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PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

A protein secreted in vivo by Echinococcus granulosus inhibits elastase activity and neutrophil chemotaxis.

A cDNA encoding the carboxy-terminal of the 12-kDa subunit of antigen B of Echinococcus granulosus has been cloned and sequenced. In addition, an amino acid sequence has been generated for the amino-terminal which is tentatively contiguous with the open reading frame of the DNA-derived sequence. Comparison of the inferred sequence of the 12-kDa antigen with other known sequences indicated a limited similarity to alpha-1 antitrypsin. In functional assays, gel-purified native 12-kDa antigen from natural infections inhibited elastase but not trypsin or chymotrypsin, providing further evidence that this antigen is a parasite protease inhibitor. Possibly unrelated to its anti-protease activity but a potentially important function of the 12-kDa antigen was its ability to inhibit recruitment of neutrophils. These functions may be important to the viability of the parasite in the face of the host immune response. In addition, the match between the DNA-derived sequence and the protein sequence was imperfect, with some residues having, according to the amino acid sequencing, two alternatives in approximately equal concentrations, and four DNA-derived residues failing to match with the protein sequence at all. The 12-kDa antigen may be expressed as isoforms from a polymorphic gene and, as far as aware, this observed sequence polymorphism has not, to date, been described for any other flatworm antigen.

Amino Acid Sequence↗
PubMed1993

A developmentally regulated gene of Echinococcus granulosus codes for a 15.5-kilodalton polypeptide related to fatty acid binding proteins.

A stage-specific expressed gene has been isolated from a cDNA expression library of Echinococcus granulosus protoscolices. The isolated clone contains the complete coding sequence. The corresponding protein (EgDf1) has a molecular weight of 15.5 kDa and is expressed at the tegumental level in the protoscolices, being undetectable in the germinal layer of the metacestode. This protein shares an important homology with a family of low-molecular weight proteins involved in the binding of hydrophobic ligands. This family includes a protein of Schistosoma mansoni (Sm 14) that has immunoprotective activity in rodents. Histochemical and metabolic data already reported for E. granulosus suggest that EgDf1 could be a molecular marker for early events in the process of protoscolex differentiation.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and characterization of actin genes from Echinococcus granulosus.

An Echinococcus granulosus genomic library has been screened with a mouse beta-actin cDNA probe. Two clones carrying DNA fragments of about 15 kb, possibly derived from the same genome region, have been isolated. This 15-kb genomic region includes 2 actin-related sequences (EgactI and EgactII) separated by about 4 kb. The nucleotide sequences of both genes were determined. The EgactI sequence presents no introns, but an intron of 591 bp was observed in the EgactII sequence. The genes potentially encode 375 and 376 amino-acid-long actins, respectively, with a homology of 85.3%. The deduced amino acid sequences from both genes were compared to the actin sequences from other organisms, showing similarities ranging from 63.5% to 90.6%. The nucleotide sequence of a partial actin cDNA clone has been determined. The deduced amino acids sequence showed a homology of 90.3% and 88.0% in relation to the EgactI and EgactII sequences respectively, suggesting the existence of at least one more actin gene in E. granulosus. This hypothesis is reinforced by the number of bands detected in the Southern blot analysis. Experiments based on the amplification of DNA segments using 3'-specific actin primers indicate that the EgactI gene is transcribed in protoscoleces.

Actins↗
PubMed1995

Identification and characterization of myophilin, a muscle-specific antigen of Echinococcus granulosus.

A muscle-specific gene of Echinococcus granulosus has been identified and characterized. A lambda gt11 clone (10P1), containing an incomplete copy of the gene, was originally isolated from a larval E. granulosus cDNA library by serum antibodies from dogs infected with the parasite. The full-length cDNA sequence was obtained by PCR amplification of cDNA from an adult E. granulosus lambda gt22A library. Southern blot analysis indicated the presence of the gene as a single copy in the genome of E. granulosus and also detected homologous genes in genomic DNA of E. multilocularis and Taenia saginata. The 21.2-kDa protein deduced from the complete cDNA sequence contains two regions of 12 amino acids with similarity to the EF-hand motif of calcium binding proteins. Antibodies raised against the purified 10P1-GST fusion protein detected a 22-kDa antigen in the E. granulosus developmental stages examined. Immunoelectron microscopy localized the native protein in the muscle of the parasite. The amino-acid sequence of the E. granulosus protein shows significant homology to the muscle proteins mp20 of Drosophila melanogaster, chicken SM22 alpha and mammalian calponin, and also to the neuronal protein NP25 of rats. A conserved carboxy-terminal motif of 17 amino acids is present in all the homologous proteins and is proposed to be the characteristic feature of a novel protein family. The term myophilin is proposed for the E. granulosus protein due to its localization and homology to other muscle proteins.

Amino Acid Sequence↗
PubMed1996

Molecular cloning, expression and characterization of a recombinant glutathione S-transferase from Echinococcus multilocularis.

We report the identification and characterization of the first cestode glutathione S-transferase (GST) cDNA sequence. A fragment of an Echinococcus multilocularis glutathione S-transferase cDNA was isolated by the polymerase chain reaction. Subsequently, a Lambda zap cDNA library prepared from mRNA from protoscolices of E. multilocularis was screened with this PCR fragment. A complete cDNA clone was isolated and the nucleotide sequence determined. Analysis of the E. multilocularis GST-deduced amino acid sequence indicates that it is clearly related to the mammalian mu-class GSTs. The E. multilocularis GST cDNA was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The 25.5-kDa enzyme subunit was purified to homogeneity using glutathione-sepharose chromatography. Gel filtration demonstrated that this GST is enzymatically active as a homodimer. The recombinant enzyme had conjugating activity with organic hydroperoxides and with members of the trans,trans-2,4 alkadienal and trans-2-alkenal series, which are secondary products of lipid peroxidation.

Amino Acid Sequence↗
PubMed1992

Polycystic hydatid disease (Echinococcus vogeli). Clinical, laboratory and morphological findings in nine Brazilian patients.

Polycystic hydatid disease occurs in neotropical zones and is caused by Echinococcus vogeli. The paca, a wild rodent, is the intermediate host and the final host is the dog. Seven cases of polycystic hydatid disease autochthonous to the Brazilian Amazon region are described. The disease was polycystic in all cases and diagnosis was based on anatomopathological findings. E. vogeli was identified by the shape and dimensions of the rostellar hooks. The liver was the organ most often involved (6/7), followed by the lungs (2/7) and mesentery (2/7), spleen (1/7) and pancreas (1/7). The main clinical manifestations were abdominal pain, hepatomegaly, jaundice, weight loss, anemia, fever, hemoptysis, palpable abdominal masses and signs of portal hypertension. Hepatic calcifications were detected in four cases. Two cases from the hinterland of the State of São Paulo are also reported. Both had calcified round structures in the liver, highly suggestive of calcified polycystic hydatids. The aim of the present report was to report on this relatively unknown hydatid disorder of Tropical America and to disseminate its clinical, ultrasound and radiological features.

Adult↗
PubMed1990

Comparison of Albendazole and Praziquantel therapy of Echinococcus granulosus in naturally infected sheep.

The effects of albendazole (10 mg kg-1 day-1) and praziquantel (50 mg kg-1 day-1) for 6 weeks on naturally infected sheep with pulmonary cysts of Echinococcus granulosus of proven viability were studied. Immediately following therapy, one of three sheep treated with praziquantel had viable cysts and 7 months later one of two sheep had viable cysts. One sheep died during albendazole therapy, but 7 months following therapy only one of five sheep had viable cysts. Electron microscopy demonstrated necrotic germinal layer tissue in most albendazole-treated cysts and praziquantel also had an effect on cyst ultrastructure. These data suggest that recurrence in humans treated with albendazole may be small. Whilst praziquantel was not particularly effective in this animal model, its clear effect on the ultrastructure suggests that an increased dose and combination therapy with albendazole may be more effective.

Albendazole↗
PubMed1992

Use of Echinococcus granulosus worm antigens for immunodiagnosis of E. granulosus infection in dogs.

Echinococcus granulosus worm excretory/secretory antigens (WES) were used in ELISA for diagnosis of E. granulosus infection in dogs and compared with protoscolex somatic antigens (PSM). Sera from 224 dogs were tested. There was no correlation between ELISA absorbance values and E. granulosus worm burdens using either antigen. There was a significant linear relationship between absorbance values of sera tested in the ELISA using WES (W-ELISA) and the ELISA using PSM (P-ELISA). However, there was a small but significant difference between the absorbance values of the sera tested against the two antigens. Western blot analysis of WES using sera from E. granulosus-infected and uninfected dogs revealed antigenic components of relative molecular mass (Mr) larger than 94,000, Mr 94,000-68,000 and Mr 43,000-39,000 in worms, and these were specific for E. granulosus and not identified in PSM; these antigenic differences may be responsible for differences in reactivity in ELISA. The sensitivities of W-ELISA and P-ELISA were 80.8% and 75.6%, respectively. The specificities of W-ELISA and P-ELISA were 93.7% and 97.9%, respectively. The reduced specificity in W-ELISA was mainly attributable to increased background reactivity of sera from Taenia hydatigena-infected dogs. Despite the reduction in specificity, both ELISAs are valuable epidemiological tools to determine the prevalence of antibody to E. granulosus in dog populations and to monitor the success of hydatid control campaigns.

Animals↗
PubMed1996

Cytochemical study of the germinal membrane of the Echinococcus granulosus cyst.

The present cytochemical study was undertaken to provide more information on the localization of enzymatic and glycoconjugates in the germinal membrane of the Echinococcus granulosus cyst. The distinctive distribution of binding sites for two lectins (peanut agglutinin and Dilochos biflorus agglutinin) in the germinal membrane are described. An investigation is made of the distribution and specific activity of adenosine triphosphatase, alkaline phosphatase and acid phosphatase. The results suggest that cells located in the deeper layer of the germinal membrane are intrinsic in the cellular differentiation process. The dissimilarities detected in both the enzymatic activity and the lectin-binding receptors could be associated with metacestode development or degeneration.

Acid Phosphatase↗
PubMed1996

Echinococcus granulosus: genomic and isoenzymatic study of Spanish strains isolated from different intermediate hosts.

The phenomenon on intraspecific variation in Echinococcus granulosus is already documented in Spain, where unilocular hydatidosis is an endemic disease. The first speciation studies, focused at a genomic level, showed the existence of three different strains: ovine-bovine-human, equine and swine-caprine. In the present study, the genomic identification, by random amplified polymorphic DNA technique (RAPD) of a larger number of Spanish E. granulosus isolates, using five different primers, showed the maintenance of these groups. Thus, some of these strains may not be infective for man. These conclusions were supported by a phenotypic characterization of the same isolates by zymodeme technique, showing the five isoenzyme systems used that Spanish E. granulosus strains can also be distinguished at a phenotypic level by isoenzymatic patterns. Both techniques (RAPD and zymodemes) were used for statistical analysis and for the construction of two dendrograms, which were slightly different. In addition, some intrastrain variation was detected with both techniques, a phenomenon that is directly related to the different speciation theories proposed for E. granulosus strains. The epidemiological implications of the results are discussed in the text.

Animals↗
PubMed1982

Characterization of glutathione S-transferase activity in Echinococcus granulosus.

1. Glutathione S-transferase activity has been characterized in protoscolices of Echinococcus granulosus. 2. The conjugation reaction was activated by treatment of protoscolices with phenobarbital and beta-naphthoflavone. 3. Protoscolices of E. granulosus contained the conjugation cofactor glutathione in the amount of 287 nmol/g fresh tissue. 4. Conjugation may represent an important factor in the resistance of the parasite to drugs.

Animals↗
PubMed1991

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.

Alkaline Phosphatase↗
PubMed1996

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices.

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCI or barbital buffer. The Km values of oxaloacetate and NADH were 0.400 +/- 0.018 and 0.410 +/- 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65 degrees C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a Ki value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a Ki value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.

Animals↗
PubMed1989

Plasmid vector for overproduction and export of recombinant protein in Escherichia coli: efficient one-step purification of a recombinant antigen from Echinococcus multilocularis (Cestoda).

We describe the use of the Escherichia coli plasmid vector, pVB2, for high-level expression and export of recombinant protein. The pBR322 derivative pVB2 harbors the mglB gene, which codes for the galactose-binding protein (GBP) of E. coli. GBP is exported into the periplasmic space of the bacterial cell. Gene mglB contains an EcoRI restriction site close to its 3' end which allows simple in-frame insertion of EcoRI fragments obtained from recombinant lambda gt11 phages. The pVB2 vector was used to express an antigen from Echinococcus multilocularis. The recombinant protein amounted to over 50% of total cellular protein and could be efficiently isolated from the periplasm by osmotic shock. The application of the purified antigen in an ELISA enabled a clear and specific detection of anti-Ec. multilocularis antibodies in human patients' sera, which had been immunosorbed with a periplasmic extract (containing wt GBP) before investigation. These data show the general usefulness of pVB2 as an expression vector for producing in E. coli diagnostically relevant antigens from any infective organism.

Animals↗
PubMed1994

Gangliosides of Echinococcus multilocularis metacestodes.

Gangliosides, glycosphingolipids with sialic acid, were found in metacestodes of Echinococcus multilocularis in low quantities. All gangliosides were resolved after preparative high-performance thin layer chromatography into four fractions. Cholera toxin was specifically bound to the major ganglioside, allowing the identification of it as a GM1. Precise structure of the four fractions was determined by sequential degradation by exoglycosidases, gas chromatography, electron impact mass spectrometry and liquid secondary ion-mass spectrometry. Beside GM1, the other fractions were GM3, GD1a and, at a lesser percentage, GM2, all belonging to the same a-ganglio-series. The ceramide part of these parasite gangliosides contained sphingosine associated to unsaturated n24, saturated n24 and n16 fatty acids.

Animals↗
PubMed2004

Genetic identification and host range of the Spanish strains of Echinococcus granulosus.

Three strains of Echinococcus granulosus have been previously identified in Spain (namely 'sheep', 'horse' and 'pig'), but these Spanish strains have not been properly genotyped yet. The aim of the present research was to identify the genotype to which they correspond to. Cyst isolates were obtained from different host species, and the strain to which each belonged was established by analysis of its random amplified polymorphic DNA (RAPD) banding patterns. These results were compared to those obtained with the analysis of two mitochondrial fragment sequences (cytochrome oxidase 1 (CO1) and NADH dehydrogenase 1 (ND1)) from each isolate. The Spanish 'sheep' strain corresponded with the genotype 1 (G1) of the parasite, infecting Spanish sheep, cattle, goat, pig, wild boar and human; the Spanish 'horse' strain corresponded with the genotype 4 (G4), only infecting Spanish horses; and the Spanish 'pig' strain corresponded with the genotype 7 (G7), infecting Spanish goat, pig and wild boar. Goat, pig and wild boar can be infected by two genotypes, G1 and G7. This circumstance, and especially the possibility of sylvatic intermediate hosts serving as reservoirs of the G1 genotype of the parasite, must be taken into consideration by authorities in order to develop and evaluate effective anti-hydatidosis programmes.

Alcohol Oxidoreductases↗
PubMed2004

Modelling the spatial distribution of Echinococcus multilocularis infection in foxes.

Alveolar echinococcosis is a rare but fatal disease in humans and is caused by the fox tapeworm Echinococcus multilocularis. The densities of fox and grassland rodent populations and the interactions between them influence E. multilocularis transmission rates in Europe. Successful rabies control has caused fox populations and E. multilocularis prevalence rates to increase in many European countries. The potential increase of the infection pressure on the human population motivates the monitoring of the infection status of foxes over space and time. Detection of E. multilocularis antigen levels in fox faecal samples collected in the field might provide a pragmatic methodology for epidemiological surveillance of the infection status in wildlife hosts across large areas, as well as providing an indication of the spatial distribution of infected faeces contaminating the environment. In this paper, a spatial analysis of antigen levels detected in faeces collected in the Franche-Comté region of eastern France is presented. In Franche-Comté, rodent outbreaks have been observed to originate in areas rich in grassland. Spatial trends in fox infection levels were modelled here as a function of the composition ratio of grassland in the landscape derived from the CORINE land-cover map. Kriging models incorporating the grassland trend term were compared to a variety of models in which five alternative trend expressions were used: the alternative trend expressions included linear and quadratic polynomials on the x and y coordinates with and without a grassland term, and a constant mean model. Leave-one-out cross-validation indicated that the estimation errors of kriging with a trend models were significantly lower when the trend expression contained the grassland index term only. The relationship between observed and predicted antigen levels was strongest when the estimated range of autocorrelation was within the home range size of a single fox. The over-dispersion of E. multilocularis in foxes may therefore account for the majority of spatial autocorrelation locally, while regional trends can be successfully modelled as a function of habitat availability for intermediate hosts.

Animals↗