The proposed thesis contributes to the knowledge about tick-borne encephalitis and its pathogenesis. The thesis describes pathogenesis and immunopathogenesis of tick-borne encephalitis, impact of host's genotype in clinical course determination, immune response of patients with acute tick-borne encephalitis, the mechanism of tick-borne encephalitis virus migration into central nervous system and virus interaction with cells of neurovascular unit as well as potential medical interventions.

Ticks acquire nutrients only by a parasitic nature of feeding on animals, including humans. During this process, a wide array of pathogens is transmitted. Ticks of the Ixodidae family receive exactly one blood meal in each active developmental. Knowing the trophic dependence of tick metabolism on the host blood meal components may enable discovering processes essential for the tick physiology and development. Exploiting a membrane system of tick feeding and whole blood fractionation, we have revealed that adult ticks need to acquire host haemoglobin-derived haem so that they can produce viable larvae, and reproduce. Haem is not further catabolised in ticks, and iron is thus acquired via independent route with the host serum transferrin as a source molecule. Using RNA-seq, we compared transcriptome compositions between guts of blood- and serum-fed ticks. We identified fifteen gut transcripts that change their levels with respect to the presence/absence of dietary red blood cells. Glutathione S-transferase, one of the identified encoded molecules, shows a clear haeminresponsive expression at both transcript and protein levels. Its apparent haem-binding properties suggest that this protein is directly involved in haem homeostasis maintenance within the tick gut. The ultimate goal of such research is to identify and verify targets that, when blocked, would render the acquisition and/or distribution system of haem in ticks nonfunctional. This would represent a novel way of anti-tick interventions in veterinary and human medicine.

Diseases can have a range of impacts on hosts and host populations. These impacts can be minimal, to the point of being considered nearly a commensal relationship. The other end of the spectrum is when a disease regulates a population or even drives it to extinction. Diseases that are directly transmitted in a density-dependent manner typically do not cause population extinction because as the population decreases so does transmission. However, there are several factors that can lead to extinction caused by diseases. These diseases can be frequency-dependent transmission (including vector-borne diseases) or diseases that infect multiple sympatric hosts. The parasite Cytauxzoon felis is a tick-borne apicomplexan that infects bobcats and domestic cats and is enzootic in the Midwest and Southeast US. Bobcats are considered the reservoir host of C. felis and typically do not show signs of disease associated with infection. This parasite is the etiological agent of cytauxzoonosis, a highly-fatal disease impacting domestic cats and occasionally other felid species. Domestic cats also can be subclinically infected. This parasite has increased its range and density within previously described enzootic areas. There are several aspects of the biology of C. felis that have not been explored and impact the epizootiology of this important veterinary parasite. The prevalence of the parasite has been studied in some locations, yet areas where the parasite has recently invaded need to be studied as populations of naïve bobcats and domestic cats may be at risk of epizootics. My research expands the knowledge of C. felis, adding information about a relatively recently-described enzootic area. The present dissertation is divided into 6 chapters. In the first chapter I provide a thorough literature review of C. felis and general information on pathogens. In my second chapter I describe the prevalence of C. felis in bobcats and ticks in southern Illinois from 2006-2017. This is the first documentation of C. felis in bobcats in Illinois. The prevalence in ticks is also the highest prevalence in ticks reported to date. These results have been published in the Journal of Parasitology. In the third chapter I provide evidence of chronic C. felis infections in bobcats. Some bobcats maintained C. felis infection for at least 2 years. I determined individual bobcats were infected with the same strain of the parasite at each capture event. This finding indicates that bobcats may carry the same strain over time. These infected bobcats could be spreading strains that are more pathogenic to domestic cats and possibly to other bobcats. Vector-borne pathogens (specifically microparasites) can show varying levels of intensity of infection in vertebrate hosts. The intensity of infection may correlate with activity of the vectors to facilitate transmission from the vertebrate to the vector. In the fourth chapter I tested if C. felis parasitemia (percent of red blood cells infected with the parasites) increased with environmental factors associated with tick activity. Cytauxzoon felis infections are increasing in domestic cats in the US in many areas. The fifth chapter describes the first study of clinical and subclinical C. felis infections in domestic cats in southern Illinois. I collaborated with veterinary clinics to obtain 642 domestic cat blood samples for this project. I also tested whether land cover types and host characteristics were related to risk of infection, and found only that feral cats were more likely to have subclinical C. felis infection. Cytauxzoon felis is transmitted through a tick vector; so direct contact between domestic cats and bobcats is not necessary for transmission to occur. For the sixth chapter I tested if the genetic populations of C. felis in domestic cats and bobcats were different (suggesting barriers to transmission). I found that there was high genetic diversity of C. felis in my samples. The within population variance accounted for nearly all variance detected. Therefore I conclude that the population of C. felis in bobcats and domestic cats in my study area is panmictic suggesting there are no barriers to transmission between these two host species.

ML (MD-2-related lipid-recognition) domain containing proteins are recognized as immune-related molecules. They do not belong among well-studied proteins in ticks although their occurence is quite often. Generally, ML proteins are involved in innate immunity processes, lipid binding and transport. Usually, expression of tick ML domain containing proteins is induced by blood feeding. Two members of the ML protein family, ML-domain containing protein and Der-p2 allergen-like protein were isolated from Ixodes ricinus and characterized for the first time.

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-22Bitstream added on 2014-06-13T20:27:43Z : No. of bitstreams: 1 demoner_lc_me_botfmvz.pdf: 668999 bytes, checksum: f0837d7e0a185cd0a8385b4105355512 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Hepatozoon canis é um hemoparasita transmitido por carrapatos que afeta cães em diversas regiões do mundo. Rhipicephalus sanguineus é considerado seu vetor biológico, entretanto, é possível que existam outros vetores. No Brasil, H. canis é descrito principalmente em áreas rurais, onde cães podem ser infestados por outras espécies, incluindo Amblyomma spp. O objetivo do presente trabalho foi infectar experimentalmente diferentes espécies de carrapatos com H. canis, a partir de cães naturalmente infectados. Para isso, foram utilizadas ninfas de A. cajennense e R. sanguineus e adultos de A. cajennense e A. ovale. Após o ingurgitamento dos espécimes em animal infectado, foi realizada pesquisa de formas evolutivas do protozoário na hemolinfa, intestino e hemocele dos ixodídeos, por técnicas citológicas e histológicas. Alguns carrapatos foram submetidos ao diagnóstico molecular pela técnica de PCR. Somente em duas fêmeas de A. cajennense foram observadas estruturas morfologicamente semelhantes a Hepatozoon spp. e uma fêmea de A. ovale apresentou oocistos na hemocele. Além disso, duas fêmeas de A. ovale e uma de A. cajennense foram positivas na PCR. Entretanto, não é possível caracterizar essas espécies como vetores de H. canis, pois foram usados muitos exemplares nas infecções experimentais e poucos foram capazes de se infectar com o parasita. É provável que R. sanguineus tenha pouca importância epidemiológica na transmissão de H. canis no Brasil, pois este estudo, assim como pesquisas anteriores, demonstraram que a espécie foi incapaz de se infectar experimentalmente com o protozoário / Hepatozoon canis is a hemoparasite that afflicts dogs in several regions of the world. Rhipicephalus sanguineus is considered to be the biologic vector of this agent, although other species may act as possible vectors. In Brazil, H. canis is described mainly in rural areas, where dogs are infested by other species, such as Amblyomma spp. The aim of this study was to transmit experimentally H. canis to different species of ticks, using naturally infected dogs. For this purpose A. cajennense and R. sanguineus nymphs and A. cajennense and A. ovale adults were used. After the repletion of specimens fed on a naturally infected animal, we searched for development forms of the protozoa in hemolymph, gut and hemocoel of the ticks by cytological and histopathologic examinations. Some ticks were also examined by molecular detection (PCR). Only two female ticks of the species A. cajennense contained structures morphologically similar to Hepatozoon spp. and oocysts were recovered from the hemocoel of an A. ovale female. In addition, two females of A. ovale and one female of A. ovale were positive through PCR. However, our results are not enough to characterize these species as vectors of H. canis mainly because many ticks were used in experimental infections and few were infected with the parasite. It is likely that R. sanguineus has little epidemiological importance in the transmission of H. canis in Brazil, since this research and previous researchers demonstrated that R. sanguineus was unable to experimentally get infected with the protozoan

Orientador: Lucia Helena O'Dwyer / Banca: Elizabeth Moreira dos Santos Schmidt / Banca: Gervásio Henrique Bechara / Resumo: Hepatozoon canis é um hemoparasita transmitido por carrapatos que afeta cães em diversas regiões do mundo. Rhipicephalus sanguineus é considerado seu vetor biológico, entretanto, é possível que existam outros vetores. No Brasil, H. canis é descrito principalmente em áreas rurais, onde cães podem ser infestados por outras espécies, incluindo Amblyomma spp. O objetivo do presente trabalho foi infectar experimentalmente diferentes espécies de carrapatos com H. canis, a partir de cães naturalmente infectados. Para isso, foram utilizadas ninfas de A. cajennense e R. sanguineus e adultos de A. cajennense e A. ovale. Após o ingurgitamento dos espécimes em animal infectado, foi realizada pesquisa de formas evolutivas do protozoário na hemolinfa, intestino e hemocele dos ixodídeos, por técnicas citológicas e histológicas. Alguns carrapatos foram submetidos ao diagnóstico molecular pela técnica de PCR. Somente em duas fêmeas de A. cajennense foram observadas estruturas morfologicamente semelhantes a Hepatozoon spp. e uma fêmea de A. ovale apresentou oocistos na hemocele. Além disso, duas fêmeas de A. ovale e uma de A. cajennense foram positivas na PCR. Entretanto, não é possível caracterizar essas espécies como vetores de H. canis, pois foram usados muitos exemplares nas infecções experimentais e poucos foram capazes de se infectar com o parasita. É provável que R. sanguineus tenha pouca importância epidemiológica na transmissão de H. canis no Brasil, pois este estudo, assim como pesquisas anteriores, demonstraram que a espécie foi incapaz de se infectar experimentalmente com o protozoário / Abstract: Hepatozoon canis is a hemoparasite that afflicts dogs in several regions of the world. Rhipicephalus sanguineus is considered to be the biologic vector of this agent, although other species may act as possible vectors. In Brazil, H. canis is described mainly in rural areas, where dogs are infested by other species, such as Amblyomma spp. The aim of this study was to transmit experimentally H. canis to different species of ticks, using naturally infected dogs. For this purpose A. cajennense and R. sanguineus nymphs and A. cajennense and A. ovale adults were used. After the repletion of specimens fed on a naturally infected animal, we searched for development forms of the protozoa in hemolymph, gut and hemocoel of the ticks by cytological and histopathologic examinations. Some ticks were also examined by molecular detection (PCR). Only two female ticks of the species A. cajennense contained structures morphologically similar to Hepatozoon spp. and oocysts were recovered from the hemocoel of an A. ovale female. In addition, two females of A. ovale and one female of A. ovale were positive through PCR. However, our results are not enough to characterize these species as vectors of H. canis mainly because many ticks were used in experimental infections and few were infected with the parasite. It is likely that R. sanguineus has little epidemiological importance in the transmission of H. canis in Brazil, since this research and previous researchers demonstrated that R. sanguineus was unable to experimentally get infected with the protozoan / Mestre

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.

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.

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.

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.