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Detection of Lyme disease spirochetes in clinical samples by PCR-based methods and optimalization of conditions borrelia cultivation conditions from samples of patients with LB symptoms
HAVRAN, Jiří
The samples under investigation were collected in Department of Pediatric Infectious Diseases of the University Hospital (Brno). Group of patients (100) was heterogeneous in terms of symptoms, age and sex. The samples were taken from patients with an LB diagnosis and from those with nonspecific symptoms. Molecular typing of LB spirochetes in clinical samples (104 blood/serum, 89 cerebro-spinal fluid and 1 synovial fluid) became necessary when the general immunological tests gave unclear results. The samples were analyzed using PCR-based and molecular biology techniques that include: nested- and spacer-PCR, specie-specific PCR, sequence, virtual hybridization, in silico RFLP analysis, similarity search. Results of conducted analysis confirmed that 51% of samples (98) were positive on B. bugrdorferi sensu lato. Using above mentioned techniques 6 spirochete species from B. burgdorferi sensu lato complex were identified; two of them weren?t detected in samples of human origin in Europe yet. Comparative analysis of two media for Borrelia cultivation from samples of human origin definitely proved the adventage of using MKP instead of traditionally used BSK-H Complete.
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Characterization of two members from the multigenic family of one-domain Kunitz-inhibitors from the tick \kur{Ixodes ricinus}
SINGEROVÁ, Barbora
Two new genes encoding proteins Monolaris 1 and Monolaris 2 were isolated from tick Ixodes ricinus. Both cDNA fragments code for 94 aminoacid residues long protein with molecular mass 8,1kDa (Monolaris 1) and 8,3kDa (Monolaris 2). The function of Monolaris 1 was tested by using RNA interference in adult females of Ixodes ricinus that were subsequently fed on guinea-pigs. Body mass, egg mass and mortality were measured to evaluate the effect of gene silencing. Recombinant protein Monolaris 1 was prepared in bacterial expression system and antibodies against this protein were raised by immunization of a rabbit. Antibodies reacted with approximately 190kDa big protein in salivary gland, ovary and gut whereas monomer of Monolaris 1 was not detected in tick saliva, salivary glands and other tissues.
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Possibility interference interest in vaccination against curse encephalitic in focus infection
VOBORSKÁ, Eliška
This diploma thesis addresses the topic of informing people of tick-borne encephalitis and vaccination against this disease. Our diploma thesis has four objectives in total. The first objective was to ascertain how many of the respondents were vaccinated against tick-borne encephalitis. The second and the third objectives were to demonstrate the reasons of vaccinated and unvaccinated respondents. The fourth objective was to analyze the attitudes of the respondents to publicization of the tick-borne encephalitis issue. As an infection with the natural focus, the disease is caused by a tick-borne encephalitis virus belonging among flaviviruses. The carrier is a sheep tick (Ixodes ricinus). Ticks live as parasites on reservoir animals, such as small rodents, ungulates and domestic animals. The clinical picture of the disease has mostly two stages and the symptomatologic form affects in particular adults and seniors. South Bohemia is a significant focus of tick-borne encephalitis with the sickness rate considerably exceeding the average of the Czech Republic. The most efficient prevention of the disease is vaccination. Two vaccines have been registered in the Czech Republic at present, namely FSME-Immun of Baxter and Encepur of Novartis. Both the vaccines are intended for children from 1 year of age. The basic vaccination scheme consists of 3 doses and the vaccinated person needs to be revaccinated every 3-5 years. It follows from the questionnaire research that 49 % of the total number of 136 respondents are vaccinated. As expected, the most frequent reason for vaccination (65 %) is the fear of becoming infected with tick-borne encephalitis. Other reasons are the interventions of the surrounding persons persuading the person to become vaccinated (19 %) and the financial contribution of the health insurance company or the employer (16 %). 35 % of the unvaccinated respondents do not plan to become vaccinated in the future. Approximately 40 % of the unvaccinated respondents feel that they do not need the vaccination or do not trust vaccination as such. 42 % of the unvaccinated persons would agree to vaccination if they received a financial contribution of the health insurance company or the employer, but nothing at all would convince 35 % of the unvaccinated respondents. It follows from the research that most respondents are satisfied with the quantity and the content of the information about tick-borne encephalitis. They do not think that anything should be changed in the system of informing of the disease. This thesis contributes to monitoring the trends in thinking of the public, and will eventuate in the finding which way the education of people about tick-borne encephalitis should be directed.
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Expression of Kunitz-type recombinant protein, potential toxin from the salivary glands of the tick \kur{Ixodes ricinus}
CERMANOVÁ, Tereza
Ticks feed on their vertebrate hosts for number of days, providing enough time for development of an effective immune response. To overcome this, tick saliva contains a complex mixture of active substances which mediate host defense mechanisms. Extremely important role at the tick-host interface is played by the protease inhibitors. In this work, we have focused on the protein named Ixocludin 2, a member of the Kunitz type/Bovine trypsin inhibitors of serine proteases, related also to the potassium channel blockers toxins. Three different expression systems (Escherichia coli, Chinese hamster ovary and Pichia pastoris) were tested to prepare an active recombinant Ixocludin 2, out of which, only bacterial system was in part successful.
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Characterization of 18,7 and 19 kDa groups of secreted proteins in the salivary glands of the castor bean tick \kur{Ixodes ricinus}
SOUČKOVÁ, Nina
The recombinant protein c90 was prepared and polyclonal antibodies against this protein were raised. The dsRNA was made for the experiments with RNA interference. The samples from dissected tissues of dsRNA silenced ticks were tested by RT-PCR and Western blot. Results suggest that protein c90 plays a role in the tick body during the reaction to injury. Finally, another experiment with injection of water, G+ and G- bacteria into the ticks was realized. It was found that the members of the 18,7 kDa protein family can create multimers. The overexpression of silenced genes was observed during RNAi experiments despite of expected inhibition of c90 production. These results together with the bioinformatics analysis could mean that these proteins are important the physiology of tick probably as a reaction to injury. However c90 protein is produced only in the first phase of feeding which could mean that it has some role in the tick-host interaction as well.
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Identification and biochemical characterization of lectins in the hemolymph of three species of tick in the genus \kur{Rhipicephalus}
FIŠER, Miroslav
Lectins are tissue specific carbohydrate binding proteins with possible functions in invertebrate immunity and pathogen transmission. The main goal of this study was to identify hemolymph lectins in three different tick species. Three proteins with molecular weights of 58 kDa, 75 kDa and 180 kDa were detected in all investigated species using antibodies directed against hemagglutination activity of Rhipicephalus appendiculatus hemolymph. These proteins were characterized by biochemical methods such as Schiff/periodate staining, lectin blotting, enzymatic deglycosylation, hemagglutination analysis, immunoblotting, and mass spectrometry.
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Vaccination Rate of Adults against Tick-borne Meningoencephalitis in České Budějovice
VOBORSKÁ, Eliška
The aim of this thesis was to map vaccination rate against tick-borne encephalitis in adults in the town of České Budějovice, the town in the focus of infection. Tick-borne encephalitis is a very malign and dangerous neurodegenerative disease. At the beginning there appear only inconspicuous flu-like symptoms. It is transmitted either by infected ticks or by eating products made from non-pasteurized milk of infected animals (goats, sheep). People living in areas with higher occurrence of ticks infected by one of the diseases (tick-borne encephalitis, borreliosis, ehrlichiois) should pay attention to prevention. In case of tick-borne encephalitis vaccination is possible. It is recommended during winter months, when ticks are not active. This precaution is done in cold weather so that in spring when ticks are active, the body has sufficient amount of antibodies against tick-borne encephalitis. During warm months it is possible to apply accelerated scheme of vaccination so that the body produces antibodies quicker, however, their survival is rather shorter. My research was carried out by means of questionnaires which were filled in by the group of randomly chosen inhabitants of České Budějovice. The form of processing was the method of quantitative research. The questionnaires were processed by the form of descriptive study. Total number of returned questionnaires was 541 (77%). The generation until 29 (68%) had the highest distribution; this age category has the highest vaccination rate (56%). The total proportion of vaccinated against non-vaccinated is 49% to 51 %. On the contrary, the lowest vaccination rate is in generations of 70 years old and older people, where 1 of 18 is vaccinated (5.5 %). In women, who created 76 % of examined sample, the vaccination rate makes up 53 % and in men, who took 24 %, there were 35 % vaccinated. The next aim of my thesis was to monitor attitudes of inhabitants to vaccination. We found out that in non-vaccinated inhabitants the main reason was opinion that vaccination is not necessary (43 %), the next was finance (20%) and further some other reasons, for example laziness. In vaccinated inhabitants the most common reason was the fear of infection and further persuasion by another person (24 %). The third place is occupied by the stay in risky environment (18 %).
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Awarness of Public of Tickborne Encefalitis Infection
VOKROUHLÍKOVÁ, Monika
In my bachelor work I monitor the awareness of the general public on the tick-borne encephalitis and the attitude of the general public to vaccination against this disease. I have found out using research that the population is aware of the tick-borne encephalitis but the population is not interested in vaccination against the disease.
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