UNIVERSITY OF NAIROBI THE BABOON (PAPIO ANUBIS) –PLASMODIUM KNOWLESI MODEL FOR PLACENTAL MALARIA BY ONDITI FAITH ISDORAH, MSc H80/98669/2015 A thesis in fulfillment of the requirement for the degree of Doctor of Philosophy in Biochemistry , School of Medicine, Biochemistry Department, University of Nairobi. 2015 1 DECLARATION This thesis is my original work and has not been presented in any other institution for any degree award or other qualifications. Onditi Faith Isdorah This thesis has been submitted with our approval as supervisors: 1. Prof. Charles O. A. Omwandho, Department of Biochemistry, University of Nairobi. 2. Dr. Hastings S. Ozwara, Department of Tropical and Infectious Diseases, Institute of Primate Research. Dr. Edward. K Muge, Chairman, Department of Biochemistry ii DEDICATION This work is dedicated to my dear husband Abraham Agumba, daughter Joy Baraka and sons Isaac Jaden and Favor Nehemiah. iii ACKNOWLEDGEMENT First and foremost, I would like to thank the Lord God Almighty for making this come to pass. I am indebted to my immediate family: my husband Pastor Abraham and children; Joy, Isaac and Favor for their unconditional support, patience and love during the course of my study. I would also like to acknowledge my parents for their encouragement and support. I thank my supervisors Prof. Charles O. A. Omwandho and Dr. Hastings S. Ozwara for their wisdom, mentorship and constant academic and moral support throughout my study. Sincere appreciation to the entire malaria team in the department of Tropical and Infectious Diseases and the Animal Sciences Department at the Institute of Primate Research (IPR). I am particularly grateful to Dr. Langoi and Dr. Akinyi who conducted the very first caesarian section in this study and Mr. Zachariah Maheli, James Ndungu, Esther Kagasi, Onkoba Nyamongo, Ruth Mumo and Victor Irungu for their support during sample collection and monitoring of experimental animals. May the Lord reward you in abundance. Last but not least, special thanks go to NACOSTI and WHO for awarding grants that supported this study. iv TABLE OF CONTENT DECLARATION ............................................................................................................................ ii DEDICATION ............................................................................................................................... iii ACKNOWLEDGEMENT ............................................................................................................. iv TABLE OF CONTENT .................................................................................................................. v LIST OF FIGURES ..................................................................................................................... xiv LIST OF TABLES ..................................................................................................................... xviii EXECUTIVE SUMMARY ......................................................................................................... xix ABSTRACT ................................................................................................................................. xxi CHAPTER 1: GENERAL INTRODUCTION ............................................................................... 1 1.1 Background information ...................................................................................................... 1 1.2 Problem statement ................................................................................................................ 4 1.3 Choice of the baboon as a study model ............................................................................... 5 1.4 Justification of the study ...................................................................................................... 6 1.5 Research questions ............................................................................................................... 7 1.6 Research hypothesis ............................................................................................................. 7 1.6.2 The null hypothesis (Ho) .................................................................................................. 7 1.6.2 The alternative hypothesis (Ha) ........................................................................................ 8 1.7 Objectives ............................................................................................................................ 8 1.7.1 General objective.............................................................................................................. 8 1.7.2 Specific objectives............................................................................................................ 8 v CHAPTER 2: LITERATURE REVIEW ........................................................................................ 9 2.1 Malaria epidemiology and disease burden ........................................................................... 9 2.2 Life cycle of Plasmodium .................................................................................................. 12 2.3 Pathogenesis of malaria ..................................................................................................... 14 2.4 Economic and social burden of malaria ............................................................................. 16 2.5 Malaria control ................................................................................................................... 17 2.6 Placental malaria (PM) ...................................................................................................... 18 2.7 Characteristics of malaria infected placenta ...................................................................... 19 2.8 Parasite adhesion in the placenta ....................................................................................... 20 2.9 The placental barrier .......................................................................................................... 21 2.10 Immunological aspects of pregnancy................................................................................. 24 2.11 The effect of pregnancy on peripheral immune responses ................................................ 27 2.11.1 T lymphocytes ................................................................................................................ 27 2.11.2 Uterine natural killer (uNK) cells................................................................................... 28 2.11.3 Macrophages .................................................................................................................. 29 2.11.4 Dendritic cells ................................................................................................................ 30 2.12 Immune responses in placental malaria ............................................................................. 30 2.13 Pathology of placental malaria........................................................................................... 32 2.14 Adverse pregnancy outcome of placental malaria ............................................................. 33 2.15 Use of non-human primate (NHP) models in biomedical research ................................... 34 2.16 Non-human primates as models for placental malaria ....................................................... 35 vi 2.17 Plasmodium knowlesi as a model for human malaria ........................................................ 36 2.18 Life cycle of Plasmodium knowlesi ................................................................................... 38 2.19 Importance of this study in placental malaria .................................................................... 39 CHAPTER 3: MATERIALS AND METHODS .......................................................................... 41 3.1 Study site ............................................................................................................................ 41 3.2 Study design ....................................................................................................................... 41 3.3 Experimental animals and parasites ................................................................................... 44 3.4 Processing baboon red blood cells for parasite culturing .................................................. 45 3.5 In vitro propagation of Plasmodium knowlesi ................................................................... 46 3.6 Cryopreservation of in vitro cultured P. knowlesi ............................................................. 46 3.7 Preparation of P. knowlesi antigens for immunological assays ......................................... 47 CHAPTER 4: HAEMATOLOGICAL AND CLINICAL INDICES OF PLACENTAL MALARIA IN NON-IMMUNE PREGNANT BABOONS......................................................... 48 4.1 Introduction ........................................................................................................................ 48 4.2 Materials and methods ....................................................................................................... 49 4.2.1 Infection of baboons with P. knowlesi H strain parasites .............................................. 49 4.2.2 Observation of parasitaemia and giemsa staining .......................................................... 50 4.2.3 Clinical monitoring of infected baboons ........................................................................ 50 4.2.4 Determination