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

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Summary 167 ____________________________________________________________________________________________ But beyond this, my son, be warned: the writing of many books is endless… Ecclesiastes 12:12 The disease, East Coast fever, and its control as it relates to the present work are set out in the General Introduction of this thesis. The introduction outlines briefly the economic implications of the disease, its epidemiology, diagnosis, control and usage of titrated sporozoite stabilates in immunising cattle. The first chapter introduces a brief history of the disease, discusses the classification of the causative agent, Theileria parva, its lifecycle within tick and mammalian hosts, disease epidemiology and control methods. Focus is placed on the Infection and Treatment (I & T) method of immunisation. Survival of the parasite in natural and experimental conditions is reviewed. The core of the chapter deals with the specifics of quantitating T. parva sporozoite stabilates used for vaccination. Although in vitro studies of theileriosis concerning characterization and immunology of the parasites abound, these techniques remain largely under-utilized in stabilate quantitation in preference for in vivo methods. Current in vitro and in vivo methodologies and their respective shortcomings are critically reviewed together with their respective data analysis methods. The objectives of the thesis are presented in chapter two. The general objective was to develop and use the in vitro tool in view of improving vaccine production, storage and field immunisation against East Coast fever. More specifically, work was set out to 1) quantitate infectivity of stabilates stored at various temperatures, 2) compare stabilate quality from two extraction methods, 3) investigate feasibility of snap freezing stabilates, 4) evaluate potentially cheaper and more readily available stabilate media as alternatives to MEM and 5) refine the Sporozoite Neutralization Assay and validate it by assaying immune sera originating from cattle experimentally infected with recombinant p67, schizonts, live or dead sporozoites In chapter three, a study set up to resolve the question "Are T. parva sporozoite stabilates produced by crushing ticks by mortal and pestle of better infectivity than those produced by tissue grinders or homogenizers" is described. Observations were that manually extracted stabilates gave lower variation between production batches and the machine extracted ones were on average as infective as the manually extracted ones. Therefore, it was recommended that preference for either method should be based on cost, available infrastructure and conformity to standards.
168 Summary ______________________________________________________________________________________________ Chapter four is an account of the quest for a cheaper and more readily available stabilate medium. The present combination of MEM/BSA is very expensive and so a few alternatives were investigated. These were: PBS, RPMI 1640 (Life Technologies # 52400-025) and plain FCS. All were supplemented with BSA except FCS. RPMI was not less than 0.63 times as efficient as MEM at 95% confidence level (0.63-1.67). PBS and FCS were 59% and 67% as efficient as MEM, respectively. The chapter also revisits refreezing of stabilates and its effect on stabilate quality. Refreezing is practiced in polyvalent stabilate production but the infectivity loss due to this process has not been quantitated. This study estimated the loss to be 35% of original infectivity. It is proposed to take into account this loss in constituting polyvalent vaccines. Chapter five presents a preliminary attempt at snap-freezing of stabilates. This ultra-rapid cooling method could preclude the formation of intracellular ice crystals which is the main factor in freeze injury. As such, it would not only improve the sporozoite resuscitation rate but also cut down on the number of equipment used because stabilates would be directly plunged in liquid nitrogen after production. Test stabilates cooled this way were 24% as infective as controls. Although this recovery rate is low, the result shows that the technique is potentially feasible for T. parva stabilates. Some recommendations for optimizing the process are given, mainly targeted at adjustments in stabilate formulation and packaging that allows greater exposure of the formulation to the freezing agent (liquid nitrogen). The sixth chapter documents trials to quantitate stabilate loss of infectivity due to storage in ice baths (4ºC) and in a domestic freezer (-20ºC). Glycerol and sucrose cryopreserved stabilates of two T. parva stocks (Katete and Chitongo) were assessed at the two temperatures. Stabilates were stored for up to 24 hours and four weeks at 4ºC and -20ºC, respectively. Storage at 4ºC resulted in infectivity loss for the two parasite stocks of 1 and 4% per hour for Katete and Chitongo, respectively. The hourly loss was not significant (P=0.45). Comparison of the effect of time on cryopreservatives showed that time was not a significant factor for glycerol (P=0.35) but so for sucrose (P=0.04). Freshly thawed stabilates indicated higher infectivity for sucrose, at 10 times that of glycerol protected stabilates (95% CI: 6.2 - 16.7). It was concluded that both Katete and Chitongo stabilates had similar loss rates when kept on ice. Sucrose could be a better cryopreservative as seen from the base infectivities. However, this needs in vivo confirmation as effects of glycerol on lymphocytes were not assessed and glycerol stabilates sometimes gave false negative results in the least diluted stabilate series. For storage at -20ºC, sucrose and glycerol stabilates lost 98% and 61% of initial infectivity respectively, after one week of storage. It was speculated that intracellular water re-crystallisation
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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 178: Curriculum Vitae
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