“Interactions between pathogenic and non- pathogenic Rickettsiales and the tick host” Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by ALAA AL-KHAFAJI October 2018 Table of contents Abstract ..................................................................................................................................... i List of Figures ............................................................................................................................ ii List of Tables ............................................................................................................................. v List of Abbreviations ............................................................................................................... vi Acknowledgments .................................................................................................................. vii Author’s Declarations ............................................................................................................. ix Chapter 1 – General Introduction ........................................................................................... 1 1. Introduction ................................................................................................................. 1 1.1 Tick Biology .............................................................................................................. 4 1.1.1 Tick classification.................................................................................................... 4 1.1.2 Tick morphology and feeding patterns ................................................................. 5 1.2 Ixodes ricinus ........................................................................................................... 7 1.2.1 Classification of the Ixodes ricinus complex ................................................... 7 1.2.2 The geographical distribution of I. ricinus ...................................................... 8 1.2.3 Ixodes ricinus morphology .............................................................................. 9 1.2.4 I. ricinus life cycle .......................................................................................... 11 1.3 Amblyomma variegatum ...................................................................................... 13 1.3.1 Amblyomma variegatum classification ........................................................ 13 1.3.2 The geographical distribution of Amblyomma variegatum ........................ 13 1.3.3 Morphology and life cycle of Amblyomma variegatum .............................. 14 1.4 Tick-borne diseases transmitted by I. ricinus and A. variegatum ....................... 16 1.5 Focusing on Anaplasma phagocytophilum, an exemplar pathogen of the Rickettsiales ....................................................................................................................... 20 1.5.1 Anaplasma phagocytophilum transmission and life cycle .......................... 21 1.5.2 Anaplasma phagocytophilum in the mammalian host ................................ 23 1.5.3 Anaplasma phagocytophilum compared with Anaplasma marginale ....... 26 1.5.4 Biological process involved in tick – Anaplasma phagocytophilum interaction ..................................................................................................................... 27 1.5.4.1 Host sensing/initial invasion ......................................................................... 29 1.5.4.2 Inhibition of cell apoptosis............................................................................ 30 1.5.4.3 Manipulation of the immune response ........................................................ 36 1.5.4.4 Anaplasma phagocytophilum infection benefits ticks ................................ 37 1.6 Tick-microbiome interaction ................................................................................. 39 1.6.1 Ticks and endosymbiotic relationships ........................................................ 39 1.6.2 Relationships between symbionts and pathogens ...................................... 43 1.7 Overall aims and objectives of the research reported in this thesis ................... 48 CHAPTER TWO ....................................................................................................................... 49 Quantification of co-infecting pathogenic and symbiotic Rickettsiales in two different tick species ................................................................................................................................... 49 2.1 Introduction ........................................................................................................... 49 2.1.1 Prevalence of the symbiont Candidatus Midichloria mitochondrii and pathogenic Rickettsiales in Ixodes ricinus from different parts of the UK .................. 49 2.1.1.1 Candidatus Midichloria mitochondrii, a unique endosymbiont of ticks ..... 50 2.1.1.2 Pathogens transmitted by Ixodes ricinus ..................................................... 52 2.1.1.2.1 Anaplasma phagocytophilum ................................................................... 53 2.1.1.2.2 Candidatus Neoehrlichia mikurensis ........................................................ 55 2.1.1.2.3 Tick-borne Rickettsioses ........................................................................... 56 2.1.2 Rickettsia africae and Ehrlichia ruminantium in Amblyomma variegatum 60 2.1.3 The aims of the work described in this chapter ........................................... 61 2.2 Materials and Methods ......................................................................................... 61 2.2.1 Ixodes ricinus sampling ................................................................................. 61 2.2.2 DNA extraction and quantification ............................................................... 63 2.2.3 Ca. M. mitochondrii qPCR ............................................................................. 65 2.2.4 Rickettsia qPCR .............................................................................................. 69 2.2.5 A. phagocytophilum and Ca. N. mikurensis qPCR ........................................ 71 2.2.6 Statistical analysis of bacterial densities ...................................................... 73 2.2.7 Amblyomma variegatum: Tick collection, DNA extraction and quantification ................................................................................................................ 74 2.2.8 Rickettsia africae qPCR ................................................................................. 74 2.2.9 Ehrlichia ruminantium pCS20 PCR ................................................................ 75 2.2.10 Generation of the pCS20 plasmid standard curve to calculate Ehrlichia ruminantium copy numbers ......................................................................................... 78 2.2.11 Ehrlichia ruminantium qPCR ......................................................................... 78 2.3 Results ................................................................................................................... 79 2.3.1 Confirmation of species identification and DNA yield ................................. 79 2.3.2 Ca. Midichloria mitochondrii density in different Ixodes ricinus life stages from Wales. ................................................................................................................... 79 2.3.3 Ca. Midichloria mitochondrii density in Ixodes ricinus nymphs from England 81 2.3.4 Pathogenic bacterial quantification ............................................................. 82 2.3.5 Rickettsia africae and E. ruminantium quantification in A. variegatum ..... 84 2.4 Discussion .............................................................................................................. 87 CHAPTER 3 ............................................................................................................................. 98 Co-evolutionary analyses of the symbiosis between Ixodes ricinus and the symbiont “Candidatus Midichloria mitochondrii” by multi-locus sequence typing ........................... 98 3.1 Introduction ........................................................................................................... 98 3.1.1 Phylogenetic analysis of populations of Ixodes ricinus in the UK and Europe 98 3.1.2 Phylogenetic analysis of Ca. Midichloria, the endosymbiont in Ixodes ricinus ticks .................................................................................................................. 101 3.1.3 The aims of the work described in this chapter ......................................... 102 3.2 Materials and Methods ....................................................................................... 102 3.2.1 Sample collection for MLST ......................................................................... 102 3.2.2 MLST design ................................................................................................. 105 3.2.3 PCR assays ................................................................................................... 106 3.2.3.1 Ca. Midichloria mitochondrii PCR assay ..................................................... 106 3.2.3.2 Tick MLST assay ........................................................................................... 107 3.2.4 Sequencing and sequence analysis ............................................................