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PubMed2005

Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection.

Two IgM murine monoclonal antibodies (MAbs), EgC1 and EgC3, were produced against the excretory/secretory (E/S) products of Echinococcus granulosus adult worms. Immunoblotting revealed that both predominantly recognized a 50 kDa antigen in the somatic extract and an 85 kDa component in the E/S products. Immunolocalization showed that both MAbs reacted with the tegument of the parasite, and additionally EgC3 reacted with parenchyma and the tegument lining the external surface of the reproductive organs. A coproantigen capture ELISA was developed using a rabbit polyclonal antibody against E/S products from adult tapeworms as catching antibodies, and each one of MAbs as detecting antibody. The assays detected seven out of eight (EgC1), and eight out of eight (EgC3) experimentally infected dogs (worm burdens ranging from 61 to 57,500), using heat-treated samples obtained at prepatent period, and none (n=8) of helminth-free samples. Time course analysis showed that, after a 12-25 days lag, coproantigen levels rose above cut off O.D. values and typically peaked around 30 days post-infection (DPI) at the end of the experiment. One dog experimentally infected with Taenia hydatigena metacestodes was slightly detected as positive at different time points after 30 DPI. Both MAbs showed a similar pattern of recognition, but T. hydatigena antigens were undetectable for a longer period, and reached lower O.D. values with EgC1. Interestingly, fecal samples from two experimentally infected dogs with Echinococcus multilocularis were not recognized by the EgC1 assay, suggesting a potential value as species-specific diagnostic tool.

Animals↗
PubMed2005

Echinococcus granulosus in Australia, widespread and doing well!

Echinococcus granulosus is the only member of the Genus Echinococcus to occur in Australia. The major biomass of E. granulosus occurs in wildlife. The wildlife transmission cycle is predominantly perpetuated via a predator/prey interaction between wild dogs (dingoes and dingo/domestic dog hybrids) and macropodid marsupials (wallabies and kangaroos). Other wildlife hosts include foxes, wombats and feral pigs. This wildlife reservoir for E. granulosus "spills over" to help maintain a domestic cycle through E. granulosus-infected wild dogs defecating on pasture, transmitting infection to livestock and some farmers and hunters feeding hydatid-infected offal of macropodids or feral pigs to domestic dogs. The potential transmission risk to humans using public picnic and camping areas in parks and forests, especially in the southeastern Australia, could be substantially reduced through regular distribution of baits containing praziquantel. Encroachment of wild dogs and foxes into urban centers presents a new potential path of transmission from wildlife to humans.

Animals↗
PubMed2003

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

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

Animals↗
PubMed1997

Expression and immunological characterisation of Echinococcus granulosus recombinant antigen B for IgG4 subclass detection in human cystic echinococcosis.

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.

Animals↗
PubMed2002

Further molecular discrimination of Spanish strains of Echinococcus granulosus.

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.

Animals↗
PubMedDate not supplied

Screening for different genotypes of Echinococcus granulosus within China and Argentina by single-strand conformation polymorphism (SSCP) analysis.

Single-strand conformation polymorphism (SSCP) analysis was employed for the direct visual display of genetic variability in mitochondrial DNA (mtDNA) fragments within and among populations of Echinococcus granulosus from the People's Republic of China and from Argentina. Fragments of the NADH dehydrogenase I gene (NDI) and the cytochrome c oxidase subunit I (COI) were individually amplified from parasite DNA by polymerase chain reaction, denatured and subjected to SSCP analysis. Using NDI and COI fragments, samples representing different genotypes could be readily identified based on characteristic SSCP profiles. The results demonstrate the utility of SSCP for the direct visual display of nucleotide variation in mtDNA of E. granulosus prior to DNA sequence analysis. The approach compares favourably with existing genotyping procedures and provides a reliable and technically reproducible method for the routine laboratory identification of Echinococcus isolates.

Animals↗
PubMed2002

Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences.

The tissue-dwelling larval stages of the cestode Echinococcus granulosus are intimately associated with the host, implying that a range of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought through a transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain high levels of mitochondrial transcripts, as well as host (bovine) genomic DNA. In particular, 60% of a conventional protoscolex stage cDNA library corresponds to the large subunit (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' end of full-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG-bearing mRNA, three cDNA libraries were made by PCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had been excluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newly described trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrial and genomic contributions were again excluded. In both cases, a majority of cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG donated by the SL, and in only 6% was the SL separated from an endogenous putative start site by >60 nt. Sequence analysis of randomly selected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E. granulosus mRNAs appear to be insensitive to the enzymatic treatments used to 'oligo-cap' unspliced mRNAs. The oligo-capped and SL strategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and, possibly, from related parasitic flatworms.

5' Untranslated Regions↗
PubMed2003

Echinococcus granulosus in Finland.

Echinococcus granulosus is shown to occur in eastern Finland in a sylvatic cycle involving wolves (Canis lupus) as the definitive host and reindeer (Rangifer tarandus tarandus) and probably also elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus) as intermediate hosts. Even though the prevalence of E. granulosus in reindeer has increased in recent years, it is still very low (<0.013%). The results suggest, however, that the reindeer is a good sentinel animal to show the contamination of soil with Echinococcus eggs. This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The parasite seems to be quite common in the Finnish wolf population, with a prevalence of approximately 30% in both intestinal and faecal samples. The present results and previous ones indicate that E. granulosus infection has not spread to dogs in the reindeer herding area.

Animals↗
PubMed1996

Improvement of a polymerase chain reaction assay for the detection of Echinococcus multilocularis DNA in faecal samples of foxes.

A polymerase chain reaction (PCR) method was developed in order to permit a sensitive and specific identification of Echinococcus multilocularis DNA from fox faecal specimens. In an attempt to overcome problems related to the presence of endogenous PCR inhibitors in faecal extracts, we investigated a DNA extraction technique using an anion binding resin (Gene-Fizz). This simple and rapid procedure was applied to 61 faecal samples. Compared with the traditional microscopic examination, the sensitivity of PCR using Gene-Fizz was 82.3% and the specificity was 95.5%. No PCR signal was obtained for 20 Echinococcus granulosus isolates, showing that this method allowed discrimination between E. multilocularis and E. granulosus. Large-scale epidemiological surveys of fox excrements may be possible by using Gene-Fizz treatment prior to PCR amplification reactions.

Animals↗
PubMed1996

Cestodes. Echinococcus.

This article deals with cystic echinococcosis (CE) and alveolar echinococcosis in humans caused by the cestode parasites Echinococcus granulosus and Echinococcus multilocularis. The life cycles of these parasites and their epidemiologic aspects are briefly discussed, and a detailed review is presented on clinical aspects of the diseases, including diagnosis and therapy. Considerable progress has been made in chemotherapy but the results are not yet satisfactory. A new alternative for treatment of CE is puncture of cysts, aspiration of fluid, injection of ethanol, and reaspiration of fluid (PAIR).

Animals↗
PubMed2001

Further studies on an intermediate host murine model showing that a primary Echinococcus granulosus infection is protective against subsequent oncospheral challenge.

We describe the use of a murine model to evaluate resistance against subsequent challenge following a primary infection with oncospheres of Echinococcus granulosus. Mice (Kunming strain) were infected with hatched oncospheres of Echinococcus granulosus; 21 days later a second challenge was given by a different route of infection. A primary infection by intraperitoneal (i.p.) injection stimulated 100 and 90.5% protection in terms of reduced cyst numbers against a secondary infection given subcutaneously (s.c.) or intravenously (i.v.), respectively. A primary infection given s.c. followed by i.p. or i.v. challenge resulted in 84.0 and 100% protection, respectively. Intravenous infection followed by i.p. or s.c. challenge resulted in 98.5 and 69.4% protection, respectively. With the i.v. route of infection, almost all resultant cysts were present in the lungs. The data show that a primary infection with oncospheres can induce total or a high degree of protection against a subsequent challenge and confirms that natural (concomitant) immunity can be stimulated in the intermediate host as the result of a primary infection. This may explain the decline in hydatid infection in sheep older than 2 years in hyper-endemic areas such as those found in Xingjiang, China. These older sheep may have been earlier infected and have subsequently self-cured, with the primary infection stimulating an immune response that protects the intermediate host animals from further infection.

Animals↗
PubMed2001

Binding properties of Echinococcus granulosus fatty acid binding protein.

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.

Amino Acid Sequence↗
PubMed1980

Taeniasis, cysticercosis and echinococcosis/hydatidosis in Nigeria: IV--prevalence of Echinococcus granulosus infection in stray dogs.

Echinococcus granulosus (Batsch, 1786; Rudolphi, 1801) infections in stray dogs were recorded in the Sudan and Northern Guinea zones at a prevalence rate of 6.2% and 1.2% respectively. The parasite was not recorded in any stray dog obtained from the Bauchi Plateau zone, although fewer dogs were examined from this zone. The high prevalence of echinococcosis in dogs reported in the Sudan zone bears a true epidemiological relationship with the high prevalence of cystic hydatidosis recorded for slaughtered livestock within the same locality. There is therefore a strong possibility of a high rate of faecal contamination of the environments under consideration with Echinococcus eggs, which places the human population at a high risk.

Animals↗
PubMed1983

Echinococcus granulosus equinus: an ultrastructural study of the laminated layer, including changes on incubating cysts in various media.

The microfibrillate component of the laminated layer of Echinococcus granulosus equinus contains, except for the zone adjacent to the germinal layer, aggregates of electron-dense bodies displaying a sub-structure of electron-lucent spheres. The tegumentary syncytial cytoplasm contains randomly distributed electron-dense granules, many occurring near the apical plasmalemma, although exocytosis was rarely seen. Granules, similar in size and sub-structure to the bodies of the aggregates, also occur in the internuncial connexions and tegumentary cytons, suggesting that they may be produced in the cytons and released into the laminated layer via the internuncial connexions and tegumentary cytoplasm. Cysts incubated for 0.5-2.5 h in serum- and non-serum-containing media showed differences from non-incubated cysts. The distal half of the syncytium contained a progressive increase in the number of granules (distal: proximal 1.9:1 at 2.5 h; cf. 1:1 in non-incubated cysts), and exocytosis of granules into the laminated layer adjacent to the cyst had occurred. This cannot be attributed wholly to serum proteins. Cysts incubated for 21 h appeared 'normal', suggesting re-establishment of an equilibrium. Since the matrix of the laminated layer is considered homologous to the glycocalyx of other cestodes, the possible protective role played by the granules/bodies, characteristic of Echinococcus spp., is discussed.

Animals↗
PubMed1987

Genomic cloning of human Echinococcus granulosus DNA: isolation of recombinant plasmids and their use as genetic markers in strain characterization.

A small, size selected (0.5-5.0 Kbp) genomic DNA library has been constructed in the bacterial plasmid pAT153 using DNA extracted from a human isolate (Kenyan origin) of the hydatid disease organism, Echinococcus granulosus. A panel of taeniid cestode DNAs has been used in conjunction with hybridization and restriction-enzyme analysis to identify in the library two recombinant plasmids with Echinococcus-specific inserts and a single recombinant plasmid (coded pEG18) with a DNA fragment unique for E. granulosus. These, and other recombinant plasmids with E. granulosus DNA inserts, have been used in restriction endonuclease, Southern transfer and hybridization analysis to independently and reproducibly discriminate between the UK horse and sheep strains of E. granulosus; these probes may well prove of value for characterizing isolates from other endemic areas. The feasibility of using a cloned DNA fragment as the basis of a simple field test for distinguishing eggs of E. granulosus from those of other taeniid cestodes is discussed. The recombinant insert (approximately 2.3 Kbp in size) of pEG18 has a low copy number - estimated at approximately 26 - and it may not be sufficiently sensitive for practical use as a DNA probe in the identification of small numbers of E. granulosus eggs by molecular hybridization.

Animals↗
PubMed1991

A monoclonal antibody against Echinococcus multilocularis Em2 antigen.

A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen.

Animals↗
PubMed1990

Survival, strobilation and sexual maturation of Echinococcus multilocularis in the small intestine of golden hamsters.

Echinococcus multilocularis survived, strobilated and matured sexually in the small intestine of 6-week-old male golden hamsters that were either non-treated or treated with prednisolone tertiary-butylacetate (PTBA), following oral administration of 20 000 protoscoleces. Non-treated hamsters harboured the adult worms for up to 25 days post-infection (p.i.). Hamsters treated with PTBA from the day of infection (day 0 p.i.) harboured more adults than the non-treated, and their survival periods were extended. Moreover, a remarkable improvement in worm recovery, development and survival periods was observed in hamsters additionally treated with PTBA on days -- 14 and -- 12 p.i., and the worm development including proglottisation, sexual maturation and egg formation was comparable with that in dogs. With the present results it is suggested that Echinococcus/rodent systems represented by the E. multilocularis/golden hamsters may rejuvenate the current stagnant research on host-parasite interactions of the definitive host-stage and egg-stage of Echinococcus.

Animals↗
PubMed1994

Comparative analysis of full-length antigen II/3 from Echinococcus multilocularis and E. granulosus.

The recombinant Echinococcus multilocularis antigen II/3-10 is one of the most promising tools for immunodiagnosis of alveolar echinococcosis in human patients. Its nucleic acid sequence represents a part of the E. multilocularis gene encoding the metacestode antigen II/3, the former being basically present and expressed in both E. multilocularis and E. granulosus. Most (94%) patients with alveolar echinococcosis respond to infection with a marked anti-II/3-10 IgG synthesis; in contrast, most of the cystic echinococcosis patients do not, for some reason, recognize the recombinant antigen. We tackled this problem by generating cDNA derived from both E. granulosus and E. multilocularis full length II/3 genes, performed by reverse transcription and PCR amplification. Sequence analysis revealed a very high degree of conservation of the primary sequence of the antigen II/3 in both Echinococcus species. cDNA fragments were subcloned and expressed in E. coli as fusion proteins with Schistosoma japonicum glutathione S-transferase. Recombinant proteins were affinity purified and comparatively assessed by ELISA with respect to antibody-binding characteristics. Sera from patients suffering from cystic echinococcosis showed no significant differences in reactivity with the antigens derived from either E. multilocularis or E. granulosus. Therefore, parameters other than some minor differences in the primary sequence seem to be responsible for the lack of antigen II/3 recognition in cystic echinococcosis.

Amino Acid Sequence↗