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In this study we have developed a modified CLARK and DIAMOND (1992, 1993) method using the polymerase chain reaction to amplify amoebic ribosomal RNA genes that allow either specific detection of Entamoeba histolytica s. str. or amoeba species identification. DNA was isolated from cultured protozoa or from stool samples (cysts andlor trophozoites). Cultures of xenie or axenic material (also stool samples) were treated with Easy Genomic DNA Prep or Genomic DNA Prep Plus (A&A Biotechnology, Poland) for DNA isolation. With these new procedures, DNA can be obtained effectively and with high degree of purity. 13 amoeba strains were investigated. Three strains produced an 876 bp fragment with Psp5 and Psp3 primers (specific product for E. histolytica s. str). Three strains gave a specific 876 bp product for E. dispar with NPsp5 and NPsp3 primers. Two strains were E. moshkovskii as identified by KpnI digestion of PCR products obtained with RD5 and RD3 primers. Four strains (one of them was cultured in two laboratories) were E. invadens as identified by HinfI or HhaI digestion of PCR products obtained with RD5 and RD3 primers. Characteristic RFLP patterns with these enzymes was also obtained for E. terrapinae and Blastocystis hominis. This RFLP analysis show significant promise as a medical diagnostic tool particularly useful for species identification.
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Pozamozgowe inwazje Acanthamoeba spp. u myszy

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A variety of Acanthamoeba spp. pathogenic strains were isolated from water pools in the area of Poznań. In many cases amoebae invaded in mice, beside central nervous system (CNS) also lungs, kidney, heart, liver, diafragm and eyeballs, causing significant changes in these organs. The presence of amoebae outside of CNS was found mainiy in the early period of infection. Amoebae isolated Crom different organs of dead animals did not exhibit any specificity in relation to the organs in following infections.
The eosin colour test estimating the viability of cysts was compared with the method of in vitro excystation on agar substrate (NNE). Single cysts were studied, isolated from populations stored under sterile conditions in water at 4°C for a period ranging from a few days to 16 years. It has been found that the inoculations of single cysts (805 cysts) on the NNE substrate allow a precise determination of the percentage of living cysts. All the cysts stained with eosin, incubated on such a substrate proved to be dead, while the non-staining cysts ex-cistated with varying percentage. Thus, among the non-staining cysts of Acanthamoeba after 2-4 years of storage at 4°C 89.2% excystated while the remaining ones were dead. After 16 years of storage only 29.8% nostaining cysts excystated. Very similar values (89.6 and 28.5% respectively) were obtained when determining the percentage of living cysts with the colour test. The eosin test has proved to be a good method of the estimation of the viability of cysts of Acanthamoeba.
The studies revealed that the infection with the „limax" group amoebae resulted in the severe changes in the respiratory system: in interstitial pneumonia, the injuries of the bronchi, the bronchioles and the blood vessels. The histopathological examinations showed the focal destruction of the alveoli in case of the mild amoeba invasion while the massive invasion caused the complete destruction of the respiratory epithelium, the blood vessels, the bronchi and the bronchioles. Apart from all the changes in the alveoli, in most cases the significant pulmonary hyperaemia and the numerous blood foci were observed. Moreover, the histopathological changes in the blood vessels, the bronchi and the bronchioles consisting in the epithelial destruction and the laceration of the blood vessel walls and the bronchioles were disclosed.
In order to gain better knowledge about pathomechanisms of amebosis, histological and histochemical studies were performed on large and small intestine of guinea pigs infected by PS-2 Entamoeba histolytica strain. Results of these studies showed that the presence of E. histolytica in large intestine caused also pathomorphological changes of small intestine: partial atrophy of villi, epithelium destruction, epithelial metaplasia, lacteral dilation, infiltration of mucous membrane by lymphocytes, plasmocytes and macrophages. Influence of this parasite on lowering of investigated enzyme reactions and reduction of mucus secretion in large intestine as well as in small intestine of infected animals were also shown. These result suggest influence of E. histolytica cytotoxins (studied earlier by others authors) on the enzymes activity in host tissues and next on the weakness of intracellular metabolism and on cells destruction. For induction of these changes the adhesion host cell-amoeba is not necessary.
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