Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

The role of complement in hydatid disease: In vitro studies

Kassis A. I. and Tanner C. E. 1976. The role of complement in hydatid disease: in vitro studies. International Journal for Parasitology6: 25–35. Fresh sera from normal humans, guinea pigs, sheep, cotton rats, B10.D2/n Sn mice or infected cotton rats lyse viable protoscoleces of Echinococcus granulosus and E. multilocularis in vitro. This protoscolecidal activity can be abolished by heating at 56°C, EDTA or incubating with cobra venom factor, suggesting that complement proteins participate in this lytic process. Crude unfiltered hydatid fluid, as well as complement-lysed dead protoscoleces, are anticomplementary in vitro and, as such, probably protect viable protoscoleces in vivo against the action of complement. This anticomplementary activity was found to be associated with the calcareous corpuscles. A hypothesis is presented which relates these in vitro findings to the development of the parasite in vivo. It is suggested that the use of formalin during surgery to kill the parasite should be replaced by fresh serum.     

Immunohistological localisation of two hydatid antigens (antigen 5 and antigen b) in the cyst wall, brood capsules and protoscoleces of Echinococcus granulosus (ovine and equine) and E. multilocularis using immunoperoxidase methods.

Cyst wall, brood capsules and evaginated protoscoleces of E. granulosus (ovine and equine) and E. multilocularis were fixed in 10% formol-saline, embedded in paraffin and cut at 8 micrometer. Specific rabbit antisera to antigen 5 and antigen B of hydatid cyst fluid were used with immunoperoxidase methods to localise the antigens in the histological sections. Antigen 5 was found in all parasites and was associated with cells of the subtegumental area of the protoscolex, the brood capsule wall and the germinal membrane. The labelled antigen appeared as distinct granules in all areas. It is suggested that antigen 5 may be synthesised in all of these sites and that a source of the antigen in cyst fluid may be the germinal and brood capsule membranes. The laminated membranes of E. granulosus (ovine and equine) were, except for the superficial layers, free from antigen 5. Antigen B was present in all parasites. It was distributed diffusely throughout the laminated membrane, germinal membrane and brood capsule wall. There were areas of densely labelled antigen B on the surface of the distal cytoplasm of the protoscolex tegument and the surface of calcareous corpuscles. The distribution of antigen B in relation to PAS positive material and possible complement activating substances is discussed. The laminated membrane of E. granulosus was apparently more permeable to antigen B than to antigen 5. It is suggested that differences in the diffusion of these antigens through the laminated membranes of hydatid cysts in the same or different host species may account for variable serological responses during infection.     

Stereological investigation on the increase in surface area due to the microtriches of the hydatid cyst in different organs and in different hosts

Bortoletti G. and Diaz G. 1978. Stereological investigation on the increase in surface area due to the microtriches of the hydatid cyst in different organs and in different hosts. International Journal for Parasitology8: 433–436. The increase in surface area of the germinal membrane due to the microtriches has been morphometrically investigated in Echinococcus granulosus cysts developed in three different intermediate hosts. The results, achieved by Stereological methods, indicate that the development of the microtriches: (a) is more or less homogeneous all over the germinal membrane of the cysts; (b) is greater in human than in pig and sheep cysts; (c) is also greater in lung than in liver cysts within the same host and it is not related to the fertility or sterility of the parasite.     

Ultrastructural aspects of fertile and sterile cysts of Echinococcus granulosus developed in hosts of different species

Bortoletti G. and Ferretti G. 1978. Ultrastructural aspects of fertile and sterile cysts of Echinococcus granulosus developed in hosts of different species. International Journal for Parasitology8: 421–431. Research was carried out on fertile and sterile cysts of Echinococcus granulosus taken from hosts of different species. Since the animals were bom and bred in Sardinia and had undergone spontaneous infection, this entails two reservations: firstly the stunted development of cysts in certain hosts, i.e. bovines, may be peculiar to a possible ‘Sardinian strain’ of E. granulosus; secondly, certain alterations in the germinal membrane may be due to parasite ageing. In some cases, the parasite may be dead despite the cyst appearing macroscopically normal because of the presence of the laminar layer.The germinal membrane ‘thrives’ to remarkably different degrees which do not, however, correlate with cyst fertility. The most thriving conditions are to be found in human cysts which are fertile; the germinal membrane may be thriving in pig cysts which are sterile; in sheep, the germinal membrane develops quite well and cysts are generally fertile; bovine cysts are generally sterile with stunted germinal membranes.There seems to be a direct correlation between cyst development and laminar layer thickness, whereas no correlations emerged with the organ in which cysts are located.     

Echinococcus granulosus: Diagnosis of human hydatid disease by the indirect haemoagglutination reaction with antigens from hydatid fluid and scoleces

Tassi Carmelo, Dottorini Silvio, Scalise Giorgio and Geranio Nadia. 1981. Echinococcus granulosus: diagnosis of human hydatid disease by the Indirect Haemoagglutination reaction with antigens from hydatid fluid and scoleces. International Journal for Parasitology11: 85–88. Various antigenic fractions were prepared from sheep hydatid fluid and scoleces of Echinococcus granulosus by ammonium sulphate salting out. Sheep red blood cells were sensitized with all antigenic preparations and tested, by Indirect Haemoagglutination reaction, against 63 sera from humans with hydatid disease and 163 controls. The greatest sensitivity was obtained with the ‘0·8 M fraction’ and ‘Band 7’ from hydatid fluid. The specificity was excellent for all antigens examined.     

Echinococcus granulosus: induction of T-independent antibody response against protoscolex glycoconjugates in early experimental infection

In this work CD4-knockout mice were used as a model to analyse the role of CD4⁺ T cells in the antibody response against Echinococcus granulosus immunization or experimental infection. Results obtained with mice immunized with protoscolex antigens indicated that these contain T-independent antigens. After infection, CD4-knockout mice and C57Bl/6 mice showed similar titres of specific antibodies indicating that T-independent antibody production was quantitatively important in early infection. We have also identified an antigenic fraction from protoscoleces (E4⁺) which induces CD4 T cell independent antibody response in early stages of infection.In conclusion, the results presented here directly support the existence of T-independent immunogens in E. granulosus protoscoleces and suggest that T-independent antibody response may be quantitatively important in early infection.     

Molecular Characterization of Echinococcus granulosus Sensu Lato from Farm Animals in Egypt

Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.     

Ultrastructural changes associated with UV irradiation in the "germinal plasm" of Xenopus laevis

To detect structural changes following UV irradiation in the “germinal plasm,” ultrastructure of the “germinal plasm” was studied in normal and UV-irradiated eggs of Xenopus laevis at the following stages: prior to fertilization, early 2-cell, 32-cell, and late blastula. It was revealed that ultrastructural features of the “germinal plasm” were essentially common between Xenopus laevis and Rana pipiens. That is, the “germinal plasm” is composed primarily of a large aggregation of mitochondria and distinctive electron dense bodies (germinal granules). Irregularly shaped cylinderlike granules (giant germinal granules), having the same internal characteristics as the germinal granules, were found in the “germinal plasm” of all eggs examined.Comparison between normal and UV-irradiated eggs has demonstrated that UV irradiation causes swelling and vacuolation of mitochondria and fragmentation of germinal granules. The suggestion is that the integrity of certain UV-sensitive factor(s), which is involved in maintaining normal structure of germinal granules, is indispensable for the determination of the primordial germ cells.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: general procedures for the determination of water,sodium, potassium and chloride in protoscoleces

Reisin I.L. and Rotunno C.A. 1981. Water and electrolyte balance in protoscoleces of Echimcoccus granulosus incubated in vitro: General procedures for the determination of water, sodium, potassium and chloride in protoscoleces. International Journal for Parasitology11: 399–404. Protoscoleces of E. granulosus (sheep strain) were incubated in vitro at 37°C in Ringer Krebs solution (RKS) for up to 3 h. When they were briefly washed in sucrose 0.3 M at 4°C, the water and electrolyte contents were: 1.768 ± 0.034 mlg^?1 d.w. for water content and 123 ± 2, 209 ± 2 and 78 ± 2 μmolg^?1 d.w. for Na⁺, K⁺ and Cl^? respectively. When protoscoleces were not washed in sucrose solution but were spun down from RKS, the K⁺ content suffered a very small change but larger values for Na⁺ and Cl⁺ contents were obtained. These higher Na⁺ and Cl^? contents are attributed to the RKS ions retained in the trapping space. The steady state distribution of Na⁺ and K⁺ in the protoscoleces incubated at 37°C indicates the activity of an active transport mechanism.     

Ultrastructural immunocytochemical localization of two hydatid fluid antigens (antigen 5 and antigen B) in the brood capsules and protoscoleces of ovine and equine Echinococcus granulosus and E. multilocularis.

The unlabelled antibody method was used in the ultrastructural localization of two hydatid fluid antigens, antigen 5 and antigen B, in brood capsules and protoscoleces of Echinococcus granulosus and E. multilocularis. Antigen 5 was found in the parenchyma cells of the protoscolex and brood capsule wall and to a lesser extent in the walls of the flame cells and collecting ducts of the excretory system and in the surrounding interstitial material. It is suggested that, while some excretion of this antigen may occur from the protoscolex, it could also be liberated into the cystic cavity by degeneration of protoscoleces and parenchymal cells of the brood capsule wall. Antigen B was found mainly in the distal cytoplasm and perinuclear cytoplasm of the tegument anterior to the suckers. It is apparently secreted to the outside and was present in the brood capsule contents; it adheres to the anterior surface and the posterior periodic acid-Schiff (PAS)-positive glycocalyx of the protoscolex and to the inner surface of the brood capsule wall. The protoscolex tegument posterior to the suckers was negative. The parenchyma cells of the protoscolex and brood capsule wall were also positive although the intensity of the reaction product was variable.     

Natural Infection of the Ground Squirrel (Spermophilus spp.) with Echinococcus granulosus in China

Background

Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.

Methodology/Principal Findings

Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.

Conclusions/Significance

This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.     

Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: Effect of metabolic inhibitors

Reisin I.L., Rabito C.A. and Cantiello H.F. 1981. Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: effect of metabolic inhibitors. International Journal for Parasitology 11: 405–410. The effects of metabolic inhibitors on the Na⁺, K⁺, Cl^? and water balance of protoscoleces of Echinococcus granulosus (sheep strain) were studied in vitro. The protoscoleces were incubated at 37°C in Ringer Krebs solution for 3 h in the presence of iodoacetate, 3 mM (IA); potassium cyanide, 3 mm (KCN); 2?4 dinitrophenol, 0.2 mm (DNP); ouabain, 10^?M or ethacrynic acid 0.5 mm. The effects of IA and/or KCN on the water and electrolyte balance were tested at high (0.95 × 10⁵Pa) and low (0.05 × 10⁵ Pa) oxygen tensions. Inhibitors produced a decrease in K⁺ as well as an increase in Na⁺ contents. At both high and low O₂ tensions the Na⁺ balance was greatly altered by IA, the action of which could be already observed during the first hour of treatment. The cations did not reach a steady state balance during 3 h of incubation. At high oxygen tension Na⁺ and K⁺ balance was also altered by KCN or DNP though their actions were not as marked as that of IA. Ouabain affected the Na⁺ and K⁺ contents that reached new steady state distribution between 1.5 and 3 h of treatment while water and electrolyte contents were not modified by ethacrynic acid. In all the experiments no changes in Cl^? and water contents were observed. It is concluded that the energy required to maintain the Na?K balance mechanisms within protoscoleces is largely provided by the anaerobic glycolytic pathway and that the aerobic oxidative pathway contribution to the energy balance is only accessory.     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.     

Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation

BackgroundThe proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA)-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs) upon the induction of strobilar development.Conclusions/SignificanceAfter the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies.     

Echinococcus granulosus protoscoleces promotes proliferation and invasion of hepatocellular carcinoma cells

There may exist a connection between Echinococcus granulosus infection and cancer development. Here, it is aimed to investigate specific effects of E. granulosus protoscoleces (PSCs) on the proliferation and invasion capacities of hepatocellular carcinoma (HCC) cells in vitro and ex vitro. HepG2 cells were cultured with different quantities of E. granulosus PSCs in vitro. MTT analysis was used to evaluate effects of E. granulosus PSCs on the proliferation of HepG2 cells. Besides, scratch and transwell assays were respectively used for the detection of HepG2 cells migration and invasion capacities after co-culture with E. granulosus PSCs. Then, HepG2 cells were subcutaneously transplanted into nude mice with or without E. granulosus PSCs. From the 25th day of transplantation, the volume of subcutaneous lesions was measured every four days. At the 37th day, subcutaneous lesions were removed and their weight was evaluated. H&E staining was used for detecting basic pathological changes. HepG2 cells grew well without obvious morphological changes. Proliferation rate and migration capacity of HepG2 cells were higher in the co-culture group than the control group, which was closely associated with quantities of E. granulosus PSCs and co-culture time length. Moreover, HepG2 cells co-cultured with E. granulosus PSCs had stronger invasion ability than the control HepG2 cells. Importantly, there existed significant differences in the volume and weight of subcutaneous lesions after transplanting HepG2 cells with E. granulosus PSCs than the control group. HepG2 cells were also more pathologically heterogeneous in morphology after transplantation with E. granulosus PSCs. Thus, E. granulosus PSCs may promote proliferation and invasion of HCC cells.