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Molecular detection of cercarial dermatitis causative agents
Procházka, Jan ; Leontovyč, Roman (advisor) ; Baláž, Vojtech (referee)
Human cercarial dermatitis (CD), also called Swimmer's itch appears as a skin rash caused by skin penetration by larval stages of avian schistosomes. In the Czech Republic, the most common causative agens is genus Trichobilharzia. Since 2021, monitoring of CD is a part of a mandatory testing of bathing water quality. The official method used for CD detection is collecting and examination of aquatic snails under a light source. However, this method has many disadvantages, such as time demands, low sensitivity and difficult parasite determination. These disadvantages can be overcome by usage of alternative approaches, such as molecular methods based on environmental DNA (eDNA) filtration and isothermal amplification. Hence, the main goal of this thesis was development of molecular method based on eDNA filtration and cLAMP isothermal amplification for detection of CD causative agent in water. Based on molecular analysis a conserved region of target DNA was selected to create a cLAMP assay specific for genus Trichobilharzia. The cLAMP assay was confirmed to specifically amplify the DNA of at least four species of the genus Trichobilharzia. The laboratory experiments showed a sufficient sensitivity of cLAMP assay for eDNA detection, based on DNA dilution series and cercariae filtration from water. The...
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The skin immune response of mice infected with avian schistosomes
Revalová, Alena ; Macháček, Tomáš (advisor) ; Kolářová, Libuše (referee)
Invasion of the mammalian skin by cercariae of avian schistosomes of the genus Trichobilharzia is associated with the skin pathology called cercarial dermatitis (CD). Although the manifestation of CD, characterized by the presence of maculo-papular rash and pruritus, is believed to be linked with the previous sensitization, the role of the infection dose and the specific causative agent is rarely considered. Therefore the skin immune response in mouse pinnae infected by 100, 1000 or 4x100 (repeated infections) cercariae of two Trichobilharzia species - T. regenti and T. szidati - was examined in this work. To compare the effect of these factors (the infection dose and species), a complex approach was ensured by employing both in vivo and in vitro methods. Firstly, histopathological changes in the pinnae were examined by classical histology and infiltrated leukocytes were characterized by flow cytometry. Sera of mice were used to detect the systemic production of cytokines and parasite-specific antibodies. In vitro cultivation of mouse pinnae was used to detect the local production of cytokines. The most remarkable observation was that the infection dose largely impacted the course of the immune response, both locally and systematically. In primary infected mouse pinnae, 100 cercariae of both...
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Molecular and serologic diagnosis of infections caused by Trichobilharzia
Vaščiková, Michaela ; Horák, Petr (advisor) ; Ditrich, Oleg (referee)
Cercariae of the genus Trichobilharzia can penetrate not only the skin of definitive hosts (ducks), but they are also able to penetrate the skin of accidental hosts (mammals). As a result of the penetration, the inflammatory response known as cercarial dermatitis appears. The goal of our thesis is to detect parasite DNA in the serum and cerebrospinal fluid of infected ducks, and also in the serum of infected mice. By using PCR with primers designed for a tandem repeated sequence, we were able to detect 1 femtograms of parasite DNA isolated from sera of infected ducks. We were able to amplify parasite DNA only from 16 samples of sera and cerebrospinal fluid of the infected ducks, but we were not able to do so with the serum of mice. Sera of infected mice were also tested by ELISA and Western blot. The homogenates of T. regenti (TRhc), T. szidati (TShc) and S. mansoni (SMhc) cercariae were selected as an antigen. The results showed progressive increase in the level of IgM antibody from 10 days after 1st infection and also increase of the level of IgG from the 2nd infection. 10 days after the 4th infection, the level of IgM and IgG gradually declined, but the level of antibodies 100 days after the 4th infection was still higher if compared to uninfected mice. Results from Western blot analysis...
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Features and functions of glycocalyx of trematode cercariae
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Trematodes are parasites from phylum Platyhelminthes which have compex life cycles involving two to four hosts. This work focuses especially on trematodes of the family Schistosomatidae. Their cercariae which leave the snail intermediate host, actively penetrate the skin of definitive hosts and transform into schistosomula. This is accompanied by detachment of cercarial tail and emptying of penetration glands. During transformation, cercarial bodies undergo extensive ultrastructural and molecular changes. One of these changes is the loss of surface glycocalyx which represents a protective coat in the aquatic environment. In glycocalyx shedding, participation of proteolytic enzymes from cercarial penetration glands is expected during invasion of the host. Glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. This work describes the origin, ultrastructure, saccharide composition, function and shedding mechanism of cercarial glycocalyx.
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Characterisation of recombinant cathepsins B of the bird schistosome Trichobilharzia regenti
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
This study focuses on the recombinant cysteine peptidases - cathepsin B originating in the bird schistosome Trichobilharzia regenti that is unique across the whole family for its ability to migrate through the nerve tissue to the final localization. For invasion, migration, degradation of nutritional proteins and/or evasion of host immune responses, schistosome employs peptidases. This study follows the research done by researchers of Department of parasitology, Faculty of Natural Sciences, Charles University. The main goal of this study was to deepen the characteristics of recombinant cathepsins B originating in T. regenti. In T. regenti, two cysteine peptidases - cathepsins B1 (TrCB1) and B2 (TrCB2) - have been previously characterized. TrCB1 is located in the gut of schistosomula and involved in digestion. TrCB2 occurs in post-acetabular penetration glands of cercariae and probably facilitates penetration. The recombinant pro-cathepsin B (isoforms TrCB1.1, TrCB1.4 and also TrCB2) were expressed in Pichia pastoris yeast system. An attempt was made to produce in P. pastoris the recombinant isoform TrCB1.6, in which the active site cysteine is substituted by glycine. While TrCB2 underwent self-processing in the expression medium, TrCB1.1 and TrC1.4 zymogens were effectively activated only after the...
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Molecular diagnostics of bird schistosomes during the infection of natural and accidental hosts
Šteiger, Vladimír ; Kašný, Martin (advisor) ; Dvořák, Jan (referee)
Bird schistosomes of the genus Trichobilharzia are known as causative agents of hyper-immune skin reaction called cercarial dermatitis (swimmer's itch). They use pulmonary water snails from family Lymnaeidae as the intermediate host and mostly anatid birds as the definitive host. The first larva, miracidium, actively moves in water environment, penetrates the snail and develops to the mother sporocyst. Then the daughter sporocysts are formed and migrate to the hepatopancreas of the snail where the high number of cercariae is assexually produced. Cercariae leave the intermediate host, actively move in a water and penetrate the skin of definitive host. Within a host body they mature and lay eggs. Cercariae can penetrate also the mammalian skin, including human, where they are immediately eliminated by the immune system of the host, which is followed by inflammatory reaction. Until now, for humans, there is no effective method enabling to differ cercarial dermatitis from other hyper-immune skin reactions and for birds the reliable diagnostic method of trichobilharziasis is missing. The main aim of this thesis was to use the molecular methods for diagnostic of bird schistosomes infection in natural (ducks) and accidental hosts (mice, human). For optimization, the conventional PCR was used for detection...
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Influence of bird schistosome Trichobilharzia regenti on haemocyte activity of lymnaeid snails
Skála, Vladimír
Gastropod molluscs are naturally exposed to various pathogens such as bacteria, or multicellular parasites that include digenetic trematodes (digeneans) which develop in snails. To combat these pathogens gastropods have evolved a sophisticated internal defence system that is composed of humoral and cellular arms. Lectins are probably the most important humoral components, whereas haemocytes represent the main effector cells. Immunity is one of the important factors determining compatibility/non-compatibility of gastropods and pathogens (particularly snails and trematodes). The introductory part of this thesis includes a review of literature focused on the components of the gastropod immune system and their reactions against pathogens represented by bacteria and digeneans. Additionally, selected immunomodulations caused by compatible digenean species are reviewed. Experimental work (presented in publications) focused mainly on the influence of the bird schistosome Trichobilharzia regenti on haemocyte activities of two lymnaeid snail species, Radix lagotis and Lymnaea stagnalis that are susceptible or refractory to the parasite, respectively. This schistosome parasite causes neuromotor disorders in specific definitive hosts (waterfowl), but it also causes cercarial dermatitis in accidental hosts...
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Analysis of secretome from Trichobilharzia regenti cercariae and characterisation of selected peptidases
Konečný, Lukáš ; Kašný, Martin (advisor) ; Horn, Martin (referee)
(English): Trichobilharzia regenti is a neurotropic parasite of birds from the family Schistosomatidae. Cercariae, the invasive stages of these trematodes actively penetrate the host skin employing excretory- secretory products (ESPs), which contain proteolytic enzymes able to disrupt host tissues and thus reach the successful transmission. The most abundant secreted enzyme responsible for cercarial penetration of the human schistosome S. mansoni is a cercarial elastase. This serine peptidase is well known for the degradation of skin proteins such as elastin, keratin, collagen or laminin. However, the active expression of the orthologue of this enzyme has never been found in the genus Trichobilharzia. For this reason, it was firmly believed, that cercaria of T. regenti uses mainly cysteine peptidases for the invasion of the host, particularly cathepsins, which were repeatedly identified in this life stage. To strengthen this hypothesis, we incubated T. regenti cercariae in the apparatus with the excised duck skin stimulating the release of their glands' content. The collected ESPs were further analysed by shotgun mass-spectrometry and for the first time, the protein form of cercarial elastase was identified. Unfortunately, we failed to produce its active recombinant protein in yeast and bacterial...
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