been shown that two related protein from other parasitic platyhelminths (Fh15 from Fasciola hepatica and Sm14 from Schistosoma mansoni) are able to confer protective inmunity against experimental infection in animal models. A three-dimensional structure would help establishing structure/function relationships on a knowledge based manner. 3D structures for EgDf1 protein were modelled by using myelin P2 (mP2) and intestine fatty acid binding protein (I-FABP) as templates. Molecular dynamics techniques were used to validate the models. Template mP2 yielded the best 3D structure for EgDf1. Palmitic and oleic acids were docked inside EgDf1. The present theoretical results suggest definite location in the secondary structure of the epitopic regions, consensus phosphorylation motifs and oleic acid as a good ligand candidate to EgDf1. This protein might well be involved in the process of supplying hydrophobic metabolites for membrane biosynthesis and for signaling pathways.

Amino Acid Sequence↗

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

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

Animals↗

myo-Inositol hexakisphosphate is a major component of an extracellular structure in the parasitic cestode Echinococcus granulosus.

myo-Inositol hexakisphosphate (IP(6)) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP(6) in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2 mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP(6), was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP(6) in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP(6) was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP(6) was not detected in any other structures or stages of the parasite. Our results imply that IP(6) is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP(6), and the possible implications beyond the biology of E. granulosus are discussed.

Animals↗

Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family.

Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.

Amino Acid Sequence↗