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Arthropod-Borne Infections in the United Kingdom and Saudi Arabia

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Arthropod-Borne Infections in the United Kingdom and Saudi Arabia Arthropod-borne infections in the United Kingdom and Saudi Arabia Bassam Alharbi Supervised by: Dr. Kevin Bown School of Environment and Life Sciences University of Salford, Salford, UK Submitted In Partial Fulfilment of the Requirement of the Degree of Doctor of Philosophy, March 2018. TABLE OF CONTENTS Table of tables. .................................................................................................................... VI Table of Figures. ................................................................................................................ VII Declaration............................................................................................................................ X Acknowledgment. ................................................................................................................ XI Abstract. ............................................................................................................................. XII Chapter One ........................................................................................................................... 1 Introduction and the aims of this thesis ................................................................................. 1 1. Introduction. ...................................................................................................................... 2 1.2. Protozoan parasites. .................................................................................................... 3 1.2.1 .Trypanosomiasis................................................................................................... 3 1.2.1.1. Host range of Trypanosoma. ......................................................................... 3 1.2.1.2. Importance of Trypanosomes. ....................................................................... 4 1.2.1.3. Transmission of human African trypanosomiasis and Chagas disease. ........ 6 1.2.1.4. The life cycle of Trypanosoma cruzi. ............................................................ 7 1.2.1.5. Animal Trypanosomiasis. .............................................................................. 8 1.2.1.6. Important trypanosomes in the United Kingdom. ......................................... 8 1.2.2.Theileria. ............................................................................................................. 12 1.2.2.1. Theileria life cycle. ...................................................................................... 14 1.2.2.2. Clinical manifestations of Theileriosis. ....................................................... 15 1.2.3. Babesia. .............................................................................................................. 16 1.2.3.1. Human Babesiosis. ...................................................................................... 17 1.2.3.2. Babesia life cycle......................................................................................... 18 1.2.3.3. Babesiosis in animals. ................................................................................. 19 1.2.3.4. Symptoms of Babesia infection. .................................................................. 21 1.2.4. Bartonella. .......................................................................................................... 21 1.2.4.1. Animal Bartonellosis. .................................................................................. 23 1.2.4.2. Host specificity. ........................................................................................... 27 1.2.5. Candidatus midichloria ...................................................................................... 28 1.3. Vector transmission. ................................................................................................. 29 1.3.1. Ticks. .................................................................................................................. 29 1.3.1.1. Hard Ticks. .................................................................................................. 29 1.3.1.2. The importance of the tick population on vertebrate health. ....................... 34 II 1.4. Investigated host. ...................................................................................................... 35 1.4.1. Bank vole (Myodes glareolus). .......................................................................... 35 1.4.2. Wood mice (Apodemus sylvaticus). ................................................................... 36 1.4.3. Libyan jird (Meriones libycus) ........................................................................... 37 1.4.4. Desert hedgehogs (Paraechinus aethiopicus). ................................................... 38 1.4.5. Red foxes (Vulpes vulpes). ................................................................................. 39 1.4.6. Greater white toothed shrew (Crocidura russula). ............................................ 40 1.4.7. Pygmy shrew (Sorex minutus) ................................................................................... 41 1.5. Aims and Objectives ................................................................................................. 42 Chapter Two ........................................................................................................................ 43 Materials and methods ......................................................................................................... 43 General Methods. ................................................................................................................ 44 2.1. Sample collection. ..................................................................................................... 44 2.2. DNA extraction. ........................................................................................................ 45 2.2.1. DNA extraction from rodent and red fox blood samples using a genomic DNA kit. ................................................................................................................................. 45 2.2.3. DNA extraction from FTA cards for Saudi jirds and hedgehogs. ...................... 45 2.3. Polymerase chain reaction. ....................................................................................... 46 2.3.1. Mammalian Tubulin PCR. ................................................................................. 46 2.3.2. Polymerase chain reaction (PCR). ..................................................................... 47 2.3.3. Real time PCR. ................................................................................................... 51 2.4. Gel electrophoresis. .................................................................................................. 53 2.5. Purification of PCR products. ................................................................................... 53 2.6. DNA concentration. .................................................................................................. 54 2.7. Bioinformatics software. ........................................................................................... 54 2.7.1. Finish TV............................................................................................................ 54 2.7.2. Blast Tool. .......................................................................................................... 55 2.7.3. Clustal omega. .................................................................................................... 55 2.7.4. Statistical analysis. ............................................................................................. 55 2.7.5. Phylogenetic tree. ............................................................................................... 55 2.8. Next generation sequencing for tick samples. .......................................................... 56 2.8.1. DNA extraction. ................................................................................................. 56 2.8.2. 16S rRNA Amplicon PCR conditions. ............................................................... 56 2.8.3. First PCR Clean-up. ........................................................................................... 57 III 2.8.4. Labelling of purified PCR products. .................................................................. 58 2.8.5. Purification of labelled 16SrRNA libraries (second clean up). .......................... 59 2.8.6. Quantification of 16S rRNA libraries. ............................................................... 59 2.8.7. Library Denaturing and Mi-seq sample loading. ............................................... 60 2.8.7.1. Denaturation and dilution of DNA. ............................................................. 60 2.8.7.2. Denaturation of diluted library. ................................................................... 61 2.8.7.3 .Denaturation and dilution of PhiX control and Amplicon Library combination. ............................................................................................................. 61 2.9. Bioinformatics analysis. ............................................................................................ 61 Chapter Three ...................................................................................................................... 62 Comparison of vector-borne protozoan infections in rodents from England and Ireland: the effect of invasion. ...............................................................................................................
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