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Diplomarbeit DIPLOMARBEIT Titel der Diplomarbeit „Microscopic and molecular analyses on digenean trematodes in red deer (Cervus elaphus)“ Verfasserin Kerstin Liesinger angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag.rer.nat.) Wien, 2011 Studienkennzahl lt. Studienblatt: A 442 Studienrichtung lt. Studienblatt: Diplomstudium Anthropologie Betreuerin / Betreuer: Univ.-Doz. Mag. Dr. Julia Walochnik Contents 1 ABBREVIATIONS ......................................................................................................................... 7 2 INTRODUCTION ........................................................................................................................... 9 2.1 History ..................................................................................................................................... 9 2.1.1 History of helminths ........................................................................................................ 9 2.1.2 History of trematodes .................................................................................................... 11 2.1.2.1 Fasciolidae ................................................................................................................. 12 2.1.2.2 Paramphistomidae ..................................................................................................... 13 2.1.2.3 Dicrocoeliidae ........................................................................................................... 14 2.1.3 Nomenclature ................................................................................................................ 14 2.2 Phylogeny and systematics .................................................................................................... 15 2.2.1 Phylogeny of helminths ................................................................................................. 15 2.2.2 Phylogeny of trematodes ............................................................................................... 16 2.2.2.1 Fasciolidae ................................................................................................................. 20 2.2.2.2 Paramphistomidae ..................................................................................................... 20 2.2.2.3 Dicrocoeliidae ........................................................................................................... 21 2.3 Biology .................................................................................................................................. 22 2.3.1 Biology of helminths ..................................................................................................... 22 2.3.2 Biology of trematodes ................................................................................................... 22 2.3.2.1 Comparative features of selected trematode species ................................................. 25 2.4 Medical relevance .................................................................................................................. 26 2.5 Veterinary relevance .............................................................................................................. 31 2.6 Aim of the study .................................................................................................................... 33 3 MATERIAL & METHODS .......................................................................................................... 35 3.1 Samples ................................................................................................................................. 35 3.1.1 Sampling sites ................................................................................................................ 35 3.1.2 Sample drawing ............................................................................................................. 38 3.2 Microscopy ............................................................................................................................ 38 3.2.1 Sedimentation after Benedek (modified) ....................................................................... 38 3.2.1.1 Calculation of eggs per gram ..................................................................................... 39 3.2.1.2 Calculation of positivity rates .................................................................................... 39 3.2.2 SAF method ................................................................................................................... 39 3.3 Liver inspection ..................................................................................................................... 40 3.4 Molecular biology ................................................................................................................. 41 3.4.1 Sample selection ............................................................................................................ 41 3.4.2 Homogenization ............................................................................................................ 42 3.4.3 DNA extraction ............................................................................................................. 42 [3] Contents 3.4.3.1 DNA extraction with tissue kit (control samples) ..................................................... 42 3.4.3.2 DNA extraction with stool kit ................................................................................... 43 3.4.3.3 DNA extraction with soil kit ..................................................................................... 43 3.4.4 Spectrophotometer ......................................................................................................... 44 3.4.5 Primer design ................................................................................................................. 44 3.4.5.1 Universal primers for trematodes .............................................................................. 44 3.4.5.1 Specific primers for Fascioloides magna .................................................................. 46 3.4.1 PCR setup ...................................................................................................................... 48 3.4.1.1 Trem_F/Trem_R ........................................................................................................ 48 3.4.1.1.1 Specificity testing ................................................................................................ 48 3.4.1.1.2 Sensitivity testing ................................................................................................ 50 3.4.1.1.3 PCR establishment .............................................................................................. 51 3.4.1.2 Magna_F/Magna_R ................................................................................................... 54 3.4.1.2.1 Specificity testing ................................................................................................ 54 3.4.1.2.2 Sensitivity testing ................................................................................................ 55 3.4.1.2.3 PCR establishment .............................................................................................. 56 3.4.2 Primers for F. hepatica .................................................................................................. 58 3.4.2.1.1 Specificity testing ................................................................................................ 59 3.4.2.1.2 Sensitivity testing ................................................................................................ 60 3.4.3 Gel electrophoresis ........................................................................................................ 61 3.4.4 Purification of agarose gel bands .................................................................................. 62 3.4.5 Sequencing .................................................................................................................... 62 3.4.5.1 Sequencing PCR ........................................................................................................ 62 3.4.5.2 Purification of the sequencing PCR product ............................................................. 63 3.4.5.3 Analysis of sequences................................................................................................ 63 4 RESULTS ...................................................................................................................................... 64 4.1 Samples ................................................................................................................................. 64 4.1.1 Overall distribution and origin of samples .................................................................... 64 4.1.2 Geographical distribution of collected samples ............................................................. 65 4.1.3 Geographical distribution of provided samples ............................................................. 66 4.1.4 Seasonal distribution of samples ................................................................................... 66 4.2 Microscopy ............................................................................................................................ 67 4.2.1 Eggs per gram ................................................................................................................ 68 4.2.2 Trematode frequencies .................................................................................................
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