Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).     

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).     

Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host

Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host‐parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.     

Echinococcus granulosus: host lymphocyte transformation by parasite antigens.

Lymphocyte transformation, measured by in vitro tritiated thymidine incorporation, and indirect hemagglutination tests were carried out on hydatid patients and normal individuals using sheep and human hydatid fluid or scolex antigens. The hydatid patients showed statistically significant lymphocyte transformation with human and sheep hydatid fluid or scolex antigens when compared to normal individuals. The indirect hemagglutination tests resulted in high titers of antibody with sheep or human hydatid fluid antigens, while very low titers were obtained with scolex antigens. Unlike in the indirect hemagglutination test, the source of the antigen, scolex or fluid, was not of consequence in the lymphocyte transformation test. Furthermore, there was no correlation between the results of the serologic and lymphocyte transformation tests, since some patients with very high lymphocyte stimulation indices produced low indirect hemagglutination titers and vice versa. Similar results were obtained from rabbits which were immunized with sheep hydatid fluid or scolex extracts. The skin tests were of the immediate type of hypersensitivity reactions. Delayed skin reactions did not occur in spite of the presence of sensitized lymphocytes in the blood of the immunized rabbits.     

The migration of oncospheres of Taenia pisiformis, T. serialis and Echinococcus granulosus within the intermediate host

Heath D. D., 1971. The migration of oncospheres of Taenia pisiformis, T. serialis and Echinococcus granulosus within the intermediate host. International journal for Parasitology, 1: 145–152. The oncospheres of Taenia pisiformis and T. serialis hatched, became activated and penetrated the tips of the villi in the jejunal area of the rabbit small intestine. Similar results were obtained forE. granulosus, T. hydatigena and T. ovis in sheep. Most species of oncospheres appeared to progress down the villus beneath the columnar epithelium until a venule of diameter sufficient to allow passive transport to the liver was penetrated. The relatively large villus lacteal of ruminants, and the large diameter ofE. granulosus oncospheres, appeared to provide an opportunity for these organisms to penetrate the lacteal and translocate in the lymph. Parenteral inoculations of activated oncospheres indicated that T. pisiformis and E. granulosus oncospheres probably reach the liver in the portal vein. E. granulosus oncospheres infecting the lung may reach that organ in the lymph. T. serialis oncospheres were able to pass through both the liver and lungs in order to reach the muscles. A stimulus may exist in the liver causing cessation of movement and initiation of postoncospheral development for T. pisiformis and E. granulosus, but not T. serialis.     

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.     

Immunity and vaccine control of Echinococcus granulosus infection in animal intermediate hosts

Much progress has been made with characterisation of the EG95 vaccine which can be used to prevent hydatid infection in animal intermediate hosts of Echinococcus granulosus. The vaccine comprises a single recombinant oncosphere antigen and the adjuvant Quil A. It induces complement-fixing antibodies that kill the invading oncosphere early in an infection. In the majority of vaccinated animals, no hydatid cysts occur following a challenge infection. However, a small number of viable cysts may occur in some vaccinated animals. The vaccine has proved effective in vaccine trials carried out in sheep in New Zealand, Australia, Argentina, Chile and China as well as in goats and cattle. Investigations of the genetic diversity of the gene encoding EG95 have identified no unequivocal variation within the G1 strain parasites; however DNA sequence diversity within the EG95 family of genes has been found in G6/G7 parasites. GMP production scale-up of the vaccine has been undertaken in New Zealand and China and it is expected that the vaccine will be become available through these sources for implementation as part of hydatid control programs worldwide.     

Genetic differences between cysts of Echinococcus granulosus from the same host

Isozyme differences were found between protoscoleces derived from different cysts in three sheep and three macropod marsupials. Isozymes were interpreted as the products of different alleles at corresponding enzyme loci, indicating that the same host may contain cysts derived from genetically different embryos. Genetic variation on this scale may cause confusion in epidemiological studies, if protoscoleces from several cysts are pooled prior to strain-typing.     

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ultrastructural study of in vitro larval development of Echinococcus granulosus protoscoleces

Through ultrastructural study of the morphological forms developed in vitro during protoscolex culture, we describe larval E. granulosus histogenesis. The transformation of the spined microtriches in the protoscolex into truncated microtriches that develop within the hydatid cyst is discussed. The paper also describes the mitochondria location change that occurs during the evolution; the mitochondria pass from the most internal area of the distal cytoplasm along the cytoplasmic extensions into the cytoplasm of tegumental cells. The ultrastructures of both the vesiculated protoscolex and the posterior bladder demonstrate that each state corresponds to the initial step on one of the two paths of in vitro vesicular development.     

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.     

Structural analysis of an Echinococcus granulosus actin-fragmenting protein by small-angle x-ray scattering studies and molecular modeling

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.     

Echinococcus granulosus: possible formation of a shelled egg in vitro.

What appeared to be the early stages in the formation of a single egg with a striated embryophore was observed in an in vitro culture of Echinococcus granulosus protoscoleces isolated from sheep hydatid cysts in North Jordan. The 'egg' measured 19 x 19 microns in diameter and was formed in an intermediate vesicular/monozoic form which was never previously reported from a culture. This is the first report of an apparently shelled egg forming in an in vitro culture, but although promising, cannot be regarded as being unequivocal and will require confirmation by further work.     

Echinococcus granulosus: evaluation of purified antigens immunoreactivity

A new method is presented for the isolation of purified Echinococcus granulosus antigens from sheep hydatid fluid. Echinococcus granulosus antigens were separated from the host's serum contaminants by absorbing sheep serum proteins with specific immunoabsorbents.No net sensitivity gain was obtained by using these purified antigens rather than crude sheep hydatid fluid in hemagglutination and immunoelectrophoretic tests.The presence of two major antigens (≥ 400,000 and 150,000 MW) was confirmed, the larger component being clearly the most immunoreactive.Evidence of a third slightly antigenic fraction of low molecular weight is presented.The effectiveness of labeled major Echinococcus granulosus antigens in radioimmunoprecipitation test is reported.