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Genotyping of \kur{Giardia intestinalis} isolates
ŠRÁMOVÁ, Eliška
The aim of this work was assemble isolates of Giardia intestinalis from humans and other mammals. Stools samples were examined for presence of cysts by concentration settling method. Consequently sequencing of 532 bp parts of the TPI gene after previous amplification by the nested PCR was performed. In vitro cultures of selected isolates were established using experimental model hosts, gerbils.
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Blastocystis in domestic birds
HRDLIČKOVÁ, Jaroslava
During 2010, faeces samples for parasitology research oriented on Blastocystis were collected from an anonymous farm. A total of 55 samples were collected (of them for 10 faeces samples from hens, ducks and geese and 25 from pigeons). The samples were cultured in Dobell-Leidlaw medium and viewed by light microscopy. However, this method led to only two foundings of Blastocystis (one from hen, the other from pigeon). Thus, PCR and nested-PCR with specific primers were later used for better detection of Blastocystis. The samples for PCR detection were not collected from the aforementioned farm, but they originated from a collection of isolated DNA samples that was available on the Parasitology institute of AS CR. The results of PCR were checked after electrophoresis and verified by sequenation. The obtained sequences of bird-isolated Blastocystis were phylogenetically analysed and as described to subtype 7.
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Isolation of the antimicrobial peptide gene (defensin) from the hard tick \kur{Ixodes ricinus}, challenged by the pathogen infection
SKLADANÁ, Veronika
Antimicrobial peptides are major components of the innate immune response of epithelial cells. In hematophagous organisms, which acts as vectors of parasitic diseases, in particular Lyme borreliosis, the gut pathogen induce the expression of defensin, that provide the first barier of host defence. This raises the posibility that defensin may play a key role in the development of parasitic infection. The gene expressed in midgut was isolated from cDNA of the hard tick, Ixodes ricinus. The gene is coding the protein, that is produced and secreted during tick infection, and is known as defensin. Expression of the gene for defensin (224 bp) was induced by the pathogen. The gene was cloned into bacterial expression system.
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The Study of the genetic variability of phytoplasmas
ROHÁČKOVÁ, Helena
Phytoplasmas are bacterial intracellular plant pathogens that cause devasting yield losses in diverse crops worldwide. Phytoplasmas were detected in clover and Catharanthus roseus plants, pear, apple and apricot trees. SecA and 16S rRNA genes, spacer region and 23S rRNA gene of five phytoplasma isolates were sequenced.
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Stady of apple proliferation phytoplasma diversity in the Czech Republic.
STREJČKOVÁ, Monika
Phytoplasmas are prokaryote organisms of the Mollicutes class. So far they have been described on more than 800 plant species. Apple proliferation phytoplasma, which classifies in the ribosomal subgroup 16Sr X-A (Apple proliferation, AP, ?Candidatus Phytoplasma mali?), belongs amongst the agriculturally most important phytoplasmas. AP is considered a quarantine organism in Europe and in North America. Common symptoms caused by AP are as follows: notably enlarged stipules, which are morphologically like real leaves ? they have petiole and lamina; the edge of the stipules is irregularly dentate small fruits with fewer seeds and reduced flavour quality premature reddening of leaves, witches' broom appearance, abnormal growth of shoots in the autumn, repeated blossoming DNA isolated from trees suspected to have AP present was tested in the presented thesis. The trees originated from the gene pool, mass production planting as well as from planting of small producers. To detect phytoplasma, the polymerase chain reaction (PCR) was used with the subsequent analysis of restriction fragment length polymorphism (RFLP). To detect and differentiate AP phytoplasma subtypes, direct PCR with primers rpAP15f/rpAP15r amplifying the DNA of ribosomal proteins rpS3 and rpl 22 with subsequent restriction breakdown by AluI enzyme.
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Detection and identification of phytoplasmas infecting pear trees
ROHÁČKOVÁ, Helena
Phytoplasmas are bacterial intracellular plant pathogens that can cause devasting yield losses in diverse crops worldwide. Phytoplasmas were detected using molecular methods in pear trees. RFLP analysis revealed the presence of pear decline, apple proliferation, clover phyllody and stolbur phytoplasmas in examined samples singly or in mixed infection.
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Possibility of utilization of molecular methods for study of population genetics of noble crayfish Astacus astacus
ŠABATA, Jakub
The noble crayfish (Astacus astacus) is one of two native species of crayfish living in this country who are all over our country strictly protecte, because they are identified as critically endangered species. In Europe it is one of five native species of crayfish, reported by IUCN as an endangered species that needs protection management. Its population was dramatically reduced due to crayfish plagues, which carry non-native crayfish species from North America, who were introduced in the past in Europe and later to the Czech Republic. In the past, have been isolated and described microsatellite markers for crayfish (Koiv et al., 2008a). As part of this work was tested using the eight microsatellite markers on samples obtained from the czech population of crayfish. Testing was performed on 53 samples from six populations of crayfish. Test samples were subjected to isolation of DNA from tissues of the third walking legs using DNA Lego Kit. Then test isolate DNA electrophoresis on agarose gel. Testing the temperature cycle of PCR amplification based on the original publication Koiv et al., (2008a), followed by PCR cycles were adjusted according to the quality of PCR products obtained in our laboratory. Primer annealing temperature 60°C was chosen as the best for six tested loci i.e. Aas 3666, Aas 3115, Aas 790, Aas 1198, Aas 3950 and Aas 766, for two other loci Aas 2489 and Aas 3040 was chosen 55°C annealing temperature. The resulting PCR products were tested on agarose gel and subsequently fragment analysis on an automatic sequencer Beckman Coulter CEQ8000 determining the lenght of PCR products in multiplex consisting of several loci. In the individual loci were in our 53 samples found from 1 to 13 alleles ? one of the loci was monomorphic in all samples analyzed. The moravian population in the Boskovice tank showed the greatest variability, the average number of alleles per locus 3.86, then the north bohemian population from Jaroměř 3.43 alleles per locus. Zelenohorská population of 3 alleles at a locus and Světlohorská population of 2.57 alleles per locus. The lowest average number of alleles per locus had a population from Landštejn 2.43. The aim of this bachelor thesis was to develop a literature search of methods use in molecular biology studies of crayfish and also validate the use of eight microsatellite markers have described for the noble crayfish. In laboratory conditions were succesfully optimized using seven microsatellite markers from the eight described. These microsatellite markers should be used for population studies, or for determining parentage (paternity) in breeding experiments. The verification of the applicability of microsatellite markers have been evaluate and some fundamental characteristics of the population, usability testing and optimization of microsatellite markers, however, was done only on a very small number of samples, because these characteristics have only a very limited explanatory power.
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Germination ecology in orchids
TĚŠITELOVÁ, Tamara
Germination ecology of four Epipactis species (E. albensis, E. atrorubens, E. helleborine, E. purpurata) was studied. Habitat preferences of adult plants were analyzed using phytosociological relevés from the Czech Phytosociological Database. A field experiment was carried out to determine course of germination of Epipactis seeds sown in different habitat types. Relationship between ecological preferences and germination ecology, and spatial aspects of seed dispersal and seedling recruitment are discussed.
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