In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

VII
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………….………….……………......…..…V
GENERAL INTRODUCTION..........................................................................................................................11
CHAPTER 1 – QUANTITATION OF THEILERIA PARVA SPOROZOITES FOR USE IN
VACCINATION AND SPOROZOITE NEUTRALIZATION ASSAY: LITERATURE REVIEW...........17
1.1. INTRODUCTION ...................................................................................................................................19
1.1.1. The history of East Coast fever .....................................................................................................19
1.1.2. Biology of T. parva........................................................................................................................20
1.1.3. Pathogenesis and pathology of T. parva .......................................................................................25
1.1.4. Immunity........................................................................................................................................26
1.1.5. Epidemiology ................................................................................................................................27
1.1.6. Diagnosis ......................................................................................................................................28
1.1.7. Control ..........................................................................................................................................29
1.2. THE PARASITE SURVIVAL....................................................................................................................33
1.2.1. Sporozoites....................................................................................................................................33
1.2.2. Schizonts........................................................................................................................................34
1.3. QUANTITATION OF T. PARVA ...............................................................................................................34
1.3.1. Direct quantitation........................................................................................................................36
1.3.2. In vivo quantitation.......................................................................................................................37
1.3.3. In vitro quantitation ......................................................................................................................38
1.3.4. Applications of in vitro quantitation .............................................................................................40
CHAPTER 2 - OBJECTIVES............................................................................................................................41
2.1. GENERAL OBJECTIVE..........................................................................................................................43
2.2. SPECIFIC OBJECTIVES .........................................................................................................................43
CHAPTER 3 –COMPARISON OF TWO THEILERIA PARVA EXTRACTION METHODS ..................45
3.1. INTRODUCTION ...................................................................................................................................47
3.2. MATERIALS AND METHODS................................................................................................................48
3.2.1. Preparation of T. parva sporozoite stabilates ...............................................................................48
3.2.2. Isolation and activation of PBMC.................................................................................................49
3.2.3. In vitro titration.............................................................................................................................49
3.2.4. Statistical analysis.........................................................................................................................50
3.3. RESULTS .............................................................................................................................................51
3.4. DISCUSSION ........................................................................................................................................52
CHAPTER 4 - INFECTIVITY OF THEILERIA PARVA SPOROZOITES FOLLOWING
CRYOPRESERVATION IN FOUR SUSPENSION MEDIA AND MULTIPLE FREEZING ...................55
4.1. INTRODUCTION ...................................................................................................................................57
4.2. MATERIALS AND METHODS................................................................................................................58
4.2.1. Media ............................................................................................................................................58
4.2.2. Stabilate preparations...................................................................................................................59
4.2.3. Refreezing cycles...........................................................................................................................60
4.2.4. In vitro titrations ...........................................................................................................................60
4.2.5. Statistical analysis.........................................................................................................................61
4.3. RESULTS .............................................................................................................................................62
4.4. DISCUSSION ........................................................................................................................................65
CHAPTER 5 - SNAP FREEZING OF THEILERIA PARVA STABILATES................................................67
5.1. INTRODUCTION ...................................................................................................................................69
5.2. MATERIALS AND METHODS................................................................................................................70
5.2.1. Stabilate preparations...................................................................................................................70
5.2.2. Freezing ........................................................................................................................................70
5.2.3. In vitro titrations ...........................................................................................................................71
5.2.4. Statistical analysis.........................................................................................................................71
5.3. RESULTS .............................................................................................................................................71