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

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Chapter 2: Objectives 43 ______________________________________________________________________________________________ If A is success in life, then A equals x plus y plus z. Work is x; y is play; and z is keeping your mouth shut. Albert Einstein (1879 - 1955) 2. CHAPTER 2 2.1. General Objective To develop and to use the in vitro tool in view of improving Theileria parva live vaccine production, storage and immunisation against East Coast fever. 2.2. Specific Objectives To quantitate T. parva (types Katete and Chitongo) stabilate infectivity loss due to storage conditions. The conditions to be investigated are storage at 4ºC (on ice) for up to 24 h and at -20ºC for up to four weeks using glycerol and sucrose as cryoprotecting agents. It was envisaged that if stabilate could be stored for at least a week in domestic freezers without appreciable infectivity loss, it would reduce the cost of storing it in liquid nitrogen during immunisation campaigns. To quantitate and compare infectivity of T. parva stabilates produced by manual and homogenizer methods of sporozoite extraction. Though OIE recommends use of homogenizers, different labs prefer one or the other method. However, no comprehensive study has been conducted that quantitatively compares the infectivities of the resultant stabilates ceteris paribus. It is envisaged that using a homogenizer would ease and improve the production of stabilate for immunisation purposes. To quantitate infectivity of T. parva stabilates after snap freezing, multiple refreezing and lyophilisation attempts. It is envisaged that snap freezing of stabilates would ease stabilate production. Determining the infectivity loss of refrozen stabilates would allow for adjustment of doses during the combination stage of polyvalent vaccine production. Lyophilised stabilate would be less expensive to store and therefore cheaper to deliver to farmers. To quantitate and compare the effects of suspending sporozoites in MEM (the standard stabilate medium), RPMI 1640, PBS and FCS as stabilate media. RPMI 1640 is more commonly used in cell culture laboratories than MEM whereas PBS is the most common buffer. FCS is a potentially cheaper alternative to the routinely used MEM complemented with bovine serum albumin.
44 Chapter 2: Objectives To optimize a Sporozoite Neutralisation Assay protocol and validate it by assessing neutralisation potential of various immune sera originating from cattle inoculated with recombinant p67 antigen, live sporozoite stabilate, dead sporozoites and schizont-infected lymphoblasts. The assumption was that cattle immunized with schizonts or dead sporozoites could induce production of anti-p67 antibodies detectable by the assay.
Page 1 and 2: Victor Mbao In vitro quantitation o
Page 3 and 4: II Front cover photograph Copyright
Page 6 and 7: V ACKNOWLEDGEMENTS This work was ma
Page 8 and 9: VII TABLE OF CONTENTS ACKNOWLEDGEME
Page 10: IX APPENDIX I .....................
Page 14 and 15: General Introduction 13 ___________
Page 16 and 17: General Introduction 15 ___________
Page 18: Chapter 1 - Quantitation of Theiler
Page 21 and 22: 20 Chapter 1: Quantitation of Theil
Page 46: Chapter 3 -Comparison of two Theile
Page 49 and 50: 48 Chapter 3: Theileria parva extra
Page 58 and 59: Chapter 4: Suspension media and mul
Page 68: Chapter 5 - Snap freezing of Theile
Page 71 and 72: 70 Chapter 5: Snap freezing of Thei
Page 78 and 79: 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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Chapter 9: General Discussion 119 _
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APPENDICES
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SUMMARY
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Curriculum Vitae
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180 Carriculum Vitae ______________
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List of Abbreviations 183 _________
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