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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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Vliv klíštěcích slin na fagocytózu borelií dendritickými buňkami
MARŠÁLKOVÁ, Eliška
In this study we examined the effect of the tick saliva from I. ricinus and the effect of recombinant protein IRS-2 from the saliva of I. ricinus on dendritic cells derived from the mice bone marrow. We studied their effect on the production of cytokines by dendritic cells after the stimulation by B. burgdorferi, their effect on the expression of genes, that participate in phagocytosis, and the impact of the tick saliva on phagocytosis of B. burgdorferi by dendritic cells.
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Dog Lyme Disease
Grittnerová, Erika ; Kubík, Štěpán (advisor) ; Vynikalová, Lucie (referee)
Lyme borreliosis is a multi-organ disease caused by spirochetes of the Borrelia burgdorferi sensu lato group. These bacterias are transmitted by ticks of genus Ixodes, in the Czech Republic by Ixodes ricinus. The main reservoirs including the ticks are rodents, insectivores and deers. Lyme disease is the most diagnosed infection transmitted by ticks in the temperate zone of the northern hemisphere. It is supposed that due to climate change in recent years it will appear more often. In the Czech Republic it is reported 3500 to 4000 cases per year in recent years. The greatest risk of infection is in deciduous and mixed forests, dense and tall vegetation and at locations along watercourses. Borrelia gets into the host body with a saliva of the tick and spreads through blood or lymph to target organs such as the nervous system, joints, myocardium, skeletal muscles, eyes and skin. In dogs the lyme disease manifests usually asymptomatically or by non-specific symptoms that can simulate a variety of other diseases. The most common clinical signs are lameness, fever, joint and muscle pains, lethargy, loss of appetite and swollen lymph nodes. Diagnosis of the lyme borreliosis is relatively complicated and it is based on an assessment of clinical signs, epidemiological history and laboratory tests. For detection of borrelia in the organism it is used direct or indirect methods. The most commonly used direct method is a polymerase chain reaction (PCR) that examines the presence of borrelia DNA in a sample. Indirect methods examine the presence of specific antibodies in a blood serum. Combination of ELISA test and Western blot seems to be the most reliable of them. Treatment consist in antibiotic therapy for several weeks. The basis of prevention is early removal of tick and measures to prevent its bite. These measures consist in using various acaricidal preparations. Currently for dogs it is available vaccine that however is not included in the basic vaccination schema.
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Tick-borne encephalitis - life with the consequences
ŠTIBINGEROVÁ, Jana
This thesis charts the course of the tick-borne encephalitis since the beginning to the eventual consequences that the illness had left and that the patient must live with. Ixodes ricinus, tiny parasites carrying the disease called encephalitis, is no longer present only in the wild, but also in city parks or in our gardens. Every year, many cases of this disease occur across groups of all ages. It is an inflammatory disease of the central nervous system caused by viruses. Disease is usually divided into two phases, where the first stage is similar to spring-summer influenza with different length of quiescent phase, in the second stage exacerbation occurs. At this stage, the patient is struggling with high temperatures, headaches, severe pain in muscles and joints, vomiting and disorders of consciousness. Correct diagnosis is specified after sampling of cerebrospinal fluid through a lumbar puncture. Patients with confirmed tick-borne encephalitis are hospitalized in isolation wards, not because of the spread of the disease, but for the experience of doctors. Here they are isolated in the rooms, which can have a detrimental effect on their psyche. Treatment follows after discharge from the hospital, and often takes several months. The aim of this study was to map the progression of the disease from its beginning, through time spent in a hospital bed, followed by home recovery up to the present. A very important part in the return-to-health process has the nurse, and therefore this paper tries to describe her role in treatment of patients with tick-borne encephalitis. Given that there exists a specific prevention in form of vaccination, the next objective was to verify the vaccination on samples of the population in the Česke Budějovice and Gmünd, Austria, and to compare these two groups of respondents. The survey of the thesis was carried out from August 2015 to April 2016 using a combination of qualitative and quantitative methods. Qualitative data were collected through semi-standardized interviews with nine respondents, who were recruited through social networks with the help of gatekeepers. Quantitative data was collected via short survey distributed in Česke Budějovice and Gmünd, Austria. The results of the qualitative part were processed through coding and compiling of diagrams, from which the answers of individual respondents can be deducted. Evident is the whole course of the disease from the beginning up to the present. Data from the quantitative section were arranged into tables and then statistically evaluated. Based on the results, the frequency of vaccination of specific groups can be compared and therefore campaigns on health protection against tick-borne encephalitis can be targeted effectively.
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Serological diagnosis of borreliosis deseases
Sližová, Ivana ; Chmelař,, Dittmar (referee) ; Lochman,, Ivo (advisor)
The aim of present master’s thesis was to compare the results of serological methods for diagnosing borreliosis that are commonly used in Spadia laboratories (ELISA, immunoblots) in terms of recommendation on how and when to indicate and interpret them. The theoretical part is focusing on the characteristics and history of borreliosis, microbiological description of Borrelia, immune system and pathogenesis of the disease as well as the therapy and prevention. The experimental part is focusing on the analysis of results obtained from common examinations of antibodies to Borrelia made in Spadia Lab laboratories from January 1st 2014 to December 31st 2015. Screening of antibodies to Borrelia made by ELISA in IgM and IgG was done for all samples according to recommendation of CDC. In 2014 the ELISA screening was done using ELISA kits from Euroimmun and Evolis sample processors whereas in 2015 it was done using DiaSorin’s CLIA kits on Liaison analyser. Positive results were then confirmed by Westernblot or lineblot alternatively if the physician did not ask otherwise. It must be remembered that ELISA and Westernblot belong among serological methods that are using antibodies, i.e. substances produced by the immune system. The immune system plays the key role in protecting the body against infection and the antibodies are its important tool. Serological methods belong among immunoassay methods, which is still not standardized. Diagnosis of infections cann‘t be based only on antibody testing. It is necessary to assess the results in the context of the entire clinical picture, history and in the case of antibodies it is recommended retesting with an interval.
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Factors regulating the expression and activity of digestive enzymes in the tick \kur{Ixodes ricinus}
KONVIČKOVÁ, Jitka
The intracellular proteolysis of ingested meal plays an essential role in tick development. The thesis focuses on the factors influencing the expressions and activities of digestive enzymes in Ixodes ricinus females during the feeding and post-feeding period. We have revealed the effect of fertilization on blood feeding and digestion. The females cannot reach the rapid engorgement phase without being fertilized. The rate of mated females in the nature proved the presumption that mating can occur even off the host. Implementation of in vitro feeding technique further extended our current knowledge about tick digestive apparatus. Adult females were fed on hemoglobin-rich and hemoglobin-poor diet and the mRNA expression levels of digestive proteases were determined. In line with obtained data, we assumed that albuminolysis is conducted by the same or similar pathway as hemoglobinolysis. The gene silencing method and protein immuno-detection were used to unequivocally identify the isoforms of 'early expressed' IrCL1 and 'late expressed' IrCL3 isoform of cathepsin L.
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Effect of \kur{Ixodes ricinus} feeding duration on transmission of the spirochete \kur{Borrelia afzelii}
CHLASTÁKOVÁ, Adéla
Lyme borreliosis is a multisystem infectious disease caused by tick-borne spirochetes belonging to the \kur{Borrelia burgdorferi} sensu lato complex. It is generally believed that if a tick is attached for less than 24 hours, infection is unlikely because it requires between 36 and 48 hours of attachment for \kur{Borrelia burgdorferi} to travel from within the tick into its saliva. Nevertheless, there is evidence that transmission to a host may even occur in the early phases of tick feeding. We investigated the dynamics of \kur{Borrelia afzelii} transmission by nymphal \kur{Ixodes ricinus} ticks with special respect to the first day of tick attachment.
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Characterization and function of Factor C from the tick \kur{Ixodes ricinus}
HARTMANN, David
Factor C is a multi-domain serine protease which recognizes Gram-negative bacteria via binding to lipopolysaccharides and triggers hemolymph clotting cascade in the horseshoe crab. A closely related molecule was also found to be present in the genome of the tick Ixodes scapularis. In this work, the full sequence of Factor C ortholog from Ixodes ricinus (IrFC) was determined. IrFC is mainly expressed in tick hemocytes and the heavy chain of the activated molecules is present in tick hemolymph as confirmed by Western blotting with antibodies raised against recombinant fragments of IrFC. The function of the IrFC in tick innate immunity was assessed using its silencing by RNA interference.
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