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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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Current options of treatment for parasitic kinetoplastids
Čadková, Aneta ; Mach, Jan (advisor) ; Richterová, Lenka (referee)
Chagas disease, sleeping sickness and leishmaniasis are diseases occurring mainly in developing areas of Africa, America and Asia. They are caused by parasitic kinetoplastids from the genus Trypanosoma and Leishmania. These protozoans undergo morphologic transformations during their development leading to changes of their properties. They are characterized by complex mechanisms that allow them to evade the host's immunity. At the same time, because of these mechanisms and selection pressures, they are able to subsequently develop resistance to used drugs. The diseases caused by protozoa are classified as "neglected tropical diseases". The research and development of drugs against them is heavily underfunded. Currently used drugs are expensive, have a number of side effects, and due to lack of usage regulation develop resistance. The work first generally presents human parasites of the class Kinetoplastida. It further describes the treatment of diseases caused by the characterized species and focuses on a summary of currently used drugs, their mechanism, resistance and potential new drugs in clinical phases. Key words: kinetoplastids, Trypanosoma, Leishmania, treatment, disease, mechanism of action, resistance, nitroreductase
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Critical factors affecting pathogen development in sand flies
Jančářová, Magdaléna
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
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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Effect of abiotic and biotic factors on Leishmania development in sand fly vectors
Hlaváčová, Jana
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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Transmission and epidemiology of visceral leishmaniasis
Spitzová, Tatiana
Visceral leishmaniasis (VL) is widespread disease caused by protozoa Leishmania donovani and Leishmania infantum. Human visceral leishmaniasis caused by Le. donovani in India is considered an anthroponosis, however in East Africa, the role of animals as reservoirs remains unclear. The first part of this thesis demonstrated natural Leishmania infection in wild rodents and bats in Ethiopia. Overall, 8.2% rodents and 4.9% bats were positive for Leishmania spp. Subsequent sequencing revealed that 10% of Leishmania-positive rodents were infected by parasites from Le. donovani complex, on the other hand, no Le. donovani DNA was detected in bats. All Le. donovani-positive rodents were captured in the localities of southwest Ethiopia where human VL cases have been reported and potential sand fly vectors occur. Our findings indicate that rodents are likely to play a role in VL transmission in Ethiopia. During blood feeding, sand flies inoculate into the host skin immunogenic salivary proteins which elicit species specific antibody response. Anti-saliva antibodies could be used as a marker of host exposure to sand flies and, in leishmaniasis endemic areas, also as risk markers of Leishmania infection. In order to find out if the domestic animals (dog, goat, cow, and donkey) from north and northwest Ethiopia...
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Salivary proteins of sand flies and the immune aspects of Leishmania transmission
Kindlová, Michaela
Sand flies serve as the vectors of leishmaniasis and their saliva was shown to affect the outcome of Leishmania infection by immunomodulation of the host. On the other hand, sand fly saliva contains a large scale of farmacologically active proteins that are strongly immunogenous for bitten hosts and specific anti-saliva immunity initiated by repeated sand fly feeding provides protection against Leishmania infection. Specific cell-mediated immunity was shown to be the core of the protectivity; however, our data suggests that the protective immunity has certain limitations. In mice bitten by sand flies for prolonged periods, we observed the desenzitization in term of abrogation of the protective immunity. Thus, we can speculate that the protective effect of immunity is linked solely with the short-term exposure. Nevertheless, our experiments showed that this aspect is also conditioned by the immediate infection after the protective short-term immunization. Taken together, it seems that these limitations may explain the circulation of leishmaniasis in endemic areas, even though humans and animals are frequently immunized by bites of uninfected sand flies. Repeated sand fly feeding on various hosts also promotes production of anti-saliva antibodies that reflect the intensity of exposure. We...
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Genotype influence on development of infections caused by Trypanosomatidae in mouse
Šíma, Matyáš
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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Role of catalase and chitinase in the life cycle of Leishmania parasites
Glanzová, Kristýna ; Sádlová, Jovana (advisor) ; Kodym, Petr (referee)
Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) which are transmitted by phlebotomine sand flies (Diptera: Phlebotominae). For the dixenous life cycle, leishmania parasites are equipped with enzymes that facilitate survival in both insect vectors and mammalian hosts. Gene for the enzyme catalase which protects cells from reactive oxygen species by the elimination of H2O2 and is present in related monoxenous trypanosomatids is, however, missing in Leishmania genome. Chitinase can be involved in the interaction of leishmania parasites with chitin-containing structures in sand flies (peritrophic matrix, stomodeal valve). The expression of the enzyme in amastigotes suggests its significant function also in the mammalian host. I tested the role of these enzymes in the life cycle of leishmania by direct comparison of L. mexicana mutants (i) with inserted catalase gene and (ii) with deleted chitinase gene with control groups. I conducted experimental infections of Lu. longipalpis including transmission of leishmania to the hosts by bite, tested the survival of leishmania in macrophages and performed experimental infections of BALB/c mice followed by xenodiagnoses. The experiments confirmed that the presence of catalase in leishmania does...
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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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