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PubMed1992

Further characterization of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Two monoclonal antibodies (24.14, 61A12) to Echinococcus granulosus Antigen 5 and two (31.15 and 39B3) to Antigen B were further characterized using modified sheep hydatid cyst fluid antigens (SHCF) in ELISA. None of these four monoclonals were directed against carbohydrate or lipid epitopes of SHCF antigens since they all reacted strongly with periodate or lipase-treated SHCF. On the other hand, they appeared to recognize SHCF determinants of protein nature as protease treatment of SHCF destroyed binding with the monoclonals. Anti-Antigen B monoclonals 31.15 and 39B3 showed strong reaction with boiled SHCF and anti-Antigen 5 monoclonal 24.14 did not. However, the second anti-Antigen 5 monoclonal 61A12 also reacted with boiled SHCF suggesting that some epitopes of Antigen 5 are heat stable. 24.14 and 61A12 may recognize a similar epitope of Antigen 5 whereas 39B3 may be against an epitope of Antigen B different from that recognized by 31.15.

Animals↗
PubMed1996

Time course of coproantigen excretion in Echinococcus multilocularis infections in foxes and an alternative definitive host, golden hamsters.

Coproantigen excretion during experimental infections of Echinococcus multilocularis in foxes and an alternative definitive host, golden hamsters, was evaluated by a sandwich ELISA using a monoclonal antibody. A sigmoidal increase of antigen excretion from the developing parasites was observed in in vitro incubation of the parasites collected on different days during the first 21 days post-infection (DPI). In hamsters, the ELISA O.D. value of faeces became positive at 4 DPI. Thereafter, the O.D. value increased in semi-sigmoidal fashion in the first 42 DPI, probably reflecting the development of the parasites. In foxes, the O.D. value became positive at 6 DPI. However, contrary to that in hamsters, after the initial steep rise, the O.D. value suddenly decreased to 1/2 the level during 15-17 DPI, indicating that a large number of worms might have been expelled. The parasite eggs were detected by the sugar centrifugal-flotation technique (Ito, Yagi & Ishige, 1989) from 29 to 84 DPI but not thereafter to 125 DPI, although mature parasites were detected at 125 DPI. In contrast, positive O.D. values were obtained almost constantly until 125 DPI, indicating that the coproantigen detection assay was more sensitive than the egg detection assay. The detection limit of the coproantigen assay was roughly estimated to be around 100 worms. These observations, along with the fact that the assay was designed to detect a heat-resistant coproantigen in heat-sterilized fecal samples, indicate that the coproantigen detection assay is a safe and useful method, not only for diagnosis in the definitive host of E. multilocularis, but also for monitoring parasite development and change in parasite burden during an experimental infection.

Animals↗
PubMed1997

Differentiation of Spanish strains of Echinococcus granulosus using larval rostellar hook morphometry.

Two multivariate statistical procedures (Principal Component Analysis and Discriminant Function Analysis) were used to analyse morphometric data from larval hooks of Spanish samples of Echinococcus granulosus previously characterized in physiological, biochemical and genetic studies. The 5 physical variables considered could be reduced to 2 functional variables ("length of hooks" and "number of hooks"). The number of hooks was found not to be a reliable character to differentiate between strains of this parasite. Three Spanish strains (sheep-cattle, pig, and horse strains) were identified; these results are compatible with those obtained using other techniques. We consider larval hook morphology as a valid criterion for identifying E. granulosus strains in Spain, with potential use for epidemiological studies.

Animals↗
PubMed1997

The homeobox-containing gene EgHbx3 from Echinococcus granulosus is expressed in the stalk of protoscoleces.

The homeobox-containing gene EgHbx3 from Echinococcus granulosus is expressed in the stalk of protoscoleces. The expression of the homeobox-containing gene EgHbx3 from E. granulosus was studied in protoscoleces by in situ hybridisation techniques. A method for performing in situ hybridisations on whole mounted hydatic material is described. Our results show a clear positive signal at the protoscolex stalk, when using the antisense EgHbx3 probe. This signal is particularly strong in the proximal end of the stalk (with respect to the protoscolex).

Animals↗
PubMed1997

Basic and applied immunology in cestode infections: from Hymenolepis to Taenia and Echinococcus.

In larval cestode infections, it is well established that the intermediate mammalian host infected with egg-derived metacestodes in the tissue becomes completely immune to reinfection with eggs, whereas autoinfection has been conceived to occur in Hymenolepis nana/mouse (and human) and Taenia solium/human systems when these hosts are initially infected with metacestode-derived adult tapeworms in the lumen. In this review paper, the first topic is immunobiology of H. nana/mouse system on the reinfection immunity in order to get critical information as to how the initially ingested parasite (eggs or metacestodes) can develop into adult worms and how autoinfection does or does not occur in immunocompetent mice, since H. nana can complete its whole life cycle in the mouse intestinal tissue and lumen. When mice are infected with eggs (= oncospheres) of H. nana, they become immune to challenge infections with eggs within a few days (early response) and with cysticercoids within two weeks (late response). The initially established adult worms are expelled later (worm expulsion response). When mice are infected with cysticercoids, either derived from beetles or mice, they become immune to challenge infection with cysticercoids but not with eggs. Therefore, autoinfection occurs in the intestinal tissue for the establishment of cysticercoids in the tissue but never occurs in the intestinal lumen for the establishment of adult worms in immunocompetent mice. The second topic is vaccination trial against challenge infection with eggs of Asian Taenia in pigs. Pigs vaccinated with frozen oncospheres of Asian Taenia from Taiwan or Korea or T. saginata showed very strong resistance, whereas pigs vaccinated with those of T. solium showed partial resistance only. It is suggested that Asian Taenia is much closer to T. saginata than T. solium from the immunobiological viewpoint. The third topic is immunodiagnosis of echinococcosis and cysticercosis. Immunoblot analysis has revealed that Em18 (18 kDa component of crude antigens of Echinococcus multilocularis protoscolex) and glycoproteins of T. solium cysticerci are highly specific or unique to alveolar echinococcosis and cysticercosis, respectively. The fourth topic is discussion on miscellaneous prospects including laboratory animal models for echinococcosis and cysticercosis.

Animals↗
PubMed1997

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

Animals↗
PubMed1997

Echinococcus granulosus myophilin--relationship with protein homologues containing "calponin-motifs".

Myophilin, a smooth-muscle protein of the tapeworm Echinococcus granulosus, was recently postulated to be a member of the calponin family of proteins. A detailed genetic analysis revealed that 17 proteins had significant homology with the amino-acid sequence of the N-terminal region of myophilin and/or possessed one or more "calponin-motifs". Comparison of the amino-acid sequences of the N-terminus showed that the homologous proteins clustered into distinct groups based on the number of calponin-motifs. The calponin-motif of myophilin was genetically more similar to that present in the muscle protein mp20 of Drosophila melanogaster than to those in any other homologous proteins of vertebrates. The existence of a distinct motif which is "conserved" in other proteins across a range of species suggests an important functional role for the motif.

Amino Acid Sequence↗
PubMed2000

Characterisation of the proximal regulatory domain of the Echinococcus granulosus homeodomain-containing gene EgHbx1.

In an attempt to understand the molecular basis of the development of Echinococcus granulosus, we have previously isolated several homeobox-containing genes (EgHbx1-5). Here we report the characterisation of the EgHbx1 proximal regulatory domain. EgHbx1 codes for an amino acid sequence presenting 90% identity at the homeodomain level with the Drosophila melanogaster NK1-S59 transcription factor. The proximal regulatory domain sequence and the transcription start site were determined. Electrophoretic mobility shift assays using nuclear extracts from protoscoleces revealed the presence of specific DNA-protein complexes. These results constitute a hint and provide new tools to decipher regulatory cascades during E. granulosus development.

Animals↗
PubMed2000

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes.

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

Animals↗
PubMed2003

Echinococcus multilocularis in two lowland gorillas (Gorilla g. gorilla).

An unusual presentation of alveolar echinococcosis was observed in two lowland gorillas (Gorilla g. gorilla). Clinical signs included progressive abdominal enlargement, apathy and anorexia. Macroscopical changes consisted of severe peritonitis and foci of hepatic necrosis with large cavities replacing most of the normal tissue. Additionally, a few structures resembling hydatid cysts were present. Histologically, some necrotic areas contained fragments of a laminated wall characteristic of echinococcal metacestodes. Only a few areas showed the multiloculated architecture typical of Echinococcus multilocularis. Serum antibodies against E. multilocularis antigen were detected in both animals, and granulomatous and necrotizing hepatitis with severe peritonitis due to E. multilocularis was diagnosed. The pathological changes in alveolar echinococcosis in gorillas appear to resemble more closely those found in human beings than those in other non-human primates.

Animals↗
PubMed2002

The molecular epidemiology of Echinococcus granulosus and cystic hydatid disease.

It is now generally recognized that the dog tapeworm Echinococcus granulosus, the cause of cystic hydatid disease, exhibits substantial genetic diversity. This variability has important implications for the design and development of vaccines, diagnostic reagents and drugs effective against this parasite. The paper describes various deoxyribonucleic acid-based approaches that have been used for accurate identification of these genetic variants and their application in molecular epidemiological surveys of cystic hydatid disease in different geographical settings.

Animals↗
PubMedDate not supplied

Combined eco-epidemiological and molecular biology approaches to assess Echinococcus granulosus transmission to humans in Mauritania: occurrence of the 'camel' strain and human cystic echinococcosis.

Mauritania lies between West-Central Africa where human cystic echinococcosis (CE) is considered extremely rare and West Maghreb where CE accounts for a real public health problem. Until 1992, Mauritania was considered as human CE-free even through CE seemed well known in livestock. In 1992, the introduction of ultrasonography led to the diagnosis of the first human CE cases. In 1997, a veterinary study revealed that dogs living around Nouakchott were commonly infected by Echinococcus granulosus. To assess E. granulosus transmission and to identify the most relevant animal reservoir responsible for human CE emerging in Mauritania, a simultaneous eco-epidemiological and molecular biology approach was performed. The fieldwork included sample collection and investigation of relationship between intermediate hosts, definitive hosts and humans. Typing of E. granulosus strains was performed using comparison of polymerase chain reaction (PCR)-amplified DNA sequences with one nuclear (BG 1/3) and 2 mitochondrial (COI, NDI) targets. Results show that the 'camel' strain is actually infectious to humans and circulates between intermediate hosts including camels and cattle. It is suggested that preventive measures at slaughtering places could reduce human contamination.

Animals↗
PubMedDate not supplied

Field diagnosis of Echinococcus granulosus infection among intermediate and definitive hosts in an endemic focus of human cystic echinococcosis.

Human, canine and ovine echinococcosis prevalence was determined in a highland community located in the central Peruvian Andes during 1997 and 1998. Human echinococcosis was determined using portable ultrasonography, chest X-ray examination, and an enzyme-linked immunoelectrotransfer blot (EITB) assay. Canine echinococcosis was determined using microscopy stool examinations and a coproantigen detection enzyme-immunoassay (EIA) for Echinococcus granulosus. Ovine echinococcosis was determined by an EITB assay for sheep echinococcosis and necropsy examination of viscera from domestic slaughtered animals. An abdominal ultrasound, a chest X-ray examination and an EITB for echinococcosis were performed on 214 subjects (45% of the village population). The frequency of presumptive liver/abdominal, lung and liver-lung hydatid cysts was 5.1% (11/214), 3.7% (8/214) and 0.5% (1/214), respectively. The overall prevalence of human cystic echinococcosis was 9.3% (20/214). The frequency of canine echinococcosis was 46% (23/50) and 32% (16/50) by the coproantigen EIA test and arecoline purging, respectively. The frequency of sheep echinococcosis was 65% (22/34) by the EITB and 38% (13/34) by necropsy. We demonstrated a high prevalence of human and animal echinococcosis in this Peruvian village. In remote areas where echinococcosis is endemic, both the coproantigen EIA and arecoline purging may be used for the study of canine echinococcosis; the EITB is useful in establishing the diagnosis of echinococcosis in sheep prior to necropsy.

Adolescent↗
PubMed1999

Control of host complement activation by the Echinococcus granulosus hydatid cyst.

Cystic hydatid disease is caused by the multicellular parasite Echinococcus granulosus. The hydatid cyst, being a long-lived, large, antigenic structure lodged in the host's internal organs, could potentially elicit major inflammatory responses. However, in practice, the cyst causes only minimal local inflammation. The complement system is a major pathway to immune-mediated inflammation. Recent results have shown that the host-exposed structure of the cyst, the hydatid cyst wall (HCW), fails to trigger the complement system strongly. We have carried out a wide survey for the mechanisms making the cyst wall relatively complement-inert. The results of those studies are summarised in this work, with emphasis on the most recently identified of the complement inhibitory mechanisms. This is based on a non-protein heat-stable, parasite inhibitor of the activation of host complement factor B.

Animals↗
PubMed2000

Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66 kDa protein of Echinococcus granulosus.

A recombinant polypeptide (referred to as EgA31), which represents a 66kDa protein, was prepared from an Echinococcus granulosus cDNA library. In order to assess its potential to induce cellular immune responses, dog popliteal and prescapular lymph nodes were sensitized with this recombinant polypeptide. Subpopulations of lymphocytes were then analyzed by flow cytometry and immunohistochemistry on lymph node sections. Five days after the sensitization, the paracortical areas of the lymph nodes appeared hypertrophic, the number of CD3+, CD4+, CD8+ and CD5+ cells increased, the number of B-cells began to augment and some secondary follicles occurred, and a number of CD4+ cells appeared in germinal centers. Many large secondary follicles and a significantly augmented number of CD5+ cells in cords of medullae were observed 10 days after the sensitization. These active cellular responses strengthen the interest for further studies on the development of a vaccine with this recombinant polypeptide.

Animals↗
PubMed1998

A mucosal IgA response, but no systemic antibody response, is evoked by intranasal immunisation of dogs with Echinococcus granulosus surface antigens iscoms.

The search for protective antigens of intestinal parasites is conditioned by the methodology used to induce a relevant local immune response against them. The present work describes the use of immuno stimulating complexes (iscoms) from tegumental antigens from protoscoleces (PSC) of the cestode Echinococcus granulosus as immunogens in dogs by the intranasal route. It also describes the evaluation of the immune response evoked at the antibody level (systemically and at a distant mucosal location) as well as at the level of antibody secreting cells in peripheral blood. Iscoms from both E. granulosus tegumental antigens and hen ovalbumin (OVA), given at 50 microg doses by intranasal route, evoked significant secretory IgA antibody responses detected in saliva. Specific IgA secreting cells in peripheral blood also increased 10-20-fold, although transiently, after primary and secondary stimulation, whereas specific IgG secreting cells in peripheral blood were only detected in some individuals after the second antigenic exposure. Generation of immune responses at a related mucosal site provides evidence of localised immunity. No significant increase in systemic antibody titers of either IgM, IgG or IgA isotype was detected in plasma as a result of the immunisation. This fact could reflect that the nasopharyngeal mucosal associated lymphoid tissue of dogs is more strictly compartmentalised than that of other mammals.

Adjuvants, Immunologic↗
PubMed2001

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.

5' Untranslated Regions↗
PubMed2003

A gene family from Echinococcus granulosus differentially expressed in mature adult worms.

Differences in mRNA expression between immature adult worms (IAW) and mature adult worms (MAW) of Echinococcus granulosus were determined using polymerase chain reaction-based differential display (DDRT-PCR). Twenty-eight putative differential cDNA fragments were isolated, cloned and sequenced. mRNAs from IAW and MAW were probed with the labelled fragments. Six cDNA fragments (coded as egM12, egM13, egM22, egM26, egM30 and egM34) were putatively determined to be specific to MAW by Northern hybridisation. The stage-specificity of egM12, egM13 and egM34 was confirmed by RT-PCR. RNAs of IAW, MAW, protoscoleces and oncospheres, probed with egM13 and egM30, showed that the mRNAs were expressed exclusively in MAW, which implied involvement in the regulation of egg development. Using the labelled fragments to screen a cDNA library of MAW, 99 clones were identified and analysed. An alignment of selected clones showed that the MAW-specific mRNAs belonged to a family. Examination of the deduced amino acid sequence of three of the corresponding cDNAs (egM4, egM9 and egM123) indicated they were cysteine-rich and contained a 24 amino acid repeat sequence, repeated four to six times. The repeat regions were predominantly alpha helical in nature with interspersed turns, forming alternating zones of positive and negative charge. The functional significance of each of the cDNAs identified is unclear as none had significant sequence similarity to genes of known function. However, polypeptides encoded by egM4 and egM123 were recognised by antibodies in a serum pool from dogs experimentally infected with E. granulosus, suggesting they could prove of value in serodiagnosis of definitive hosts.

Amino Acid Sequence↗