Rhipicephalus appendiculatus/zambeziensis complex from southern and eastern Zambia: genetic and phenotypic diversity related to the associated variation of the epidemiology of bovine theileriosis. Proefschrift ter verkrijging van de graad van doctor in de diergeneeskundige wetenschappen aan de faculteit diergeneeskunde Universiteit Gent, 2008 Jupiter MTAMBO Promotoren Prof. Dr. P. Dorny Prof. Dr. T. Backeljau Dr. M. Madder Vakgroep Virologie, Parasitologie en Immunologie Faculteit Diergeneeskunde, Universiteit Gent Salisburylaan 133, B-9820 Merelbeke ii Dedication This one is for my father (late) and mother, not because they will ever understand any of this, but because of the encouragement they gave me in “all things school” in those crucial initial years despite the fact that they themselves had little (father) or no (mother) education. If I were to live again and had a choice, I would choose you with your particular strengths and weaknesses. iii iv Acknowledgements This work comes to fruition trailing clouds of kindness of many institutions and people that deserve formal recognition for their help, support and accessibility throughout the period of my studies. Firstly, I extend a special thank you to my ITMA supervisor Dr. Maxime Madder, who gave me a lot of latitude in both the conceptualization and the execution of this study. Maxime, also had the unenviable task of going through my manuscripts, warm from the oven of my mind (read as confused), before any one else went through them! They came back expeditiously and… cool of course! For this I am grateful. A great deal of thanks must also go to Dr. Wim van Bortel and Patricia Roelants of the Entomology section of the Parasitology department and Pim de Rijk of the Mycobacteriology laboratory. Wim was especially helpful in the early stages when I was trying to come to terms with molecular techniques. He provided me access to additional web based resources (like BEN) without which some aspects of this study would have been impossible. Patricia was responsible for introducing me to the dos and don’ts of molecular biology laboratory practice. Both Wim and Patricia and indeed the whole entomology section laboratory group provided an amiable atmosphere, just what I needed especially in those initial stages when the “PCRs did not work.” Pim’s extensive experience in molecular biology laboratory techniques came in handy not a few times and I owe my first successful DNA sequence alignment to his tutelage. I cannot fully express my gratitude to my promoters Profs. Pierre Dorny and Thierry Backeljau. Prof. Dorny is thanked for facilitating my “return” to Antwerp, his helpful suggestions during the course of studies and his natural calming and assuring influence when things were bad. Prof. Backeljau is especially thanked for his patience in reading every single component of this thesis and his invaluable comments, contributions and useful remarks. I am further grateful to him for securing for me those difficult-to-find software packages and references. May I also express my sincere appreciations to all members of staff of the Animal Health Department of the ITMA for their support in many and diverse ways. Viviane and Monique are thanked for their patience with my late knocking off and always reminding me of public holidays. Daniele De Bois deserves hearty thanks for ably v handling all the administrative aspects of my study. Bjorn and his lab crew are thanked for their efficiency in ordering stuff and their friendship. I thank all the teaching staff for their contributions, remarks and suggestions especially during presentations. Special thanks to Dr. Tanguy Marcotty for his incredible commitment to the success of what came to be called the “Zambian PhD group”. Even in the face of personal calamity your commitment was unwavering! Thanks are also due to Dr. N. Speybroeck (St.Niklass) for that treasure trove of references you availed to me. You were really God sent! This work would not have seen the light of day had it not been for the determination and foresight of Prof. ir. Dirk Berkvens and Dr. Mulumba. I thank you both for all the time you spent on the ASVEZA project in general and the Zambian PhD programme in particular and for believing in me personally and consequently granting me the opportunity to take a shot at this PhD study. This definitely was not a birdie but I hope I did not, as it were, abuse your trust. I extend my thanks to Dr. Peter Gilbert Sinyangwe the Director of the Department of Veterinary and Livestock Development for his encouragement in the course of the study by periodically checking on our (my colleagues and I) progress and facilitation of my study leave. I should also additionally thank my PhD friends and colleagues (past and present) in the department and the Institute in general for their camaraderie, i.e. Chola Shamputa and Anthony Ablordey (Mycobacterium lab), Paul Fandamu, Baba Soumare, Justin Masumu (chief), Victor Mbao, Christopher Kubi, Nynke Deckers, Amel Abdel and Akoda G. Komlan. I would like to acknowledge, Chaka George, Mataa Liywalii, Mostin Laurent, Penne Koert, Billiouw Michel, Elyn Rik, Mulenga Patrick, Henry Chungu (late), Bubala Desmond, Keembela Valentine, Nyambe Robert Ita, Hadobe Oliver , Chikandi Evans, Chimbala Gerald, Banda Victor, Syandebwe Dixon and many others from the former ASVEZA project for their generous assistance in research and field work. I equally lift my hat off to the farmers especially Mr. Bbuku of Nteme and the farmer group at Halubilo who allowed us (ASVEZA project) to use their herds for research. My social and spiritual life would have been pathetic if it were not for the many friends I met at Antwerp Christian Fellowship. I shall always treasure the friendship vi of the Rev. William Martin and his wife Deborah, Steve and Linda Petra, family Seth van Herck and Gracina, Craig and Heather Wallis, Denise Gormley, Agnita Souman, Dorothy Tanyi, Pauline Aguire, Ian MacBeath, Paul Kattepogu and Geovanna Benedictis to mention just a few. You guys meant Antwerp to me! Ann Schervenels and Helga Bodges of the student service are thanked too for their every effort made towards this work. I am grateful to the Belgium Government for funding my study through the Belgium Technical Cooperation (BTC) to the Government of the Republic of Zambia in the context of the project “Assistance to the Veterinary Services of Zambia (ASVEZA) in Southern, Central and Eastern provinces”. I thank Mr. Thierry Copin at the BTC for his able administration of the disbursement of the funds. Finally, I wish to thank “Biggie” and all members of my family for their support. Munsaka Chami Syatwiinda for her patience and love. vii viii CHAPTER 1 - GENERAL INTRODUCTION 1 1.1 Introduction 2 1.2 Theileria parva 2 1.2.1 Life cycle of T. parva 3 1.3 The disease (East Coast fever) 5 1.3.1 The mammalian host 5 1.3.2 The distribution of ECF 5 1.3.3 The epidemiology of ECF 7 1.3.4 The control of ECF 8 1.4 The vectors 9 1.4.1 Systematic relationship of R. appendiculatus and R. zambeziensis to other ticks 9 1.4.2 The Genus Rhipicephalus 11 1.4.3 General life cycle of R. appendiculatus and R. zambeziensis 12 1.5 Rhipicephalus appendiculatus and R. zambeziensis in Zambia 14 1.5.1 The vegetation of Zambia 15 1.5.2 Agro-ecological zones of Zambia 15 1.6 References 18 CHAPTER 2 - A REVIEW OF VARIATION WITHIN AND BETWEEN R. APPENDICULATUS AND R. ZAMBEZIENSIS 25 2.1 Introduction 26 2.2 Why study variation? 26 2.3 Intra-specific variation 27 2.4 Species concepts 28 2.4.1 Morphological species concept (MSC) 28 2.4.2 Phenetic species concept (PhSC) 29 2.4.3 The biological species concept (BSC) 30 2.4.4 The evolutionary species concept (ESC) 30 2.4.5 The phylogenetic species concept (PSC) 31 2.5 Speciation 32 2.5.1 Geographic speciation 32 2.6 Reconstructing speciation 35 2.6.1 Neighbour-joining 36 2.6.2 Maximum parsimony 36 2.6.3 Maximum likelihood 37 ix 2.6.4 Non-parametric bootstrapping 38 2.6.5 Bayesian inference of phylogeny 39 2.6.6 Choice of gene sequence to use in reconstruction of speciation 39 2.7 Phenotypic variation within and between R. appendiculatus and R. zambeziensis 42 2.7.1 Morphological variation 42 2.7.2 Morphological description of Rhipicephalus appendiculatus 43 2.7.3 Morphological description of R. zambeziensis 47 2.7.4 Morphological variation between R. appendiculatus and R. zambeziensis 49 2.7.5 Cellular variation 50 2.7.6 Biochemical variation 50 2.7.7 Behavioural variation 51 2.7.8 Phenology 54 2.7.9 Ecological variation 55 2.7.10 Distribution 55 2.7.11 Life cycle traits 57 2.8 Genetic variation 57 2.8.1 Source and nature of genetic variation 57 2.8.2 Genetic variation within R. appendiculatus 57 2.8.3 Genetic variation within R. zambeziensis 57 2.8.4 Genetic variation between R. zambeziensis and R. appendiculatus 58 2.9 Conclusion 58 2.10 References 59 CHAPTER 3 - OBJECTIVES OF THE STUDY 69 3.1 Introduction 70 3.2 General objectives 71 3.3 Specific objectives 71 3.4 References 72 CHAPTER 4 - PHENOTYPIC COMPARISON OF THREE RHIPICEPHALID TICK STOCKS FROM ZAMBIA 75 4.1 Introduction 76 4.2 Materials and Methods 77 4.2.1 Tick stocks 77 4.2.2 Morphological identification, Scoring and Measurements 78 4.2.3 Life cycle traits 78 4.2.4 Statistical analysis 78 x 4.3 Results 79 4.4 Discussion 84 4.5 References 88 CHAPTER 5 - INTRASPECIFIC VARIATION IN RHIPICEPHALUS APPENDICULATUS FROM EASTERN AND SOUTHERN PROVINCES 91 5.1 Introduction 92 5.2 Materials and Methods 93 5.2.1 Sampling 93 5.2.2 Identification of ticks and DNA extraction 95 5.2.3 PCR amplification, DNA sequencing and alignment 95 5.2.4 Phylogenetic analysis 96 5.3 Results 96 5.3.1 PCR amplification and sequence alignment statistics 96 5.3.2 Phylogenetic analysis 97 5.4 Discussion 98 5.5 References 102 CHAPTER 6 - CORRELATING GENETIC AND ECOLOGICAL VARIATION IN RHIPICEPHALUS APPENDICULATUS FROM ZAMBIA WITH R.