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PubMed2004

Cystic echinococcosis in Argentina: evolution of metacestode and clinical expression in various Echinococcus granulosus strains.

Echinococcus granulosus hydatid cysts were examined in 41 patients from Neuquén and Tucumán provinces in Argentina. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) revealed in 19 patients common sheep strain (G1), in 6 patients Tasmania sheep strain (G2), in 1 patient cattle strain (G5), and in 15 patients camel strain (G6). In Argentina the only known is the domestic cycle that affects dogs and herbivorous, including ovine, swine, cattle and goats. These strains produced a total of 58.6% of primary liver infections, 29.2% primary in lung, 2.4% primary in spleen and 9.8% were multiorgan abdominal infections. The metacestode was classified using the evolutive stages proposed by WHO-IWGE (from CE1 to CE5). We estimated that CE1 cyst has a duration of about 22 years, CE2 of 14 years, CE3 of 10 years, CE4 of 19 years and CE5 was not determined. The active types CE1 and CE2 reached 75% of all cases from all strains. In 36 patients with cysts from G1, G5 and G6 strain, there were only two asymptomatic cases. The strains of the E. granulosus complex do not present important clinical differences; only G6 seems to have higher growth rate.

Adolescent↗
PubMedDate not supplied

Echinococcus granulosus cattle strain identification in an autochthonous case of cystic echinococcosis in central Mexico.

Echinococcosis is a frequent hepatic parasitic disease in several countries but it is practically absent in Mexico. A cattle strain of Echinococcus granulosus was identified by RAPD, PCR-RFLP and mitochondrial CO1 gene analysis in an autochthonous case. The parasite was obtained after a laparoscopic excision of a liver cyst from a patient that was symptomatic for 6 years but mis-diagnosed before hospitalization.

Adult↗
PubMed2006

Development and sexual maturation of Echinococcus granulosus adult worms in the alternative definitive host, Mongolian gerbil (Meriones unguiculatus).

In the present study for the first time an alternative definitive host model has been established for the complete development of Echinococcus granulosus in Meriones unguiculatus (Mongolian gerbils) up to shelled eggs production, as happened in the natural host. Animals treated with methylprednisolone acetate were orally inoculated with approximately 3000 protoscoleces and the development of parasites in the small intestine observed up to 46 days post-infection (p.i.). The cestodes survive, strobilate, mature sexually and complete their development in the intestine up to production of shelled embryonated eggs containing fully formed six-hooked oncospheres, similarly to the natural canid host (stage 7 by Smyth). Shelled eggs are first observed in the rodent/E. granulosus model at 44 days p.i. Somatic growth in gerbils appears to follow a similar trend to dogs, length increasing to over 3mm at 44 days p.i., while, compared to the natural host, organogenesis is observed only to progress at a slower rate in the early developmental stages. Parasite burden was found to vary among non-inbred animals and developmental stages of worms recovered from the same animal differed widely. Worm localisation follows the pattern observed in the natural host, the parasites appearing for the most part distributed in the proximal third of the small intestine. The development of alternative definitive hosts may facilitate research in this field, overcoming legislative impediments that restrict the use of dogs for experimental purposes.

Animals↗
PubMed2006

Antigens for the immunodiagnosis of Echinococcus granulosus infection: An update.

The taeniid tapeworm Echinococcus granulosus is the causative agent of the echinococcal disease, an important zoonosis with worldwide distribution. Accurate immunodiagnosis of the infection requires highly specific and sensitive antigens to be used in immunodiagnostic assays. The choice of an appropriate source of antigenic material is a crucial point in the improvement of the diagnostic features of tests, and must be based on the developmental stage of the parasite and the host. The most common antigenic sources used for the immunodiagnosis of echinococcal disease are hydatid cyst fluid, somatic extracts and excretory-secretory products from protoscoleces or adults of E. granulosus. Hydatid cyst fluid is the antigenic source of reference for immunodiagnosis of human hydatidosis, which is mainly based on the detection of antigens B and 5. Somatic extracts have been widely used in the serodiagnosis for E. granulosus infection in dogs and ruminant intermediate hosts, although in the last few years the detection of excretory-secretory products of the worm in faeces (coproantigens) have become the most reliable method for the detection of the parasite in the definitive host. This review emphasizes recent advances in the identification and characterization of novel antigens with potential for the immunodiagnosis of echinococcal disease. Progress in recombinant technologies and synthetic peptides are also discussed. The paper highlights the need to search for new antigenic components with high diagnostic sensitivity and specificity, a fact that remains a crucial task in the improvement of the immunodiagnosis of the disease.

Animals↗
PubMed2006

Detection of Osteopontin in the pericyst of human hepatic Echinococcus granulosus.

It aims at investigating the expression and distribution of the Osteopontin (OPN) in the pericyst of human hepatic Echinococcus granulosus and their related significances. Sixty pericysts excised by "sub-adventitial cystectomy" were studied. OPN was detected in 80% (48/60) of cysts by Western blotting and distributed in the side of "exocyst" layer directing to the parasite, also macrophages were identified in the vicinity of OPN by immunohistochemistry staining. The coexpression of OPN and CD68 was observed by immunofluorescence double labeling and analyzed by Image-Pro Plus 5.1; with special stain techniques, variable degrees of calcium deposits were observed in 80% (48/60) cysts, and the calcium deposits concurrencely found with the OPN expression. The selective distribution of OPN, calcium in the "exocyst" provides a new pathological evidence for the "sub-adventitial cystectomy" we developed. The pericyst of hepatic E. granulosus consists of two detachable layers with different formative mechanisms: the "exocyst" layer directing towards the cyst of parasite was the result of granulomatous reaction; also the results suggest OPN is one regulator in the granulomatous reaction and calcification of "exocyst".

Animals↗
PubMed2006

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers.

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.

Amino Acid Sequence↗
PubMed2004

Synthetic studies on glycosphingolipids from Protostomia phyla: total syntheses of glycosphingolipids from the parasite, Echinococcus multilocularis.

Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with benzoyl ceramide in the presence of N-iodosuccinimide (NIS)/TfOH, which gave the desired oligosaccharide derivatives. The fully protected glycosides 13, 20, 22 and 25 were deprotected to give four target glycosphingolipids (1-4).

Animals↗
PubMed2004

Echinococcus granulosus in Jordan: assessment of various antigenic preparations for use in the serodiagnosis of surgically confirmed cases using enzyme immuno assays and the indirect haemagglutination test.

The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB), and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation (r = 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24 KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen B fractions of CSHF or AAB are detected.

Adult↗
PubMedDate not supplied

Echinococcus granulosus: oncosphere-specific transcription of genes encoding a host-protective antigen.

A recombinant antigen vaccine has been developed which is effective in preventing the hydatid parasite, Echinococcus granulosus from infecting its animal intermediate hosts. The vaccine antigen, designated EG95, is expressed by a cDNA cloned from E. granulosus oncosphere mRNA. The gene encoding EG95 belongs to a small gene family where six of the members are transcribed in the oncosphere. Conditions were established which allowed specific RT-PCR amplification of mRNA for each gene family member and these conditions were applied to determine transcription patterns for each gene family member in gravid adult worms, oncospheres, and protoscoleces. The four eg95 gene family members which encode an identical EG95 protein, were transcribed only in the oncosphere. In contrast, two gene family members that encode variant EG95 proteins did not have transcription patterns confined to the oncosphere. These findings suggest a common biological role for four genes in the gene family and a separate role for the other, more variant gene family members.

Animals↗
PubMedDate not supplied

The Echinococcus granulosus antigen B shows a high degree of genetic variability.

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.

Animals↗
PubMed2005

Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice.

We describe a method for obtaining improved secondary infections of Echinococcus granulosus that involves culturing protoscolex larvae in vitro prior to inoculation into mice. This approach provides a far superior method for obtaining secondary echinococcosis infections in mice compared with the traditional method of direct inoculation of protoscoleces (PSC) where the majority of parasites are killed by the host. We obtained a high rate of recovery both in terms of secondary cyst numbers and their viability. After 50 weeks post-infection (p.i.), brood capsules were formed and the first PSC developed in each of the capsules. After 56 weeks p.i., the fastest developing brood capsule contained four PSC. The approach will prove valuable for investigating parasite development and the host-parasite interaction in secondary echinococcosis.

Animals↗
PubMed2006

Hepatic atrophy-hypertrophy complex due to Echinococcus granulosus.

Obstruction of a major hepatic vein, or major portal vein, or biliary tree branch causes atrophy of the related hepatic region, and frequently, hypertrophy in the remaining liver-the atrophy-hypertrophy complex (AHC). Whether hydatid cysts can cause AHC is controversial. The records of 370 patients who underwent surgery for hepatic hydatid disease between August 1993 and July 2002 were evaluated retrospectively. Excluding six patients with previous interventions on the liver, AHC had been recorded in the operative notes of 16 patients (4.4%); for all patients, a cyst located in the right hemiliver had caused atrophy of the right hemiliver and compensatory hypertrophy of the left hemiliver. The computed tomography images of seven patients were suitable for volumetric analysis. The median (range) right and left hemiliver volumes were 334 (0-686) ml and 1084 (663-1339) ml, respectively. The median (range) cyst volume was 392 (70-1363) ml. AHC due to Echinococcus granulosus was confirmed by objective volumetric analysis. The presence of AHC should alert the surgeon to two implications. First, pericystectomy may be hazardous due to association with major vascular and biliary structures. Second, in patients with AHC, the hepatoduodenal ligament rotates around its axis; this should be considered to avoid vascular injury if a common bile duct exploration is to be performed.

Adult↗
PubMed2003

Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis.

Receptor tyrosine kinases (RTKs) are crucially involved in the development of metazoan organisms and possible mediators of cell-cell communication that occurs between eukaryotic parasites and hosts. We have now cloned and characterized the complete complementary DNA (cDNA) molecule encoding a novel receptor tyrosine kinase, EmER, of the human parasite Echinococcus multilocularis. EmER shared significant amino acid sequence homologies with members of the epidermal growth factor (EGF) receptor family of different phylogenetic origin, exhibited a domain structure which is typical for this group of membrane receptors and contained all catalytically important residues at the corresponding positions. Highest homologies were detected between EmER and a previously identified receptor kinase, SER, from the parasitic trematode Schistosoma mansoni. The EmER encoding gene, emer, spans a chromosomal region of 39 kb and is composed of 23 exons. Structural comparisons indicated that emer and the EGF receptor encoding genes from mammals derive from a common ancestor. DNA/DNA hybridization experiments demonstrated that emer is present as a single copy locus in the parasite. Transcriptional analyses on in vitro cultivated parasite larvae revealed that emer is expressed in both the metacestode and the protoscolex stage, although about 10-fold higher transcription levels were detectable for the protoscolex. Northern blot experiments further indicated that emer is expressed as a single 5.2-kb transcript in parasitic larvae during an infection of the intermediate host. These results suggest an involvement of EmER in echinococcal differentiation processes towards the protoscolex stage during natural infections and provide, for the first time, structural information on an epidermal growth factor receptor-like kinase from a cestode.

Amino Acid Sequence↗
PubMed2004

Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage.

A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible.

Animals↗
PubMed2004

Contingent, non-neutral evolution in a multicellular parasite: natural selection and gene conversion in the Echinococcus granulosus antigen B gene family.

Recent studies have demonstrated that the Echinococcus granulosus antigen B (AgB) interferes with the intermediate hosts' immune response and is encoded by a multigene family. The number of members within the family is still uncertain, but there are several evidences of a large genetic variability. The E. granulosus AgB genomic sequences available in nucleotide databases can be grouped into four clades, corresponding to genes EgAgB1, EgAgB2, EgAgB3 and EgAgB4. In the present study, we use PCR amplifications followed by cloning and sequencing to evaluate the genetic variability for AgB isoforms. Two pairs of primers were independently used for PCR amplification. Both PCR reactions from each of three isolated protoscolex (larvae) were cloned in a plasmid vector and the plasmid inserts of 30 colonies from each cloning experiment were sequenced. Using phylogenetic tools, the 113 EgAgB clones are classified as follows: 25 are related to EgAgB1, 24 to EgAgB2, 9 to EgAgB3 and 39 to EgAgB4. The remaining 16 clones form a separate cluster, which we name EgAgB5, more closely related to EgAgB3 than to any of the other genes. Within each gene group, a number of variant sequences occur, which differ from one another by one or few nucleotides. One EgAgB3 clone has a premature stop codon (pseudogene) and an EgAgB2 clone lacks the region corresponding to the intron. The overall variation cannot be explained by differences among the asexual protoscoleces, or by experimental artifacts. Using Echinococcuss AgB genes from other species/strains as outgroups, neutrality is rejected for EgAgB2, and balancing selection is detected for EgAgB5, which also seems to be involved in gene conversion. We suggest that EgAgB1-EgAgB5 represent a family of contingency genes, that is, genes that are variably expressed, so that some but not others are expressed in each individual parasite. Contingency genes are common in parasitic protozoa and other microparasites, but the EgAgB family is the first set identified in a multicellular parasite.

Alleles↗
PubMed2004

Processes leading to a spatial aggregation of Echinococcus multilocularis in its natural intermediate host Microtus arvalis.

The small fox tapeworm (Echinococcus multilocularis) shows a heterogeneous spatial distribution in the intermediate host (Microtus arvalis). To identify the ecological processes responsible for this heterogeneity, we developed a spatially explicit simulation model. The model combines individual-based (foxes, Vulpes vulpes) and grid-based (voles) techniques to simulate the infections in both intermediate and definite host. If host populations are homogeneously mixed, the model reproduces field data for parasite prevalence only for a limited number of parameter combinations. As ecological parameters inevitably vary to a certain degree, we discarded the homogeneous mixing model as insufficient to gain insight into the ecology of the fox tapeworm cycle. We analysed five different model scenarios, each focussing on an ecological process that might be responsible for the heterogeneous spatial distribution of E. mulitlocularis in the intermediate host. Field studies revealed that the prevalence ratio between intermediate and definite host remains stable over a wide range of ecological conditions. Thus, by varying the parameters in simulation experiments, we used the robustness of the agreement between field data and model output as quality criterion for the five scenarios. Only one of the five scenarios was found to reproduce the prevalence ratio over a sufficient range of parameter combinations. In the accentuated scenario most tapeworm eggs die due to bad environmental conditions before they cause infections in the intermediate host. This scenario is supported by the known sensitivity of tapeworm eggs to high temperatures and dry conditions. The identified process is likely to lead to a heterogeneous availability of infective eggs and thus to a clumped distribution of infected intermediate hosts. In conclusion, areas with humid conditions and low temperatures must be pointed out as high risk areas for human exposure to E. multilocularis eggs as well.

Animals↗
PubMed2005

Echinococcus multilocularis in red foxes (Vulpes vulpes) of the Italian Alpine region: is there a focus of autochthonous transmission?

Alveolar echinococcosis, caused by the metacestode of Echinococcus multilocularis, is a zoonosis with a wider distribution area than described in the past. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes (Vulpes vulpes) were detected in the Bolzano province (Trentino Alto Adige region) near the Austrian border. The aim of this work was to evaluate the prevalence of infection in red fox populations from five Italian regions. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples. Amplicons were confirmed by sequencing. Of 500 faecal samples from foxes shot in Valle d'Aosta (n=57), Liguria (n=44), Lombardy (n=102), Veneto (n=56), and Trentino Alto Adige (n=241) regions, 24 animals, all from the Trentino Alto Adige region, were found positive. Twenty-two positive animals originated from the Bolzano province and two positive animals from the Trento province. Several localities of the Bolzano province, in which positive foxes were detected, are the same as those where alveolar echinococcosis had been described in humans in the second half of the 19th century, suggesting an old endemicity for the investigated area, which is adjacent to endemic areas of Austria. Therefore, the question arises if we are observing an increase and expansion of foci, or if the new records are due to the more sensitive and specific methods used to detect the worm DNA.

Animal Diseases↗
PubMed2005

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats.

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.

Animal Diseases↗