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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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Acid peptidases of schistosomes and haematophagous monogeneans
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Blood is a complex nutrient-rich mixture. No wonder that haematophagy has been adopted as a feeding strategy by many invertebrates, including many parasitic helminths. In general, processing of haemoglobin (and other blood proteins) in blood-feeding helminths relies on an evolutionary conserved network of cysteine and aspartic peptidases (e.g., cathepsins L, B and D). However, some helminth taxa have been neglected from this point of view - very little information has been available about the occurrence of these enzymes in haematophagous monogeneans. Therefore, the presented thesis focuses on the molecular and biochemical characteristics of peptidases that maybe potentially involved in blood processing by the monogenean Eudiplozoon nipponicum (Heteronchoinea, Diplozoidae), an ectoparasite inhabiting gills of common carp. We show that the most abundant haemoglobinolytic endopeptidase activities in soluble protein extracts and excretory/secretory products of E. nipponicum belong to the cysteine and aspartic classes, with cathepsin L-like activity predominating over cathepsin B-like activity and supplemented with cathepsin D-like activity (paper 1). Additionally, we found that E. nipponicum adults express a variety of cathepsins L with different structural characteristics and probably different...
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Avian schistosomes and cercarial dermatitis in Czech Republic: distribution, diverzity and factors influencing their occurrence
Pokrupová, Zuzana ; Bulantová, Jana (advisor) ; Soldánová, Miroslava (referee)
Cercarial dermatitis (CD) is manifested as a strong itchy rash that usually occurs after bathing in the natural water bodies where it makes recreation uncomfortable. As a consequence, the natural swimming areas and be closed because of inconvenient water quality, what subsequently leads to the financial losses. The infection can also affect people working in the natural water bodies as lifeguards (at the natural swimming areas), people monitoring water organisms or water quality etc.). Nowadays, CD in Czech Republic occurs more frequently than in the past. For this reason, the topic of avian schistosomes and CD is very attractive not only for scientists, but newly also for employees of hygienic stations and health institutes. Based on the Act No. 258/2000 Coll. on protection of public health and related executive Decree No. 238/2011 Coll. approved this year their duty will be regular monitoring of official natural swimming areas for causative agents of CD. For the comprehensive overview about the occurrence of the avian schistosomes and CD, up to now, at the localities in the Czech Republic the specialized overview map with the marked catches of avian schistosomes and CD was created in the program ArcGIS Online. This map was made with use the records obtained from the scientific articles, final...
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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš
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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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš
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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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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Interactions of the eggs and miracidia of Trichobilharzia regenti with the duck nasal mucosa
Vlčková, Linda ; Horák, Petr (advisor) ; Chanová, Marta (referee)
Trichobilharzia regenti is a nasal avian schistosome which has during the initial phase of infection an affinity to the nervous system. Larvae migrate through the central nervous system to the nasal mucosa of waterfowl, where they mature and reproduce. Until now this infection phase has been described only marginally. Adults are located in the nasal mucosa approximately from 13th to 24th day post infection. During this life phase, they migrate through the vascularized connective tissue and lay eggs, the presence of which has been detected in the tissue only. Maturation and hatching of miracidia occur in the tissue (unlike human schistosomes). The parasite causes inflammation, and the tissue is infiltrated with immune cells. Lymphocytes, granulocytes, macrophages, plasma cells and giant multi-nuclear cells were described by histological methods. The thesis is focused on a more detailed description of cellular immune response and histopathological changes of the tissue by means of histological stains, and antibody/lectin probes. The flukes were observed more frequently in the blood vessel lumen, together with a higher number of immune cells compared to the healthy duck. Infiltration by a high number of lymphocytes occurred in the tissue, the macrophages were frequently observed in clusters around the...
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