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PubMed1999

Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels.

Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology93, 171-180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with anti-hsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV + ALB produced less damage as manifested by fewer changes in the aforementioned damage parameters but PZ and PZ + ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression.

Albendazole↗
PubMed1987

Studies on the adhesion of protoscoleces from Echinococcus multilocularis and E. granulosus to artificial substrates and human endothelial cells in vitro.

This paper describes experiments in vitro to investigate the interaction between isolated protoscoleces from Echinococcus granulosus and E. multilocularis and human endothelial cells in monolayer culture. During a maximum coculture period of 48 h neither protoscoleces nor endothelial cells showed evidence of cytotoxicity. However, protoscoleces adherent to either endothelial cells or artificial substrata developed a glycocalyx-like coat. Adherence of protoscoleces to endothelial cells requires serum (pooled human serum or foetal calf serum) in a dose-dependent fashion. However, protoscolex adhesion to artificial substrates such as glass with or without gelatin coating was markedly inhibited by serum. Thus, even 0.001% serum reduced adhesion by 46% compared with the control value. Whilst the chemical nature of the serum components have not been identified, these experiments show that serum contains both inhibitory and promoting factors for protoscolex adhesion, a finding of possible significance for the therapeutic prevention of systemic spread of established Echinococcus infections.

Animals↗
PubMed1983

Proliferation and metastases formation of larval Echinococcus multilocularis. I. Animal model, macroscopical and histological findings.

Using surgical techniques, 70 Meriones unguiculatus were infected by implantation of 0.15-0.20 g of larval Echinococcus multilocularis tissue into the subcutis of the neck region. In 64 of 65 animals necropsied, the transplants had increased in size and weight and reached an average weight of 4.1 g at the end of the experiment 12 weeks post infection (p.i.). Metastatic Echinococcus lesions developed in the regional lymph nodes and in the lungs; in 18 animals the parasite proliferated into the thoracic cavity. Of 41 animals examined 10 and 12 weeks p.i., 88% had multiple or single metastatic lung lesions, the first being observed 6 weeks p.i. Typical cysts and protrusions ("buds") of the germinal layer were detected in samples of parasite tissue before and after transplantation to experimental animals, as well as in lymph nodes and lungs in which metastases had developed. Similar structures were found in liver sections of two patients with alveolar echinococcosis. It is concluded that the spread of the parasite from the subcutis of the neck region to the draining lymph nodes and the lungs had taken place via the lymph and blood system. The possible role of the "buds" in metastases formation is discussed.

Animals↗
PubMed1995

Comparison of the patterns of codon usage and bias between Brugia, Echinococcus, Onchocerca and Schistosoma species.

Patterns of codon usage and bias were compared among taxa of the genera Brugia, Echinococcus, Onchocerca and Schistosoma by metric multidimensional scaling and three commonly used indices of bias: Nc, GC3S and B. The overall codon usage for each taxon was compared, as was the codon usage for each individual gene within the taxa. Differences in the patterns of codon usage observed between taxa were dependent on the overall base composition of the genes analysed. The codon usage of Echinococcus was distinct from that of the other taxa. Furthermore, the pattern of codon usage detected by the average codon usage summed across all genes for each taxon was not shown by all genes from that taxon.

Animals↗
PubMed1992

Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Echinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with either E. granulosus or E. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n = 37) or infected with non-Echinococcus cestodes (n = 76) or with various nematodes (n = 42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring greater than 1,000 E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing greater than 200 E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring less than 1,000 E. multilocularis and 9 of 10 samples from dogs or dingoes bearing less than 200 E. granulosus tested negative. Experimental prepatent infections of dogs with E. granulosus revealed positive ELISA reactions within the prepatent period (10-20 days post-infection) for six animals bearing greater than 1,000 E. granulosus each; a low worm burden (less than 1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected with E. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.

Animals↗
PubMed1976

[Diagnosis and therapy of liver echinococcus (author's transl)].

From 1970 to April 1975 42 patients were treated for Echinococcus of the liver at the Bonn University Dept. of Surgery. There were 33 cases of E. cysticus and 9 cases of E. alveolaris. These two types of Echinococcus, different in parasitology and epidemiology present different clinical manifestation of disease with different course and prognosis. Angiography with celiaco- and superselective hepaticography are decisive for diagnosis. Therapy can only be surgical with total removal of the parasites. In E. cysticus this is almost always possible by enucleation-resection or pericystectomie following evacuation of the cyst and instillation of 20% sodium-chloride or formaldehyde. Such radicality is the exception in E. alveolaris. Here partial resections, biliodigestive and hepatodigestive anastomoses as palliative measures are carried out predominantly to ensure bile passage.

Adult↗
PubMed1987

Value of isotypic characterization of antibodies to Echinococcus granulosus by enzyme-linked immuno-filtration assay.

In order to establish prognostic criteria for patients with hydatid cysts and to identify functional antigenic components of Echinococcus granulosus 87 sera from 36 patients were studied by enzyme-linked immuno-filtration assay (ELIFA) to characterize the four main classes of specific immunoglobulins and to determine compared immunological profiles (CIP) between samples or subjects. This method uses filtration with labelled antibodies to reveal precipitating systems preformed by immuno-electro-diffusion on cellulose acetate strips. IgG antibodies were demonstrated in the arc 5 and in 11 others bands. Specific IgA, IgE and IgM to Echinococcus granulosus was found in 19%, 53% and 69% respectively of patients with hydatids. The presence of IgM and/or IgE antibodies one year after treatment always correlated with disease. Specific IgA was detected more often in patients with pulmonary cysts. Pre- and post-operative monitoring by CIP-ELIFA proved simple and rapid to perform and of decided prognostic value. Two antigens, corresponding to arc 5 and arc X, were frequently associated with the poly-isotypic immune response.

Animals↗
PubMed1993

Molecular variation in Echinococcus.

In the past decade, molecular genetic approaches have revolutionised the study of population biology and taxonomy and have provided a vast accumulation of DNA sequence and other genetic data. DNA techniques, however, have only recently been applied to the study of helminth parasite populations yet, already, valuable information about population structure, genetic variation and phylogeny of the Echinococcus organisms is emerging. Some of this recently acquired molecular information is reviewed here, and its implications in terms of our understanding of variation and taxonomy within the genus Echinococcus emphasised. In particular, the current strain status of some geographically isolated populations of E. granulosus is discussed and the issue of inter- and intra-strain variability within E. granulosus examined.

Animals↗
PubMed1990

Echinococcus: biology and strain variation.

Biology and strain variation in the causative agent of hydatid disease is reviewed with emphasis on developmental and genetic aspects. In vitro cultivation experiments have made a significant contribution to current knowledge of the developmental plasticity of Echinococcus. However, the mechanisms which regulate and determine developmental strategies in the parasite, as well as the characteristics, source and cytodifferentiation of germinal cells, are not understood. The nature, significance and origin of strain variation in Echinococcus are examined. Before we can fully appreciate the phenotypic consequences of genetic differentiation between populations, we need to know something about the genetic and environmental components of variation in traits such as development rate, host preference, host specificity, virulence and drug resistance. There is an urgent need for research on the developmental pathways by which genetic differences within and between strains of E. granulosus are translated to phenotypic differences in these traits.

Animals↗
PubMed1993

NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus.

Nucleotide sequences of a 471 bp region of the mitochondrial NADH dehydrogenase 1 gene were obtained for 59 Echinococcus isolates including representatives of each of the 4 recognised species. Ten distinct genotypes were detected among these isolates, including 6 within E. granulosus. This information complements and extends knowledge of inter- and intraspecific variation within Echinococcus and should prove useful in phylogenetic studies.

Animals↗
PubMed1989

The isolation, by differential antibody screening, of Echinococcus multilocularis antigen gene clones with potential for immunodiagnosis.

A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multilocularis protoscolex mRNA. Differential screening of the library with pools of E. multilocularis and Echinococcus granulosus human infection sera has revealed 13 potentially immunodiagnostic clones. On the basis of plaque immunoassays and lysogen characteristics, two of these clones, designated EM2 and EM4, have been further characterised. The recombinant fusion-peptides have been purified and their potential as immunodiagnostic reagents has been assessed by immunoblotting and, in the case of one fusion-peptide (EM4), by enzyme-linked immunosorbent assay (ELISA). Furthermore, the native parasite antigens coded for by these clones have been identified. EM2 corresponds to a 70 kDa protein and epitopes coded for by EM4 have been found on three antigens of 62, 49 and 44 kDa. The native antigens of both clones are present in the protoscolex and those corresponding to EM4 appear to be excreted/secreted products. They are not recognised in ELISA by a variety of human parasitic infection sera other than sera taken from patients infected with E. multilocularis. Nevertheless, the native antigens for both clones are present in E. granulosus protoscoleces and Taenia solium cysticerci. These antigens are not detectable in E. granulosus cyst fluid, and this may, in part, explain the lack of immune response to them in human E. granulosus and T. solium infections.

Animals↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of an echinococcal microtrichal antigen immunoreactive in Echinococcus multilocularis disease.

A cDNA expression library of the larval stage of the cestode worm Echinococcus multilocularis has been established in the phage lambda ZAPII system. By immunoscreening with pooled sera from patients with alveolar echinococcosis an immunoreactive clone, termed pEM13, was isolated. EM13 was expressed using the expression vector pGEX-3X, resulting in the synthesis of a glutathione S-transferase fusion protein. 82% of the sera from 28 patients suffering from E. multilocularis disease had antibodies against EM13, whereas none of the 55 sera obtained from Echinococcus granulosus-infected patients and none of the 15 sera from patients with other helminthic infections reacted with recombinant EM13. By use of a polyclonal rabbit anti-EM13 hyperimmune serum native EM13 protein could be detected only in the protoscolices of E. multilocularis, but not in E. granulosus larvae or hydatid fluid. Immunoelectron microscopy suggested that EM13 is located in the microtriches on the surface of the larvae. In contrast, EM13 mRNA could be detected by Northern blot analysis in both E. multilocularis and E. granulosus larval RNA preparations. Nucleotide and amino acid sequence analysis of a cDNA clone coding for the corresponding antigen of E. granulosus larvae, termed EG13, revealed a 21-bp insertion, a 51-bp deletion and additional 22 nucleotide exchanges resulting in a 96.3% identity at the nucleotide sequence level and a 96.6% identity at the amino acid sequence level to the coding region of the cDNA pEM13. Cross-reactivity of the polyclonal anti-EM13 serum with the recombinant EG13 indicates a posttranscriptional regulation mechanism, resulting in an EG13 negative phenotype in E. granulosus.

Amino Acid Sequence↗
PubMed1995

Echinococcus granulosus, E. multilocularis and mammalian liver-type alkaline phosphatases: a comparative study.

The alkaline phosphatases (EC 3.1.3.1) from Echinococcus granulosus and E. multilocularis (Cestoda) were compared to each other and to a liver-type enzyme. The purified proteins (210 and 220 kDa, respectively) had a tetrameric structure composed of 4, 56/53 kDa subunits. Enzymatic removal of their N-linked sugar moieties abolished the differences in their apparent molecular weight under reducing conditions. After phase separation in Triton X-114, the E. multilocularis enzyme was the most amphiphilic, and treatment with PI-P1C reduced the amount of the parasite alkaline phosphatases that were in a hydrophobic form by about 50%. Both parasite enzymes were highly resistant to heat denaturation and insensitive to the inhibitors L-phenylalanine and L-leucine. In addition, L-homoarginine, levamisole and ZnCl2 can be used to differentiate the parasite and mammalian liver-type enzymes from each other. The Echinococcus alkaline phosphatases have original biochemical properties when compared to the mammalian liver-type enzyme.

Alkaline Phosphatase↗
PubMed2003

Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals.

Helminth antigens were investigated in the search for accessible heterologous antigens capable to discriminate different helminthiases, by the enzyme linked immunosorbent assay (ELISA) and the immunoblot assay (IB). Antigens used were: Taenia solium cysticercus total saline (Tso); Taenia crassiceps cysticercus vesicular fluid (Tcra-VF); T. crassiceps cysticercus glycoproteins (Tcra-GP and Tcra-(18-14)-GP); Toxocara canis larva excretory-secretory (TES); Schistosoma mansoni adult total saline (Sm) and Echinococcus granulosus hydatid fluid (Eg). The assayed sera were from patients with: cysticercosis (n = 18); toxocariasis (n = 40); schistosomiasis (n = 19) and hydatidosis (n = 50) with proven clinical and laboratory diagnosis, and sera from rabbits immunized with Tso, Tcra-VF, TES and Eg. Cross-reactivity occurred mostly between infections caused by Taenia and Echinococcus or in immunized rabbits, by ELISA. Moreover, the cross-reactivity among helminthiases was found with the use of antigens belonging to phylogenetically related parasite species, Eg, Tso and Tcra-VF, by sharing same antigenic components. Lower cross-reactivities were obtained by IB technique, when only peptides were considered as antigens, and the use of T. crassiceps purified glycoproteins demonstrated high sensitivity and specificity in the diagnosis of human cysticercosis, similarly to that using homologous antigen (Tso) by the same technique.

Animals↗
PubMed2005

Double-antibody sandwich ELISA using biotinylated antibodies for the detection of Echinococcus granulosus coproantigens in dogs.

Here we present the diagnostic evaluation of an improved double-antibody sandwich ELISA for detecting Echinococcus granulosus antigens in dog faecal samples (coproantigens). A purified rabbit IgG fraction against protoscolex excretory-secretory products was used as primary antibody, and the same fraction conjugated with biotin as secondary antibody. In order to validate the sandwich ELISA, intra- and inter-assay precision, linearity, and recovery percentages were calculated. The diagnostic evaluation of the method was carried out by investigating faecal samples from 37 dogs naturally infected with E. granulosus, 15 Echinococcus-free dogs infected with Taenia spp., 82 dogs with non-taeniid helminths and 66 dogs free of helminth infections. An overall sensitivity of 78.4% and specificity of 93.3% were determined. Positive and negative predictive values were 72 and 95%, respectively, and the diagnostic efficiency was 90.5%. In addition, the sandwich ELISA detection limit was estimated in 5.12 ng ml(-1). These results are highly satisfactory, allowing the use of this methodology in surveillance and control programs for intestinal echinococcosis in dogs.

Animals↗
PubMed2006

Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits.

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.

Amino Acid Sequence↗
PubMed2005

Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and plateau pika in China.

The taeniid cestode Echinococcus shiquicus n. sp. was found from the Tibetan fox Vulpes ferrilata and the plateau pika Ochotona curzoniae in the Qinghai-Tibet plateau region of China. In the adult stage, E. shiquicus from the foxes is morphologically similar to Echinococcus multilocularis. However, the new species is differentiated by its smaller rostellar hooks, fewer segments, distinct position of genital pore in the mature segment and fewer eggs in the gravid segment. Hydatid cysts of E. shiquicus found in the livers from the pikas were essentially unilocular but an oligovesicular cyst was also found. The data of mitochondrial and nuclear DNA sequences proved E. shiquicus to be a valid taxon.

Amino Acid Sequence↗