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PubMed1989

Echinococcus multilocularis: inhibition of murine neutrophil and macrophage chemotaxis.

Resident peritoneal neutrophils and macrophages from mice infected with 50 +/- 5 cysts of Echinococcus multilocularis were collected at 2, 4, 6, 8, 10, and 16 weeks postinfection. Their ability to respond and migrate to purified parasite larval antigens or endotoxin-activated mouse serum (EAMS) in comparison to normal peritoneal cells from uninfected mice was tested in vitro using Boyden chambers. Early in the infection, both cell types responded to the specific and nonspecific chemoattractants as the control group. However, at 8 and 10 weeks postinfection, the neutrophils and macrophages lost their response to parasite antigens but retained their ability to migrate to EAMS. Toward the 12th and 16th week postinfection, both cell types lost their ability to migrate to the specific as well as the nonspecific factors. The data presented suggest that the cellular mechanisms of recognition and chemotaxis in mice infected with alveolar hydatid cysts are impaired.

Animals↗
PubMed1992

Echinococcus granulosus: study of the in vitro complement activation by protoscoleces by measuring the electric potential difference across the tegumental membrane.

Complement activation by protoscoleces of Echinococcus granulosus was studied by analyzing the damage to their tegumental membrane produced by incubation in both normal and hydatid human sera. The state of the apical tegumental membrane was evaluated by measuring the electric potential difference with microelectrodes. Protoscoleces incubated in Ringer-Hepes or in heat-decomplemented normal human serum in the presence or absence of specific antibodies did not show significant variations in the electric potential difference throughout the experiment (P > 0.4 in all cases) and their mean values were -46 +/- 3, -43 +/- 4, and -56 +/- 5 mV, respectively. In contrast the potential difference of protoscoleces incubated in 1:2 diluted normal human serum showed a significant variation (P < 0.001), reaching -10 +/- 6 mV after 30 min, and the median depolarization time was estimated to be 21 +/- 3 min. The capacity of normal human serum to depolarize the tegumental membrane of protoscoleces was abolished by treatment at 50 degrees C during 20 min or by 10-fold dilution. In addition, protoscoleces incubated in 1:10 diluted hydatid human serum plus 1:10 diluted normal human serum or Factor B-inactivated normal human serum showed a significantly faster depolarization (0.01 < P < 0.02 and P < 0.001, respectively): the potential difference reached -13 +/- 5 mV after 15 min and the median depolarization times were 9 +/- 5 and 5 +/- 3 min, respectively. Our results suggest that following the time course of the potential difference is a useful tool for studying complement activation in the host-parasite interface and they show that the tegumental membrane of protoscoleces can activate the alternative pathway of human complement.

Animals↗
PubMed1995

Diagnosis of Echinococcus multilocularis infection by reverse-transcription polymerase chain reaction.

Alveolar echinococcosis is a life-threatening parasitosis occurring in countries in the Northern hemisphere. The diagnosis of an Echinococcus multilocularis infection is routinely performed by radiological techniques and by the detection of specific antibodies in the sera of infected patients. However, because serodiagnosis fails in 5%-10% and radiological techniques are difficult to interpret in some cases, we developed a polymerase chain reaction for the detection of echinococcal-specific messenger RNA from fine-needle biopsy specimens. This technique was applied to the diagnosis of alveolar echinococcosis in a 20-year-old seronegative woman. Detection of messenger RNA not only allowed the diagnosis of echinococcal disease but also proved to be a reliable measure for determining the efficacy of an antiparasitic therapy.

Adult↗
PubMed1989

In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces.

The in vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces were studied by means of light and electron microscopy. Both drugs had a protoscolicidal activity that increased proportionally with increasing concentrations of the drugs. Ivermectin showed the more rapid effects and caused contraction and paralysis of protoscoleces. A paralyzing effect was also observed with levamisole, followed by irreversible tissue vacuolation leading to death.

Animals↗
PubMed1990

A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs.

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.

Animals↗
PubMed1990

Extraintestinal strobilar development of immature Echinococcus multilocularis in laboratory rodents following intratracheal inoculation of the protoscoleces.

Intratracheal inoculation of Echinococcus multilocularis protoscoleces was performed in prednisolone tertiary-butylacetate(PTBA)-treated and untreated golden hamsters and AKR/J mice. Repeated PTBA treatments prior to the day of inoculation prevented the influx of neutrophils against the cestode. More than 1 month after infection, living immature adult forms of E. multilocularis, showing some somatic prolongation, were recovered from the pulmonary alveolar space of PTBA-treated golden hamsters, in the absence of any cystic development.

Animals↗
PubMed1990

Immunology and molecular biology of Echinococcus infections.

Echinococcus spp. are the etiological agents of hydatid disease in man and other intermediate hosts. Many questions regarding the factors which determine susceptibility/resistance to hydatid disease, and the factors which influence the viability and fertility of hydatid cysts, remain to be answered. Recent research into the effects of hydatid infection on the immune system of the host has provided some insights into the host-parasite relationships. Immunochemical and recombinant DNA techniques are being applied to improve diagnosis of hydatidosis in man and E. granulosus infection in dogs, and also in the development of vaccines against infection with taeniid cestode larvae. The successes which have been achieved in these areas are likely to provide valuable tools for the control of cystic hydatidosis in man. These recent studies in the areas of immunobiology, serological diagnosis and vaccination are reviewed.

Animals↗
PubMed1991

An established cell line of larval Echinococcus multilocularis.

A cell line of larval Echinococcus multilocularis has been established from an echinococcal cyst excised surgically from a patient with alveolar hydatid disease. A standardized procedure established for the preparation and continuation of primary cultures was applied to isolate the E. multilocularis cells from the tissue fragments. Trypsin was used for the enzymatic release of the monodispersed cells from the tissue fragments and for dispersing monolayers. The culture medium was RPMI 1640 with 10% fetal calf serum. Cell supports were collagen-coated plastic dishes and flasks. The morphological features of the cultured cells showed spindle-like cells during the first few subcultures, and then polygonal or star-like cells. Population doubling time at passage 34 was approximately 23 h and plating efficiency at the same passage was 15%. Chromosome numbers obtained from 70 metaphase plates at passage 40 ranged between 14 and 104 and cells with 91-100 chromosomes were clearly predominant. The chromosomes could be morphologically classified into telocentric, subtelocentric, and metacentric types. Over 90% of the chromosomes were of the telocentric type. Cells collected at passage 57 were intraperitoneally inoculated into two cotton rats (Sigmodon hispidus) at a cell concentration of 10(7) and the rats were sacrificed 100 days later. It was found that the two rats had echinococcal cyst masses in the peritoneal cavity. This result indicates that our isolated cells are germinal cells with ability to differentiate into cystic structures in vivo.

Animals↗
PubMed1991

Echinococcus granulosus: development of an intermediate host mouse model for use in vaccination studies.

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.

Animals↗
PubMed1991

The origin of a new focus of infection with Echinococcus granulosus in Tasmania.

Hydatid cysts were discovered in cattle on King Island, north of Tasmania, where Echinococcus granulosus was thought to have been eradicated. Using enzyme electrophoresis, isolates from King Island were compared genetically with isolates from Tasmania and the mainland of Australia. The genetic distinctness of the King Island isolates make it unlikely that they originated from a recent introduction from either Tasmania or mainland Australia. Alternative possibilities, that the infection resulted from a recent introduction from another source or from previously undetected persistence of E. granulosus on King Island, could not be distinguished from available data.

Animals↗
PubMed1992

Effects of cyclosporin A on the course of murine alveolar echinococcosis and on specific cellular and humoral immune responses against Echinococcus multilocularis.

The effects of cyclosporin A (CsA) on Echinococcus multilocularis (E. multilocularis) metacestode growth, and on the specific immune responses of the hosts, were examined in AKR mice. Mice were intra-peritoneally infected with a metacestode homogenate. CsA (40 mg kg(-1) day(-1)) was injected subcutaneously from the 45th day after infection (Group 1), and from the day before infection (Group 2) until the day of autopsy (days 125 and 80, respectively). Results showed that unlike ths situation with some other helminthiases, CsA had no antiparasitic effect, although it lengthened the maturation time of protoscoleces in Group 1. The parasitic burden, unmodified in Group 1, was significantly enhanced in Group 2. This enhancement was associated with a decrease in antibody levels, whereas the delayed-type hypersensitivity was decreased in the two groups. These results confirm the role of cellular immunity in controlling the first stages of the larval development of E. multilocularis and indicate the necessity for a careful follow-up of any recurrence of alveolar echinococcosis in patients treated with CsA after liver transplantation.

Animals↗
PubMed1992

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.

Animals↗
PubMed1993

Anthelmintic effects of cyclosporin A on protoscoleces and secondary hydatid cysts of Echinococcus granulosus in the mouse.

Cyclosporin A (CsA), employed primarily as an immunosuppressant during the management of organ and graft transplants, exhibits anthelmintic properties. However, its efficacy against tapeworm infections in laboratory models is variable. A preliminary investigation has been undertaken to assess the action of CsA on the establishment and growth of protoscoleces and secondary hydatid cysts of ovine Echinococcus granulosus in mice. Administration of CsA in five consecutive daily doses, beginning 2 days prior to infection, resulted in significant reduction in cyst establishment (measured in terms of cyst masses, cyst numbers and cyst wet weights), when mice were autopsied 20 weeks post-infection. None of these parameters were significantly reduced when the drug was administered 18 weeks post-infection, although wet weight decreased by 42%. Ultrastructural examination of the germinal membrane and laminated layer of late-treated E. granulosus revealed abnormalities in all cysts studied whereas control and early-treated hydatids were normal. A case is made for the consideration of a clinical use for CsA for post-operative control of secondary hydatidosis and its efficacy against hydatid cysts is discussed.

Animals↗
PubMed1995

In vitro quantitative assessment of Echinococcus multilocularis metacestode viability after in vivo and in vitro maintenance.

The aim of this study was to apply the enzymatic MTT (3,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide--formazan colorimetry for quantifying the viability of Echinococcus multilocularis whole cysts, after maintenance in vivo or in vitro. The enzymatic activities of young cysts freshly removed from rodents were linearly correlated with the parasite cyst weight. A comparative evaluation of the MTT assay and the in vitro viability assessments showed that the number of animals used for drug-screening purposes would be reduced by 35.8%. In this way, the use of different parasite samples removed from the same host is required, because of their different ages and their subsequent different abilities to reduce MTT. Cysts removed from mice exhibited higher colorimetric values than those removed from jirds. Thus, small entire cysts obtained from mice were maintained in the CMRL 1066 culture medium. Their enzymatic activities were evaluated at different times. The results indicate that, in such conditions, the optimal period of time for testing the effect of drugs against the metacestodes is limited to the 10 days following their transfer from mouse to culture flasks. The MTT assay encourages further studies to improve the viability of the whole cysts in vitro, using other standardizable culture conditions.

Animals↗
PubMed1995

Transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in Uruguay.

A base-line survey was carried out on the transmission dynamics of Echinococcus granulosus, Taenia hydatigena and Taenia ovis in sheep in the Department of Florida, Uruguay. Mean life expectancy at birth of the sheep population in this Department was estimated at 3.5 and 4.8 years for male and female sheep, respectively. Both intensity and prevalence of E. granulosus infection increased with age providing no evidence that immunity was acquired in the population. This revealed that E. granulosus was only relatively stable and in an equilibrium endemic steady state with an estimate of the basic reproduction ratio (Ro) of about 1.2. The mature larval population in sheep was over-dispersed with parameter k, from fitting a negative binomial, being 0.199 and 0.556 in 42- and 54-month-old sheep, respectively. The mean number of cysts increased by 1.05 cysts per year and the infection pressure was calculated as 0.174 infections per year, implying that each infection consisted of 693.6 eggs to produce 6.03 cysts. Neither intensity nor prevalence of infection with T. hydatigena or T. ovis increased with age. This indicated that immunity was acquired to reinfection by both parasites and they were in an equilibrium hyperenedemic steady state with an estimated Ro of 4.2-5.8. Viability and fertility of E. granulosus, and viability of T. hydatigena cysts in the older age groups were low. The effects of the different transmission levels of the parasites on potential control strategies are discussed.

Animals↗
PubMed1994

Expression and analysis of the diagnostic value of an Echinococcus granulosus antigen gene clone.

A pool of 9 sera from Echinococcus granulosus infected patients (PSP) was used to screen an E. granulosus cDNA library constructed in the expression vector lambda gt11. Ten reactive phage clones were isolated and 8 were confirmed in spot-lysis arrays probed with PSP. The insert of 1 of these clones (lambda AgEg4) previously characterized as an E. granulosus cytosolic malate dehydrogenase encoding gene was subcloned into the plasmid vector pGEX-1 and expressed as a fusion with glutathione S-transferase. The fusion peptide (Ag4-GST) was produced in Escherichia coli and its antigenicity was confirmed in colony immunoassay and in immunoblot using nondenaturing conditions. The lack of antigenicity of Ag4-GST in immunoblot using denaturing conditions suggests that the recognized epitopes are conformational. Ag4-GST was purified by affinity chromatography and tested in ELISA and immunodots to access its sensitivity and specificity in the diagnosis of human cystic hydatid disease. An overall sensitivity of 53.6% was obtained. Cross-reactions were observed with some sera from patients infected with Schistosoma mansoni and Wuchereria bancrofti. Ag4-GST was not recognized by any of the sera from Taenia solium infected patients tested. These preliminary results suggest that Ag4-GST could be useful as an accessory antigen to discriminate some cross-reactions with sera from cysticercosis patients, especially in regions like southern Brazil, where schistosomiasis and filariasis are not prevalent.

Animals↗
PubMed1994

The effects of albendazole and albendazole sulphoxide combination-therapy on Echinococcus granulosus in vitro.

Protoscoleces of Echinococcus granulosus were incubated in vitro with decreasing concentrations of either albendazole (ABZ) or albendazole sulphoxide (ABZ.SO) (50, 10, 1 and 0.1 micrograms ml-1), and in combination. Viability was assessed by the methylene blue exclusion test and establishment of infection in mice. Protoscolex ultrastructure was determined by means of transmission and scanning electron microscopy. ABZ and ABZ.SO, when used separately had protoscolicidal activity after a longer incubation period (30 days) than when used as combined compounds. When incubated in the presence of ABZ + ABZ.SO, protoscolex viability dropped rapidly. That is, protoscoleces were all non-viable at 12 days of exposure, with no cyst developing following their inoculation into mice. The ultrastructural changes induced by ABZ or ABZ.SO alone, were: (a) rostellar disorganization, (b) formation of numerous blebs on the tegument, (c) loss of the microtriches, (d) increased vesiculation within the tegumentary cytons together, (e) an increase in lipid deposits and (f) depletion of glycogen reserves. After incubation with combined ABZ and ABZ.SO the tegument contained numerous blebs which became detached, leaving debris only, some intact nuclei being discernible in the protoscolex parenchyma.

Albendazole↗