The health of hedgehogs in relation to tick infestation and transmission of pathogens by the hedgehog tick, Ixodes hexagonus in vitro. Khyara van Hummel April 2014 – July 2014 P a g e | 1 The health of hedgehogs in relation to tick infestation and transmission of pathogens by the hedgehog tick, Ixodes hexagonus in vitro. Author: K.M.L.B. van Hummel Location: Utrecht Centre for Tick-borne Diseases (UCTD) Faculty of Veterinary Medicine Utrecht University Timeslot: April 2014 – July 2014 Supervisor: Prof. Dr. F. Jongejan The image on the front was taken by the author. P a g e | 2 Index Abstract .............................................................................................................................................5 Introduction ......................................................................................................................................6 Ixodes hexagonus ............................................................................................................................................... 6 In vitro feeding ................................................................................................................................................... 6 Objective of this research ................................................................................................................................... 7 Materials and method ........................................................................................................................8 Questionnaire ..................................................................................................................................................... 8 Ticks .................................................................................................................................................................... 8 Blood ................................................................................................................................................................... 8 Membranes ........................................................................................................................................................ 9 Feeding units ...................................................................................................................................................... 9 Bonbons ............................................................................................................................................................ 10 Attachment stimuli and odour test .................................................................................................................. 10 Water bath........................................................................................................................................................ 10 In vitro feeding procedure ................................................................................................................................ 10 DNA extraction ................................................................................................................................................. 11 PCR .................................................................................................................................................................... 11 Reverse Line Blot hybridization ........................................................................................................................ 12 Results ............................................................................................................................................. 13 Week 1 .............................................................................................................................................................. 13 Week 2 .............................................................................................................................................................. 13 Week 3 .............................................................................................................................................................. 14 Week 4 .............................................................................................................................................................. 16 Week 5 .............................................................................................................................................................. 16 Week 6 .............................................................................................................................................................. 18 Week 7 .............................................................................................................................................................. 19 Week 8 .............................................................................................................................................................. 21 Week 9 .............................................................................................................................................................. 22 Week 10 ............................................................................................................................................................ 24 Questionnaire ................................................................................................................................................... 26 .......................................................................................................................................................................... 27 P a g e | 3 RLB .................................................................................................................................................................... 27 Discussion ........................................................................................................................................ 28 Test feeding ...................................................................................................................................................... 28 Feeding 1 .......................................................................................................................................................... 28 Feeding 2 .......................................................................................................................................................... 28 Feeding 3 .......................................................................................................................................................... 28 Feeding 4 .......................................................................................................................................................... 28 Feeding 5 .......................................................................................................................................................... 28 Feeding 6 .......................................................................................................................................................... 28 Feeding 7, 8 and 9 ............................................................................................................................................ 29 Odour test ......................................................................................................................................................... 29 Questionnaire ................................................................................................................................................... 30 RLB .................................................................................................................................................................... 30 Conclusion ....................................................................................................................................... 31 References ....................................................................................................................................... 32 Appendix ......................................................................................................................................... 34 Appendix A ........................................................................................................................................................... 34 Enquête opvangcentra ..................................................................................................................................... 34 Appendix B ............................................................................................................................................................ 35 DNA EXTRACTION FROM TICKS ....................................................................................................................... 35 DNA EXTRACTION FROM BLOOD PROCEDURE ............................................................................................... 37 PCR RLB PROCEDURE ....................................................................................................................................... 39 REVERSE LINE BLOT HYBRIDIZATION PROCEDURE ......................................................................................... 41 IN VITRO FEEDING UNIT PREPARATION PROCEDURE ....................................................................................... 43 IN VITRO FEEDING BLOOD PREPARATION PROCEDURE ..................................................................................