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Trials to induce protective immunity in mice and sheep by application of protoscolex and hydatid fluid antigen or whole body antigen of Echinococcus granulosus.

In the current study, soluble proteins prepared from 200 mature Echinococcus granulosus and protoscolices of sheep hydatid cysts were applied to immunize sheep and mice respectively. The samples were mechanically homogenized in a blender, sonicated and the final yield was maintained at -20 degrees C until analysis. Hydatid fluid was isolated from liver or lung of sheep under sterile conditions. In the first experiment, 15 mice were randomly allocated to three groups of five mice each. Each mouse in groups 1 and 2 was immunized with 100 microg of hydatid fluid and protoscolex proteins in 100 microl of phosphate-buffered saline (PBS) and emulsified with an equal volume of Freund's complete adjuvant (FCA) respectively. The mice of group 3 were immunized with adjuvant in PBS. The mice were boosted 4 weeks after the first vaccination with the same preparation except that FCA was replaced by Freund's incomplete adjuvant (FIA). In the second experiment, eight male or female lambs 4-6 months of age, were allocated to two groups of four lambs each. Each lamb in the test group was vaccinated subcutaneously in the neck with a 2-ml dose of vaccine (1 mg of whole body protein of E. granulosus dissolved in 1 ml of PBS plus 1 ml of FCA). Control lambs were vaccinated with adjuvant in PBS. Lambs were boosted the same way as in the first experiment. Three weeks after the second vaccination, each mouse and lamb received a challenge infection with 2000 protoscolices intraperitoneally and each lamb additionally received 10 gravid E. granulosus. All mice and sheep were killed after 7 months and examined for hydatid cysts. In these studies, protective immunity was induced in mice with protoscolex protein and with hydatid fluid, and in sheep with whole-body homogenate of E. granulosus and the levels of protection afforded were found to be 72.1, 82.6 and 90.9% respectively.

Animals↗

Echinococcus multilocularis in Austrian foxes from 1991 until 2004.

The prevalence rates of Echinococcus multilocularis in foxes (n=5600) evaluated in several Austrian surveys conducted between 1991 and 2004 were analysed for spatial and temporal differences. Data from early studies (1993-1997) in which the intestinal scraping technique (IST) was utilized were compared with data from recent (1999-2004) investigations, which made use of the shaking in a vessel technique (SVT), and it was assessed whether or not the infection rates of Austrian foxes had increased between the investigated intervals. In total, data from 85 districts are presented and both the retrospective and recent data are available from 39 of these districts. A Bayesian hierarchical model of parasite prevalences is presented which (i) accounts for differences in the sensitivity of IST and SVT, (ii) incorporates spatial auto-correlation between neighbouring districts, (iii) investigates the possibility of a temporal shift in the infection status of foxes, and (iv) quantifies uncertainty at each level of the model. The national average prevalence rates in the mid-1990s and at the turn of the millennium were 2.4% (95% confidence intervals 1.1-4.8) and 3.9% (95% confidence intervals 1.5-8.4) respectively. Above average prevalence rates were observed in the western and the northern parts of the country. Evidence is also presented for a temporal augmentation of the prevalence rates in some districts in the northern and eastern parts of the country. These findings are in concordance with several investigations in other European states where both newly emerged areas and elevated levels of transmission in existing endemic areas have been found. None of the districts investigated here showed significant evidence of a drop in prevalence.

Animals↗

Detection of Echinococcus multilocularis in foxes: evaluation of a protocol of the intestinal scraping technique.

The intestinal scraping technique (IST) is widely used for the detection of definitive hosts infected with Echinococcus multilocularis. The sensitivity of the method has been questioned in recent years. Several variations of the technique are used that may differ in their performance. We therefore estimated the diagnostic sensitivity and specificity of the IST protocol used in our lab by examining the small intestines of 210 E. multilocularis-infected foxes and 294 foxes that had tested negative for this parasite. To this end, IST was first performed on 48 and 294 intestines from infected and uninfected foxes respectively, followed by an examination of the entire remaining mucosa of the small intestine to discover any infections with E. multilocularis that had not been detected by IST. The point estimate for diagnostic sensitivity was 100%. Five different classes of infection intensities were formed. The lower limits of the 95% confidence intervals ranged between 76.8% and 92.6% depending on the number of samples analysed per class. When the small intestines of another 162 infected foxes were examined by IST and the results recorded separately for the anterior, middle and posterior third of the small intestine, a strong preference of E. multilocularis for the posterior third was observed. For epidemiological purposes, it may thus be possible to restrict routine investigations to the posterior portion of the small intestine. By contrast, a reduction of the number of slides examined per infected animal may lead to a considerable loss in sensitivity in animals with a low intensity of infection.

Animals↗

Primers designed for amplification of Echinococcus multilocularis DNA amplify the DNA of Encephalitozoon-like spores in the polymerase chain reaction.

Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis. The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi. The Encephalitozoon-like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.

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Characterization of myo-inositol hexakisphosphate deposits from larval Echinococcus granulosus.

The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.

Animals↗

Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens.

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

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In vitro activities of itraconazole, methiazole, and nitazoxanide versus Echinococcus multilocularis larvae.

Albendazole (ABZ) and mebendazole are the only drugs licensed for treatment of human alveolar echinococcosis. In order to augment the armamentarium against this deadly disease, we tested a series of drugs for their efficacy against Echinococcus multilocularis larvae. E. multilocularis larvae grown intraperitoneally in Mongolian gerbils were transferred into tissue culture. Vesicles budded from the tissue blocks and after 6 weeks, drugs were added, and the effect on the vesicles was observed. We tested the following drugs at various concentrations: ABZ, artemether, caspofungin, itraconazole (ITZ), ivermectin, methiazole (MTZ), miltefosine, nitazoxanide (NTZ), rifampin, and trimethoprim-sulfamethoxazole. ABZ, ITZ, MTZ, and NTZ effectively destroyed parasite vesicles in this in vitro culture system. At high NTZ doses of 10 microg/ml, disintegration of all vesicles was observed after 7 days and was significantly more rapid than with ABZ at equal concentrations (21 days). After drug discontinuation, regrowth of vesicles occurred between 7 and 14 days for all four drugs, indicating a parasitostatic effect. Combination treatment with NTZ-ABZ at concentrations between 1 and 10 microg/ml for either 3 weeks, 3 months, or 6 months yielded no vesicle regrowth during 8 months after drug discontinuation. The treated larval tissue was injected intraperitoneally into gerbils, and no regrowth of larval tissue was observed, suggesting a parasitocidal effect after combined treatment. ITZ, MTZ, and NTZ are potent inhibitors of larval growth, although they proved to be parasitostatic only. The combination of NTZ plus ABZ was parasitocidal in vitro. Animal experiments are warranted for studies of dose, toxicity, and drug interactions.

Animals↗

Effects of multiple doses of isoprinosine on Echinococcus multilocularis metacestodes.

Isoprinosine was given at daily doses of 0.5, 1, 2, and 4 g kg-1 of body weight to jirds that were infected for 3 months with Echinococcus multilocularis metacestodes. The effects of the different drug doses on metacestodes were studied by transmission electron microscopy and biochemical methods. At lower doses, increases in uric acid and adenosine deaminase activity were noted. At 4 g kg-1 of body weight, marked ultrastructural damage with metabolic perturbations was observed.

Adenosine Deaminase↗

In vitro activities of benzimidazoles against Echinococcus multilocularis metacestodes.

Alveolar echinococcosis, caused by the larval (metacestode) stage of the tapeworm Echinococcus multilocularis, is a lethal parasitosis of the liver prevalent in the Northern Hemisphere. For chemotherapy the benzimidazole derivatives mebendazole and albendazole were introduced, and their use has resulted in a significant improvement in the survival rates. However, data from experiments with animals and clinical observations indicate that these drugs elicit only parasitostatic activity and in most cases are not able to completely eliminate the parasitic metacestode tissue. In the present study, we applied a culture system for the in vitro growth and proliferation of E. multilocularis metacestodes to analyze the parasitostatic and parasitocidal potential of mebendazole. Here, we demonstrate for the first time that at concentrations of >0.1 microM, i.e., at concentrations used for therapy of human alveolar echinococcosis, this antihelminth drug is parasitocidal in vitro. Viability assessment was performed by infection experiments with Meriones unguiculatus and mebendazole-treated metacestode tissue and by reverse transcription-PCR for the detection of E. multilocularis mRNA. The E. multilocularis in vitro model proved to be a valuable tool for the analysis of the potential of antihelminth drugs.

Animals↗

Efficacies of albendazole sulfoxide and albendazole sulfone against In vitro-cultivated Echinococcus multilocularis metacestodes.

The metacestode stage of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a parasitic disease affecting the liver, with occasional metastasis into other organs. Benzimidazole carbamate derivatives such as mebendazole and albendazole are currently used for chemotherapeutic treatment of AE. Albendazole is poorly resorbed and is metabolically converted to its main metabolite albendazole sulfoxide, which is believed to be the active component, and further to albendazole sulfone. Chemotherapy with albendazole has been shown to have a parasitostatic rather than a parasitocidal effect; it is not effective in all cases, and the recurrence rate is rather high once chemotherapy is stopped. Thus, development of new means of chemotherapy of AE is needed. This could include modifications of benzimidazoles and elucidiation of the respective biological pathways. In this study we performed in vitro drug treatment of E. multilocularis metacestodes with albendazole sulfoxide and albendazole sulfone. High-performance liquid chromatography analysis of vesicle fluids showed that the drugs were taken up rapidly by the parasite. Transmission electron microscopic investigation of parasite tissues and nuclear magnetic resonance spectroscopy of vesicle fluids demonstrated that albendazole sulfoxide and albendazole sulfone had similar effects with respect to parasite ultrastructure and changes in metabolites in vesicle fluids. This study shows that the in vitro cultivation model presented here provides an ideal first-round test system for screening of antiparasite drugs.

Albendazole↗

Echinococcus multilocularis alkaline phosphatase as a marker for metacestode damage induced by in vitro drug treatment with albendazole sulfoxide and albendazole sulfone.

Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. The disease affects the human liver and occasionally other organs and is fatal if treatment is unsuccessful. The present chemotherapy of AE is based on the administration of benzimidazole carbamate derivatives, such as mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some cases, parasitostatic rather than parasiticidal, and the recurrence rate is rather high. Therefore, chemotherapy usually involves the lifelong uptake of massive doses of albendazole and new treatment options are urgently needed. In order to avoid costly and time-consuming animal experimentation, a first step in searching for novel parasiticidal compounds could be the in vitro drug screening of novel compounds by employing metacestode cultivation. However, presently used techniques (e.g., transmission electron microscopy) for determination of parasite viability involve costly equipment and time-consuming preparation of rather large amounts of parasite material. We therefore searched for a parasite marker which can be easily traced and the presence or absence of which is indicative of parasite viability. In this study we show that the increase of E. multilocularis alkaline phosphatase activity in culture supernatants during in vitro drug treatment with albendazole derivatives correlates with the progressive degeneration and destruction of the metacestode tissue. The inexpensive and rapid assay presented here will serve as an ideal tool for performing first-round in vitro tests on the efficacy of a large number of antiparasitic compounds.

Albendazole↗

In vitro parasiticidal effect of Nitazoxanide against Echinococcus multilocularis metacestodes.

When humans serve as inadvertent intermediate hosts for Echinococcus multilocularis, disease (alveolar echinococcosis [AE]) may result from the expanding parasite metacestode in visceral organs, mostly in the liver. Benzimidazole carbamate derivatives such as mebendazole and albendazole are used for chemotherapeutic treatment of AE. However, these treatments are, in most cases, parasitistatic rather than parasiticidal. As treatment is discontinued, a recurrence of parasite growth has been observed in many AE patients with nonradical resections. The only curative treatment for AE is radical surgical resection of the parasite tissue and support by chemotherapy. As there is a need for new treatment options for AE, the in vitro efficacy of nitazoxanide (NTZ), a broad-spectrum drug used against intestinal parasites and bacteria, was investigated. We showed that in vitro treatment of E. multilocularis metacestodes with NTZ induced high levels of alkaline phosphatase activity in the medium. Concurrently, distinct morphological and ultrastructural alterations were detected. Most significantly, two distinct types of alterations were observed as soon as after 3 h of NTZ treatment. At first, the drug induced a peripheral output of membranous vesicles from the tegumental membrane into the laminated layer. Simultaneously, germinal layer-associated undifferentiated cells produced large vacuoles filled with lipid-like and often electron-dense membranous segments. Other alterations were observed at later time points, including vacuolization of the germinal layer, accumulation of lipid droplets, and lastly, loss of microtriches and separation of the laminated and germinal layers. The pattern of damage induced by NTZ was different from the alterations earlier observed in albendazole sulfoxide-treated vesicles. The nonviability of NTZ-treated metacestodes was confirmed through bioassay, i.e., inoculation of treated and untreated parasites into mice. These experiments demonstrate the in vitro parasiticidal effect of NTZ on E. multilocularis metacestodes.

Alkaline Phosphatase↗

Effect of amphotericin B on larval growth of Echinococcus multilocularis.

Alveolar echinococcosis is caused by the parasitic cestode Echinococcus multilocularis. Benzimidazoles, namely, mebendazole and albendazole, are the only drugs available for the treatment of inoperable alveolar echinococcosis. At present, no therapeutic alternative is available for patients with progressive disease under treatment or for patients who are unable to tolerate the side effects of the benzimidazoles. In addition, benzimidazoles are only parasitostatic for E. multilocularis. Thus, new therapeutic options are of paramount importance. In the present study we examined the in vitro effect of amphotericin B on E. multilocularis larvae. E. multilocularis metacestodes grown in the peritoneal cavities of Mongolian gerbils were transferred into a culture system. Vesicles budded from the tissue blocks and increased in number and size during the first 5 weeks. After 6 weeks drugs were added and deleterious effects on the vesicles were observed macroscopically and microscopically. By use of this in vitro tissue culture model we demonstrated that amphotericin B effectively inhibits the growth of E. multilocularis metacestodes. This destructive effect was significantly more rapid with amphotericin B than with the benzimidazoles. Cyclic treatment was effective in suppressing parasite growth. However, amphotericin B appears to be parasitostatic for E. multilocularis larvae, and regrowth occurs even after extended periods. In summary, amphotericin B constitutes the first promising alternative for the treatment of alveolar echinococcosis in cases of intolerance or resistance to benzimidazoles. It holds promise as an effective treatment option for otherwise fatal courses of disease.

Amphotericin B↗

Mitochondrial ribosome as the target for the macrolide antibiotic clarithromycin in the helminth Echinococcus multilocularis.

The mitochondrial rRNA of the tapeworm species Echinococcus multilocularis carries an adenine at sequence position 2058 (numbering according to that for Escherichia coli) of the large-subunit rRNA (lsrRNA), while the nucleus-encoded rRNA, as determined in this study, is characterized by 2058G. This indicates a dichotomy in the drug susceptibilities of ribosomes: cytoplasmic ribosomes are predicted to be resistant to macrolide antibiotics, while mitochondrial ribosomes lack the most common chromosomal resistance determinant, lsrRNA 2058G. Upon incubation with the macrolide clarithromycin, the formation of vesicles from metacestode tissue was reduced in a dose-dependent manner. Electron microscopy revealed distinct morphological alterations both of the mitochondria and of the vesicle wall (e.g., loss of microtriches) in drug-treated vesicles. Adult worms lost their motility and displayed morphological changes (shortening and constriction of proglottids and the presence of vacuoles) upon incubation with clarithromycin. Our findings demonstrate that macrolides have distinct in vitro effects on E. multilocularis, endorsing the use of sequence-based in silico approaches for exploitation of available ribosomal drugs as anthelmintic agents.

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Usefulness of hydatid cyst fluid of Echinococcus granulosus developed in mice with secondary infection for serodiagnosis of cystic Echinococcosis in humans.

The aim of this work was to assess the usefulness of hydatid cyst fluid (HCF) of Echinococcus granulosus, obtained from mice experimentally infected with hydatid cyst tissue homogenates, for the serodiagnosis of cystic echinococcosis (CE) in humans. The sensitivity and specificity of HCF obtained from mice for the detection of immunoglobulin G (IgG) antibodies in the sera of CE patients were compared with those of HCF from sheep and/or from a human CE patient by using immunoblotting (IB) and an enzyme-linked immunosorbent assay (ELISA). HCFs obtained from three different host species all were highly useful for immunoblotting, and sera from 19 (95%) of 20 CE patients equally recognized the antigen B subunit (approximately 8 kDa). HCF from mice showed a cross-reaction with 9 of 20 alveolar echinococcosis (AE) sera (45%), whereas HCFs from two other host species cross-reacted with 14 of the AE sera (70%). Although 2 (10%) of 20 sera from neurocysticercosis (NCC) patients were false positive with HCF from both sheep and humans, none of these sera showed a positive reaction with HCF from mouse origin. ELISAs with HCFs from both mouse and sheep origins detected all 20 CE and AE sera; however, these ELISAs showed 45% (9 of 20) and 60% (12 of 20) false-positive reactions with 20 NCC sera, respectively. The presence of nonspecific human IgG in HCF obtained from a CE patient prevented us from applying it to the ELISA. HCF of E. granulosus, obtained from laboratory mice with a secondary infection with hydatid cyst tissue homogenates, appears to be highly useful for the serodiagnosis of CE in humans and may be useful in domestic animals.

Animals↗

Echinococcus granulosus antigen B impairs human dendritic cell differentiation and polarizes immature dendritic cell maturation towards a Th2 cell response.

Despite inducing a strong host cellular and humoral immune response, the helminth Echinococcus granulosus is a highly successful parasite that develops, progresses, and ultimately causes chronic disease. Although surgery remains the preferred therapeutic option, pharmacological research now envisages antihelminthic strategies. To understand the mechanisms that E. granulosus uses to escape host immunosurveillance and promote chronic infection, we investigated how two hydatid cyst components, purified antigen B (AgB) and sheep hydatid fluid (SHF), act on host dendritic cell (DC) differentiation from monocyte precursors and how they influence maturation of DC that have already differentiated. We evaluated the immunomodulatory potential of these antigens by performing immunochemical and cytofluorimetric analyses of monocyte-derived DCs from healthy human donors. During monocyte differentiation, AgB and SHF downmodulated CD1a expression and upregulated CD86 expression. Compared with immature DCs differentiated in medium alone (iDCs), AgB- and SHF-differentiated cells stimulated with lipopolysaccharide included a significantly lower percentage of CD83(+) cells (P < 10(-4)) and had weaker costimulatory molecule expression. When stimulated with AgB and SHF, iDCs matured and primed lymphocytes towards the Th2 response typical of E. granulosus infection. SHF and particularly AgB reduced the production of interleukin-12p70 (IL-12p70) and tumor necrosis factor alpha in lipopolysaccharide-stimulated iDCs. Anti-IL-10 antibodies increased the levels of IL-12p70 secretion in AgB- and SHF-matured DCs. AgB and SHF induced interleukin-1 receptor-associated kinase phosphorylation and activated nuclear factor-kappaB, suggesting that Toll-like receptors could participate in E. granulosus-stimulated DC maturation. These results suggest that E. granulosus escapes host immunosurveillance in two ways: by interfering with monocyte differentiation and by modulating DC maturation.

Animals↗

Protection of cotton rats against experimental Echinococcus multilocularis infections with BCG cell walls.

Previous works has indicated that cotton rats treated with Mycobacterium bovis strain BCG are effectively protected against an infection with the metastatically proliferating metacestodes of Echinococcus multilocularis. In an attempt to induce a similar protection in the absence of tubercular granulomatous lesions, cotton rats were treated with BCG cell walls. A single injection of 150 micrograms of cell walls, emulsified in mineral oil-Tween-saline, 2 weeks before the inoculation of the parasite completely protected the animals against infection with E. multilocularis. This protection was correlated with an increase in the numbers of monocytes and, as judged by acid phosphatase activity, an activation of these cells. This study shows that BCG cell walls are as effective in protecting animals against E. multilocularis as the viable organism.

Animals↗