Longitudinal Study Regarding Borrelia burgdorferi sensu lato Populations in Defined Habitats in Latvia von Mercy Akinyi Okeyo Inaugural-Dissertation zur Erlangung der Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians- Universität München Longitudinal Study Regarding Borrelia burgdorferi sensu lato Populations in Defined Habitats in Latvia von Mercy Akinyi Okeyo aus Suba, Kenia München 2020 Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Bakteriologie / Mykologie Arbeit angefertigt unter der Leitung von: Univ.-Prof. Dr. Reinhard K. Straubinger, Ph.D. Angefertigt am Nationalen Referenzzentrum für Borrelien, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit Oberschleißheim (LGL) Mentoren: Dr. rer. nat. Gabriele Margos Dr. med. Volker Fingerle Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Reinhard K. Straubinger, Ph.D. Berichterstatter: Univ.-Prof. Dr. Reinhard K. Straubinger, Ph.D. Korreferent/en: Univ.-Prof. Dr. Markus Meißner Tag der Promotion: 25. Juli 2020 To my loving partner Stefan and adorable daughter Alice Table of Contents List of Abbreviations ...................................................................................... 1 Publications ................................................................................................. 3 Introduction ............................................................................ 5 Causative Organism .............................................................. 7 2. 1 Borrelia Taxonomy .................................................................. 8 2. 2 Disease and Epidemiology .................................................... 12 2. 3 Vectors and Hosts ................................................................. 14 2. 4 Population Structure of B. burgdorferi s.l. ............................. 20 2. 5 Genome Organization and Protein Expression ..................... 23 2. 6 Molecular Typing of B. burgdorferi s.l. Species ..................... 26 2. 7 Multilocus Sequence Typing (MLST) .................................... 27 2. 8 DNA Sequencing ................................................................... 28 Sanger Sequencing Method (Sanger et al., 1977) ........ 28 Next Generation Sequencing (NGS) ............................. 29 Aims and Objectives ........................................................... 31 Materials and Methods ........................................................ 33 4. 1 Tick Samples ......................................................................... 33 4. 2 DNA Extraction Methods ....................................................... 34 Handling of Ticks from Oberschleißheim ....................... 35 DNA Extraction by Ammonium Hydroxide Solution (NH4OH) ......................................................................... 36 Page I Table of Contents DNA Extraction Using Commercially Available Kits ...... 36 4. 3 Polymerase Chain Reaction (PCR) ....................................... 37 Tick Cytochrome C Oxidase Subunit I PCR .................. 38 Real-time PCR to Screen for the Presence of Borrelia . 38 Nested PCR for Multilocus Sequence Typing (MLST) .. 39 Outer Surface Protein C (OspC) PCR ........................... 40 4. 4 Polyethylenglycol (PEG) DNA Precipitation .......................... 40 4. 5 Gel Electrophoresis ............................................................... 41 4. 6 Sequencing ............................................................................ 41 4. 7 Comparison of Nextera XT DNA Libraries and Nextera DNA Flex Libraries ......................................................................... 42 4. 8 Computational Sequence Analysis ........................................ 43 CLC Genomics Workbench ........................................... 43 Alignments and Phylogenies Construction .................... 43 Analysing GATC Sequences and Determining STs and Species .......................................................................... 44 4. 9 Statistical Analysis ................................................................. 44 Results .................................................................................. 47 5. 1 Comparison of Methods for Economic and Efficient Tick and Borrelia DNA Purification ....................................................... 47 5. 2 Comparison of Sequencing and Library Production Methods .......................................................................................... 54 5. 3 Infection Prevalence of B. burgdorferi s.l. in Questing Ticks from Latvia ............................................................................. 58 5. 4 Population Structure of B. burgdorferi s.l. in Latvia ............... 69 Spatial Distribution of Borrelia spp. in the Sampling Sites ....................................................................................... 69 Page II Temporal Distribution of B. burgdorferi s.l. in Latvia...... 71 Species Diversity ........................................................... 76 Temporal Distribution of Sequence Types in the Sampling Sites ............................................................... 77 Phylogenetic Analysis of B. burgdorferi s.l. based on MLST Housekeeping Genes .......................................... 82 Analysis of Relationships Among Sequence Types by goeBURST ..................................................................... 86 Detection of Potential Recombination Events ............... 91 Phylogenetic Analysis of OspC Gene Sequences ......... 93 Determination of OspC Major Groups ........................... 95 Comparison of Sequence Types and OspC type Strains ....................................................................................... 97 Discussion ......................................................................... 101 6. 1 Methods for Borrelia DNA Extraction and MLST Sequencing ........................................................................................ 101 6. 2 Sequence Acquisition for MLST/MLSA ............................... 104 6. 3 Infection Prevalence of B. burgdorferi spp. in Questing Ticks from Latvia ........................................................................... 105 6. 4 Species Diversity – Spatial and Temporal........................... 111 6. 5 Population Structure of B. burgdorferi s.l. in Latvia ............. 112 Temporal and Spatial Distribution of STs in the Collection Regions ........................................................................ 113 6. 6 Comparison of MLST and OspC Phylogenetic trees of B. burgdorferi s.l. from Latvia .............................................. 115 Summary - Zusammenfassung ........................................ 119 7. 1 English version .................................................................... 119 Page III Table of Contents 7. 2 Deutsche Version ................................................................ 121 Table of Figures .......................................................................................... 123 List of Tables ............................................................................................. 125 Bibliography ............................................................................................. 127 Appendix ............................................................................................. 153 Appendix 1: List of primers used in this study ......................................... 153 Appendix 2: Total number of ticks analyzed in the whole study .............. 156 Appendix 3: Samples used in this study with at least one MLST gene positive ................................................................................ 157 Appendix 4: Total number of samples used for spatio-temporal distribution analysis ................................................................................ 177 Appendix 5: Temporal distribution of B. burgdorferi species in Babite .... 177 Appendix 6: Temporal distribution of B. burgdorferi species in Jaunciems ........................................................................................ 178 Appendix 7: Temporal distribution of B. burgdorferi species in Kemeri ... 178 Appendix 8: OspC major groups of B. afzelii ........................................... 179 Appendix 9: OspC major groups of B. burgdorferi s.s. ............................ 184 Appendix 10: OspC major groups of B. garinii ......................................... 185 Appendix 11: OspC major groups of B. valaisiana .................................. 188 Appendix 12: Gadgetry used ................................................................... 189 Appendix 13: Consumables ..................................................................... 192 Appendix 14: Kits ..................................................................................... 193 Appendix 15: Chemicals .......................................................................... 194 Appendix 16: STs Analysed in this study. ................................................ 195 Acknowledgements .................................................................................... 199 Page IV List of Abbreviations µl Microliter 12s rRNA 12s ribosomal RNA ACA Acrodermatitis Chronica Atrophicans s.l. sensu lato s.s. sensu stricto COI Cytochrome C Oxidase Subunit I DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetate acid ELISA Enzyme-linked Immunosorbent