Serological differentiation between cystic and alveolar echinococcosis by use of recombinant larval antigens.

Two recombinant antigens of the larval stages of Echinococcus granulosus and Echinococcus multilocularis, termed EG55 and EM10, respectively, were applied for serodiagnosis and serological differentiation between parasitic infections caused by the metacestode tissue of both tapeworms. Antigen EM10 is synthesized by E. multilocularis larvae. Antigen EG55 represents the recombinant form of the low-molecular-weight subunit of antigen B, which is an Echinococcus genus-specific antigen. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins. A sandwich enzyme-linked immunosorbent assay with monoclonal antibodies against EM10 and EG55 as capture reagents for the recombinant antigens was established and was evaluated with 74 serum samples from patients with histologically confirmed alveolar echinococcosis and 63 serum samples from patients with histologically confirmed cystic echinococcosis. A sensitivity of 93.2% and a specificity of 96.8% were achieved for the serodiagnosis of alveolar echinococcosis. Cystic echinococcosis could be detected with a sensitivity of 89.1% and a specificity of 98.6%.     

Molecular identification of Echinococcus species from eastern and southern Qinghai, China, based on the mitochondrial cox1 gene

The Qinghai-Tibetan Plateau (QTP, in western China), which is the largest and highest plateau on Earth, is a highly epidemic region for Echinococcus spp. We collected 70 Echinococcus samples from humans, dogs, sheep, yaks, plateau pikas, and voles in eastern and southern Qinghai and genotyped them using the mitochondrial DNA marker cytochrome oxidase subunit I gene and maximum parsimony and Bayesian reconstruction methods. Based on the 792-bp sequence matrix, we recorded 124 variable sites, of which, 115 were parsimony-informative. Thirty-four haplotypes (H1-H34) were detected, of which H1-H15, H16-H17, and H18-H34 belonged to Echinococcus shiquicus, Echinococcus multilocularis, and Echinococcus granulosus, respectively. Within 26 human isolates, three were identified as E. multilocularis and 23 were E. granulosus. We also detected a dual infection case in a dog with E. multilocularis and E. granulosus. The intraspecific haplotype (Hd ± SD) and nucleotide (Nd ± SD) diversity of E. shiquicus (0.947 ± 0.021; 0.00441 ± 0.00062) was higher than that for E. granulosus (0.896 ± 0.038; 0.00221 ± 0.00031) and E. multilocularis (0.286 ± 0.196; 0.00036 ± 0.00025). Moreover, the haplotype network of E. shiquicus showed a radial feature rather than a divergent feature in a previous study, indicating this species in the QTP has also evolved with bottleneck effects.     

Specific detection of Echinococcus spp. from the Tibetan fox (Vulpes ferrilata) and the red fox (V. vulpes) using copro-DNA PCR analysis

There are three Echinococcus species, Echinococcus granulosus, E. multilocularis, and E. shiquicus, which are distributed on the vast area of pastureland on the eastern Tibetan plateau in China. Tibetan foxes (Vulpes ferrilata) have been determined to be the main wild definitive host of E. multilocularis and E. shiquicus, but little information is available on the prevalence of these two parasites in Tibetan foxes. Consequently, the copro-prevalence of these parasites in foxes from the eastern Tibetan plateau was evaluated in this study. For each copro-DNA sample extracted from fox feces, a 133-bp segment of EgG1 Hae III was used to screen for infection with E. granulosus. Multiplex nested polymerase chain reaction (PCR) analysis was used to target an 874-bp segment of the mitochondrial COI gene to distinguish E. multilocularis and E. shiquicus. Among 184 fecal samples, 120 were from Tibetan foxes and six from red foxes (Vulpes vulpes). Of the fecal samples from Tibetan foxes, 74 (giving a copro-prevalence of 62?%) showed the presence of Echinococcus spp.: 23 (19?%) were found to contain E. multilocularis, 32 (27?%) E. shiquicus, and 19 (16?%) showed mixed infection with both E. multilocularis and E. shiquicus. Two fecal samples from red foxes were found to be infected with E. multilocularis. No fox feces were found to be infected with E. granulosus. Tests on zinc finger protein genes and a 105-bp fragment of the Sry gene found no significant difference in the prevalence of the two parasites between sexes. The efficiency of our multiplex nested PCR methods were compared with previous polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) methods and some problems associated with the copro-PCR were discussed.     

Molecular and immunological diagnosis of echinococcosis.

Echinococcosis is an infectious disease of humans caused by the larval (metacestode) stage of the cestode species Echinococcus granulosus (cystic echinococcosis or hydatid disease) or Echinococcus multilocularis (alveolar echinococcosis or alveolar hydatid disease). Clinical manifestations depend primarily on localization and size of hepatic lesions and may include hepatomegaly, obstructive jaundice, or cholangitis. Prognostically, alveolar echinococcosis is considered similar to liver malignancies, including a lethality rate of 90% for untreated cases. Diagnosis is based on imaging techniques coupled with immunodiagnostic procedures. Antibody detection tests for E. multilocularis have markedly improved with the use of affinity-purified Em2 antigen and recombinant antigen II/3-10 in enzyme immunoassays. Antigens of corresponding quality for E. granulosus are still unavailable. The detection of circulating antigens and immune complexes in the sera of patients with cystic echinococcosis, the demonstration of in vitro lymphocyte proliferation in response to stimulation with Echinococcus antigens, and the discrimination of serum immunoglobulin isotype activity to various Echinococcus antigens in both cystic and alveolar echinococcosis have been suggested for diagnostic purposes as well as for monitoring patients after treatment. New diagnostic molecular tools include DNA probes for Southern hybridization tests and polymerase chain reaction for the amplification of E. multilocularis and E. granulosus species-specific DNA fragments.     

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.     

Identification of the Echinococcus (hydatid disease) organisms using cloned DNA markers

Cloned DNA fragments of the ribosomal RNA gene of Schistosoma mansoni hybridise strongly to Echinococcus DNA following restriction endonuclease and Southern transfer analysis. Individuals within a strain of E. granulosus exhibit identical patterns of hybridisation. However, the hybridisation patterns show significant differences between E. granulosus and E. multilocularis, and between the horse and sheep strains of E. granulosus. This technique represents a powerful, additional method for the identification and characterisation of new isolates of E. granulosus and E. multilocularis.     

The diagnostic value and molecular characterisation of an Echinococcus multilocularis antigen gene clone

A diagnostic Echinococcus multilocularis antigen gene (EM4) has been expressed using the Escherichia coli expression vector pGEX-1 resulting in the high level synthesis of a readily purifiable, soluble, non-denatured peptide fused to the 26-kDa glutathione-S-transferase of Schistosoma japonicum. This recombinant antigen, on testing by enzyme-linked immunosorbent assay (ELISA) with heterologous human antisera, demonstrated 100% E. multilocularis specificity. Specific anti-EM4 antibody immunoprecipitated a single 66-kDa protein from protoscolex total RNA directed in vitro translation products indicating the probable involvement of post-translational modification in the production of the native EM4 antigens. Southern blotting analysis suggests that the EM4 native antigens are coded for by a single-copy gene and that the genomic organisation of the EM4 related genes in other parasites is not conserved. The nucleotide sequence of the cloned EM4 cDNA molecule has been obtained and the derived amino acid sequence shows no significant homology with other existing protein sequences.     

Isolation and characterization of a secretory component of Echinococcus multilocularis metacestodes potentially involved in modulating the host-parasite interface

Echinococcus multilocularis metacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction in E. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface.     

Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus

Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.     

<Emphasis Type="Italic">14-3-3</Emphasis> gene characterization and description of a second 14-3-3 isoform in both<Emphasis Type="Italic"> Echinococcus granulosus</Emphasis> and<Emphasis Type="Italic"> E. multilocularis</Emphasis>

Members of the 14-3-3 protein family have been identified as regulatory molecules in intracellular signaling pathways and cell cycle control. Previously, the first Echinococcus 14-3-3 isoform (E14-3-3.1) was isolated from E. granulosus and E. multilocularis metacestode stages. Hyperexpression of this isoform was claimed to be associated with non-restricted tumor-like growth of the E. multilocularis metacestode. In this report, we describe the characterization of a 14-3-3 cDNA from E. granulosus and E. multilocularis corresponding to a second isoform of this family, E14-3-3.2. The characterized 14-3-3 gene was interrupted by two introns whose sequence and positions were conserved in both Echinococcus species. The deduced amino acid sequence of E14-3-3.2 showed 88% identity to the E14-3-3.1 isoform and 52% identity to a third Echinococcus isoform (E14-3-3.3) described by other authors. These findings, coupled to Southern blot analysis, suggest the presence of more than one 14-3-3 gene in Echinococcus. Phylogenenetically, the Echinococcus 14-3-3.1 and 14-3-3.2 isoforms appeared to cluster with zeta-type ("pro-tumorigenic") 14-3-3 isoforms from closely related organisms, whereas the E14-3-3.3 isoform grouped with 14-3-3 epsilon isoforms. The presence of more than one 14-3-3 isoform might indicate isoform-specific roles in the different parasite stages of Echinococcus.     

Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern

Echinococcosis in humans is a zoonotic infection caused by larval stages (metacestodes) of cestode species of the genus Echinococcus. Cystic echinococcosis (CE) is caused by Echinococcus granulosus, alveolar echinococcosis (AE) is caused by E. multilocularis, and polycystic forms are caused by either E. vogeli or E. oligarthrus. In untreated cases, AE has a high mortality rate. Although control is essentially feasible, CE remains a considerable health problem in many regions of the northern and southern hemispheres. AE is restricted to the northern hemisphere regions of North America and Eurasia. Recent studies have shown that E. multilocularis, the causative agent of AE, is more widely distributed than previously thought. There are also some hints of an increasing significance of polycystic forms of the disease, which are restricted to Central and South America. Various aspects of human echinococcosis are discussed in this review, including data on the infectivity of genetic variants of E. granulosus to humans, the increasing invasion of cities in Europe and Japan by red foxes, the main definitive hosts of E. multilocularis, and the first demonstration of urban cycles of the parasite. Examples of emergence or reemergence of CE are presented, and the question of potential spreading of E. multilocularis is critically assessed. Furthermore, information is presented on new and improved tools for diagnosing the infection in final hosts (dogs, foxes, and cats) by coproantigen or DNA detection and the application of molecular techniques to epidemiological studies. In the clinical field, the available methods for diagnosing human CE and AE are described and the treatment options are summarized. The development of new chemotherapeutic options for all forms of human echinococcosis remains an urgent requirement. A new option for the control of E. granulosus in the intermediate host population (mainly sheep and cattle) is vaccination. Attempts are made to reduce the prevalence of E. multilocualaris in fox populations by regular baiting with an anthelmintic (praziquantel). Recent data have shown that this control option may be used in restricted areas, for example in cities, with the aim of reducing the infection risk for humans.     

Isolation and partial characterization of species-specific and cross-reactive antigens of Echinococcus granulosus cyst fluid

Two parasite antigens have been isolated from Echinococcus granulosus hydatid cyst fluid using hydrophobic interaction chromatography, anion exchange chromatography and gel filtration chromatography. Initial characterization of the antigens indicates that both are glycoproteins, of approximately 20 and 48 kDa (Eg20 and Eg48). When the two antigens were tested with a battery of antisera from patients with heterologous parasitic infections, only Eg20 was found to be specific for E. granulosus. The Eg48 antigen cross-reacted with the sera of 33% of E. multilocularis patients. In both antigens, some of the epitopes recognized by antibodies in the sera of hydatid patients were periodate-sensitive. This suggests the involvement of carbohydrates in at least some of the antigenic determinants. Due to the abundance of the Eg48 antigen in the hydatid cyst fluid, it would be the more practically useful antigen for disease diagnosis, especially in countries where only E. granulosus is endemic.     

Human alveolar echinococcosis in Slovenia

Between January 2001 and December 2005, 1263 patients suspected of having echinococcosis were screened serologically by indirect haemagglutination assay (IHA). IHA-positive patient sera were then retested by western blot for confirmation and differentiation between Echinococcus granulosus and Echinococcus multilocularis infection. Of 43 sera confirmed as Echinococcus-positive, nine appeared to be specific for alveolar echinococcosis (AE) caused by E. multilocularis. AE-positive serological results corresponded to the clinical and/or imaging findings concerning the patients' liver cysts. The detected incidence of AE was 0.45/10(5) inhabitants, which suggests that clinicians and health authorities in Slovenia should give greater attention to AE in the future.     

Immunodiagnosis of Echinococcus infections: confirmatory testing and species differentiation by a new commercial Western Blot

The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis.     

Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction

The nucleotide sequence of the cloned Echinococcus multilocularis DNA probe pAL1 was determined in order to simplify and improve the sensitivity of a diagnostic assay through the application of the polymerase chain reaction (PCR). The insert-specific oligonucleotides BG1 and BG2 define a 2.6-kb fragment in the genomic DNA of E. multilocularis, while BG1 and BG3 define a 0.3 kb fragment. A PCR study including 14 independent E. multilocularis isolates in addition to Echinococcus granulosus. Echinococcus vogeli, Taenia spp. and other cestodes revealed that the 2.6-kb fragment was amplified from genomic DNA of all E. multilocularis isolates tested (originating from Switzerland, Alaska, Canada, France, Germany and Japan), but from genomic DNA of none of the other cestode species. PCR with BG1 and BG2 furthermore uniquely resulted in the synthesis of a 0.55-kb fragment specific for Taenia saginata and a 0.6-kb fragment specific for T. taeniaeformis. In contrast to the species specificity of the 2.6-kb BG1/BG2 product, the 0.3 kb (BG1/BG3) product demonstrated genus specificity: the 0.3-kb product was amplified from genomic DNA of all E. multilocularis, E. granulosus and E. vogeli isolates tested, but from genomic DNA of none of the other cestode species. The diagnostic sensitivity of PCR using both primer sets was determined to be 50 pg parasite DNA, suggesting the practical utility of this simple assay in demonstrating parasite DNA in specimens from a variety of sources. At the basic level, the pAL1-derived oligonucleotides may also prove useful in assessing strain variation, RFLPs or other manifestations of genetic variation in E. multilocularis.     

IgE-dependent humoral immune response in Echinococcus multilocularis infection: circulating and basophil-bound specific IgE against Echinococcus antigens in patients with alveolar echinococcosis.

Clinical symptoms of immediate-type hypersensitivity (ITH) and specific IgE against Echinococcus granulosus antigens are frequently present in patients with hydatid cysts. In alveolar echinococcosis (AE) due to E. multilocularis, clinical manifestations related to ITH have never been reported. The IgE-dependent humoral immune response was evaluated in 30 patients with AE. Circulating specific IgE (sIgE) were determined with two different methods of radio-allergo-sorbent test. Serum sIgE were determined sequentially in 18 patients over 15 months. Specific IgE bound to circulating basophils were assessed with two tests in vitro, measuring specific degranulation and histamine release. The respective abilities of E. granulosus and E. multilocularis antigens to reveal bound and circulating IgE antibodies were also assayed. Despite the absence of clinical symptoms of ITH and the frequent lack of circulating sIgE, an immunological response involving IgE was always present in human AE: basophil-bound sIgE were revealed in every patient by histamine release and degranulation tests; these tests were constantly negative in control subjects. Echinococcus granulosus extracts were more effective for detecting circulating sIgE; however E. multilocularis antigenic preparation induced a histamine release significantly higher than E. granulosus extracts. These results suggest that IgE-dependent humoral immune response could play a role in the host-parasite relationship in AE. Moreover, the sensitivity of the tests used to detect basophil-bound sIgE was higher than that of the usual serological tests, and the basophil degranulation test could be used to confirm diagnosis of AE in endemic countries.     

Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen.

Alveolar echinococcosis (AE) in humans is generally a fatal disease when not diagnosed early enough to provide curative treatment such as radical surgery. Immunodiagnosis for early detection of AE was improved by the isolation of an affinity-purified metacestode Em2 antigen and by the synthesis of recombinant Echinococcus multilocularis antigen II/3-10. Both antigens were individually assessed by enzyme-linked immunosorbent assay (ELISA) and demonstrated high specificities and diagnostic sensitivities, although both missed approximately 4 to 11% of diagnostic cases of AE. To provide an optimal serodiagnostic test, we investigated the two purified antigens by using a test employing a mixture of both purified antigens (designated Em2plus antigen) in one assay. For comparative purposes, crude E. multilocularis and Echinococcus granulosus metacestode antigens were investigated as well. The Em2plus ELISA proved to be the optimal diagnostic test with the highest diagnostic sensitivity, 97%, in serum samples from 140 patients with AE and an overall specificity of 99% for infections due to other Echinococcus and non-Echinococcus parasites. The new test combination (Em2plus ELISA) is suggested for the serodiagnosis of AE in patients and for seroepidemiological surveys.     

Subunit composition and specificity of the major cyst fluid antigens of Echinococcus granulosus

The subunit composition and specificity of the major Echinococcus granulosus cyst fluid antigens were determined by immunochemical analysis using murine monoclonal antibodies against Antigen 5 and Antigen B and human sera. Immune complexes cut out from immunoelectrophoresis gels and murine hybridomas were used as a source of specific anti-Antigen 5 and anti-Antigen B antibodies. Immunoprecipitation and Western blot analyses in sodium dodecyl sulphate-polyacrylamide gels using these reagents identified Antigen 5 to be a heterodimer composed of 24-kDa and 38-kDa subunits linked by disulphide bonding. Antigen B comprised a regularly spaced group of molecules with the smallest subunit estimated to be 8 kDa and the other components each differing in size by approximately 8 kDa, i.e., 16 kDa, 24 kDa, 32 kDa etc.; all possibly derived from the 8-kDa monomer. The relative abundance of the Antigen B subunits decreased asymptotically with increasing molecular weight. Neither the Antigen 5 nor the Antigen B subunit was specific for E. granulosus. Both antigens generated readily detectable levels of specific antibody in the sera of patients with Echinococcus multilocularis, Echinococcus vogeli or E. granulosus infection. Relatively high levels of antibody to Antigen 5 were also detected in the sera of patients infected with Taenia solium. The presence of phosphorylcholine epitope(s) on Antigen 5 was confirmed.     

A gene family expressing a host-protective antigen of Echinococcus granulosus.

Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.