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Macrophages and nitric oxide in leishmania - sandfly - host interactions
Kratochvílová, Tereza ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
Leishmania reside fagolysosome of macrophages immediately after their entry to host where they multiply and consequently infect other macrophages or eventually other cells. A synthesis of a reactive reactant of oxygen and nitrogen is one of the mechanisms that some mammal cells are equipped with and that also contributes to eradication of leishmania. Nitric oxide rising during a metabolic change of L-arginine under the catalysis of NO synthase is of a large importance. Beyond cytotoxic function, nitric oxide is involved in signalling pathways for a neurotransmission (nNOS) and vasorelaxation (eNOS). Not all types of macrophages have ability to produce NO (iNOS). It is a heterogeneous group differing in immunological function and also in physiology. A group of classical activated macrophages represents an effective APC capable of efficient killing of intracellular pathogens. In addition to NO, they also secrete an inflammatory cytokines, which evolve an immune reaction towards to Th1. Contrary to this, a group of alternative activated macrophages is not capable of any efficient antigen presentation and nitric oxide production but produces L-ornithine, which is a precursor of polyamines, which leishmania utilizes for its own intracellular growth. For the mouse model, status of resistance and/or...
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Genotype influence on development of infections caused by Trypanosomatidae in mouse
Šíma, Matyáš ; Lipoldová, Marie (advisor) ; Krulová, Magdaléna (referee) ; Kolářová, Iva (referee)
Parasitic protists of genera Trypanosoma and Leishmania are members of Trypanosomatidae family. In our studies, we investigated genetic influence on infections caused by these parasites in a mouse model. These diseases are on genetic level controlled by quantitative trait loci (QTLs), when the resulting phenotype is controlled by set of genes with small individual effect. As a mouse model for mapping of QTLs controlling these infections, we used recombinant congenic strains (RCS). Each RCS carry unique set of 12.5% of the genome from donor parental strain on genetic background of other parental strain. For mapping of QTLs controlling infections caused by Trypanosoma brucei brucei (T. b. brucei) and Leishmania tropica (L. tropica) and eosinophil infiltration into inguinal lymph nodes after Leishmania major (L. major) infection, we used RCS from CcS/Dem series, where STS is donor strain and BALB/cHeA is strain of genetic background. First, it was necessary to find suitable model strains for mapping. In all three studies, we selected RCS, which exceeded range of monitored phenotype parameters in comparison with any other tested RCS or parental strains. Mice of RCS CcS-11 showed shorter survival after T. b. brucei infection and strain CcS-9 exhibited higher eosinophil infiltration after L. major infection. For...
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Sex in Trypanosomatids
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Čepička, Ivan (referee)
3 Abstrakt Rody Leishmania a Trypanosoma jsou původci vážných lidských onemocnění: leishmaniózy a trypanosomózy. Dlouhá léta nebyly u těchto parazitů nalezeny přesvědčivé důkazy o genetické výměně, a proto byly rody Trypanosoma a Leishmania považovány za klonálně se rozmnožující, a to binárním štěpením jako většina prvoků. Výzkum ztěžovaly i skutečnosti, že pohlavní dimorfismus není patrný a chromosomy nekondenzují, tudíž nejsou viditelné. Nicméně klonální model začaly zpochybňovat pozorování přirozeně se vyskytujících hybridních druhů. Nejdříve byla existence sexu popsána u trypanosom a to prvním přímým důkazem hybridů T. brucei, získaných po společném přenosu rodičů mouchou tsetse. U leishmanii byl důkaz poskytnut na základě dvojitě rezistentních hybridů a sexuální výměna podstupovala stejný meiotický proces jako T. brucei. Byli pozorovaní přirozeně se vyskytující hybridi Nového i Starého světa jak u rodu Viannia, tak i u rodu Leishmania. Otázkou dalších výzkumů bylo, jaký je mechanismus genetické výměny, ale odpověď dodnes není jasná. Klíčová slova: genetická výměna, Trypanosoma, Leishmania, klonalita, meióza, GFP, přenašeč Abstract Genera Leishmania and Trypanosoma are agents of serious human diseases: leishmaniasis and trypanosomózy. For many years these parasites were considered clone-replicating by...
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Macrophages in leishmania - sand fly - host interaction
Kratochvílová, Tereza ; Kolářová, Iva (advisor) ; Fialová, Anna (referee)
Sand flies (order Diptera) are vectors of Leishmania parasites (Trypanosomatida), which are inoculated into the host skin together with the vector saliva. Sand fly saliva plays the important role in the Leishmania transmission; in naive host it supresses the host immune response assisting Leishmania to establish the infection, while in repeatedly bitten host it elicits a protective immune response. The submitted thesis focuses on the effect of sand fly saliva on macrophages, the key cells in the infection control. In the first part of the thesis we established a laboratory model L. major - P. papatasi - Balb/c to describe the protective effect of saliva immunization on Leishmania infection development. Immunized mice were protected against Leishmania infection which was reflected in the ear lesion size, parasite load in the ear dermis and draining lymph nodes but also in cytokine production. On the contrary, produced lower amount of nitric oxide, while arginase activity was comparable with nonimmunized group. The IgG antibodies against saliva served as a marker of exposure to sandflies while IgG antibodies against Leishmania antigens served as a marker of infection severity. The experiments were aimed on the possibility of cross-protectivity in Balb/c mice against L. major between closely related...
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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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Leishmania development in sand flies during bloodmeal digestion
Pružinová, Kateřina ; Volf, Petr (advisor) ; Bates, Paul Andrew (referee) ; Valenzuela, Jesus G. (referee)
This thesis is focused on the bloodmeal digestion of phlebotomine sand flies and its effects on Leishmania development within their midguts. In the first part, we studied various parameters of bloodmeal digestion in four sand fly species differing in susceptibility to Leishmania donovani to evaluate the effects on vector competence. Both proven vectors of L. donovani (Phlebotomus orientalis and P. argentipes) showed lower trypsin activity and slower formation of the peritrophic matrix (PM) than refractory species (P. papatasi and Sergentomyia schwetzi). Remarkably, P. orientalis and P. argentipes strikingly differed from each other in a time course of bloodmeal digestion. Phlebotomus orientalis females showed very slow bloodmeal digestion with low peaks of proteolytic activities and defecated around day five post bloodmeal. In contrast, P. argentipes females digested faster with a very high peak of chymotrypsin activity, their PM was present for only a short time and defecation was finished by day three post bloodmeal. We presume that the period between the degradation of the PM and defecation (i.e. time frame when Leishmania bind to the midgut to avoid expulsion with bloodmeal remnants), is one of crucial parameters affecting the establishment of Leishmania in the sand flies. In both natural...
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Development of a new sand fly exposure test to evaluate vector control tools
Willen, Laura Adrienne André ; Volf, Petr (advisor) ; Kopecký, Jan (referee) ; Valenzuela, Jesus G. (referee)
In the Mediterranean basin, human visceral leishmaniasis caused by the protozoan parasite Leishmania infantum is a zoonotic disease that gives rise to 1,200 to 2,000 new cases annually. The domestic dog constitutes its main reservoir, of which some may suffer from a severe chronic disease, canine leishmaniasis (CanL). The sand fly Phlebotomus perniciosus is considered to be the principle vector. Saliva of bloodfeeding vectors of diseases has been used in the past to assess host exposure to vector bites and to evaluate vector control tools. This Ph.D. focused on saliva of P. perniciosus to identify exposure markers that could be used in the preparation of a new vector exposure tool. The first part of this Ph.D. aimed at validating the use of a recombinant salivary protein of P. perniciosus - rSP03B - in endemic settings of CanL. During a cross-sectional study, no significant differences between the antibody (Ab) response against whole saliva or the rSP03B were observed between different regions across the Mediterranean basin. Furthermore, the rSP03B was shown to resemble the native protein. During a subsequent study this protein was used to assess the seasonal dynamics of the canine Ab response to P. perniciosus in an endemic area of L. infantum. This study elucidated that also in a heterogeneous...
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Effect of abiotic and biotic factors on Leishmania development in sand fly vectors
Hlaváčová, Jana ; Volf, Petr (advisor) ; Alten, Bülent (referee) ; Traub-Cseko, Yara Maria (referee)
The thesis deals with Leishmania development in sand flies focusing on factors that could affect the vectorial part of their lifecycle. First, we examined an effect of temperature on Leishmania development in sand flies since the global warming was suggested to be one of the most important factors affecting dissemination of leishmaniasis. Development of three Leishmania species were studied at two different temperatures (20 and 26řC); L. infantum in natural vectors Lutzomyia longipalpis and Phlebotomus perniciosus, and two closely related Viannia species L. braziliensis and L. peruviana in Lu. longipalpis. Leishmania peruviana thrived well only at the lower temperature tested. At the higher temperature, most of infections were lost between days 2 and 8 post feeding, i.e. time of blood-meal remnants defecation. This suggests that this Andean species is adapted to slower metabolism of vectors living at lower temperatures. On the other hand, L. infantum and L. braziliensis successfully developed in sand flies at both temperatures tested, which might be substantial for their further spread to new areas. Next, an impact of gregarine Psychodiella sergenti on L. tropica in Phlebotomus sergenti was studied. Gregarines are commonly found in sand flies, and in mosquitoes, they can affect development of other...
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Types of Leishmania development in phlebotomine species
Nováková, Julie ; Votýpka, Jan (advisor) ; Sádlová, Jovana (referee)
The aim of this thesis is to depict the means of Leishmania development in sand flies (Phlebotominae). Three main Leishmania evolutionary branches (subgenera Leishmania, Viannia and Sauroleishmania) have a slightly different development in the vector, the Sauroleishmania development being the least known. The vector-bound development of the subgenus Leishmania is localized in the midgut and foregut of the sand fly solely, whereas species of the Viannia subgenus occur in the hindgut (although the necessity of this phase for a successful development and infection of the vertebrate host is a matter of doubt) and the subgenus Sauroleishmania development is according to literature confined to the hindgut. Quite little is known about the genus Endotrypanum, according to phylogenetic data set inside the Leishmania group. In my bachelor thesis, I review up-to-date literature on the topic of different types of Leishmania development inside the sand flies and their probable reasons.
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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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