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C:\Dokumente Und Einstellungen\Nikola Vorwerk Aus dem Institut für Parasitologie der Stiftung Tierärztliche Hochschule Hannover ___________________________________________________________________________ Etablierung und Validierung einer Real-Time-PCR zur Detektion der Oozysten von Toxoplasma gondii INAUGURAL-DISSERTATION Zur Erlangung des Grades einer Doktorin der Veterinärmedizin (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover Vorgelegt von Nikola Vorwerk aus Soltau Hannover 2008 Wissenschaftliche Betreuung: Apl. Prof.’in Dr. Astrid M. Tenter 1. Gutachter: Apl. Prof.’in Dr. Astrid M. Tenter 2. Gutachter: Apl. Prof.’in Dr. rer.nat. Irene Greiser-Wilke Tag der mündlichen Prüfung: 21.11.2008 Für meine Eltern und meine Schwester Die Menschheit lässt sich grob in zwei Gruppen einteilen: in Katzenliebhaber und in vom Leben Benachteiligte. Francesco Petrarca Inhaltsverzeichnis 5 INHALTSVERZEICHNIS 1. EINLEITUNG.............................................................................................................11 2. LITERATURÜBERSICHT........................................................................................13 2.1 TOXOPLASMA GONDII.................................................................................................13 2.1.1 Taxonomie ....................................................................................................13 2.1.2 Lebenszyklus und Morphologie der Entwicklungsstadien..............................14 2.1.3 Wirtsspektrum, Übertragungswege und geografische Verbreitung .................17 2.1.4 Genetische Divergenz....................................................................................20 2.1.5 Bedeutung von T. gondii als Zoonoseerreger .................................................20 2.1.6 Bedeutung der verschiedenen Infektionsstadien.............................................21 2.1.6.1 Tachyzoiten...............................................................................................22 2.1.6.2 Zysten .......................................................................................................22 2.1.6.3 Oozysten ...................................................................................................23 2.1.7 Epidemiologie ...............................................................................................24 2.1.7.1 Bedeutung der Katzen................................................................................24 2.1.7.2 Bedeutung der Oozysten für die Infektion von Zwischenwirten .................26 2.2 ANDERE EINZELLIGE EUKARYOTEN IM KATZENKOT ...............................................26 2.3 DIAGNOSTISCHE METHODEN....................................................................................29 2.3.1 Indirekter Erregernachweis............................................................................29 2.3.1.1 Sabin-Feldman-Test...................................................................................29 2.3.1.2 Indirekter Fluoreszenzantikörpertest..........................................................30 2.3.1.3 Enzyme-linked Immunosorbent Assay.......................................................31 2.3.1.4 Aviditäts-ELISA........................................................................................32 2.3.1.5 Agglutinationstests ....................................................................................33 2.3.1.6 Komplementbindungsreaktion ...................................................................34 2.3.2 Direkter Erregernachweis ..............................................................................35 2.3.2.1 Koproskopie ..............................................................................................35 6 Inhaltsverzeichnis 2.3.2.2 Bioassays...................................................................................................36 2.3.2.3 Mikroskopischer und immunhistochemischer Nachweis im Gewebe..........38 2.3.2.4 Nachweis von zirkulierendem Antigen ......................................................38 2.3.2.5 Hybridisierungsassays ...............................................................................39 2.3.2.6 Gelbasierte Polymerase-Ketten-Reaktion...................................................39 2.3.2.6.1 Prinzip .................................................................................................39 2.3.2.6.2 PCR in der Diagnostik von T. gondii....................................................41 2.3.2.7 Quantitative Real-Time-Polymerase-Ketten-Reaktion................................57 2.3.2.7.1 Prinzip .................................................................................................57 2.3.2.7.2 Real-Time-PCR in der Diagnostik von T. gondii..................................58 3. MATERIAL UND METHODEN...............................................................................66 3.1 MATERIAL ................................................................................................................66 3.1.1 Reagenzien und Lösungen.............................................................................66 3.1.2 Geräte und Verbrauchsmaterialien.................................................................66 3.2 METHODEN...............................................................................................................67 3.2.1 Toxoplasma gondii ........................................................................................67 3.2.1.1 Tachyzoiten...............................................................................................67 3.2.1.1.1 In-vitro-Kultur von T.-gondii-Tachyzoiten...........................................67 3.2.1.2 Oozysten ...................................................................................................69 3.2.1.2.1 Infektion der Mäuse.............................................................................69 3.2.1.2.2 Infektion der Katzen ............................................................................70 3.2.1.2.3 Gewinnung der Oozysten.....................................................................70 3.2.1.2.4 Extraktion genomischer DNA aus T.-gondii-Oozysten.........................71 3.2.1.3 DNA..........................................................................................................72 3.2.2 Andere Parasiten ...........................................................................................74 3.2.3 Wirts-DNA....................................................................................................75 3.2.3.1 Extraktion genomischer DNA aus Katzenleber ..........................................75 3.2.4 Auswahl der Zielsequenzen für die Real-Time-PCR......................................76 3.2.4.1 B1-Gen......................................................................................................76 3.2.4.2 529-bp-Repeat-Region von T. gondii.........................................................78 3.2.4.3 Agarosegelelektrophorese..........................................................................79 Inhaltsverzeichnis 7 3.2.4.4 Sequenzierung der PCR-Produkte..............................................................80 3.2.4.4.1 Reinigung der PCR-Produkte...............................................................80 3.2.4.4.2 Klonierung der PCR-Amplifikate.........................................................80 3.2.4.4.3 Isolierung von Plasmid-DNA...............................................................81 3.2.4.4.4 Sequenzierung der PCR-Produkte........................................................82 3.2.5 Real-Time-PCR.............................................................................................83 3.2.5.1 Durchführung der Real-Time-PCR ............................................................83 3.2.5.2 Auswertung der Real-Time-PCR ...............................................................84 3.2.5.3 Bestimmung der Sensitivität und der Spezifität der Real-Time-PCR ..........84 3.2.5.3.1 B1-Gen................................................................................................84 3.2.5.3.2 529-bp-Repeat-Region.........................................................................86 3.2.6 Statistik .........................................................................................................87 4. ERGEBNISSE.............................................................................................................88 4.1 REAL-TIME-PCR MIT DEM B1-GEN ALS TARGET ....................................................88 4.1.1 Optimierung der Real-Time-PCR ..................................................................93 4.1.2 Spezifität der Real-Time-PCR mit Primerpaar TgB1b ...................................95 4.1.3 Sensitivität der Real-Time-PCR mit Primerpaar TgB1b.................................99 4.1.3.1 Titration von klonierter T.-gondii-DNA...................................................100 4.1.3.2 Titration von genomischer T.-gondii-DNA mit und ohne Hintergrund .....101 4.1.3.3 Titration von Tachyzoiten und Oozysten..................................................107 4.2 REAL-TIME-PCR MIT DER 529-BP-REPEAT-REGION ALS TARGET ........................112 4.2.1 Optimierung der Real-Time-PCR ................................................................112 4.2.2 Spezifität der Real-Time-PCR mit Primerpaar Tg529c ................................115 4.2.3 Sensitivität der Real-Time-PCR mit Primerpaar Tg529c..............................119 4.2.3.1 Titration genomischer T.- gondii-DNA mit und ohne Hintergrund ...........119
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