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PubMed1993

Effects of mebendazole, albendazole, and praziquantel on succinate dehydrogenase, fumarate reductase, and malate dehydrogenase in Echinococcus granulosus cysts harbored in mice.

Echinococcus granulosus cyst wall possess high biochemical activities of malate dehydrogenase (MD) and fumarate reductase (FR), but low activity of succinate dehydrogenase (SD), suggesting that the cyst wall may utilize a partial reverse tricarboxylic acid cycle. When infected mice were given intragastrically with mebendazole, 25-50 mg.kg-1.d-1, albendazole 300 mg.kg-1.d-1 or praziquantel 500 mg.kg-1.d-1 for 7-14 d, no apparent effects on SD and FR activities of the cyst wall were found, while the MD activity was suppressed by all the 3 drugs, the inhibition rates being 34.6-61.6%, 59.8%, and 50.6%, respectively. The results suggested that MD may not be an important target for the antihydatidosis drugs.

Albendazole↗
PubMed1993

[Screening antihydatid drugs using cultivated germinal cells of Echinococcus granulosus].

Germinal cells isolated from Echinococcus granulosus cysts harbored in mice have been maintained in an in vitro culture system containing RPMI 1640 supplemented by 20% calf serum, and used as a model for screening anti-hydatid drugs. When the germinal cells were maintained in the medium for 6 days, the cell proliferation rate was rather high in the first four days but declined in the last two days. In screening drugs, 1.4 x 10(6) germinal cells were exposed to known effective drugs against metacestodes of E. granulosus in mice, such as mebendazole (Meb), albendazole (Alb) or praziquantel (Pra) at various concentrations. One to three days after exposure, cell counts were made daily in 3 samples of each drug concentration. The mean cell number of each group was compared with that of the control and the inhibition rate of the cell was then calculated. The results showed that the minimal effective concentrations of Meb, Alb and Pra, were 1.0 (48 h), 2.5 (24 h) and 10.0 (72 h) micrograms/ml, respectively, while the inhibition rates of the cell were 34.1, 55.7 and 18.5%. Interestingly, the in vitro effects of Meb, Alb and Pra were consistent to those obtained from the in vivo tests, ie Meb > Alb > Pra. Nevertheless, after exposure of germinal cells to Meb at 2.5 micrograms/ml for 24 h, the cells appeared in roughness, indistinction, shrunk or swelling, collapse, deformation and hole-like feature detected by light microscopy and scanning electron-microscopy, while the ultrastructure alterations of the cells noted by transmission electron-microscopy were lysis in cytoplasm, disruption or disappearance of nucleus and even darkness of the whole cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Albendazole↗
PubMed1993

[ELIB of specific antigens in in vitro cultivated germinal cells of Echinococcus granulosus].

Specific antigens are detected in cyst fluid and cyst wall of Echinococcus granulosus, as well as germinal cells cultivated in vitro by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme linked immunotransfer blot technique (ELIB), using the sera from mice infected with protoscoleces of E. granulosus for at least 10 months. A specific reaction band of 52 kDa or 38 kDa was detected in soluble protein of germinal cells and cyst fluid, respectively, but these two reaction bands were present concurrently in the cyst wall. The sera from 7 normal controls, 7 hydatidosis patients and 3 cysticercosis patients were used to study the specific antigen from the germinal cells. The results noted that the reaction band of 52 kDa was seen in all the sera from hydatidosis patients, while the sera from normal human controls and cysticercosis patients showed no reaction band.

Animals↗
PubMed1993

Morphological characterization of adult Echinococcus granulosus as a means of determining transmission patterns.

Host induced changes in morphological characters of the rostellar hooks of Echinococcus granulosus were used to determine the origin of infection in definitive hosts in rural areas of southeastern Australia where wild and domestic cycles of transmission may interact. The morphological characters studied vary depending on the species of intermediate host (macropod marsupials, sheep) in which protoscoleces develop, and these characters are retained in adult worms. It was therefore possible to determine whether definitive hosts (dingoes and foxes) acquired infection by consuming protoscoleces of E. granulosus from macropods or from sheep. The results correlated well with the known distribution of intermediate hosts and illustrate the practical value of such morphological markers in epidemiological studies.

Animals↗
PubMed1993

Echinococcus multilocularis identified in Indiana, Ohio, and east-central Illinois.

Echinococcus multilocularis was identified in a coyote in Indiana in January 1990. Subsequently, 300 wild canids (red foxes, gray foxes, coyotes) were collected from Indiana, Ohio, Illinois, Michigan, and Kentucky and examined for this parasite. Of these, 41 (13.7%) were infected with E. multilocularis. Infected animals were from northern and central Indiana, northwestern Ohio, and east-central Illinois. Twenty-nine of 162 (17.9%) animals from northern and central Indiana were positive, versus 0 of 83 from southern Indiana. Infected animals from northern and central Indiana included 16 of 71 (22.5%) red foxes and 13 of 70 (18.6%) coyotes. In Ohio, 6 of 22 (27.3%) red foxes were infected, and in Illinois, 6 of 17 (35.3%) coyotes were positive. Mean intensity of infection for red foxes was 372 worms (range, 2-3, 640), and for coyotes, 6,579 worms (range, 1-52,000). These findings constitute new state and distribution records for E. multilocularis and a significant range extension for this parasite species in central North America.

Animals↗
PubMed1993

[Effects of mebendazole on glucose, glycogen, lactic acid, and lactate dehydrogenase in Echinococcus granulosus cyst wall].

The glucose, glycogen, and lactic acid contents in Echinococcus granulosus cyst wall were 3570 +/- 623, 1276 +/- 126, and 267 +/- 29 micrograms.g-1, respectively. The lactate dehydrogenase (LDH) activity in the cyst wall was 4307 +/- 297 U/(min.mg protein). When infected mice were treated ig with mebendazole (Meb) 25 or 50 mg.kg-1.d-1 for 7-14 d, the glucose and glycogen contents in the cyst wall decreased markedly. No apparent changes in lactic acid and LDH activity were seen. The results suggested that Meb had no effect on glycolysis of the cyst wall, but it exhibited an inhibitory effect on the transport of exogenous glucose from the host to the cyst wall, which might further promote the decomposition of endogenous glycogen for energy supply and resulted in a decrease of endogenous glycogen content in the cyst wall.

Animals↗
PubMed1995

Use of a monoclonal antibody against the antigen B of Echinococcus granulosus for purification and detection of antigen B.

A monoclonal antibody directed against the 8 kDa subunit of antigen B of Echinococcus granulosus was raised. This antibody was used for purification of antigen B by affinity chromatography. SDS-PAGE and immunological analysis of the purified antigen B demonstrated that the 8 kDa antigen B subunit was purified to homogeneity. The purified antigen retained its strong immunoreactivity in ELISA using human hydatid sera. Furthermore, a sandwich ELISA was established for detection of antigen B from hydatid cysts. The usefulness of this test system was demonstrated by the detection of antigen B in human hydatid cyst fluids, thus confirming the diagnosis of an echinococcal disease.

Animals↗
PubMed1995

Effect of antihydatid drugs on carbohydrate metabolism of metacestode of echinococcus granulosus.

A biochemical some enzymes of glycolysis and a partial reversed tricarboxylic acid cycle together with hydrolytic enzymes in the cyst wall of Echinococcus granulosus was carried out. Lactate dehydrogenase (LDH), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and adenosine triphosphatase (ATPase) showed their high level of activity, suggesting that the proliferation of E. granulosus cyst wall is an energy-dependent process and the major pathways for glucose metabolism is glycolysis. Treatment of E. granulosus-infected mice with mebendazole and albendazole resulted in marked inhibition of PK, PEPCK and ATPase of E. granulosus cyst wall, whereas praziquantel had no effect, indicating that PK, PEPCK, and ATPase might be chemotherapeutic targets and the differences in the inhibitory effects might account for the efficacy of the three antihydatid drugs.

Adenosine Triphosphatases↗
PubMed1996

The effects of ivermectin on the hydatid cyst of Echinococcus granulosus after direct injection at laparotomy.

The effect of ivermectin on the hydatid cysts of Echinococcus granulosus was studied in vivo. Jirds were infected with secondary hydatid cysts of E. granulosus by intraperitoneal passage. One to one and a half years postinfection a laparotomy was performed on each of 20 jirds and the hydatid cysts located. Ivermectin or the vehicle (1% dimethyl sulfoxide in sterile physiological saline) was injected into a single hydatid cyst in each animal (10 with ivermectin; 10 with vehicle as control). The jirds were killed 44-58 days postsurgery and necropsied. The integrity of each injected cyst was assessed and tissue samples were taken for microscopic examination. Treated cysts were discolored, collapsed, and had no viable protoscolices. The germinal layer of treated hydatid cysts was necrotic; the laminated layer was intact. The effects were restricted to the ivermectin-treated cysts. Treated cysts were not infective to naive jirds by intraperitoneal passage.

Animals↗
PubMed1995

Possible formation of new brood capsule by the previously formed brood capsule in Echinococcus multilocularis metacestodes.

In Echinococcus multilocularis metacestodes obtained from the peritoneal cavity of an experimentally infected jird, cellular accumulations were found not only on the inner surfaces of germinal layers but also on the outer surfaces of brood capsules. These cellular accumulations are believed to represent at incipient form of the brood capsule. It has been thought that brood capsules are produced by the germinal layer, but we speculate that the brood capsule itself, as well as the germinal layer, may have the potential to produce new brood capsules.

Animals↗
PubMed1996

Parenteral strobilar development of Echinococcus multilocularis in scid mice.

Parenteral strobilation of Echinococcus multilocularis was observed in acid (severe combined immuno-deficient) mice after intracerebral, subcutaneous and intraperitoneal inoculation with protoscoleces. Evaginated protoscoleces and segmented worms were obtained at the inoculated sites. Most worms recovered from peritoneal cavity of scid mice were encapsulated by connective tissue and granulocytes but showed a maximum of 3 proglottids, elongation of genital primordia and vesiculation. Viability of worms recovered from the subcutaneous tissue and peritoneal cavity of scid mice were higher (69.1-91.4%) than those from the immunologically normal C.B-17 (4.0-48.0%) control mice. However, viabilities of worms from the cerebrum of both scid and C.B-17 were almost the same (87.7-94.4%). Worms recovered from scid mice showed further development of reproductive organs when transplanted into the small intestine of prednisolone treated golden hamster. These findings suggest that the parenteral milieu of scid mice allows adult development of E. multilocularis protoscoleces.

Animals↗
PubMed1977

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↗
PubMed1996

[PCR for detection and characterization of parasites (Leishmania, Echinococcus, Microsporodia, Giardia)].

The application of PCR for the diagnosis and the characterisation of parasites is discussed using four examples. (1) A PCR assay that was developed for the detection of Leishmania was shown to be as highly sensitive as in vitro cultivation of the parasites from lymph node aspirates and bone marrow biopsies. (2) Single eggs of Echinococcus multilocularis can principally be detected and identified by PCR. However, a cumbersome sample preparation is inevitable in order to remove PCR-inhibitory substances present in fox faeces. (3) Analysis of the amplified SSU rRNA gene of Encephalitozoon-like isolates from humans, rabbits and farm foxes confirmed for the first time that E. cuniculi-isolates from humans and animals are indistinguishable and that they are of importance as opportunistic parasites in HIV-infected patients. (4) Swiss isolates of Giardia originating from humans, calves, sheep and a dog could be allocated into three distinct genetic groups by analysing several PCR-amplified genes that encode for variant-specific surface proteins. There was no evidence for the presence of host-specific genotypes.

AIDS-Related Opportunistic Infections↗
PubMed1977

Host serum proteins in Echinococcus multilocularis: complement activation via the classical pathway.

The presence of host serum proteins, including IgG and IgM, within the cyst membranes and on the surface of protoscoleces of Echinococcus multilocularis was demonstrated by the use of HRPO-conjugated purified antisera. Viable protoscoleces incubated either in EDTA- or EGTA-fresh human sera were not lysed; the addition of calcium restored the protoscolecidal activity of the EGTA serum. It is concluded that the complement-mediated lysis of this metazoan organism proceeds via the classical pathway of complement activation. The results are discussed in relation to the ability of this parasite to survive in the immunologically hostile environment of the host.

Animals↗
PubMed1996

Genetic variability in Italian sheep isolates of Echinococcus granulosus.

The possible polymorphism of distinct Italian Echinococcus granulosus isolates was evaluated by RAPD-PCR using five arbitrarily chosen random primers. Sheep DNA was used as control in each RAPD-PCR to ascertain the host DNA contamination. Distinct amplification patterns were detected for each isolate revealing identical bands, with the same or different intensity, as well as distinct bands. The results of this study confirm the usefulness of RAPD-PCR technique in genetic typing of parasites and show the existence of genetic variability within the Italian E. granulosus sheep strain.

Animals↗
PubMed1977

Echinococcus granulosus: identification of subunits of the major antigens.

This paper describes a procedure for obtaining the two "major" Echinococcus granulosa antigens (antigens 4 and 5) from sheep hydatid fluid. Purified antigen 4 (m.w. 400,000 and over) and antigens 5 (m.w. 150,000) were separated in order to identify molecular subunits of each. SDS 12.5% polyacrylamide gel electrophoresis of antigen 4 gave a single band for which a m.w. of 67,000 was calculated. When 5% 2-mercaptoethanol was added to the antigen 4 preparation, SDS 12.5% polyacrylamide gel electrophoresis gave two bands (m.w. 47,000 and 20,000). SDS 12.5% polyacrylamide electrophoresis of antigen 5, with or without 2-mercaptoethanol, gave at least three bands with m.w. ranging from 10,500 to 20,000. Low m.w. subunits of both antigens were eluted in a single peak from a preparatory SDS 7.5% acrylamide gel electrophoresis; these subunits were found to be immunogenic and antigenic.

Animals↗
PubMed1996

Diagnosis of the Echinococcus multilocularis infection in final hosts.

In view of the considerable public health significance of Echinococcus multilocularis, the causative agent of the highly lethal human alveolar echinococcosis, there is an urgent need for reliable and simple techniques for the diagnosis of the infection in populations of final hosts (foxes, dogs, cats) and also in individual dogs and cats. The standard technique presently used is parsitological examination of the small intestine at necropsy. This reliable technique requires high expenditure and special safety precautions. An alternative approach is coproantigen detection. Recently, in our laboratory an ELISA was evaluated using rabbit and chicken polyclonal antibodies against E. multilocularis antigens (affinity purified coproantigens and somatic adult worm antigens). The specificity of this test (evaluated in 20 foxes and 661 dogs with helmintic infections other than E. multilocularis) was very high (95%-99.5%). Average sensitivity in 35 foxes infected with E. multilocularis was 80%, but reached 93% in foxes with individual worm burdens over 55. A Polymerase Chain Reaction (PCR) was used for detecting DNA of E. multilocular is in faecal samples of foxes after the parasite eggs had been isolated by a sieving procedure. In a total of 55 foxes specificity was 100% and sensitivity 94%. For field application the coproantigen ELISA has the potential of replacing parasite detection at necropsy, and PCR is a valuable method for confirmation of positive coproantigen results and for diagnosis in individual animals. Detection of circulating anti-Em2 antibodies by ELISA may be useful for primary screening of fox populations but antibody prevalence rates do not correlate with prevalence rates of the intestinal infection with E. multilocularis.

Animals↗
PubMed1997

Complement evasion by Echinococcus granulosus: sequestration of host factor H in the hydatid cyst wall.

Hydatid disease, which affects humans and various livestock species, is caused by the larval stages of the cestode parasite Echinococcus granulosus. The disease involves the growth of a fluid-filled cyst in the host's internal organs. The hydatid cyst wall is generally thought to preclude the access of host immune cells to the cyst, while allowing passage of macromolecules. Thus, evasion of humoral effector mechanisms, in particular the complement system, may be crucial to parasite survival. Evasion of complement might involve inhibition of the C3b deposition step, which can in principle be achieved through parasite-derived or host-derived and parasite-bound regulatory molecules. In this work, extracts from bovine hydatid cysts were analyzed for the presence of factor H-like function, namely the cofactor activity for the breakdown of amidated bovine C3 by bovine factor I. Both this activity and antigenically detectable bovine factor H were found in most extracts. The cofactor activity was retained by an anti-bovine factor H Ab column; as no factor H-cross-reactive parasite molecules were detected in the active extracts, the activity was indeed due to host factor H. The pattern of extraction of host factor H from the parasite tissue indicated that at least part of it was selectively bound to the cyst wall, and that a strong charge component was involved in the binding. Our results indicate that the selective concentration of host factor H from plasma/interstitial fluid may contribute to complement evasion by E. granulosus.

Animals↗