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Molecular characterization of selected amoeba strains with emphasis to potential pathogens
KAŇKOVÁ, Hana
Goals of my work are: preparation of literature review, which will focus on free living amoebas as pathogens of humans and animals, molecular characterization of selected collection strains of amoebas: obtaining sequences of genes used as markers for these organisms (SSU rDNA, ITS). Data got by sequencing will be phylogenetically analyzed and results of analyzes interpreted. In the theoretical part I described the basic morphology of potentially pathogenic amoebas (acantamoebas, naegleriae), their pathogenicity and briefly also treatment of these diseases (primary amebic meningoencephalitis, granulomatous amebic encephalitis, acanthamoeba keratitis). Acanthamoebas and naeglerias (Naegleria fowleri and various species of acanthamoebas) are capable of entering human organism. Especially important is their ability to penetrate into brain, where they can be cause of amebic meningoencephalitis (disease of CNS). Primary amebic meningoencephalitis (PAM) is caused by N. fowleri. The diaseas occurs in children and young adults, who bathed in a permanently warmed water, where this species is found. Amoeba enter the body through the nasal cavity. PAM has rapid course human die within 7 days from first symptoms. Granulomatous amebic encephalitis (GAE) is caused by Acanthamoeba spp. The disease has a chronic character (its duration is up to several months). It is found in immunocompromised people (diabetes, HIV/AIDS, drug addiction ect.) Acanthamoebas enter through an injury and go to CNS by hematogenous route. Another disease caused by genus Acanthamoeba is acanthamoeba keratitis (AK). AK is inflammation of cornea. Acanthamoeba come in to the eye through contaminated contact lenses or when cornea is damaged. Treatment these diseases is very complicated and success rate is not high. Differences between acanthamoebas and naeglerias concern several areas life cycle, size and pathogenic potential. Naeglerias are able to create 3 stages (trophozoite, flagellate, cyst), acanthamoebas alternate between 2 stages (trophozoit, cyst). Trophozoite of acanthamoebas (12-35 ?m) is somewhat bigger in comparsion with naegleria (15-25 ?m). Major differences are visible on cysts which are irregular in shape in acanthamoebas and spherical and smooth in naeglerias. In the practical part of my work I try to answer several questions: determination of genotypes or species of amoebas that were isolated and to ascertain more information about their pathogenity, geographic range etc. In the methodical part I described procedures of cultivation of amoebas, isolation of their DNA, course of PCR, electrophoresis and software processing of sequences. In cultivation I used classic non-nutrient agar and amoebas were harvested after their visual checking.The PCR protocol was standard: I used specific primers and termocycler. PCR products were separated by electrophoresis. After cutting proper bands from the gel, I sent the sample to be sequenced by a specialized company (Macrogen). Final sequences were checked and sequences with high quality signal were processed by programs SeqMan, ClustalX, BioEdit. For phylogenetic analysis I used programs Paup, RAxML and MrBayes. I got 9 sequences of genes for SSU rRNA of acanthamoebas and 1 naegleria´s ITS sequence. Using phylogenetic analysis I got phylogenetic trees which I inserted to chapter with results. In the discussion chapter I outlined problems during gaining of naegleria´s sequences. The next part of discussion is devoted to detailed analysis of phylogenetic trees. Some of my sequences show close association with sequences derived from acanthamoebas which were isolated from patients with AK from various regions of the world, which indicate global distribution of some lineages of amoebas. Naegleria groups are more restricted by environment, but there are exceptions.
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Molecular characterization of selected strains of amoebae of the genus Naegleria, the potential human parasites.
ZÍTKOVÁ, Klára
The genus Naegleria is a eukaryotic organism, one of those we can call "amoeba" a general name of creatures changing their shape during their locomotion. Amoebae comprise a huge number of various different genera some of which contain species able to infect humans similar to Naegleria fowleri. Two important exaples are the genera Acanthamoeba and Entamoeba, causative agents of acanthamoeba keratitis or diarrhoea, respectively, and other serious symptoms. Although superficially similar, they are not close relatives of Naegleria: they belong to Amoebozoa, whereas Naegleria is a Heterolobosea. The most important species of this speciose genus is the amoeba Naegleria fowleri. Is is the only Naegleria which is a human pathogen. After infection, it causes primary amoebic meningoecephalitis. The invasive stage of Naegleria is an amoebic trophozoite. Under certain circumstances, it can transform in another form, the flagellate bearing undullipodia. When the conditions worsen, it changes in the resistant cyst stage. Major part of my work is related to Naegleria fowleri and primary amebic meningoencephalitis, which causes human (PAM). They clarify here the source of infection and risk factors for assault. The length of incubation period and the first appearance of symptoms. The next chapter concerns the mechanism of intrusion into the body, how can this infection properly diagnose and detect and also a treatment option, resulting in early detection of symptoms. Possibilities prevention and epidemiology, including presentation of biggest epidemic, that happened in the Czech Republic. In the practical part of this thesis summarize my own study and methods used, as well as the results and their interpretation. The aim of the experimental part was the analysis of Naegleria DNA maintained in the collection of the laboratory of fish pathogens. To reach my goal and test the hypotheses of this thesis, I identified the Naegleria isolates. The final phylogenetic analysis was enriched with samples sequenced earlier, but so far not analyzed in any previous work. The first step in the workflow was the DNA isolation. The next one was to PCR amplify and sequence the ITS sequences to confirm they belong to Naegeria species, and to analyze the sequences phylogeneticaly and identify the species. The obtained sequences were edited in BioEdit software, program ClustalX was used to align them. The phylogenetic trees were computed in PAUP. Sequencing was provided by Seqme company. During the interpretation of the phylogenetic trees, I found which species were present in studied samples. They were as follows: two samples were N. americana, twelve others N. canariensis, three were N. dobsoni and there were also N. pagei and N. tihangensis one sample each. Several samples (GERK, MSED4, ALM1A, 62K4 and GG1BV) were different from any known species and it is therefore possible to consider them new, undescribed species. On the basis of this fact we can confirm the hypothesis nr. two: the species diversity is understudied and the recognition of new species probable. The data seem to support hypothesis 3: in a given locality, a single dominant species becomes dominant. In some localities, however, we could not fully exclude the opposing hypothesis 4.
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