Genetic diversity of Echinococcus vogeli in the western Brazilian Amazon

Human polycystic echinococcosis is a parasitic infection caused by the larval stage of Echinococcus vogeli, which occurs in rural areas of Central and South America. Until now, little information on the genetic variability of E. vogeli is available. Here, 32 samples from human-excised E. vogeli cysts had a 396-bp sequence of the mitochondrial cytochrome oxidase I (COI) gene sequenced and compared to another 17 COI sequences representing nine Echinococcus species. A Bayesian COI tree revealed that all E. vogeli sequences formed a monophyletic and well-supported clade with an E. vogeli reference sequence. The occurrence of geographically restricted E. vogeli COI haplotypes suggests retention of ancestral polymorphisms with little migration in Acre, Brazil.

Immunoregulatory cytokine [TGF-beta and IL-10] responses in mice inoculated with protoscoleces and major hydatid fluid antigens of cystic echinococcosis

Our objectives were to investigate whether immunomodulatory cytokines, TGF-beta and IL-10, are stimulated in response to cystic echinococcosis [CE] components in mice model, and whether major hydatid fluid antigens or live protoscoleces could equally contribute to such cytokines. Protoscoleces were obtained by aseptic puncture of fertile sheep hydatid cysts. Hydatid fluid antigens [HFAgs] and Antigen B [AgB] were prepared by partial purification and electroelution, respectively. Of the 25 Balb/c mice assigned in four groups, the first group was inoculated ip with 2000 live protoscoleces; the second and the third groups were injected ip with 50 micro g HFAgs and 50 micro g AgB in 200 micro l of PBS, respectively. Control group was only injected with PBS. The sera concentration of TGF-beta and IL-10 were determined by ELISA. Data were analyzed using One-Way ANOVA and Tukey-HSD tests to compare differences between means. The mean concentration of TGF-beta in those groups injected with protoscoleces, HFAgs and AgB were significantly higher than control group. However, in the case of IL-10 such differences were only detected in mice that were inoculated with protoscoleces [356 +/- 11 pg/ml] compared to control [207 +/- 9 pg/ml], HFAgs and AgB groups. TGF-beta and IL-10, two important immunomudulatory cytokines are induced by different molecules or components of CE, so that AgB could induce TGF-B and components of protoscolex, other than AgB and Ag5, could induce IL-10

Descripción de genotipos de Echinococcus granulosus obtenidos de especímenes de hidatidosis humana / Echinococcus granulosus genotypes in samples of human hydatidosis

Introducción: Se ha descrito variabilidad ambiental de Echinococcus granulosus (Eg). Se han reportado 10 genotipos y cierta heterogenicidad intergenotipo en estudios con material proveniente de animales. El objetivo de este estudio es describir los resultados de un protocolo de genotipificación de Eg en muestras de hidatidosis humana. Material y método: Estudio de corte transversal. Se recolectó el líquido hidatídico de una muestra consecutiva de pacientes intervenidos quirúrgicamente por hidatidosis hepática y pulmonar en hospitales de Temuco entre julio de 2004 y septiembre de 2005. Se diseñó un protocolo de extracción de ADN para Eg en muestras homogeneizadas de aspirado de quistes hidatídicos. Se emplearon 2 sistemas de reacción de polimerasa en cadena (PCR): PCREg9 y PCREg16; ambos en concentraciones de MgCl2 al 2mM. Para PCREg9, se utilizaron los primers Eg9F y Eg9R a concentraciones de 0.5 mM, empleándose 35 ciclos con temperatura de hibridación a 60°C. Para PCREg16 se utilizaron los primers Eg16F y Eg16R a concentraciones de 0,5 mM, empleándose 35 ciclos con temperatura de hibridación a 65°C. Los productos de las PCR fueron digeridos con una enzima de restricción (Rsa1) para la discriminación de los genotipos. Resultados: Se analizaron 25 muestras, 4 provenientes de quistes pulmonares y 21 de quistes hepáticos. Se logró amplificación de Eg en 22 de 25 muestras (88 por ciento). La digestión enzimática reveló la presencia de 3 genotipos posibles: en 21 de 22 muestras (95,45 por ciento) se observó un patrón de restricción correspondiente a los genotipos G1 ó G7 y en la muestra restante a los genotipos G4 ó G7. Conclusión: Con los sistemas de PCR empleados se detectó ADN de Eg.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines

Selection, recombination and history in a parasitic flatworm (Echinococcus) inferred from nucleotide sequences

Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analysis revealed several ambiguities, indicationg that the taxonomic status of the E. granulosus horse strain should be revised.

Echinococcus granulosus recombinant antigens

Echinococcus granulosus is a small parasitic platyhelminth, the larval stage of which is the causative agent of cystic hydatid disease both in man and domestic ungulates. This zoonosis constitutes major economic and public (both human and veterinary) health problems in many parts of the world, including southern Brazil, where it is hyperendemic. Serodiagnosis of cystic hydatid disease has been of great importance in clinical diagnosis, posttreatment surveillance of patients and epidemiólogical surveys, since it is the most specific of the noninvasive alternatives for detecting infections with the E. granulosus metacestode. Nevertheless, immunological tests present chronical problems associated with the quality and availability of parasite antigens. In this sense, the cloning of E. granulosus antigen-encoding genes comes forth as a valuable alternative for the production of pure and well characterized antigens. In the last few years, several research groups have cloned and characterized genes that encode E. granulosus antigens, many of which are potentially useful in the immunodiagnosis of cystic hydatid disease. Furthermore, several of these antigens represent important components of the parasite biology and may be particularly relevant to vaccination, immunotherapy, or as potential targets for chemotherapy. In this review we summarize the available data concerning the production and characterization of E. granulosus recombinant antigens. We also discuss some of the perspectives opened by the use of molecular biology techniques in the diagnosis of cystic hydatid disease as well as in the study of the biology of this parasite.

Some recent advances in the molecular characterization of Echinococcus and Taenia solium.

Some recently obtained data from our laboratory on the molecular characterization of Echinococcus and Taenia solium are described and are complimented by relevant new information obtained by other groups. Progress made in the development of satisfactory immunodiagnostic assays and in the production of recombinant molecules, suitable for application in serology of hydatid disease and cysticercosis, is highlighted. Results arising from the application of polymerase chain reaction and direct sequencing, using primers homologous to evolutionarily conserved sequences, in phylogenetic studies and for distinguishing individual taeniid species are also discussed.

Molecular cloning and characterization of the ribosomal RNA genes of the cestode Echinococcus granulosus

The analysis of total protoscolex DNA and some rDNA recombinats of Echinococcus granulosus by restriction endonuclease mapping and hybridization to rDNA probes indicated the complex organization of the ribosomal RNA genes and that some repeat units are larger than 15 kb. The nontranscribed spacer can be up to 13 kb in length in some repeat units. Restriction site polymorphisms was detected mainly in the nontranscribed spacer regions although some polymorphisms was also observed in the 28S rRNA coding region. On the basis of Southern blot hybridization using EcoRi-digested genomic DNA, we conclude that the repeat units containing an extra EcoRI site are present almost in the same proportion as the repeat units without the extra EcoRi site in the 28S rRNA coding region