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Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid.

This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.

Animals

Immune response to Echinococcus multilocularis infection in the mouse model: a review.

Echinococcus multilocularis is a cestode helminth which, along with E. granulosus, E. oligarthus and E. vogeli is a causative agent of hydatid disease in man. In the intermediate host (including man), cysts formed by the metacestode (larval stage) develop in the internal organs, causing functional impairment which often leads to the death of the host. In this review larval E. multilocularis infection in mice, the most popular experimental intermediate host, is examined, and the immune response to the organism is described in detail. Evidence is presented which suggests that cell-mediated immunity (CMI) plays a large role in suppression of larval growth. Congenitally athymic nude mice, and mice treated to remove thymocytes had high susceptibility to infection, while mice strains assessed as having high helper T lymphocyte function showed resistance to infection. The degree of antibody response shown by the host does not correlate with the susceptibility to E. multilocularis. Infection with E. multilocularis is accompanied by immunosuppression, manifested by inhibition of effector cell chemotaxis and receptor expression, suppressor macrophage and lymphocyte activity, decline in helper T-lymphocyte activity and immune-complex deposition.

Animals

[Effect of mebendazole on glucose uptake of Echinococcus granulosus cysts].

Mice infected with secondary cysts of Echinococcus granulosus were treated ig with mebendazole (Meb) 25 mg.kg-1.d-1 for 7-14 d. At 24 h after the last dose the endocysts in the treated mice were removed out for in vitro cultivation and exposed to [U-14C]glucose 11.1 kBq.ml-1 for 2 min, no apparent difference in radioactivity content in the cyst walls between the treated and control groups was observed. When [U-14C]glucose was given iv to the infected mice 24 h after they had been treated ig with Meb 25 mg.kg-1 or 50 mg.kg-1 daily for 14 d, the radioactivity content in the cyst wall and cyst fluid decreased significantly as compared to the corresponding control group. Nevertheless, no apparent change in the incorporation of radioactivity into the endogenous glycogen of the parasites was observed, although the glycogen in the cyst wall decreased markedly.

Animals

Synthesis and in vitro activity of alkylaminoalkyloxy chromones on protoscoleces of Echinococcus multilocularis (Cestoda).

Twelve derivatives with alkylaminoalkyloxy chromone structures were synthesized and tested upon protoscoleces of Echinococcus multilocularis metacestode kept alive in vitro. Assays were performed with protoscoleces attached to the germinal layer in open and in closed vesicles. Compounds IVb and IIIe at the concentration of 0.1 mmol.L-1 killed 50% of the protoscoleces in open vesicles in 48 hours and compound IVb killed 100% of the protoscoleces in open vesicles within 96 hours at the same concentration. In closed vesicles after four days compound IVb killed all protoscoleces, compounds IIIe and IVd half of them, whereas in the controls all protoscoleces were alive. Trifluoperazine (TFP) was the reference compound; none of the new compounds showed better activity than TFP.

Animals

Effects of mebendazole, albendazole, and praziquantel on alkaline phosphatase, acid phosphatase, and adenosine triphosphatase of Echinococcus granulosus cysts harbored in mice.

Mice infected with protoscoleces of Echinococcus granulosus for 12-14 months were treated ig with mebendazole (Meb) 25-50 mg.kg-1 x d-1 for 7-14 d, albendazole (Alb) 200 mg.kg-1 x d-1, cr praziquantel (Pra) 500 mg.kg-1 x d-1 for 14 d. The mice were killed 24 h after the last medication, and acid phosphatase (ACP), alkaline phosphatase (AKP), and adenosine triphosphatase (ATPase) including (Na, K, Mg)-ATPase, (Na, K)-ATPase, and (Mg)-ATPase were determined and compared with those of untreated control group. The results showed that ACP activities of cyst wall in treated groups were lower than the control group. Whereas AKP activity of cyst wall in Pra group increased markedly, this is not the case in Meb and Alb groups. Three ATPase activities of cyst wall were inhibited in both Meb and Alb groups, Meb being more potent. No apparent changes in the ATPase activities were seen in Pra group.

Acid Phosphatase

[Observation on ultrastructure of sterile cysts of Echinococcus granulosus in mice].

The fine structure of sterile cysts of Echinococcus granulosus in mice was studied by TEM. The cyst wall consists of laminated layer and germinal membrane. At ultrastructural level, the laminated layer contains microfibrillate matrix and irregularly shaped electron-dense granules. The germinal membrane comprise of tegument and cell region. Plasmalemma invagination resembling the "pinocytotic vesicle" are seen between the microtriches of the tegument, and mitochondria are seen in the basal portion of the tegument. In the cell region, there are tegumental cells, muscle cells, and glycogen-containing cells. "Nucleolar channel system" can be observed in some of the glycogen-containing cells.

Animals

Echinococcus granulosus and E. multilocularis: in vitro culture of the strobilar stages from protoscoleces.

The techniques for the in vitro culture of Echinococcus granulosus and E. multilocularis are described in detail. The sheep strain of E. granulosus was grown to sexual maturity but fertile eggs were not produced due to the failure to achieve insemination in vitro. The horse strain of E. granulosus grew only slightly in vitro and then ceases to develop, although remaining active for long periods. E. multiloculoris differentiated largely into unsegmented, sexually mature, "monozoic" forms; some organisms developed "pseudo-proglottids" with the interproglottid membrane absent or poorly formed. After prolonged culture, some strobila of E. multilocularis developed an extra scolex.

Animals

Cross-protection between the cysts of Echinococcus granulosus, Taenia hydatigena and T ovis in lambs.

Lambs were infected orally at 14 weeks of age with either Taenia hydatigena, T ovis or Echinococcus granulosus eggs. To induce high levels of resistance to oral reinfection, they were then injected intramuscularly with eggs of the homologous species four weeks and eight weeks later. The degree of immunity was then tested by oral challenge with eggs from either the homologous or an heterologous species. Autopsy results indicated that prior infection with an homologous species induced a very high degree of immunity to challenge infection. Prior infection with all combinations of heterologous species resulted in a lesser degree of immunity, which appeared to have acted on the establishment phase of development and did not influence cyst survival.

Animals

[Echinococcus multilocularis: superinfections in experimentally infected mice (author's transl)].

The influence of subcutaneous superinfections following an intraperitoneal primary infection after 4 and 14 weeks respectively has been examined in Echinococcus multiolcularis infected NMRI-mice. It could be shown that the growth of subcutaneous echinococci is inhibited in superinfected mice (372 mg) if compared with control mice (903 mg parasite weight). Other differences (composition of serum proteins and white blood cells, hemoglobin content) are exclusively due to the primary intraperitoneal infection.

Animals

Characterization of allergens in Schistosoma mansoni, Fasciola hepatica and Echinococcus granulosus.

Various antigens interacting specifically with IgE antibodies from infected humans were identified in S. mansoni, F. hepatica and E. granulosus soluble extracts by means of radioimmunoelectrophoresis. Two allergens common to adult worm and cercariae were identified in S. mansoni extracts. They proved to be distinct from genus--or species--specific antigens previously identified in the parasite. In adult F. hepatica extract a species-specific antigen and another lipoprotein were shown to interact with IgE antibodies of patients with fasciolasis. Two major allergens were also found in whole fluid of sheep E. granulosus cysts. One of them corresponded to Echinococcus genus specific antigen. These results are discussed according to immunological findings in parasitic infections.

Allergens

Survival of protoscolices of Echinococcus granulosus at constant temperatures.

A study was conducted to determine the effects of storage at constant temperatures upon the survival of protoscolices of Echinococcus granulosus from hydatid cysts removed from infected sheep. Parallel tests were conducted on intact cysts from both lung and liver, and on protoscolices stored within 1-ml samples of hydatid fluid. The longest survival times of any of the samples tested at each temperature were: -20 C, 1 hr; -10 C, 4 hr; 1 C, 16 days; 10 C, 16 days; 20 C, 8 days; 30 C, 4 days; 40 C, 2 days, and 50 C, 2 hr. In general, protoscolices survived considerably better when stored within intact cysts than when in 1 ml of hydatid fluid. At temperature ranges where putrefaction occurred, protoscolices in cysts from lung survived longer than those from liver. The ability of these protoscolices to survive extended periods of time after an infected sheep has died or been killed suggests that stringent preventive and control measures should be established in areas where hydatid disease is endemic. Animal pits at community dumping grounds where sheep carcasses might be discarded should be enclosed or covered, and all stray and roving dogs in those regions should be rigidly controlled.

Animals

Permeability studies on taenid metacestodes: I. Uptake of proteins by larval stages of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus.

Host immunoglobulins of several different classes were detected within the bladder fluids of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus. Radioiodinated proteins were taken up in vitro by larvae of both T. taeniaeformis and T. crassiceps and were shown to retain their physicochemical and antigenic characteristics. Rates of uptake were similar in the 2 species and were not related to the molecular weight of the proteins. Immunoglobulins were taken up both in vitro and in vivo by larvae of T. taeniaeformis. Absorbed immunoglobulins were shown to retain both antigen binding capacity and biologic functions associated with the Fc portion of the molecules. Not all cysts of E. granulosus contained detectable host proteins. Uptake of 125I occurred when hydatid cysts were exposed to labeled proteins in vitro, but it appeared that rapid degradation of the labeled carrier led to the appearance of dialysable fragments. This may be attributable to proteolysis within the bladder fluid. We conclude that taeniid metacestodes are capable of absorbing a variety of proteins, and that these macromolecules can retain their structural and functional integrity following transport. This absorptive capacity accounts for the presence of host serum components within bladder fluids.

Animals

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.

Antibody Formation

[Fenbendazole therapy of experimental larval echinococcosis. I. The effect of fenbendazole on worm burden and protoscolex development of Echinococcus multilocularis (author's transl)].

Application of fenbendazole (methyl-5-(phenyl-thio)-benzimidazole-2-carbamate) in Echinococcus multilocularis infected mice showed a significant efficiency on the development of the cysts; the worm burden and the number of protoscolices were significantly reduced in the treated animals in comparison with the non-treated control mice. The degree of efficiency was subject to the way of application, the duration of medication, and the parasite strain: 1. On application of f. as medicated feed (500 ppm) worm burden and protoscolex number were reduced the most an average parasite weight of 105 mg and 2 protoscolices against 1942 mg and 99 protoscolices. Treatment by oral application with emulsion of 2.5% (1 ml/mouse/day, 5 x per week over a period of 2--5.5 months) the efficiency was less expressed (845 mg, 53 protoscolices). 2. By extending the treatment from 60 to 102 and 165 days resp. the reduction of worm burden can be increased from 81 to 95%. 3. From 2 parasite strains one which was recently isolated from a fox, reacted more on the treatment than the other one: 85 mg parasite weight against 1218 mg in the nontreated controls. On the other strain continued since about 25 years by intraperitoneal inoculation, the efficiency was more reduced: The worm burden was 866 against 2666 mg in the control mice.

Animals

Antigenic characterization of Echinococcus granulosus cysts.

Limited numbers of antigens were detected in the structural components of Echinococcus granulosus cysts by double diffusion and immunoelectrophoresis tests using homologous and heterologous antisera without antibody activity to host serum contaminants. Although antigens common to the germinal membranes, laminated layers and protoscoleces were demonstrated, no evidence was found of antigen sharing between the laminated layer and hydatid fluid from fertile and sterile cysts. These fluids, however, had antigenic determinants in common with protoscoleces. Antigens responsible for the formation of a characteristic cathodic are were detected but only in the three structural cyst components. In contrast, E. granulosus arc 5 antigens were found solely in hydatid fluid. These observations suggest that these two antigen groups are of somatic and metabolic origin, respectively. The results are discussed in terms of current knowledge of the immunobiology of hydatid cysts.

Animals

Comparative antigenic characterisation of Echinococcus granulosus and Taenia hydatigena cyst fluids by immunoelectrophoresis.

Hydatid cyst fluid from Echinococcus granulosus (HCF) and cyst fluid from Taenia hydatigena (TCF) cysts were compared in reciprocal immunoelectrophoresis (IEP) tests using homologous and heterologous antisera which were free of antibodies to host serum contaminants. The antigens for the E granulosus arc 5 were demonstrated in TCF. Antibody activity to these and other antigens common to HCF and TCF was removed from homologous antisera by absorptions with the heterologous antigenic preparation. Antigens not shared by the two metacestodes fluids were then demonstrated by IEP tests. These findings are discussed in terms of their significance to phylogenetic and immunodiagnostic studies of these parasites in their immediate hosts.

Animals

Activation of complement by hydatid cyst fluid of Echinococcus granulosus.

Factors present in hydatid cyst fluid of Echinococcus granulosus were found to interact with complement from several species. This nonimmunologic fixation resulted in the depletion of hemolytically active complement from fresh guinea pig serum, the conversion of human C3 to an electrophoretically faster species and the genration of smooth muscle contracting substances, analogous to anaphylatoxins, in normal rat serum in vitro. Vascular permeability changes were produced in vivo in rats and humans after intradermal inoculation of complement interacting fractions of hydatid fluid. It is suggested that these factors may contribute to the pathogenesis of the shock syndrome which follows intravenous administration of hydatid fluid in normal animals, and to the nonspecificity of immunodiagnostic skin tests for hydatid infection in man and animals.

Anaphylatoxins