Fachbereich Veterinärmedizin



    Untersuchungen zum Vorkommen verschiedener Genotypen und Untersuchungen zum Kohlenhydratstoffwechsel von Histomonas meleagridis (2014)

    Balczulat, Stefanie (WE 15)
    Berlin: Mensch und Buch Verlag, 2014 — XV, 159 Seiten
    ISBN: 978-3-86387-454-4
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000096716
    Institut für Geflügelkrankheiten

    Königsweg 63
    14163 Berlin
    Tel.+49 30 838 62676 Fax.+49 30 838 62690

    Abstract / Zusammenfassung

    Histomonosis, blackhead disease or infectious typhlohepatitis of the gallinaceous birds is caused by the protozoen parasite or Trichomonad Histomonas meleagridis. The disease is characterized by an inflammation of the ceca and liver damages. The mortality in turkey flocks can reach 90 %.

    The first part of this thesis included investigations and analysis of different genotypes of H. meleagridis. The first studies were done on 568 samples, which had been send to the Institute for Poultry Diseases and were tested for H. meleagridis using a qPCR.
    The samples originated from 252 suspected cases of histomonosis of the years 2004 till 2010. Of the 568 samples 209 samples were positive, 357 samples negative and 2 samples questionable. Four hundred seventy three samples were organs including 193 ceca, 190 livers and 42 pools of ceca and liver. 48 samples were different organes like gizzard, rectum, cloaca, spleen, kidney, oesophagus or pancreas. 252 of the organ samples were from turkeys and 176 from chickens. Only 19 samples were from other gallinaceous birds like peacocks, pheasants, snow cocks, guinea fowls and quails and were tested with mostly negative result. The species of 29 samples was unknown. Of 46 samples of invertebrates and 42 samples of the environment only eight and four samples respectively were positive, while 38 each were negative.

    For genotyping the method of the C-Profiling was modified by designing primers specific for H. meleagridis. One hundred twenty five samples, that were positiv for H. meleagridis, were genotyped. The ITS1-region of this samples was C-profiled. Five different profiles were found (type A through E). Type A was the most frequently detected type in over half the samples. Types B and C were mostly found in turkey samples. Type D occured only in chicken samples and in one peacock sample. Type E was found in only one chicken sample.

    Furthermore 273 samples were tested for the presence of Blastocystis spp. and Tetratrichomonas gallinarum. There was a significant relationship between the occurrence of histomonosis and the concurrent absence of T. gallinarum. In contrast the relationship between H. meleagridis and Blastocystis ssp. was not significant. The second part of the thesis dealt with investigations on the metabolism of H. meleagridis. Several experiments on the cultivation were done. First the effect of the buffers MOPS and HEPES were tested. With 5, 25 or 100 mmol/L HEPES and with 50 or 100 mmol/L MOPS respectively histomonads grew well. In concentrations of 250 mmol/L MOPS or HEPES no histomonal growth was observed.

    In the following experiments the influence of different starch sources and carbohydrates instead of rice powder were tested. Starch sources were potato starch, amylose, amylopectin and glycogen. Only potato starch supported histomonal growth.
    The carbohydrates glucose, pyruvate, glucose-1-phosphat, glucose-6-phosphat and maltose were tested in different concentrations with or without the addition of rice. Cultures without rice survived at best. Often they died. Growth with the addition of rice was better. To some extent growth was achieved with pyruvate and rice.

    Histomonads were grown under different atmospheric conditions. If the flasks were opened several times the medium with the histomonads had contact with the oxygen in the air for a certain time. This as well as microaerophilic and anaerobic conditions had no negative influence on the histomonal growth.

    The expression of the genes for glycogen phosphorylase, GAPDH and alpha-amylase in histomonads which were grown with pyruvate and rice were analyzed by qPCR. Alphatubulin, beta-tubulin and 18S rRNA served as reference genes. It could be shown that histomonads digest the starch with both enzymes alpha-amylase and glycogen phosphorylase. The expression of the genes of the three enzymes mentioned above was regulated differently. The genes of GAPDH and glycogen phosphorylase were significantly upregulated whereas the gene of alpha-amylase was significantly downregulated.