RESUMO
The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3-25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.
Assuntos
Cistos/parasitologia , Cistos/ultraestrutura , Giardia/fisiologia , Giardia/ultraestrutura , Animais , Western Blotting , Cistos/patologia , Microfibrilas/patologia , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Peptídeos/metabolismo , Polissacarídeos/metabolismoAssuntos
Humanos , Amebíase/diagnóstico , Amebíase/fisiopatologia , Entamoeba histolytica/análise , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/ultraestrutura , Inquéritos Epidemiológicos , Imunoeletroforese , Imunoeletroforese/instrumentação , Testes Sorológicos , Testes de Hemaglutinação/instrumentação , Testes de Hemaglutinação/métodos , VirulênciaRESUMO
Human amebiasis is a parasitic infection which involves asymptomatic as well as intestinal and extraintestinal symptomatic stages in individuals harbouring Entamoeba histolytica. Several factors have been proposed to explain the variability in the outcome of the disease. Among these are differences in virulence of E. histolytica strains and methods such as zymodeme analysis have been used to differentiate invasive from non invasive strains of this parasite (Sargeaunt and Williams, 1978). In order to establish a possible correlation between zymodeme analysis and serological data in human cases of amebiasis, a cross-sectional epidemiological study was carried out in the community area of Cadereyta, State of Queretaro, Mexico. In this study, fecal and serum samples from individuals were also tested by Indirect Hemagglutination assay (IHA) to determine antibody titre and by a standardized Electroimmunotransfer blot assay (EITB) for recognition of E. histolytica specific antigens. Zymodemes were determined in a total of 81 samples. Of these, 27 had a pathogen zymodeme (PZ) while 54 had a non pathogen zymodeme (NPZ). Values obtained by IHA varied from negative to titres up to 1:256 in serum samples tested. Reactivity to E. histolytica antigens determined by EITB was observed in all sera. Patterns of antigen recognition were complex and showed reactivity to several parasite molecules. Among these, components with M. Wt. of 165, 119, and doublets of 98-100 and 50-52 Kd were more frequently recognized by antibodies present in the sera tested. In general, antibody titres detected by IHA did not showed a direct correlation with zymodeme analysis. Samples with PZ or NPZ had similar variable levels of reactivity to E. histolytica antigens.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/análise , Western Blotting , Entamoeba histolytica/classificação , Entamebíase/parasitologia , Testes de Hemaglutinação , Isoenzimas/análise , Proteínas de Protozoários/análise , Adulto , Animais , Antígenos de Protozoários/imunologia , Estudos Transversais , Entamoeba histolytica/enzimologia , Entamoeba histolytica/imunologia , Entamoeba histolytica/patogenicidade , Entamebíase/epidemiologia , Fezes/parasitologia , Feminino , Hemaglutininas/análise , Humanos , Lactente , Recém-Nascido , Masculino , México/epidemiologia , VirulênciaRESUMO
The complexity of the clinical spectrum in human amebiasis and the high variability in laboratory methods used to detect Entamoeba histolytica infections have impeded the collection and evaluation of reliable epidemiological data. Thus, more sensitive, specific and standardized methods are needed in order to accurately identify infections with this parasite. An important step in the development of serological diagnostic methods is the identification and isolation of specific parasite antigens which are immunogenic in the host. In this work, we have standardized an electroimmunotransfer blot technique to characterize E. histolytica antigens recognized by antibodies present during human amebic infections. An important aspect was an investigation of technical variations in the preparation of cell lysates including the use of different protease inhibitors and solubilizing agents. The highest yield of protein was achieved by homogenization of trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) as a protease inhibitor and by lysis using Triton X-100 and a mixture of protease inhibitors. Recovery of degraded vs non-degraded proteins in the cell extracts was evaluated by gradient polyacrylamide gel electrophoresis. Both quantitative and qualitative differences were noted between the different methods of preparing soluble cell extracts. A more complete set of antigenic components was obtained by homogenization and use of pHMB. Thus parasite extracts prepared by this method were selected for protein transfer. In this, the optimal protein concentration was of 120 micrograms of protein per cm of gel width and efficient transfer of proteins to nitrocellulose sheets was achieved at 100 V for 2 hrs and at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)