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The development of Leishmania in the insect vector of the genus Phlebotomus
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Bilej, Martin (referee)
The work was focused on the study of various aspects of development of Leishmania in vectors of genus Phlebotomus and can be divided into two main parts. In the first part, we studied the competition of Leishmania major and L. turanica during their development in the sand flies who are natural vectors of L. major using the experimental co-infections of fluorescently marked promastigots of these two species. While both leishmania species developed similar in the intestine of Phlebotomus papatasi, L. turanica prevailed in P. duboscqi in the late stages of infection. The fluorescent marking of Leishmania should allow us also to study possible genetic exchange between species at different stages of Leishmania infection (2nd, 9th and 14th day after the infective feeding). Using the flow cytometry (FACS) we have repeatedly identified dozens of objects emitting red and green signals in the intestinal homogenates of co-infected sand flies, however further analysis with confocal microscope disproved these objects as the hybrid promastigots of Leishmania. In the second part of this thesis we investigated the role of L. major genes HASP and SHERP, which lies on the locus LmcDNA16 and are expressed exclusively in metacyclics. We used mutant lines KO (lacking the locus LmcDNA16) and HASPB (KO line with gene...
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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš ; Horák, Petr (advisor) ; Bilej, Martin (referee) ; Schabussova, Irma (referee)
Helminth neuroinfections represent a serious health issue, but the mechanisms of the host immune response often remain neglected despite the fact they might contribute to pathogenesis. This is partly due to the unavailability of clinical samples and the lack of suitable laboratory models. Herein, I focused on the characterization of several aspects of the immune response of mice infected with the neuropathogenic avian schistosome Trichobilharzia regenti. After the percutaneous infection of mice (accidental hosts), most T. regenti schistosomula are entrapped and eliminated in the skin, but the parasite antigens initiating the protective immune reaction are not known. Our in vitro experiments revealed that T. regenti cathepsin B2, a cysteine peptidase used for the skin penetration, activates bone marrow-derived dendritic cells much stronger than the parasite homogenate, suggesting its role in initiating the mixed type1/2 host immune response. However, some schistosomula manage to escape from the skin and continue their migration to the spinal cord. Here they crawl preferentially within the white matter which we demonstrated by the robust 3D imaging techniques, ultramicroscopy and micro-CT. The invasion of the spinal cord is accompanied by striking hypertrophy of astrocytes and microglia. We showed...
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Bacteria genus Asaia and Wolbachia in sandflies
Sovová, Kristina ; Volf, Petr (advisor) ; Bilej, Martin (referee)
Phlebotomine sand flies are proven vectors of many pathogens including parasites of genus Leishmania. Leishmania develop in sand fly midgut which is colonized also by many others microorganisms, creating rich community known as gut microbiota. The presence and composition of gut microbiota affect sand fly mortality, but also development of transmitted pathogens. In contrast to mosquitoes, sand fly gut microbiota is not well studied. This thesis focuses on bacteria of the genera Asaia and Wolbachia and their potential impact on Leishmania in sand fly midgut. Thesis reports the first finding of Asaia sp. and Wolbachia sp. in sand flies from Balkan penninsula - hotspot for visceral leishmaniasis and phleboviruses. In 273 individuals from subgenera Larroussius were Asaia sp. and Wolbachia sp. detected with infection prevalence 2,5 % and 8,4 %, respectively. In addition, laboratory-reared sand flies were tested for presence of these bacteria: from twelve studied colonies, only Phlebotomus perniciosus was infected by Wolbachia sp. Then, we focussed on elimination of Wolbachia sp. from this laboratory colony with the aim to use Wolbachia-negative sand flies in future experiments with Leishmania. The final part of the thesis was dedicated to bacteria of the genus Asaia (specifically A. krungthepensis),...
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Critical factors affecting pathogen development in sand flies
Jančářová, Magdaléna ; Volf, Petr (advisor) ; Ratinier, Maxime (referee) ; Bilej, Martin (referee)
This thesis deals with barriers and factors critical for development of viruses, leishmania and gregarines in sand flies. First, we focused on life cycle of sand fly-borne phleboviruses, especially possible routes of sand fly infection. As a laboratory model we chose Massilia virus (MASV), species closely related to Toscana virus, which is main causative agent of summer meningitis in Mediterranean area. We tested different ways of infection by MASV in various developmental stages of Phlebotomus perniciosus; infection of (i) first (L1) and fourth (L4) instar larvae through larval food, (ii) females by blood meal, (iii) both sexes by sugar meal. Infection of L1 and L4 by larval food and subsequent transstadial MASV transmission to adults were not efficient; from 875 adults only three were MASV-positive. Infection through bloodmeal led to high infection rate before defecation, nevertheless, post defecation the infection rate declined and only 5 out of 27 females were MASV-positive. The most efficient infection way was through the sugar meal: 72% of females (88 out of 122) and 51% of males (58 out of 113) were detected as MASV-positive. Moreover, both males and females infected by this way released MASV particles into the drop of sugar which stayed infectious for next 24 hours for other naïve sand...
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Recombinant proteins and synthetic peptides as tools for studies on antibody response against Phlebotomus orientalis
Ferencová, Blanka ; Volf, Petr (advisor) ; Bilej, Martin (referee)
Sand fly saliva contains proteins and peptides that have an important role in bloodfeeding. Some of those proteins are antigenic and repeated sand fly bites result in a specific antibody response of the bitten host. Antigenic salivary proteins of Phlebotomus orientalis, main vector of visceral leishmaniasis in Sudan and Ethiopia, were identified using immunoblot with dog sera. The 5 most promising antigens were expressed in an E. coli bacterial system. Subsequently, these proteins were tested in ELISA with sera of domestic animals from Ethiopia naturally exposed to P. orientalis, and with sera of mice bitten experimentally by this sand fly species. Salivary gland homogenate (SGH) was used as the positive control. The best antigenic properties were detected in two recombinant proteins, Yellow-related protein PorSP24 and ParSP25-like protein PorSP65, especially in tests with sheep and dog sera. However, nonspecific binding of dog sera was also detected using both antigens. In addition, we proved that sera of mice repeatedly bitten by P. papatasi and Sergentomyia schwetzi do not crossreact with SGH and the tested recombinant proteins of P. orientalis. In a second part of this thesis we designed peptides representing epitopes recognized by specific anti-saliva antibodies. Two peptides were derived from...
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Effects of microbiota on defense system of earthworms
Dvořák, Jiří ; Bilej, Martin (advisor) ; Kopečný, Jan (referee) ; Horák, Petr (referee)
Earthworms are important soil invertebrate organisms that participate in nutrient cycling in terrestrial ecosystems and in the formation of the soil profile from the physical, chemical and also microbial point of view. Soils are considered the most microbially diverse environments on earth. All invertebrates living in soil therefore need to possess a complex immune system. Earthworms are used as a model organism in immunology for decades. Their simple body plan consists of two main body cavities: true coelom and digestive tube. Both coelomic cavity and digestive tract represent open systems with permanent contact with soil microorganisms. Eisenia andrei species is used as a standard immunological model in our laboratory for many years. E andrei earthworms live in compost, microbially abundant environment, which is reflected in their well-developed immune system. Some new mechanisms of E. andrei defense system are described in this work. Two novel pattern recognition receptors (PRRs), Toll-like receptor (TLR) and lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI) were characterized in earthworms. These molecules are expressed in coelomocytes and their production is upregulated after microbial challenge. Moreover, both receptors were detected in digestive tract...
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