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Occurrence and Genetic Determination of Giardia in Dogs from South Eastern Europe

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Occurrence and Genetic Determination of Giardia in Dogs from South Eastern Europe Occurrence and genetic determination of Giardia in dogs from South Eastern Europe Marie Franziska Sommer IN DOGS FROM SOUTH EASTERN EUROPE GIARDIA édition scientifique VVB LAUFERSWEILER VERLAG Inaugural-Dissertation zur Erlangung der Doktorwürde VVB LAUFERSWEILER VERLAG der Tierärztlichen Fakultät der STAUFENBERGRING 15 ISBN: 978-3-8359-6350-4 D-35396 GIESSEN Ludwig-Maximilians-Universität München MARIE F. SOMMER Tel: 0641-5599888 Fax: -5599890 [email protected] www.doktorverlag.de 9 7 8 3 8 3 5 9 6 3 5 0 4 édition scientifique VVB LAUFERSWEILER VERLAG VVB Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Die rechtliche Verantwortung für den gesamten Inhalt dieses Buches liegt ausschließlich bei den Autoren dieses Werkes. Jede Verwertung ist ohne schriftliche Zustimmung der Autoren oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme. 1. Auflage 2015 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Authors or the Publisher. 1st Edition 2015 © 2015 by VVB LAUFERSWEILER VERLAG, Giessen Printed in Germany édition scientifique VVB LAUFERSWEILER VERLAG STAUFENBERGRING 15, D-35396 GIESSEN Tel: 0641-5599888 Fax: 0641-5599890 email: [email protected] www.doktorverlag.de Inaugural-Dissertation zur Erlangung der Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Occurrence and genetic determination of Giardia in dogs from South Eastern Europe von Marie Franziska Sommer aus Tübingen München 2015 Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Vergleichende Tropenmedizin und Parasitologie Arbeit angefertigt unter der Leitung von Priv.-Doz. Dr. Cornelia Silaghi Gedruckt mit der Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Joachim Braun Berichterstatter: Priv.-Doz. Dr. Cornelia Silaghi Korreferent: Priv.-Doz. Dr. Monika Rinder Tag der Promotion: 18. Juli 2015 Die vorliegende Arbeit wurde nach § 6 Abs. 2 der Promotionsordnung für die Tierärztliche Fakultät der Ludwig-Maximilians-Universität München als kumulative Dissertation gestaltet. Für meine Eltern und für meinen Großvater, der ein Leben lang von einem Hochschulstudium geträumt hat (1921–2012) So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach der Zeit und den Umständen das Möglichste getan hat. JOHANN WOLFGANG VON GOETHE (1749–1832) Table of content TABLE OF CONTENT ABBREVIATIONS ..................................................................................................... X I. INTRODUCTION .......................................................................................... 1 II. LITERATURE REVIEW .............................................................................. 3 1. Giardia duodenalis .......................................................................................... 3 1.1. Taxonomy and assemblages ............................................................................. 3 1.2. Morphology ...................................................................................................... 7 1.3. Life cycle .......................................................................................................... 7 1.4. Pathogenesis and clinical symptoms ................................................................ 8 1.5. Epidemiology ................................................................................................... 9 1.6. Zoonotic potential .......................................................................................... 10 1.7. Diagnostics ..................................................................................................... 12 1.8. Treatment of Giardia infections ..................................................................... 14 2. G. duodenalis in South Eastern Europe. ..................................................... 15 2.1. Albania ........................................................................................................... 15 2.2. Bulgaria .......................................................................................................... 16 2.3. Croatia ............................................................................................................ 16 2.4. Hungary .......................................................................................................... 17 2.5. Macedonia ...................................................................................................... 17 2.6. Romania ......................................................................................................... 17 2.7. Serbia .............................................................................................................. 18 III. MATERIALS AND METHODS ................................................................ 21 1. Sample origin ................................................................................................ 21 2. Screening for Giardia positive samples ...................................................... 22 2.1. Enzyme linked immunosorbent assay (ELISA) ............................................. 22 3. Screening for Giardia cysts .......................................................................... 23 3.1. Screening with immunofluorescence assay (IFA) ......................................... 23 3.2. Screening with merthiolate iodine formalin concentration (MIFC) .............. 24 4. DNA extraction ............................................................................................. 25 5. DNA purification .......................................................................................... 25 VII Table of content 6. Quality control of extraction and quantisation of DNA ........................... 25 7. Polymerase Chain Reaction for detection of Giardia DNA ...................... 26 7.1. Nested PCR for the detection of the SSU rRNA gene ................................... 27 7.2. Nested PCR for the detection of the ITS1-5.8S-ITS2 region ......................... 28 7.3. Nested PCR for the detection of the beta giardin gene .................................. 29 7.4. Nested PCR for the detection of the glutamate dehydrogenase gene ............ 30 7.5. Nested PCR for the detection of the triosephosphate isomerase gene ........... 31 8. Visualisation of PCR products .................................................................... 32 8.1. Agarose gel electrophoresis ........................................................................... 32 8.2. Capillary electrophoresis ................................................................................ 33 9. DNA purification .......................................................................................... 33 10. Sequencing and sequence analysis: determination of assemblages ......... 33 11. Translation of nucleotide sequences into amino acids .............................. 33 12. Statistical analysis ........................................................................................ 34 IV. RESULTS ...................................................................................................... 35 1. Publication .................................................................................................... 35 2. Further results .............................................................................................. 58 V. DISCUSSION ............................................................................................... 61 VI. CONCLUSION ............................................................................................. 69 VII. SUMMARY .................................................................................................. 70 VIII. ZUSAMMENFASSUNG ............................................................................. 72 IX. REFERENCES ............................................................................................. 74 X. FIGURES ...................................................................................................... 93 XI. TABLES ........................................................................................................ 94 XII. ANNEX .......................................................................................................... 95 1. Global prevalence data of G. duodenalis .................................................... 95 2. Frequently used genes for molecular typing of G. duodenalis ................. 98 3. Nomenclature for incompletely specified bases in nucleic acid sequences .. 99 VIII Table of content 4. Sequence comparison with GenBank ....................................................... 100 5. Combined genotyping results .................................................................... 100 6. Equipment ................................................................................................... 100 7. Kits ............................................................................................................... 101 8. Chemicals .................................................................................................... 101 9. Nucleotides
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