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

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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
VIII 5.4. DISCUSSION ........................................................................................................................................73 CHAPTER 6 - STORAGE OF THEILERIA PARVA STABILATES AT 4 AND -20°C ..............................75 6.1. INTRODUCTION ...................................................................................................................................77 6.2. MATERIALS AND METHODS................................................................................................................78 6.2.1. Stabilate preparations...................................................................................................................78 6.2.2. Peripheral Blood Mononuclear Cells (PBMC).............................................................................79 6.2.3. Ice bath storage.............................................................................................................................80 6.2.4. Storage at -20°C............................................................................................................................80 6.2.5. In vitro titrations ...........................................................................................................................80 6.2.6. In vivo titrations............................................................................................................................81 6.2.7. Statistical analysis.........................................................................................................................81 6.3. RESULTS .............................................................................................................................................83 6.4. DISCUSSION ........................................................................................................................................87 CHAPTER 7 - LYOPHILISATION OF THEILERIA PARVA SPOROZOITES .........................................91 7.1. INTRODUCTION ...................................................................................................................................93 7.2. MATERIALS AND METHODS................................................................................................................93 7.2.1. Parasite material and cryoprotectants..........................................................................................93 7.2.2. Freezing protocol..........................................................................................................................94 7.2.3. Drying protocols ...........................................................................................................................94 7.2.4. Assessment of infectivity................................................................................................................96 7.2.5. Vital staining .................................................................................................................................97 7.3. RESULTS .............................................................................................................................................97 7.3.1. In vitro and in vivo infectivity assessment ....................................................................................97 7.3.2. Vital staining .................................................................................................................................98 7.4. DISCUSSION ........................................................................................................................................98 CHAPTER 8 - SPOROZOITE NEUTRALISATION ASSAYS AND REFINEMENT OF TITRATION PROTOCOL......................................................................................................................................................101 8.1. INTRODUCTION .................................................................................................................................103 8.2. MATERIALS AND METHODS...............................................................................................................104 8.2.1. PBMC storage.............................................................................................................................104 8.2.2. Exclusion of Con-A .....................................................................................................................105 8.2.3. Use of 96-wellmicrotitration plates Vs microtubes .....................................................................105 8.2.4. Use of PCR for reading titration results .....................................................................................105 8.2.5. Inoculations.................................................................................................................................106 8.2.6. Sporozoite Neutralization Assay .................................................................................................107 8.2.7. Serology ......................................................................................................................................108 8.2.8. Statistical analysis.......................................................................................................................108 8.3. RESULTS ...........................................................................................................................................109 8.3.1. PBMC storage.............................................................................................................................109 8.3.2. Exclusion of Con-A .....................................................................................................................110 8.3.3. Use of 96-well microtitration plates Vs microtubes ....................................................................111 8.3.4. Use of PCR for reading titration results .....................................................................................111 8.3.5. Sporozoite Neutralisation Assay .................................................................................................112 8.3.6. Serology ......................................................................................................................................112 8.4. DISCUSSION ......................................................................................................................................113 CHAPTER 9 - GENERAL DISCUSSION......................................................................................................117 9.1. OVERVIEW........................................................................................................................................119 9.2. QUANTITATION OF T. PARVA SPOROZOITES IN EAST COAST FEVER VACCINE STABILATES.................119 9.3. VACCINE PRODUCTION PROCESSES ...................................................................................................121 9.4. STORAGE CONDITIONS ......................................................................................................................122 9.5. SPOROZOITE NEUTRALISATION ASSAYS ...........................................................................................123 9.6. CONCLUSIONS AND RECOMMENDATIONS..........................................................................................123 REFERENCES..................................................................................................................................................127 APPENDICES...................................................................................................................................................147
Page 1 and 2: Victor Mbao In vitro quantitation o
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Chapter 5 - Snap freezing of Theile
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APPENDICES
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SUMMARY
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Curriculum Vitae
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