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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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Functional morphology of schistosome miracidia
Svobodová, Kateřina ; Horák, Petr (advisor) ; Bulantová, Jana (referee)
Trematodes of the family Schistosomatidae are important pathogens invading various species of birds and mammals, including man. Miracidium is the first larval stage in the complex life cycle of these parasites. The fate of the parasite depends on the ability of the larva to find, recognize and penetrate into a suitable snail host. Miracidium locates its snail host actively. Miracidium respond to environmental cues and also by recognition of specific glycoproteins produced by snails to the water environment. Free-living miracidium has limited energy reserves, so it has to find and penetrate into a suitable host as quickly as possible. Cilia serve to fast movement in the aquatic environment. The surface of the miracidium is covered with flat ciliated epidermal plates arranged in four tiers. These plates are separated from each other by epidermal ridges. The anterior end of miracidia is cone-shaped and is called apical papilla. Apical papilla is a key tool for penetration and it contains openings of penetration glands. Miracidia display a variety of sensory receptors which help miracidium in orientation. However, there is not much known about miracidial receptor function. Miracidial morphology can serve as a suitable characteristic for schistosome species identification. The thesis summarizes the...
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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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Laboratory cycles of schistosomes, breeding of intermediate snail hosts and their ecological requirements
Nečasová, Monika ; Mikeš, Libor (advisor) ; Horák, Petr (referee)
The Schistosomatidae family is an important group of blood flukes, including serious human and livestock animal pathogens. With regard to reproduction, they are gonochoristic parasites. Their life cycle has two stages - asexual reproduction in an intermediary host (fresh water and sea gastropods) and sexual reproduction in the definitive host (mammals and birds). With regard to their pathogenicity at the definitive hosts, the blood fluke life cycle is abundantly maintained in laboratories. To preserve the whole cycle, the intermediary host gastropods are very important, They can include, without limitation, representatives of the Lymnaeidae family. However, their sensitivity to parasitism by blood flukes varies significantly by species. Floats were classified into several genera according to various parameters in the past. The systematics was most frequently based on anatomic and morphological differences. However, the most precise taxonomies are based on molecular data and mutual phylogenetic relationships of Lymnaeidaegastropods remain a subject of research. It is a fresh water family of gastropods with specific ecological and nutritional requirements. Summary information about their ecology estimate that the float gastropods are highly sensitive to the presence of heavy metals in water, such as...
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Excretory-secretory proteins of schistosome intravertebrate stages in context of their biology
Konečný, Lukáš ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Schistosomatidae is a family of blood-dwelling trematodes which cause serious disease in humans and warm blooded animals. Human schistosomiasis affects over 258 million people worldwide. The fundamental pathological manifestations of the disease are not caused by adult worms, but by responses to antigens released from eggs, which remain trapped in the tissues. All stages of these parasites release excretory-secretory products, which mostly comprise of proteins which are, to a certain extent, species- and stage-specific. These products have many functions that play roles in successful transmission of the parasite and its interactions with the host. Various approaches to the collection and analysis of these molecules do not always accurately reflect the secretory processes in vivo, because they always employ in vitro techniques. The research of these secreted molecules discovered many mechanisms, through which the parasite successfully modulates and evades host immune system. Despite the fact, that many of these molecules were identified as potential vaccine candidates, still no sufficiently effective vaccine has been developed. With great technological advances used for the identification of these proteins, this area is becoming more and more topical. This review focuses on composition of...
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Determination of gastropods of the genus Radix, the intermediate hosts of trematodes
Mikešová, Kateřina ; Beran, Luboš (referee)
Lymnaeid snails of the genus Radix are common freshwater molluscs of Europe. Formerly, the following species have been reported from the Czech Republic according to the morphology of the shell: R. auricularia, R. peregra, R. ovata and R. ampla. However, recent studies involving molecular data revealed that species determination based on traditional approaches employing morphology of the shell and gonads can be more complicated, confusing and not fully reliable. The shape of the shell is affected by particular life conditions of the snails and proves to be highly variable. The characteristics that can be found on gonads are dependent on the reproductive phase of the snail, and sometimes also on the presence of trematode larval stages, which can cause parasitic castration. Recently, molecular taxonomy is the most reliable approach, especially when a combination of sequences of mitochondrial and nuclear DNA is used. As molecular analyses are relatively time-consuming and expensive, some authors try to find some additional morphological features that could be used for determination of Radix snails in the field, without the need of demanding equipment. Problems in taxonomy and systematics of members of the genus Radix have a practical point in studies of trematode life cycles. From the view of human and...
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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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Humoral response of specific hosts to bird schistosome antigens
Turjanicová, Libuše ; Mikeš, Libor (advisor) ; Kolářová, Libuše (referee)
This thesis focuses on humoral immune response of specific hosts to antigens of various developmental stages of bird schistosomes T. regenti and T. szidati, and follows up on previous research of antibody response in non-specific hosts (mouse, human). Sera of experimentally infected and hunted-down wild ducks were examined using the ELISA and western blot methods. The sera samples were taken in predefined intervals. Results of the ELISA analysis show the process of humoral immune response after infection by bird schistosomes. The level of specific antibodies IgY against homogenate of T. regenti cercariae increased significantly 20 d.p.i. in ducks infected by T. regenti. Such reaction wasn't observed in ducks infected by T. szidati. Slight changes in level of specific antibodies IgM against T. szidati cercariae homogenate were observed 10 d.p.i. only in fully immunocompetent ducks and in reinfected ducks. Examination of hunted-down wild ducks didn't prove infection by bird schisosomes; this conclusion was confirmed by results of the ELISA analysis. IgY antibodies from ducks infected by T. regenti demonstrated strong reactions with 2 antigens in ranges 49-47 kDa and 47-45 kDa. Other reactions, which were recognized, have not been observed in all specimen. An Western blott with homogenate from 7 days...
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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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