Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in Scandinavia Rikke Rollum Thesis for the Master’s degree in Molecular Biosciences 60 study points Department of Molecular Biosciences Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO 2014 II © Rikke Rollum 2014 Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in Scandinavia Supervisors: Vivian Kjelland (UiA), Hans Petter Leinås (UiO), Audun Slettan (UiA) http://www.duo.uio.no/ Print: Reprosentralen, University of Oslo III IV Acknowledgements This master thesis was partly funded by the ScandTick project, which is a transnational project in Scandinavia devoted to ticks and tick-borne diseases. The laboratory work was conducted at the Department of Natural Sciences at the University of Agder (UiA) as an external thesis from the University of Oslo (UiO). I want to acknowledge all the people at UiO and UiA who have guided and helped me during my thesis. Vivian Kjelland (UiA), my supervisor, who gave me the opportunity to use her lab and for always being helpful, thorough and positive, which I really appreciate. You have inspired me to explore my opportunities, build connections and to be more confident and independent – Thank you! Audun Slettan (UiA), my co-supervisor, for always having a cheerful attitude and keeping my courage up when things did not go exactly as planned. I am also grateful to Hans Petter Leinaas (UiO), my co-supervisor, who have guided me in the writing process and for not letting me get carried away in fun facts. I would also like to thank Lars Korslund (UiA), who have helped me to understand and interpret the value of my results from a statistical point of view. I specially want to thank all the people at The Norwegian Institute of Public Health (FHI) for welcoming me and letting me work in their laboratories whenever I was in Oslo. Åshild Andreassen (FHI), thank you for showing me how you work at FHI, and for sharing all your knowledge about ticks and tick-borne diseases and general laboratory work. Katrine Paulsen (FHI), master student, for helping me during DNA extraction on the Abbott m2000sp machine – I would never have gotten any samples done if it wasn’t for you. Finally, I will devote huge thanks to my parents, family, boyfriend and friends for their love and support during this period – what would I have done without you all? This work was partly supported by Interreg IV A ÖKS and Norwegian Interreg funds. V Table of contents Acknowledgements ................................................................................................................... V Summary .................................................................................................................................... 1 Abbreviations and explanations ................................................................................................. 2 1 Introduction ........................................................................................................................ 4 1.1 Lyme borreliosis .......................................................................................................... 6 1.2 Ixodes ricinus ............................................................................................................... 7 1.2.1 Developmental stages ........................................................................................... 7 1.2.2 The tick as a vector ............................................................................................... 7 1.2.3 Transmission ........................................................................................................ 8 1.2.4 Distribution of I. ricinus in Scandinavia .............................................................. 8 1.3 Borrelia burgdorferi sensu lato ................................................................................... 9 1.3.1 General characteristics ......................................................................................... 9 1.3.2 Genospecies ........................................................................................................ 10 1.3.3 Genotyping ......................................................................................................... 13 Aims of the study ..................................................................................................................... 14 2 Materials and methods ..................................................................................................... 15 2.1 Tick collection ........................................................................................................... 15 2.2 DNA extraction.......................................................................................................... 16 2.2.1 DNeasy Blood and Tissue kit from Qiagen ....................................................... 16 2.2.2 Phenol-chloroform ............................................................................................. 17 2.2.3 Abbott m2000sp ................................................................................................. 18 2.2.4 NukEx kit from Orion Diagnostica .................................................................... 18 2.2.5 Purity and total concentration ............................................................................ 19 2.3 PCR and sequencing .................................................................................................. 19 2.3.1 Detection of B. burgdorferi sensu lato ............................................................... 19 2.3.2 Genotyping B. burgdorferi sensu lato species ................................................... 20 2.3.3 Nested PCR and sequencing .............................................................................. 21 2.4 Statistical analyses ..................................................................................................... 23 3 Results .............................................................................................................................. 24 3.1 Borrelia spp. in Scandinavia ..................................................................................... 24 3.1.1 Prevalence of Borrelia spp. in ticks ................................................................... 24 VI 3.1.2 Determination of Borrelia spp. .......................................................................... 25 3.2 Comparison of tick DNA extraction methods ........................................................... 27 3.2.1 Purity and concentration ..................................................................................... 27 3.2.2 PCR and sequencing ........................................................................................... 29 4 Discussion ........................................................................................................................ 31 4.1 Borrelia spp. in Scandinavia ..................................................................................... 31 4.1.1 Prevalence of Borrelia spp. in ticks ................................................................... 31 4.1.2 Detection of B. miyamotoi .................................................................................. 37 4.1.3 Evaluation of the detection methods .................................................................. 38 4.2 Comparison of tick DNA extraction methods ........................................................... 41 4.2.1 Study design ....................................................................................................... 41 4.2.2 Purity and concentration ..................................................................................... 43 4.2.3 PCR and sequencing ........................................................................................... 45 5 Conclusions ...................................................................................................................... 48 6 Future perspectives ........................................................................................................... 49 7 References ........................................................................................................................ 50 8 Appendices ....................................................................................................................... 59 8.1 Summary results - prevalence of Borrelia spp. ......................................................... 59 8.2 Summary results - tick DNA extraction methods ...................................................... 64 8.3 Sequences of primers and probes .............................................................................. 68 8.4 Physical maps of the genes ........................................................................................ 69 8.5 Chemicals and reagents ............................................................................................. 70 8.6 Solutions .................................................................................................................... 72 VII Summary Lyme borreliosis (LB) is the most frequent human tick-borne disease in Scandinavia. This master thesis is focusing on the Borrelia burgdorferi sensu lato (s.l.) complex, which is the causative agent of LB. Today, the B. burgdorferi s.l. complex consists of 20 different genotypes, in which three (B. afzelii, B. garinii and B. burgdorferi sensu stricto) are commonly known as human pathogens. During the past decades, the abundance