Comparative chemical composition of protoscolices and hydatid cyst fluid of Echinococcus granulosus (Cestoda)

Hydatid cysts were analysed for electrolytes, nucleic acids, proteins, enzymes, nitrogenous waste products, carbohydrates and lipids. Substantial amounts of cations were detected. Potassium, magnesium and calcium were concentrated in protoscolices as compared to cyst fluid, but sodium levels were similar in both. Of the anions, chloride constituted the major bulk in the fluid anions, and was not found in protoscolices. In contrast, phosphate and bicarbonate had more significant values in the protoscolices. RNA and DNA which were found in substantial amounts in the protoscolices were nearly absent in the fluid ; so was ammonia. The reverse pattern of distribution was observed in urea, uric acid, creatinine and bilirubin. The proteins of cyst fluid were mainly albumin and globulin, the latter having always double the concentration of the former. Albumins and globulins also formed J of protoscolex protein. Among enzymes, LDH, phosphatases, GOT and GPT exhibited high activities. Cholesterol, cholesterol esters, mono-, di- and triglycerides, fatty acids and phospholipids were detected mainly in the protoscolices with phospholipids constituting the major portion. The carbohydrates of the protoscolices were glycogen, trehalose, glucose and alkali stable carbohydrates. Of particular interest was the detection of sucrose in both protoscolices and fluid.     

Echinococcus granulosus: Permeability of hydatid cysts to mebendazole in mice

Mice infected with Echinococcus granulosus were given one dose of 15 mg kg^?1 of ¹⁴C-mebendazole by gavage. Blood, hydatid cyst fluid and membranes were collected and counted at varying intervals thereafter. Radioactivity in blood peaked by 16 h and declined rapidly thereafter. Activity in hydatid cyst fluid paralleled that in blood but in amounts of only 5–10%. While levels of radioactivity in hydatid cyst membranes for the most part paralleled those of blood, in several samples they remained stable or increased from 16 to 48 h while those in blood had decreased to baseline. Protoscolices lost all signs of viability by 48 h after treatment.     

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.     

In vitro protoscolicidal effects of hypertonic glucose on protoscolices of hydatid cyst

To evaluate the protoscolicidal effects of various concentrations of hypertonic glucose, live protoscolices of sheep were exposed to 10%, 15%, 25% and 50% glucose solutions. Cetrimide (0.5%), silver nitrate (0.5%) and hypertonic saline (20%) were used as positive controls, while physiological saline was used as a negative control. After 1, 2 and 5 min, the protoscolicidal effects were determined by 1% eosin. A 25% glucose solution had no significant protoscolicidal effect. However, a 50% glucose solution revealed higher protoscolicidal effect than 0.5% silver nitrate but weaker effect than 0.5% cetrimide; the effect was comparable with that of 20% hypertonic saline. The results showed that hypertonic glucose solution is highly effective in killing protoscolices of Echinococcus granulosus in vitro.     

Echinococcus granulosus cysts: Early development in vitro in the presence of serum from infected sheep

When cultured in vitro in the presence of serum from a number of sheep infected with Echinococcus granulosus cysts, varying proportions of oncospheres died within 24 h. Of the survivors, some died during reorganization into cysts; others were able to develop normally but showed evidence of precipitates in the outer layers of the cyst. The lethal effects were removed by heating the serum to 56°C for 30 min and could be restored by the addition of freshly-collected normal sheep serum. In the presence of serum from sheep immunized against E. granulosus, most oncospheres were dead within 24 h, and few or none of the survivors were able to reorganize into cysts.     

Resistance of dogs to Echinococcus granulosus

Further studies on the immunization of dogs against E. granulosus indicated that certain dogs have a natural resistance, which is not mediated by specific antibodies or sensitized lymphocytes to tapeworm secretory antigens. In a pilot experiment with two pups, E. granulosus recovered from an immunized pup were arrested in their development and exhibited 100% inhibition of egg production compared to 100% egg production in mature worms recovered from a control pup. When the experiment was repeated with 10 pups, three of five control pups given Freud's adjuvant and Bordetella pertussis carried E. granulosus which were arrested in their development and showed 100% inhibition of egg production. Two of five immunized pups also exhibited signs of resistance. Arrested worms recovered 40 days after infection appeared to be at a stage equivalent to about 18 days of optimal development. Although immunized pups exhibiting resistance showed strong positive in vitro and in vivo immune responses, all control pups gave negative reactions to tapeworm secretory antigens.     

The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1

We describe the 1.6 Å crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease.The crystal structure reveals that EgFABP1 has the expected 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1.The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R…R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.     

The permeability of the membranes of experimental secondary cysts of Echinococcus granulosus to [14C]mebendazole

The permeability of secondary E. granulosus cysts to [¹⁴C]mebendazole was studied. The cysts were obtained by transplanting secondary cysts raised in mice into rats. The permeability to [¹⁴C]mebendazole was established by two different experiments: uptake and washout of the drug. The cyst wall permeability to [¹⁴C]mebendazole was found to be 1·33 × 10^?4 cm s^?1, which is of the same order as the diffusion permeability coefficient to water (1·88 × 10^?4 cm s^?1, Rotunno, Kammerer, Perez Esandi & Cereijido, 1974).The drug readily permeates through the cyst wall and experimental data suggest that it moves across the barrier by simple diffusion.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.     

Echinococcus granulosus: Hook-muscle systems and cellular organisation of infective oncospheres

The morphology of the oncospheres of Echinococcus granulosus has been reconstructed from thin and semi-thin serial sections. Four major types of oncospheral cells have been identified. These consist of: (1) a bi-nucleate medullary center, (2) glandular regions (eight nuclei) subdivided into three types of oncospheral glands, (3) ten germinative cells, and (4) 34 muscle cells, of which 16 are somatic and 18 are hook muscle cells. The hook muscle cells of each hook are organized functionally into the three following systems: (1) the protraction system, for hook extension, (2) the abduction system for drawing the hooks together toward the median plane of bilateral symmetry, and (3) the retraction system for pulling the hooks back into the body. The interconnections observed between different muscle fibers provide a structural basis for coordinated hook action.     

Echinococcus granulosus: Protoscolicidal effect of high intensity focused ultrasound

High intensity focused ultrasound (HIFU) is a new non-invasive technique which can cause cell death and tissue necrosis by focusing high-energy ultrasonic waves on a single location. The aim of our work is to investigate the damaging effect of HIFU on Echinococcus granulosus protoscolices, as well as its inhibitory effect on growth of hydatid cysts derived from protoscolices. The damaging effect of HIFU on protoscolices was investigated by following parasite mortality after irradiation, while the inhibitory effect was investigated by infection experiments in vivo. The results demonstrated that HIFU was able to damage protoscolices and the protoscolicidal effect was dose-dependent and showed late-onset. The growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro, and the mean weight of hydatid cysts resulting from such protoscolices in the experimental group was less than that in controls. Evidences including the protoscolicidal effect, fragmentized protoscolices and low post exposure temperatures, suggest that cavitation may contribute to the protoscolicidal effect of HIFU. In addition, the structure of the germinal membrane in cysts developing from the irradiated protoscolices was not as normal or intact as that from non-irradiated ones, and morphological changes related to degeneration were observed, suggesting that HIFU could prevent protoscolices from developing normal germinal membrane and consequently stop the proliferation of secondary hydatid cysts. HIFU demonstrated damaging effect on protoscolices, inhibited the growth of protoscolices in vitro and in vivo, and could be a possible therapeutic option for cystic echinococcosis.     

Echinococcus granulosus: Isolation and characterization of sheep hydatid fluid antigens

Five principal antigens are identifiable by immunoelectrophoresis using specific sera from rabbits immunized with sheep hydatid fluid and absorbed by polymerized sheep serum.     

Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis In vitro

Herd R. P., Ko L., Weisbrode S. E. and Heath D. D. 1984. Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis in vitro. International Journal for Parasltology14:141–149. Sequential changes (5,10, 20, 30,40, 50 min, 1, 2, 3, 4, 5, 6, 7, 8, h) were observed by light, scanning and transmission electron microscopy after 38-day-old adult Echinococcus granulosus were exposed to 50% guinea pig serum in vitro. Early changes within 3 h included contraction of worms, fusion of microtriehes, vacuolization and vesiculization of the distal cytoplasm, followed by rupture of vesicles leading to erosion and loss of the distal cytoplasm. This was most marked in the terminal proglottid but ultimately there was complete erosion of the distal cytoplasm of all proglottids and the scolex. After 3 h there was loss of definition of organelles, apparent edema of the perinuclear cytoplasm and, in some instances, rupture of the circular muscle layer with extrusion of parenchyma. Adult tape-worms exposed to heat-inactivated complement showed none of these changes. Lysis and death of the parasite was attributed to osmotic changes subsequent to the formation of trans-membrane channels induced by complement-mediated attack of the tegument after activation of the alternate pathway by factors present in the cestode tegument.     

Modelling the age variation of larval protoscoleces of Echinococcus granulosus in sheep

In autumn 2006, a study of the age-dynamics of Echinococcus granulosus cyst abundance was undertaken from an abattoir study of 1081 sheep slaughtered in Naryn Province in central Kyrgyzstan, an area endemic for echinococcosis. The results demonstrated approximately 64% of sheep were infected with the prevalence increasing markedly with age. The mean abundance was 3.8 cysts per sheep. From established models, an infection pressure of 1.33 cysts per year was estimated. In addition all cysts were recovered from infected sheep and the numbers of protoscoleces was evaluated in each cyst. A new model was developed that examined the variation in numbers of protoscoleces per infected sheep with age. This demonstrated that young sheep aged 1-2 years had very few protoscoleces, but there was a massive increase as the sheep aged. The best-fitting model assumed that the number of protoscoleces in a sheep was proportional to the volume of the cysts. In this model, the radius of the individual cyst increased linearly with the age of the cyst and hence the volume increased with the cube of the cyst age. This combined with the linear increase in numbers of cysts with age resulted in a massive accumulation of protoscoleces with the age of sheep. When the model was parameterised it demonstrated that 80% of protoscoleces were present in sheep aged 4 years and older and this represented just 28% sheep slaughtered. An average sheep at 6 or more years of age has an abundance of over 9700 protoscoleces, whilst in a young sheep of 1 year of age an average of just 16 protoscoleces could be found. The average for the sampled population across all ages was 1562 protoscoleces per sheep. The maximum number of protoscoleces in a single cyst was just 482 for sheep aged 1 year rising to 92,000 for sheep aged 6 years or older. The mean volume of cysts containing protoscoleces increased from approximately 0.7 ml at 1 year of age to 8.8 ml at 6 years of age. Cysts containing protoscoleces ranged from a diameter of 0.5-8 cm with a volume of fluid ranging from 0.2 to 50 ml. It is hypothesised that removal of old sheep through a culling programme could substantially improve the control of cystic echinococcosis.     

Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.     

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.     

Distribution of glycolipids and phospholipids in Pteridium aquilinum

The glycolipids and phospholipids in fronds and rhizomes of Pteridium aquilinum were determined. The total quantity of polar lipid decreased towards the base of the frond, but increased in the storage rhizome. The monogalactosyl diglyceride/digalactosyl diglyceride ratio was 1.8 in the pinnae, 1.0 in the lower petiole and 0.3 in the storage rhizome.     

Effect of cold shock and freezing on loss of total lipids and phospholipids of buffalo spermatozoa (Bubalusbubalis)

Buffalo spermatozoa were subjected to cold shock and freezing treatments to assess loss of lipids and phospholipids. Cold shock and freezing resulted in significant loss of 15.8% and 34.6% of total lipids and 6.4% and 19.1% of phospholipids, respectively.     

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000 bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.     

Primary cell culture of Echinococcus granulosus developed from the cystic germinal layer: Biological and functional characterization

Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives.