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Echinococcus granulosus strain typing in Bulgaria: the G1 genotype is predominant in intermediate and definitive wild hosts.

Addressing the genetic variability in Echinococcus granulosus is epidemiologically important, as strain characteristics may influence the local transmission patterns of zoonotic cystic echinococcosis. To classify the genotype(s) present in intermediate (pig, cattle and sheep) and definitive (jackal and wolf) hosts in Bulgaria, a DNA-based approach was used to assess parasite protoscoleces or strobiles. Genes corresponding to coding and non-coding regions of the nuclear and mitochondrial genome ( ND-1, HBX, Act II, AgB-1) were amplified by PCR and subsequently sequenced. The sequences resolved were all found to be identical to those published for the common sheep strain of E. granulosus, indicating that the G1 genotype is predominant in Bulgaria. One microvariant for ND-1 was found in the pig isolates; however no epidemiological significance was attributed to this finding.

Animals↗

Scraping or shaking--a comparison of methods for the quantitative determination of Echinococcus multilocularis in fox intestines.

A modified sedimentation technique (the "shaking in a vessel" technique; SVT) to examine intestines for smaller helminths such as Echinococcus multilocularis is described and compared with the intestinal scraping technique (IST). Out of 356 foxes, 26 were E. multilocularis positive, 19 using IST (sensitivity 73.1%) and 25 with SVT (sensitivity 96.2%) after IST. Especially low infection intensities went undetected with IST. The result is comparable to the established sedimentation and counting technique (SCT). SVT is recommended for the routine examination of fox intestines as an alternative to SCT.

Animals↗

Humoral and cytokine response during protection of mice against secondary hydatidosis caused by Echinococcus granulosus.

Infection of BALB/c mouse with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis and the associated immune response in immunization and infection trials. The aims of this study were to induce a protective immunity against secondary hydatidosis using conventional vaccination approaches and to analyse the immune responses that accompany this protection. Mice immunized with antigen B (AgB), a component of crude sheep hydatid fluid (CSHF), showed a significant level of protection as indicated by a 98.3% reduction in cyst load. This reduction in cyst development was accompanied by a high concentration of interferon gamma secreted by antigen-stimulated spleen cells, as compared with those secreted by cells of mice immunized with CSHF or protoscoleces homogenate (PSH) antigens. In contrast, interleukin-4 was significantly higher in the supernatants of cells stimulated with CSHF or PSH compared with AgB (191.5, 195.7 and 127.5 pg, respectively). Kinetic analysis of immunoglobulin subclasses showed persistently high levels of IgG1 and IgG2a subclasses in immunized infected animals until 6 months of infection, whereas IgG3 showed a significant decline after 1 month of infection. In infected non-immunized control mice, all IgG subclasses showed a gradual increase after the first month of infection until the experiment termination (8 months after infection).

Animals↗

In vitro effects of flubendazole on Echinococcus granulosus protoscoleces.

The aim of the present work was to determine the in vitro protoscolicidal effect of flubendazole (FLBZ) against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with FLBZ at concentrations of 10, 5 and 1 microg/ml. The first signs of FLBZ-induced damage were observed 3 days post-incubation. A clear protoscolicidal effect, reducing the vitality of protoscoleces to 35.6+/-0.7%, was observed after 18 days of incubation. After 25 days of FLBZ incubation (5 microg/ml), the percentage of vital protoscoleces was 13.9+/-5.9%. Protoscolex mortality was 100% (10 and 1 microg/ml) and 0.7+/-0.7% (5 microg/ml) after FLBZ incubation for 30 days. Results of vitality tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of FLBZ against E. granulosus protoscoleces.

Animals↗

Echinococcus multilocularis: immunity response to purified alkaline phosphatase in BALB/c mice.

The study of purified alkaline phosphatase and crude extract antigen immunogenicity from Echinococcus multilocularis was carried out on BALB/c mice. The animals were immunized, then infected with E. multilocularis metacestode. The immune response against purified alkaline phosphatase was studied. Flow cytometry analysis of the CD4+ and CD8+ lymphocyte populations showed a predominance of CD4+ populations in infected immunized mice. The specific humoral response to purified alkaline phosphatase was analyzed by enzyme-linked immunosorbent assay method. We noted a stimulation of an immunoglobulin IgG response. The isotypic profile showed a prevalence of IgG1 and IgG3 in immunized infected mice compared to IgG2a and IgG2b. In addition, analysis of the profiles of the in vitro secreted cytokines, after stimulation of the splenocytes from immunized mice, was performed. The cytokine profile was a mix of Th1/Th2 types in the infected and uninfected immunized mice. The results of this study suggest a humoral mixed Th1/Th2 response, with a high predominance of Th2 response. A similar study was conducted in mice immunized with crude total antigen. The comparison of the immune response showed an important immune response in mice immunized with purified alkaline phosphatase compared to mice immunized with the crude total antigen.

Alkaline Phosphatase↗

Molecular characterization of Echinococcus granulosus strains in Sardinia.

Investigations were undertaken to determine the genotypes of the parasite Echinococcus granulosus that were present in livestock animals on the island of Sardinia. Liver, lung, and spleen samples were obtained from 770 sheep, 229 cattle, and 277 pigs slaughtered in Sardinia between January 2003 and April 2005, and the number and fertility of hydatid cysts were determined. Protoscoleces and/or germinal layer were collected from individual cysts, DNA was extracted from 91 samples, and polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods were used for identification of the strain genotype for each sample (G1, G5, G6/G7). Fragments of the mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase I were sequenced. Hydatid disease prevalence of 75.3, 41.5, and 9.4% were found in the organs collected from sheep, cattle, and pigs, respectively. Molecular analysis showed that 89 of 91 ovine, bovine, and swine cysts belonged to the G1 genotype (common sheep strain) of E. granulosus. Parasite isolates from two pigs were identified to belong to the G7 genotype (pig strain). Our results confirm the high prevalence of E. granulosus infection in livestock animals in Sardinia and reveal the presence of at least two parasite genotypes in Sardinia.

Animals↗

Modified cellular immune responses in dogs infected with Echinococcus multilocularis.

Parasite-specific antigen responses and lymphocyte blastogenesis in dogs orally inoculated with Echinococcus multilocuralis metacestodes were examined. Serum IgG1 (Th2-oriented) and IgG2 (Th 1-oriented) levels against somatic and excretory-secretory (ES) antigens of protoscoleces and adult worms increased from 7 days post-infection (DPI), with the highest responses against protoscolex excretory-secretory antigen (PES). Specific blastogenesis of peripheral blood mononuclear cells (PBMC) against the parasite antigens was not observed during the 21-day infection period, but Peyer's patches cells from one out of two dogs at 21 DPI showed blastogenesis against PES (stimulation index: 4.65). Interestingly, only at 7 DPI were concanavalin A (ConA)-induce proliferative responses of PBMC reduced. Moreover, ConA-induced proliferative responses of lymphocytes from various origins were suppressed by the addition of parasite antigens, especially with PES. These data suggest that although both Th1- and Th2-oriented humoral immune responses were observed in E. multilocularis infected dogs, the parasite antigens, especially PES, may have incompletely suppressed lymphocyte responses in these dogs.

Animals↗

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in gerbils: modified lymphocyte responses due to the parasite antigen.

Immune responses to oral infection with Echinococcus multilocularis protoscoleces in Mongolian gerbils were investigated. Gerbils not treated with prednisolone expelled most of the parasites within 3 days post-infection and induced parasite-specific intestinal IgA secretion after the oral inoculation with protoscoleces. In contrast, prednisolone-treated gerbils harbored notable numbers of parasites, and the parasite-specific intestinal IgA secretion was lower. In gerbils not treated and orally inoculated with protoscoleces (infected group), parasite-specific antibody levels in sera and intestinal washings were elevated, but blastogenesis against protoscolex antigens was observed only in cells from Peyer's patches at 14 days post-infection. Concanavalin A-induced proliferative lymphocytes from both infected and naive gerbils were suppressed by adding protoscolex somatic antigen. These data indicate that while parasite-specific antibody responses were observed, lymphocyte proliferative responses were suppressed by parasite infection/antigens in the Mongolian gerbil, prednisolone-untreated rodent definitive host model.

Administration, Oral↗

Immunogenicity of two Echinococcus granulosus antigens EgA31 and EgTrp in mice.

Here, we investigate in mice the immunogenicity of two antigens EgA31 and EgTrp which are expressed by the larval stage of Echinococcus granulosus. These recombinant proteins were used alone or as a mixture (EgA31-EgTrp) to immunize BALB/c mice. By flow cytometry, we have shown that the ratio CD4+/CD8+ of splenocytes were significantly higher in the antigen-immunized groups. The specific antibody in the sera and cytokine producing splenocytes was evaluated by enzyme-linked immunosorbent assay. EgA31, EgTrp or EgA31-EgTrp elicited high antibody titer of IgG and IgA. Among IgG isotypes, IgG1 was predominant for each antigen tested alone or combined. The production of IL-12, IFN-gamma, IL-10 and IL-6 cytokines was significantly higher in mice immunized with recombinant proteins. Our results suggest that, in BALB/c mice, a mixed Th1/Th2, response to EgA31, EgTrp and EgA31-EgTrp is obtained. The use of both antigens separately or in combination as candidate vaccine proteins is discussed.

Animals↗

Echinococcus granulosus (Cestoda, Taeniidae) in the Iberian wolf.

The intestinal contents of 27 Iberian wolves (Canis lupus signatus) from Spain were screened for the presence of the taenid cestode Echinococcus granulosus. Four animals were found positive (15% prevalence). The intensity of parasitation was variable (mean 71, range 1-147 E. granulosus per host). Gravid individuals were found in all wolves positive with the parasite. Molecular characterization of the parasite material showed that the wolf strain belongs to the G1 genotype. According to the results, we conclude that the Iberian wolf takes part in the maintenance of the life cycle of this zoonotic parasite in Spain and that this fact could have public health relevance.

Animals↗

A relevant enzyme in granulomatous reaction, active matrix metalloproteinase-9, found in bovine Echinococcus granulosus hydatid cyst wall and fluid.

In addition to the ability of matrix metalloproteinases (MMP) to degrade components of the extracellular matrix and their involvement in pathology-related processes of tissue remodeling, they were recently reported to enhance inflammation by activation of proinflammatory cytokines, or their release from the cell surface. In the work reported here, proteolytic activity previously found for hydatid cysts was further characterized as MMP-9. Active host MMP-9 was found in walls and fluids of bovine hydatid cysts of Echinococcus granulosus in the environment of granulomatous reaction. Pooled walls and fluids of hydatid cysts obtained from infected cattle were processed. Strong proteolytic activity was detected by zymography. The proteolytic fraction was purified by anion exchange and gelatin-agarose affinity chromatography. Major proteinases of the purified fraction were subjected to mass spectrometry and their identities were further confirmed by Western blotting using commercial anti-human MMP-9 monoclonal antibodies. Two proteinases were characterized as latent and active forms of host MMP-9. Using the same antibody for immunoblot, activity was localized, in paraffin-embedded sections of the parasite and the local host environment, to epithelioid and giant multinucleated cells. It is proposed here that MMP-9 is secreted by specialized host cells of monocytic lineage (epithelioid/giant cells) as an effector, in an attempt to digest the persistent foreign body. In vivo activation of MMP-9 suggests its involvement in inflammatory reaction and in the chemotaxis of inflammatory cells to the cyst. However, E. granulosus can deal efficiently with MMP-9. Research is suggested into possible immune evasion mechanisms, including the secretion of an inhibitory molecule.

Amino Acid Sequence↗

Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries.

The taxonomic and phylogenetic status of Echinococcus granulosus strains are still controversial and under discussion. In the present study, we investigated the genetic polymorphism of E. granulosus isolates originating from three countries of Africa, including a region of Algeria, where the common G1 sheep and the camel G6 strains coexist sympatrically. Seventy-one hydatid cysts were collected from sheep, cattle, camels, and humans. Two mitochondrial markers (cox1 and nad1) were used for strain identification. Two nuclear markers (actII and hbx2) were used to study the possible occurrence of cross-fertilization. Despite the heterogeneity observed among the G1 isolates, they were all localized within one robust cluster. A second strong cluster was also observed containing all of the G6 isolates. Both strains appeared as two distinct groups, and no cases of interbreeding were found. Thus, the attribution of a species rank can be suggested. We also found the Tasmanian sheep G2 strain for the first time in Africa. Because of the slight variations observed between the common sheep and the Tasmanian sheep strains, further studies should be carried out to elucidate the epidemiological relevance of this genetic discrimination.

Algeria↗

Effects of protoscoleces and AgB from Echinococcus granulosus on human neutrophils: possible implications on the parasite's immune evasion mechanisms.

The factors affecting the innate susceptibility to Echinococcus granulosus infections are largely unknown. We assessed the interaction of healthy human neutrophils with protoscoleces (PSC) and antigen B (AgB) of E. granulosus by analysis of CD11b upregulation and H(2)O(2) production by flow cytometry. PSC induced neutrophil activation, but their viability was not affected. In contrast, no effects were observed with AgB in both assays. Neutrophil-enriched fractions were also incubated with PSC or AgB, and interleukin 8 (IL-8) production was measured by ELISA. Significant increment in IL-8 production was detected only in supernatants from neutrophil-enriched fractions cultured with PSC. The possible effect of a prior incubation with AgB on the phorbol myristate acetate-induced activation was also evaluated. No changes were observed in CD11b expression, but the H(2)O(2) production was significantly reduced in platelet-activating factor (PAF)-primed neutrophils. These results suggest a possible AgB-mediated mechanism of evasion of the host immune response, which would operate upon events of spillage of the fertile hydatid cyst content.

Adult↗

In vitro and in vivo effects of flubendazole on Echinococcus granulosus metacestodes.

The aim of the present work was to determine the efficacy of flubendazole (FLBZ) against Echinococcus granulosus metacestodes by using in vitro and in vivo models. Groups of 50 microcysts developed in vitro, and groups of 10 peritoneal cysts were obtained from Balb C mice with experimental secondary infections of 8 months. The cysts were placed in Leighton tubes containing 10 ml of culture medium. FLBZ was added to the medium resulting in final concentrations of 5 and 1 microg/ml for mycrocysts treatment and 10, 5, and 1 microg/ml for murine cysts treatment. In vivo treatment was performed on 20 mice that developed an experimental secondary hydatid disease over a period of 11 months. FLBZ was given (1.5 mg/kg) by the oral route once a day for 50 days. A loss of turgidity was detected in all in vitro drug treated cysts irrespective of the drug concentration or parasite origin. Inspection of treated cysts by scanning electron microscopy (SEM) revealed that the germinal layer lost it characteristic multicelular structure. These results were confirmed on the ultrastructural level by transmission electron microscopy (TEM), treated metacestodes had undergone considerable degenerative changes after the in vitro treatment. The results obtained after the in vivo treatment with FLBZ showed no significant difference between the control and treated groups related to the weight of cyst masses. However, the ultrastructural study at TEM of cysts that developed in mice from the treated group revealed alterations in the germinal layer with the presence of numerous vacuoles. With regard to the ultrastructural study at SEM, only cellular debris of the germinal layer could be seen. In conclusion, the data obtained clearly demonstrate that in vitro and in vivo treatment with FLBZ is effective against E. granulosus metacestodes.

Animals↗

The immunodiagnostic potential of Echinococcus granulosus adult-worm antigens in human cystic echinococcosis.

Echinococcus granulosus adult-worm antigens were characterised for assessment of their immunodiagnostic potential for human cystic echinococcosis (CE). The analysis of worm extracts by enzyme-linked immunosorbent assay (ELISA) showed a sensitivity of 83% for CE, which is comparable with data obtained for cyst-fluid-based serodiagnostic tests. Immunoprecipitation of in vitro-translated E. granulosus worm mRNA revealed a range of low-molecular-weight antigenic proteins (12-45 kDa) recognised by human CE sera. E. granulosus adult worms may provide an additional or alternative source to metacestode material for the isolation of both native and recombinant antigens to be considered for the serodiagnosis of human echinococcosis.

Animals↗

Echinococcus granulosus infection of farm dogs of Iran.

The prevalence and distribution of Echinococcus granulosus in sheepdogs was studied in 13 provinces of Iran, where 90% of the Iranian sheep and goat populations and, thus, sheepdogs are found. Worms were found in 27.17% of 390 dogs successfully purged with 4 mg/kg arecoline hydrobromide. The highest prevalence was detected in dogs from the rural areas of Isfahan (central part of Iran) and the lowest, in dogs from those of Sistan (Southeast Iran). The frequency distribution of E. granulosus was overdispersed, with only a few animals harboring heavy infections.

Animals↗

Development and morphological variability of Echinococcus granulosus.

Characteristics are presented of Echinococcus granulosus strobila from dogs experimentally infected with protoscolices from a single cyst isolated from the liver of a domestic pig in a central Slovakian district. Adult cestodes are characterized mainly by 28-32 hooks highly variable in shape and size; 26-51 testes situated mostly throughout the proglottis (1 or 3 rows behind the vitelline gland); a lobate ovary; the shape of the uterus, which in 10% of proglottides is more or less spherical, in 62% is saccular with lateral sacculations of variable size, in 27% is saccular without lateral sacculations and in 1% is more or less tubular or strongly ramified; and an intermediately rapid rate of development lasting until between 40 and 44 days p.i. The present report analyzes the high degree of variability of the clone studied, referring to the knowledge on the characteristics of the pig, sheep, and cattle strains of E. granulosus and E. multilocularis species, with which some taxonomic characters of the clone representatives overlap.

Animals↗

Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80-84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radiolabeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited.

Amino Acid Sequence↗