Echinococcus granulosus: development of resistance to albendazole in an animal model

Gerbils with well developed peritoneal cysts of Echinococcus granulosus were randomized to albendazole 50 mg/kg/day or untreated control. Treated animals had less disease at post mortem after 3 months of treatment. Cysts were then taken from both albendazole-treated and control animals and cultured in vitro either with or without albendazole sulphoxide (Alb Sx) 500 micrograms/L for 14 days. Viability of cysts was then established by implantation of whole cysts into gerbils. Whilst naive cysts were affected by Alb Sx (only 2 cysts developed/gerbil) cysts from animals treated with albendazole were not sensitive to further therapy (6.4 cysts/gerbil).     

The action of albendazole on hydatid cysts in sheep experimentally infected with eggs of Echinococcus granulosus

According to the Brazilian Agricultural Ministry, the prevalence of cystic echinococcosis in the state of Rio Grande do Sul, Brazil has decreased between 1990 and 1998, possibly due to the common use of albendazole to control parasites in sheep. Therefore, albendazole (ABZ) action was tested on hydatid cysts of sheep experimentally infected with eggs of Echinococcus granulosus in Uruguaiana County (RS) with the aim of evaluating the observed changes in the parasite's prevalence. The sheep were divided into three groups of 15 animals each and treated as follows. Group 01 was a control (without treatment), groups 02 and 03 received 7.5 mg/kg and 15 mg/kg, respectively, of ABZ orally for 12 months, at intervals between 36 and 45 days. At the end of the studied period the animals were necropsied and the viability of the hydatid cysts was evaluated. Lung and hepatic cysts of sheep treated with ABZ were reduced in size. Degeneration and evident calcification were seen in 87.3% (group 02) and 75.7% (group 03), which were significantly different (P < 0.01) from the 17.3% demonstrated in the control group. It could be concluded that ABZ used early and over long periods, even in small doses, is capable of promoting lesions that devitalize the cysts. This leads to a decrease in fertility of E. granulosus in sheep and could be the reason why the prevalence of cystic echinococcosis has decreased in recent years.     

Viability of Echinococcus granulosus cysts in mice following cultivation in vitro.

The viability of hydatid cysts developed in vitro for 90 days was assessed by implantation into mice. Cysts removed from mice at 270 days post-infection (p.i.) increased their size 13.5-fold and contained several brood capsules containing protoscoleces. Thus, cysts remain viable after prolonged in vitro culture. The implantation in mice of 15 cysts developed in vitro yielded an average of 10 cysts per mouse, which is indicative of a high survival rate in these experimental infections. The ultrastructural study of cysts recovered from mice 270 days p.i. showed that the germinal membrane was more compact than before implantation and several layers of tegumental cells had developed. Observations of cysts removed from mice indicated that the plasma membrane surrounding microtriches had prolongations opening into the laminated layer.     

Echinococcus granulosus infection in urban areas

In a previous survey, carried out in 100 dogs in Rome by Tassi and Widenhorn (1977), two animals, which always lived in the urban area, were found infected by Echinococcus granulosus. An urban infection was therefore hypothized. During a following experiment 6 three-month old Beagle dogs were fed for nine months with raw meat sold in the butcheries of Rome as "food for pets". Two dogs of the same breed and origin were fed for the same period with canned food and were kept as control animals. At the end of the experiment three of the six dogs were found infected by E. granulosus, while the renmant three and the two controls resulted negative. An additional similar trial was carried out in Bologna. Three dogs were fed for seven months with the same kind of raw meat sold in three butcheries of Bologna, and a fourth dog was kept as control. At the end of the seven months also one of the three dogs was found infected by E. granulosus. These results seem to confirm the hypothesis of an urban infection by E. granulosus in dogs. Various possibilities of contamination of raw meat sold in butcheries as food for animals are discussed.     

Presence of cholinesterases in Echinococcus granulosus protoscolices

Cholinesterases were detected in protoscolices of Echinococcus granulosus spectrophotometrically and electrophoretically. To characterize these activities as acetylcholinesterases or pseudocholinesterases, BW284C51 and the organophosphate anthelmintic Neguvón were assayed as specific inhibitors of acetylcholinesterases, while Iso-OMPA was employed as specific inhibitor of pseudocholinesterases. We concluded that these cholinesterase (ChE) activities would be considered as possible targets in chemotherapy.     

Echinococcus granulosus in the wolf in Italy

During the period 1987-1999, 119 wolf cadavers were examined and checked for the presence of Echinococcus granulosus. All the animals were retrieved along the whole Apennines range of distribution of the species in Italy and most of them were illegally killed. Eighteen wolves resulted positive (15%). The mean intensity was 697.5. The force of infection for prevalence was 8.2 year(-1). The prevalence of the parasite was significantly and positively influenced by the local prevalence of cystic echinococcosis (CE) in sheep. Mean intensity was significantly and positively influenced by both the age of the wolf and the prevalence of CE in sheep. A deterministic model was used in order to simulate a purely theoretical sylvatic cycle of the parasites having the wolf as the only definitive host with 15% of prevalence. The expected prevalence of CE in wild intermediate species ranges between 10% and 25%. This prevalence overlaps the one observed in sheep. Even if both the wolf and the wild ungulate populations are increasing, the wolf still acts as a part of the main dog-sheep cycle of the parasite.     

The diagnosis of Echinococcus granulosus in dogs

The problem of diagnosing Echinococcus granulosus in dogs has still only been partially resolved, even after the advent of biotechnology. The eggs of taeniid Cestoda are extremely similar, and thus identification by microscopic examination of the faeces is risky and non-specific. For this reason, Echinococcus granulosus was traditionally diagnosed in dogs ante mortem after an arecoline hydrobromate purge. The faeces were examined macro and microscopically to establish if the adult tapeworm or its proglottids were present. Although this method is 100% specific, it is bio-hazardous and time-consuming, requires trained personnel, and its sensitivity varies. In the 1990s copro-antigens were discovered and characterised. These are released by the adult worm in the faeces. This made it possible to use enzyme-linked immune-adsorbent assay (ELISA) for in vitam diagnosis of Echinococcus granulosus. In recent years several PCR protocols have been published on the identification of Echinococcus granulosus DNA from eggs or from adult parasites and new ways of diagnosing this cestode have been developed.     

Ribonucleic acid of Echinococcus granulosus protoscolices

Echinococcus granulosus protoscolices RNA has been characterized in terms of its nucleotide composition, its behavior in sucrose density gradients and methylated albuminkieselguhr columns, its template activity, its biosynthesis kinetics, and the effect of actinomycin D.     

An experimental in vitro model for evaluating drugs against protoscoleces of Echinococcus granulosus

Pharmacological studies carried out on protoscoleces in vitro to standardize conditions that would permit a preliminary estimate of the efficacy of drugs with potential activity against Echinococcus granulosus are reported. Media such as PBS and Hanks solution, maintenance temperature, different pH values and concentrations of various solvents have been tested to check the effects on protoscolex survival in tubes in vitro. Mebendazole has been used as the pharmacological standard reference. Changes in the viability of protoscoleces have been used to demonstrate pharmacological activity. Best conditions were obtained employing Hanks solution and propylene glycol at low concentrations. Mebendazole was not completely effective at the concentrations achievable in human therapy. Linear, reproducible results demonstrated that Hanks solution provides an ideal medium for pharmacological studies. Among tested solvents, propylene glycol and dimethyl sulphoxide showed no lethal activity at low concentrations. At concentrations similar to those normally obtained in human sera, mebendazole, as in vivo, demonstrated only partial lethality for protoscoleces. The present study represents a new experimental approach to chemotherapy of hydatid disease.     

Echinococcus granulosus protoscolex formation in natural infections

Echinococcus granulosus is a parasitic platyhelminth that is responsible for cystic hydatid disease. From the inner, germinal layer of hydatid cysts protoscoleces are generated, which are are the infective forms to the dog. Systematic studies on the cell biology of E. granulosus protoscolex formation in natural infections are scarce and incomplete. In the present report we describe seven steps in the development of protoscoleces. Cellular buds formed by a clustering of cells emerge from the germinal layer of hydatid cysts. The buds elongate and the cells at their bases seem to diminish in number. Very early on a furrow appears in the elongated buds, delimiting anterior (scolex) and caudal (body) regions. Hooks are the first fully-differentiated structures formed at the apical region of the nascent scolex. In a more advanced stage, the scolex shows circular projections and depressions that develop into suckers. A cone can later be seen at the center of the hooks, the body is expanded and a structured neck is evident between the scolex and the body. During protoscolex development this parasitic form remains attached to the germinative layer through a stalk. When fully differentiated, the stalk is cut off and the infective protoscolex is now free in the hydatid fluid.     

2-Deoxy-D-glucose and combined 2-Deoxy-D-glucose/albendazole exhibit therapeutic efficacy against Echinococcus granulosus protoscoleces and experimental alveolar echinococcosis

2-Deoxy-D-glucose (2-DG) is a glucose analog used as a promising anticancer agent. It exerts its effects by inhibiting the glycolytic energy metabolism to deplete cells of energy. The larval stage of Echinococcus relies on glycolysis for energy production. Therefore, in this study, we investigated the in vitro and in vivo efficacy of 2-DG against the larval stage of Echinococcus granulosus and E. multilocularis. 2-DG exhibited significant time- and dose-dependent effects against in vitro cultured E. granulosus protoscoleces and E. multilocularis metacestodes. A daily oral administration of 500 mg/kg 2-DG in E. multilocularis-infected mice effectively reduced the weight of metacestodes. Notably, the combination treatment, either 2-DG (500 mg/kg/day) + albendazole (ABZ) (200 mg/kg/day) or 2-DG (500 mg/kg/day) + half-dose of ABZ (100 mg/kg/day), exhibited a potent therapeutic effect against E. multilocularis, significantly promoting the reduction of metacestodes weight compared with the administration of 2-DG or ABZ alone. Furthermore, the combination significantly promoted apoptosis of the cells of metacestodes and inhibited glycolysis in metacestodes, compared with the administration of 2-DG or ABZ alone. In conclusion, 2-DG exerts an effective activity against the larval stage of Echinococcus. Thus, it may be a promising anti-Echinococcus drug, and its combination with ABZ may provide a new strategy for the treatment of echinococcosis in humans.     

Metalloproteinases in the larvae of Echinococcus granulosus.

Proteolytic activity in hydatid cyst fluid, cyst membranes and protoscoleces of E. granulosus was analyzed by electrophoresis in gelatin-containing polyacrylamide gels, including characterization with a set of protease inhibitors. All contained metalloproteinases in the range 60-120 kDa, with neutral/alkaline pH optima. Major activity was observed in hydatid fluid and the membranes (five bands) with both exhibiting similar electrophoretic patterns. The samples prepared from protoscoleces shared only some of these bands.     

Partial characterisation of carbohydrate-rich Echinococcus granulosus coproantigens

Coproantigen ELISA based tests for diagnosis of canine echinococcosis provide high specificity and sensitivity. However, the antigenic molecules present in faeces from infected dogs have not yet been characterised. While initial attempts to determine the molecular weights of Echinococcus granulosus coproantigens by SDS-PAGE and Western blotting with coproantigen reactive capture antibodies were equivocal, they suggested presence of a significant carbohydrate component. Periodate treatment of coproantigen positive faecal supernatants resulted in a significant reduction (53%) in ELISA activity, suggesting that carbohydrates are involved in the antigenic structure of E. granulosus coproantigens. Protease treatment of antigenic molecules resulted in an 11% reduction in absorbance in ELISA, indicating that protein components were also present which affected by enzyme activity. Lectin-binding ELISA assays indicated strong affinity of E. granulosus coproantigens to concanavalin agglutinin and Lens culinaris agglutinin, and moderate binding to wheat-germ agglutinin and peanut agglutinin. No binding was detectable to Ulex europaensis agglutinin-I, Bandeiraea simplicifolia or Dolichos biflorus agglutinin. These data indicate that E. granulosus coproantigens from infected dog faeces possibly contained alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues. To verify the role of carbohydrate moieties in coproantigens, faecal samples were treated with exoglycosidase and tested in the coproantigen ELISA. Treatment with beta-galactosidase or N-acetyl-beta-glucosamine reduced ELISA activity by 44 and 30%, respectively. Incubation with a panel of other specific exoglycosidases including alpha-galactosidase as well as alpha-L-fucosidase, alpha-mannosidase, beta-mannosidase, alpha-glucosidase, beta-glucosidase, beta- fructosidase, or neuraminidase, did not alter coproantigen detection in ELISA. The results indicate that coproantigens present in faeces from E. granulosus naturally infected dogs were highly glycosylated and contain beta- galactose and N-acetyl-beta-glucosamine. The putative relationship of antigenic molecules with the tapeworm glycocalyx is discussed.     

In silico studies of Echinococcus granulosus FABPs

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14–15?kDa with a characteristic β-barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4?ns molecular dynamics simulations, and their protein–ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasite's metabolism.