DIPLOMARBEIT Amoebae of medical relevance in reptiles Establishment of a PCR detection system for parasitic amoebae angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag. rer.nat.) Verfasserin / Verfasser: Verena PECAVAR Matrikel-Nummer: 9805884 Studienrichtung /Studienzweig Anthropologie (lt. Studienblatt): Betreuerin / Betreuer: Univ. Doz. Mag. Dr. Julia Walochnik Univ.-Prof. Dr. Sylvia Kirchengast Wien, im September 2009 1 Contents 1 ABBREVIATION ............................................................................................ 6 2 INTRODUCTION ............................................................................................ 7 2.1 History ................................................................................................................................ 7 2.1.1 Acanthamoeba spp. ........................................................................................................ 7 2.1.2 Balamuthia mandrillaris .................................................................................................. 8 2.1.3 Naegleria spp. ................................................................................................................. 9 2.2 FLA: Phylogeny ................................................................................................................. 9 2.2.1 Phylogeny of "amoebae" ................................................................................................ 9 2.2.1.1 Acanthamoeba spp. ............................................................................................. 12 2.2.1.2 Balamuthia mandrillaris ........................................................................................ 12 2.2.1.3 Naegleria fowleri .................................................................................................. 13 2.2.2 Distribution .................................................................................................................... 14 2.2.2.1 Acanthamoeba spp. ............................................................................................. 14 2.2.2.2 Balamuthia mandrillaris ........................................................................................ 15 2.2.2.3 Naegleria fowleri .................................................................................................. 15 2.3 FLA: Physiology and morphology ................................................................................ 16 2.3.1 FLA comparative characteristics .................................................................................. 16 2.3.2 Acanthamoeba spp. ...................................................................................................... 17 2.3.2.1 Life cycle .............................................................................................................. 17 2.3.2.2 Trophozoites ........................................................................................................ 18 2.3.2.3 Cysts .................................................................................................................... 19 2.3.3 Balamuthia mandrillaris ................................................................................................ 20 2.3.3.1 Life cycle .............................................................................................................. 20 2.3.3.2 Trophozoites ........................................................................................................ 21 2.3.3.3 Cysts .................................................................................................................... 22 2.3.4 Naegleria fowleri ........................................................................................................... 22 2.3.4.1 Life cycle .............................................................................................................. 22 2.3.4.2 Trophozoites ........................................................................................................ 23 2.3.4.3 Flagellate .............................................................................................................. 24 2.3.4.4 Cysts .................................................................................................................... 24 2.4 FLA: Medical relevance .................................................................................................. 25 2.4.1 Acanthamoeba spp. ...................................................................................................... 25 2.4.1.1 Acanthamoeba keratitis ....................................................................................... 26 2.4.1.2 Granulomatous amoebic encephalitis (GAE) ....................................................... 28 2.4.1.3 Cutaneous Acanthamoebosis .............................................................................. 29 2.4.2 Balamuthia mandrillaris ................................................................................................ 30 2.4.3 Naegleria fowleri ........................................................................................................... 33 2 Contents 2.4.4 Free-living amoebae in animals .................................................................................... 34 2.5 Entamoeba invadens ....................................................................................................... 36 2.5.1 History ........................................................................................................................... 36 2.5.2 Phylogeny ...................................................................................................................... 36 2.5.3 Biology ........................................................................................................................... 38 2.5.4 Life cycle ....................................................................................................................... 38 2.5.5 Physiology and morphology .......................................................................................... 39 2.5.5.1 Trophozoites ......................................................................................................... 39 2.5.5.2 Cysts ..................................................................................................................... 40 2.5.6 Medical relevance for animals ....................................................................................... 41 2.6 Aims: ................................................................................................................................. 43 3 MATERIAL AND METHODS ....................................................................... 44 3.1 Animals ............................................................................................................................. 44 3.2 Sample drawing ............................................................................................................... 45 3.3 Culture .............................................................................................................................. 45 3.3.1 Aerobic amoebas .......................................................................................................... 45 3.3.2 Anaerobic amoebae: ..................................................................................................... 46 3.3.3 Nematode cultivation ..................................................................................................... 47 3.4 Molecular biology ............................................................................................................ 47 3.4.1 Primer design ................................................................................................................ 47 3.4.2 DNA isolation ................................................................................................................. 50 3.4.3 Primer evaluation .......................................................................................................... 51 3.4.3.1 Balamuthia mandrillaris Primer specificity testing ................................................ 51 3.4.3.2 Primer sensitivity testing ....................................................................................... 52 3.4.4 Nested PCR establishment ........................................................................................... 55 3.4.5 Nematodes PCR ........................................................................................................... 56 3.4.6 Gelelectrophoresis ........................................................................................................ 56 3.4.7 Sequencing ................................................................................................................... 57 4 RESULTS .................................................................................................... 59 4.1 Culture .............................................................................................................................. 59 4.1.1 Snakes .......................................................................................................................... 59 4.1.1.1 Snakes: first sample drawing (6th of November 2007) ........................................ 59 4.1.1.2 Snakes: second sample drawing (13th of December 2007) ................................ 60 4.1.1.3 Summary snakes: ................................................................................................