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Molecular characterization of selected strains of amoebae of the genus Acanthamoeba, the potential human parasites.
ŠTAUBEROVÁ, Kamila
The genus Acanthamoeba belongs to a group of organisms generally called "amoebae". These amoebae can cause fatal disease in humans, being able to enter e.g. through skin in bloodstream and then spreading through the body. In the body they further parasitize in various tissues including the brain there they cause a little known disease called "granulomatous amoebic encephalitis". The disease is most prevalent in people previously ill with, e.g., systemic lupus erythematosus. Granulomatous amoebic encephalitis leads especially to a brain inflammation, which evolves in brain tissue necrosis. Most prominent symptoms are headaches, nausea and vomiting, aphasia and ataxia. This work mentions also some other amoebae as Neagleria fowleri, Balamuthia mandrillaris, Sappinia diploidea a Entamoeba histolytica. Neagleria fowleri is known as a causative agent of an illness called primary amoebic encephalitis. First, I had isolated DNA using Genomic Mini Kit from five amoeba strains. The next step was PCR amplification it is a method depending on alternation of high and lower temperatures in thermocycler. It was followed by electrophoresis, where the presence of bands in agarose gel, which I had prepared myself, showed the presence of absence of amoebic DNA fragments. In case of successful amplification (presence of PCR product) the fragment was excised from gel with scalpel and transferred in a test-tube. The DNA was then cleaned via extraction. The extraction was done with Gel/DNA Fragments Extraction Kit. An intermediate step before sequencing, we tried to clone the product it became obvious that this way is not very effective. The sequencing was provided by the company Seqme. Following work with sequences was conducted in programs BioEdit and alignment in ClustalX. Then, phylogenetic trees were computed in the program PAUP and viewed in TreeView. The results were interpreted in the terms of relationships between the amoebae majority of them belonged to the most common genotype, T4, but their relations within the genotype were not so tight. Concerning the clinical samples available to me (both from human eyes), one of them (O1) belonged to T4 genotype, whereas the other one (O2) belonged to the genotype T3. Another result was evaluation of the effectivity of different primers: specific primers AcaJDP1 and AcaJDP2 were much more efficient than originally used "eukaryotic" primers ERIB1 and ERIB10.
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