EPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF ANAPLASMA AND EHRLICHIA SPECIES INFECTING DAIRY CATTLE IN SMALLHOLDER FARMS IN PERI-URBAN NAIROBI, KENYA Dr. Shepelo Getrude Peter (BVM, MvetMed) A thesis submitted in fulfillment of requirements for Doctor of Philosophy Degree in Veterinary Clinical Studies (Medicine) Department of Clinical Studies Faculty of Veterinary Medicine University of Nairobi November 2020 DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Signature Date 19th November 2020 Shepelo Getrude Peter (BVM, MVetMed) SUPERVISORS This thesis has been submitted for examination with our approval as the University supervisors: Signature Date 19th November 2020 1. Prof. Daniel Waweru Gakuya (BVM, MSc, PhD) Signature Date 19th November 2020 2. Dr. Gabriel Oluga Aboge (BVM, MSc, PhD) Signature Date 19th November 2020 3. Prof. Ndichu Maingi (BVM, MSc, PhD) Signature Date 19th November 2020 4. Prof. Charles Matiku Mulei (BVM, PhD) i DEDICATION I dedicate this thesis to my late mother Peyasei Kuraru may her soul rest in peace, my father Peter Simintei, my husband Polycarp Matara and my daughters; Anatolia Nyaboke and Victoria Nanyori for the incredible love, patience and encouragement they showed me through my PhD journey. ii ACKNOWLEDGEMENT Every good and perfect gift comes from above; it comes down from the father of all light with Him there is no such thing as alteration or shadow caused by change (James 1:17). Trusting and claiming His Word, I give all glory and thanksgiving to the Almighty and powerful God for the special graces and favour He has given me through this PhD journey. Special gratitude to my supervisors; Prof. D.W. Gakuya, Dr. G.O. Aboge, Prof. N. Maingi and Prof. C.M. Mulei who believed in me and dedicated their valuable time and wisdom to the success of this research. I appreciate your great counsel and guidance without which this research work would not have been a success. I appreciate Dr. Dennis Makau who patiently guided me through the epidemiological analysis of my research data. You did not tire in the many times we had to repeat the analysis. Special appreciation to Hellen Kariuki and Dr. Esther Kanduma, who articulately and patiently took me through the DNA sequence analysis for this research. You were definitely God sent ladies, may you be blessed abundantly. To my soul-mate and friend Dr. Christine Mbindyo thank you for sharing every challenge and success I experienced through this doctoral journey. I am grateful for the support I received from my colleagues at the Department of Clinical Studies, especially Prof. James Mwangi-Nguhiu, who constantly encouraged me and believed I would deliver quality research and even when things got tough, he took to my side to guard my work. Additionally, I appreciate the laboratory assistance from Richard Omulo Otieno- Veterinary Parasitology Laboratory, Nixon Simani and Alice Kinyua-Hematology and Biochemistry Laboratory respectively and Alfred Mainga-Molecular Laboratory. iii Sincere appreciation to the University of Nairobi for waivering my school fees and providing a conducive environment that enabled me complete my PhD. I acknowledge the financial and mentorship support from CARTA (Consortium for Advanced Research Training in Africa) and Deutscher Akademischer Austauschdienst (DAAD). The special model of pedagogy taught through the CARTA program molded the researcher I am today through the guidance of the wonderful faculty across Africa and the North. I am forever indebted to their support and guidance. Special thanks to CARTA-cohort 8 fellows for creating a cheerful team of doctoral students that made research so exciting. To my mentor Prof. Peter Ngure, thank you for believing in me and challenging me to explore my full potential. To my wonderful and loving husband Polycarp Matara, you were there to listen and bear the heaviness of my challenges and the joy of my successes all through this doctoral journey, I sincerely appreciate you. Thank you for running errands on our behalf as I worked on this PhD. Special thanks to my beautiful PhD girls; Anatolia Nyaboke and Victoria Nanyori for the radiant love you always showed me despite being away from you for long hours and at times for months. To my baby-minder, Elizabeth Nashipae, thank you for being the sister that I never had. iv TABLE OF CONTENT DECLARATION ...................................................................................................................... I DEDICATION ......................................................................................................................... II ACKNOWLEDGEMENT .................................................................................................... III TABLE OF CONTENT .......................................................................................................... V LIST OF TABLES ................................................................................................................ IIX LIST OF FIGURES ............................................................................................................... XI APPENDICES ................................................................................................................. XIIIIII LIST OF ABBREVIATIONS ............................................................................................. XIV ABSTRACT .......................................................................................................................... XV CHAPTER 1: INTRODUCTION ........................................................................................... 1 1.1 Background ........................................................................................................................... 1 1.2 Problem statement ................................................................................................................ 6 1.3 Objectives ............................................................................................................................. 8 1.3.1 Overall objective ........................................................................................................ 8 1.3.2 Specific objectives ..................................................................................................... 9 1.3.3 Hypothesis .................................................................................................................. 9 1.4 Justification ........................................................................................................................... 9 CHAPTER 2: LITERATURE REVIEW ............................................................................. 11 2.1 Etiology and transmission of anaplasmosis and ehrlichiosis ............................................. 11 2.2 Prevalence of anaplasmosis and ehrlichiosis ...................................................................... 14 2.3 Risk factors for anaplasmosis and ehrlichiosis ................................................................... 15 2.4. Pathogenicity and clinical signs of anaplasmosis and ehrlichiosis.................................... 17 v 2.5. Diagnosis of anaplasmosis and ehrlichiosis ...................................................................... 21 2.6. Treatment and control of anaplasmosis and ehrlichiosis ................................................... 23 2.7. Molecular classification of Anaplasma and Ehrlichia pathogens ..................................... 24 CHAPTER 3: MATERIALS AND METHODS .................................................................. 26 3.1 Study area ........................................................................................................................... 26 3.2 Sample size calculation ...................................................................................................... 28 3.3 Study design ....................................................................................................................... 29 3.4 Sample collection ............................................................................................................... 31 3.4.1 Blood collection and serum harvesting .................................................................... 31 3.4.2 Tick collection and identification ............................................................................. 32 3.5 Laboratory analysis ............................................................................................................ 32 3.5.1 Blood smear preparation .......................................................................................... 32 3.5.2 Serological screening for Ehrlichia ruminantium .................................................... 32 3.5.3 DNA extraction from blood ..................................................................................... 33 3.5.4 DNA extraction from ticks ....................................................................................... 35 3.5.5 Primer design ........................................................................................................... 35 3.5.6 Amplification of Anaplasma and Ehrlichia DNA from blood and ticks ................. 36 3.5.7 Purification and sequencing of Anaplasma and Ehrlichia DNA from blood and ticks ........................................................................................................................................... 37 3.6 Data analysis ......................................................................................................................