Fachbereich Veterinärmedizin



    Entwicklung eines Zellkultur-basierten Testverfahrens zur Evaluierung potentieller Wirkstoffe gegen Cryptosporidium parvum (2015)

    Schupfner, Maja (WE 13)
    Berlin: Mensch und Buch Verlag, 2015 — III, 152 Seiten
    ISBN: 978-3-86387-603-6
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000100122
    Institut für Parasitologie und Tropenveterinärmedizin

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35, 22, 23
    14163 Berlin
    Tel.+49 30 838 62310 Fax.+49 30 838 62323

    Abstract / Zusammenfassung

    Cryptosporidiosis is a common gastro-intestinal infection in both, humans and animals, worldwide caused by the zoonotic protozoan C. parvum and other members of the same genus. The clinical course of C. parvum infections is highly dependent on the immune status of the individual host. While in immunocompetent individuals Cryptosporidium infections most commonly result in acute but self-limiting gastroenteritis, in immunodeficient or immunosupressed individuals cryptosporidiosis can become a chronic and life-threatening diarrhoeal disease and also (additional) extraintestinal infections may occur. Not only in human medicine but also in veterinary medicine C. parvum ranges among the most relevant parasitic enteritis pathogens. Due to their immature immune system neonates are highly susceptible for infections with this parasite and routinely get infected by oral uptake of the ubiquitary oocysts which are very resistant against environmental changes and chemicals. As one of the major pathogens causing neonatal diarrhoea in calves, C. parvum is responsible for fatalities and retarded weight gain in calf rearing and therefore the cause of significant economical losses in agriculture. Even though approved agents for the treatment of cryptosporidiosis and for pro- and metaphylactic use are available in human as well as in veterinary medicine, fully effective drugs in all cases of cryptosporidiosis are still missing but urgently needed. In the present work a method was established to evaluate the efficacy of putative anticryptosporidial compounds which had not been tested yet. The so-called ”C. parvum inhibition assay“ is a combination of in vitro cultivation of C. parvum in HCT-8 cells and quantitative real-time PCR (qPCR). The qPCR-based method used here allowed the relative quantification of the development of C. parvum under drug exposure, therefore the efficacy of the tested compound could be determined. For the detection of the parasite DNA a target sequence of 255 bp within the cryptosporidial 18S rDNA gene was used. The amount of this target sequence was normalised to an internal control for each sample. As internal control a sequence with a size of 189 bp from the human host cell 18S rDNA gene was amplified in a second qPCR. Overall 51 compounds were tested in at least three different concentrations. Two promising compounds, a heterocyclic substituted 1,2,4-triazinedione (BAY-AB24992) and a substituted benzimidazole (BAY-AF76184), were examined in more detail. Both compounds were tested in eight different concentrations for their ability to interfere with C. parvum development in vitro. Furthermore the same concentrations were also used in a lactate dehydrogenase and a WST-1 assay to analyse potential cytotoxic and anti-proliferative effects on the chosen host cell line, respectively. The drug BAY-AF76184 displayed a good concentration-dependent inhibition of C. parvum growth with a maximum inhibition of 97%. The concentration response curve had an EC50 value of 2.37 μM. Although this compound also showed concentration-dependent antiproliferative and cytotoxic effects on HCT-8 cells, further in vivo tests at low dosages are advisable. Even a concentration of 4.46 μM, which was associated with low cytotoxicity and no cytostatic effect, reached an inhibition of the growth of C. parvum in vitro of approximately 78%. Therefore BAY-AF76184 can be considered a promising/putative compound despite its unwanted side effects. Future in vivo testing of BAY-AB24992 also appears to be recommendable. This compound did not inhibit C. parvum growth in a concentration-dependant manner but also showed good in vitro efficacy against C. parvum. At least the four highest concentrations yielded an inhibition of more than 60% (approx. 71-88%). Additionally this drug had neither antiproliferative nor cytotoxic effects. Overall the established C. parvum inhibition assay displays a fast and reliable method for assessing drug efficacy against the protozoan C. parvum. Consequently it is suitable to at least partially replace expensive and labour-intensive animal experiments which are also of ethical concern. Although there is of course no guarantee that drugs with good in vitro activity will also perform in vivo, a cell culture-based assay nevertheless improves prediction for overall success of consecutive in vivo tests.