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

More documents

Recommendations

Info

Chapter 5: Snap freezing of T. parva stabilates 73 ______________________________________________________________________________________________ Snap 2-Step Programmed A 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 B 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -8 -7 -6 -5 -4 -3 1 1 1 0.9 0.9 0.9 0.8 0.8 0.8 0.7 0.7 0.7 C 0.6 0.5 0.6 0.5 0.6 0.5 0.4 0.4 0.4 0.3 0.3 0.3 0.2 0.2 0.2 0.1 0 -8 -7 -6 -5 -4 -3 0.1 0 -8 -7 -6 -5 -4 -3 0.1 0 -8 -7 -6 -5 -4 -3 Stabilate dilution (ln t.e.) Figure 11: Titration curves (logistic prediction) of Theileria parva stabilates cryoprotected with (A) glycerol, (B) sucrose, (C) trehalose and frozen by snap-freezing, 2-step slow freezing and programmable freezer (control). Y-axis is the proportion of positive wells. The points are observed averages 5.4. Discussion Snap-freezing of stabilates in the cryoprotectant formulations used induced significant infectivity losses, especially in the glycerol stabilates. It is speculated that the low recovery of viable sporozoites could be due to the packaging of the stabilates in 1.5 ml vials. This may have resulted in a non uniform cooling due to the diameter of the column with the material at the periphery of the tube freezing first and protecting the deeper material, that cooled more slowly from outside in. Ideally, straws should be used to increase the surface area to volume ratio. Also, a limited range of
74 Chapter 5: Snap freezing of Theileria parva stabilates ______________________________________________________________________________________________ cryoprotectant concentrations were used and the concentration of sucrose and trehalose could have been too low for snap-freezing. Solutions used for vitrification are at least 1 M (James, 2004). The loss of infectivity of the glycerol stabilate could have been either due to low concentration or the low starting infectivity of the stabilate. It is worth exploring the use of different concentrations and combinations of cryopreservatives. This was a preliminary investigation that followed protocols of cryopreservative concentration and stabilate packaging that are used currently in T. parva stabilate production in our laboratory and internationally (OIE, 2005). Stabilate infectivities obtained for sucrose and trehalose of 27% and 23%, respectively of that of the control stabilate show that snap-freezing results in the recovery of viable T. parva sporozoites. This is a valuable finding suggesting that further exploration and optimisation of the snap-freezing process could yield an effective and economical method for cryopreservation of T. parva stabilates. It has been shown that when slow freezing of parasites is successful, vitrification has increased the surviving proportion at recovery from storage (James, 2004). We therefore conclude that, with improvements in the protocol, snap-freezing offers a potentially cheaper and easier means of cryopreserving T. parva stabilates.
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 23 and 24: 22 Chapter 1: Quantitation of Theil
Page 44 and 45: Chapter 2: Objectives 43 __________
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 73: 72 Chapter 5: Snap freezing of Thei
Page 78 and 79: Chapter 6: Storage of Theileria par
Page 90: Chapter 6: Storage of Theileria par
Page 94 and 95: Chapter 7: Lyophilisation of Theile
Page 100: Chapter 7: Lyophilisation of Theile
Page 104 and 105: Chapter 8: SNA and refinement of ti
Page 116: Chapter 8: SNA and refinement of ti
Page 120 and 121: Chapter 9: General Discussion 119 _
Page 122 and 123: Chapter 9: General Discussion 121 _
Page 124 and 125:
Chapter 9: General Discussion 123 _
Page 126:
Chapter 9: General Discussion 125 _
Page 130 and 131:
References 129 ____________________
Page 132 and 133:
References 131 ____________________
Page 134 and 135:
References 133 ____________________
Page 136 and 137:
References 135 ____________________
Page 138 and 139:
References 137 ____________________
Page 140 and 141:
References 139 ____________________
Page 142 and 143:
References 141 ____________________
Page 144 and 145:
References 143 ____________________
Page 146 and 147:
References 145 ____________________
Page 148:
APPENDICES
Page 151 and 152:
150 Appendices ____________________
Page 153 and 154:
152 Appendices ____________________
Page 155 and 156:
154 Appendices ____________________
Page 157 and 158:
156 Appendices ____________________
Page 159 and 160:
158 Appendices ____________________
Page 161 and 162:
160 Appendices ____________________
Page 163 and 164:
162 Appendices ____________________
Page 166:
SUMMARY
Page 169 and 170:
168 Summary _______________________
Page 171 and 172:
170 Summary _______________________
Page 174 and 175:
Samenvatting 173 __________________
Page 176 and 177:
Samenvatting 175 __________________
Page 178:
Curriculum Vitae
Page 181 and 182:
180 Carriculum Vitae ______________
Page 184 and 185:
List of Abbreviations 183 _________
show all

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

Create successful ePaper yourself

Delete template?

Save as template?