Genotypic diversity and distribution of Giardia – Central Europe versus the Caribbean Doctoral thesis at the Medical University of Vienna for obtaining the academic degree Doctor of Philosophy Submitted by Ms. Mellesia Lee Supervisor: Associate Professor Dr. Julia Walochnik, MSc, PhD Institute of Specific Prophylaxis and Tropical Medicine Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna Kinderspitalgasse 15 A-1090 Vienna, Austria Vienna, March 2017 1 2 Table of Contents TABLE OF CONTENTS ........................................................................................................................... 3 I. DECLARATION ..................................................................................................................................... 7 II. LIST OF FIGURES ............................................................................................................................... 8 III. LIST OF TABLES ................................................................................................................................ 9 IV. SUMMARY ........................................................................................................................................ 10 V. ZUSAMMENFASSUNG ..................................................................................................................... 12 VI. PUBLICATIONS ARISING FROM THE DOCTORAL THESIS .......................................................... 14 VII. ABBREVIATIONS ............................................................................................................................ 17 VIII. ACKNOWLEDGEMENTS ............................................................................................................... 19 CHAPTER ONE: INTRODUCTION......................................................................................................... 20 1.1. OVERVIEW ..................................................................................................................................... 20 1.2. DISEASE BURDEN ........................................................................................................................... 20 1.3. GIARDIA TAXONOMY ....................................................................................................................... 21 1.3.1. Generic and species names ................................................................................................... 22 1.3.2. Classification by morphological characteristics ....................................................................... 23 1.3.3. Sub-classification of G. duodenalis ........................................................................................ 24 1.4. BIOLOGICAL CHARACTERISTICS OF GIARDIA: MORPHOLOGICAL FORMS ............................................... 26 1.4.1. Interphasic nuclei and cellular features .................................................................................. 28 1.4.2. Cytoskeletal structure: flagella ............................................................................................... 30 1.4.3. Cytoskeletal structure: adhesive disc ..................................................................................... 30 1.4.4. Cytoskeletal structure: median body ...................................................................................... 30 1.4.5. Cytoskeletal structure: microfilaments .................................................................................... 30 1.4.6. Trophozoite surface antigens: variant surface proteins .......................................................... 31 1.4.7. Occurrence and significance of antigenic variation ................................................................. 32 1.4.8. Surface antigens: Cyst Wall Proteins ..................................................................................... 32 3 1.4.9. Enzymes and energy metabolism .......................................................................................... 33 1.5. LIFE CYCLE .................................................................................................................................... 34 1.6. TRANSMISSION CYCLES .................................................................................................................. 35 1.6.1. Foodborne transmission ......................................................................................................... 36 1.6.2. Waterborne outbreaks ............................................................................................................ 37 1.6.3. Assemblages associated with waterborne and foodborne outbreaks ..................................... 38 1.7. GIARDIA IN ENVIRONMENTAL WATER AND MATRICES .......................................................................... 38 1.8. PATHOGENESIS AND CLINICAL MANIFESTATION ................................................................................. 39 1.8.1. Giardia in children .................................................................................................................. 39 1.8.2. Giardia in immunocompromised persons ............................................................................... 40 1.9. HOST IMMUNE RESPONSE ............................................................................................................... 40 1.10. TARGET GENES FOR MOLECULAR ANALYSIS .................................................................................... 40 1.11. PREVALENCE OF GIARDIASIS IN HUMANS ........................................................................................ 42 1.12. PREVALENCE OF GIARDIASIS IN ANIMALS ........................................................................................ 43 1.13. GIARDIASIS IN AUSTRIA ................................................................................................................. 43 1.14. GIARDIASIS IN JAMAICA ................................................................................................................. 44 1.15. DISTRIBUTION OF ASSEMBLAGES IN HUMANS AND ANIMALS .............................................................. 45 1.16. DISTRIBUTION OF THE A SUB-ASSEMBLAGES .................................................................................. 47 1.17. ZOONOTIC POTENTIAL ................................................................................................................... 49 1.18. TREATMENT OF GIARDIASIS ........................................................................................................... 49 1.19. VACCINATION AGAINST GIARDIA .................................................................................................... 50 1.20. LABORATORY DIAGNOSIS OF INFECTIONS ....................................................................................... 50 1.20.1. Microscopy ........................................................................................................................... 51 1.20.2. Direct smear ......................................................................................................................... 51 1.20.3. Concentration techniques ..................................................................................................... 51 1.20.4. Enzyme-Linked Immunosorbent Assay ................................................................................ 52 1.20.5. Immunofluorescence assay .................................................................................................. 52 1.20.6. Nucleic acid detection .......................................................................................................... 52 1.21. DNA EXTRACTION AND PCR AMPLIFICATION OF GIARDIA CYSTS FIXED IN FORMALIN ......................... 53 1.22. AIMS ........................................................................................................................................... 55 4 1.22.1. Publication I ......................................................................................................................... 55 1.22.2. Publication II ........................................................................................................................ 55 1.22.3. Publication III ....................................................................................................................... 55 CHAPTER TWO: MATERIALS AND METHODS ................................................................................... 58 2.1. ETHICS STATEMENT ........................................................................................................................ 58 2.2. SAFETY ......................................................................................................................................... 58 2.3. SAMPLE COLLECTION ...................................................................................................................... 58 2.4. MICROSCOPIC ANALYSIS ................................................................................................................. 58 2.5. MOLECULAR INVESTIGATION ............................................................................................................ 59 2.5.1. DNA extraction ....................................................................................................................... 59 2.5.2. Conventional PCR.................................................................................................................. 59