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

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Summary 169 ______________________________________________________________________________________________ might have occurred as stabilates were raised to this temperature resulting in freeze-injury of the sporozoites. Due to the high infectivity loss, this method of storage is not recommended. Chapter seven is an account of stabilate lyophilisation attempts aimed at repeating and improving a documented protocol. Lyophilisation is potentially a cheaper method than cryopreservation for stabilate storage. Authors of the documented attempt noted that although cattle inoculated with the product developed patent infections, less than 1% of sporozoites had survived the freeze-drying procedure. The rationale was that if it was repeatable, optimization of the procedure would raise the surviving proportion of sporozoites. Katete and Chitongo T. parva stabilates cryopreserved with sucrose or trehalose were tested. In addition, whole infected ticks, tick salivary glands and top layers of stabilate debris left after centrifugation at production were lyophilised. No sporozoite viability was evident in the lyophilised materials despite various modifications of the protocol and parasite presentations. Possible sources of failure are discussed. In chapter eight, refinements to the Sporozoite Neutralization Assay are outlined and the improved protocol is used to assay various sera. Presently, SNA analyses are based on counting infected cells which is laborious and prone to high variability. The idea of scoring wells positive or negative in plates is proposed. It is more user-friendly and presents binary data that are easily analysed by logistic regression. The chapter also argues for the less cumbersome use of microtitration plates instead of microtubes for stabilate in vitro titrations. By shaking plates for a minute and keeping the target cells in the refrigerator before infection instead of 37ºC, the system as a whole was still as efficient as the original protocol. In addition, a quicker way of reading the culture plates by PCR was explored. The in vitro protocol was shortened by 48 hours by skipping the step of activating Peripheral Blood Mononuclear Cells with Con-A. Sera collected at weekly intervals from cattle inoculated with schizonts and sporozoites were assayed in parallel with a positive (anti-p67) and negative (FCS) control. The tested sera were also subjected to PIM-ELISA, p67-ELISA and a SELISA using infected lymphoblasts as antigen. No significant sporozoite inhibition was evident from all three sera. Albeit, the assay demonstrated a clear distinction between the controls. Serum from the live sporozoite inoculation was positive on all three serology tests and that from the schizont inoculation was positive both on SELISA and PIM-ELISA. The lack of sporozoite neutralisation with serum from the animal receiving repeated schizont inoculation lends support to the documented observation of low p67 molecule expression in the schizont stage. This result also indicates inefficiency of sporozoite neutralisation by anti-PIM antibodies in vitro and that SNA is specific for anti-p67 antibodies.
170 Summary ______________________________________________________________________________________________ Chapter nine summarizes and discusses the main results of the thesis with overall conclusions. It offers recommendations for further research in related areas to clarify some of the questions and speculations raised in the present work.
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Victor Mbao In vitro quantitation o
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II Front cover photograph Copyright
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V ACKNOWLEDGEMENTS This work was ma
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VII TABLE OF CONTENTS ACKNOWLEDGEME
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IX APPENDIX I .....................
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General Introduction 13 ___________
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General Introduction 15 ___________
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Chapter 1 - Quantitation of Theiler
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20 Chapter 1: Quantitation of Theil
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Chapter 2: Objectives 43 __________
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Chapter 3 -Comparison of two Theile
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48 Chapter 3: Theileria parva extra
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Chapter 4: Suspension media and mul
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Chapter 5 - Snap freezing of Theile
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70 Chapter 5: Snap freezing of Thei
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Chapter 6: Storage of Theileria par
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Chapter 7: Lyophilisation of Theile
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Chapter 8: SNA and refinement of ti
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Page 120 and 121: Chapter 9: General Discussion 119 _
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Page 178: Curriculum Vitae
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Page 184 and 185: List of Abbreviations 183 _________
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