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

More documents

Recommendations

Info

Chapter 3: Theileria parva extraction methods 49 ________________________________________________________________________________________________ at 50 g for 5 min in 10 ml centrifuge tubes in a cooled (4°C) centrifuge. Supernatant fractions were harvested using sterile Pasteur pipettes, leaving the large tick debris behind, and transferred to a beaker to which an equal volume of cold glycerol (150 g/l) MEM/BSA was added drop-wise: first 5 min 1 drop every 2 sec and thereafter 1 drop/sec. The stabilates were stirred continuously in an ice bath throughout this process. Four batches of 50 ml stabilate each, giving a 4 tick-equivalent (t.e.) per ml concentration were produced. They were aliquoted into appropriately labelled 1.5 ml cryogenic vials (Nalgene1). The tubes were placed in an ultra-freezer (-80°C) for 24 h and then transferred to permanent storage in liquid nitrogen. 3.2.2. Isolation and activation of PBMC The method of Peripheral Blood Mononuclear Cells (PBMC) isolation is based on Goddeeris and Morrison (1988) modified by Marcotty et al. (2004). In brief, blood from the jugular vein was collected in Alsever's solution (Alsever and Ainslie, 1941) from a Friesian heifer. PBMC were isolated by density gradient and suspended at 6x10 6 /ml in culture medium comprising RPMI-1640 (Life Technologies # 52400-025) with 25 mM HEPES, FCS (15%), gentamycin (50mg/ml), 2- mercaptoethanol (0.5 M) and L-glutamine (0.2 M). The cells were kept at 37°C in a CO 2 incubator overnight (see Appendix I.A). 3.2.3. In vitro titration The T. parva stabilates were titrated in vitro as described by Marcotty et al. (2004), with some modifications. In brief, T. parva stabilates were thawed and diluted serially (6 series by 1.5-fold dilution) in 96-well flat-bottomed microplates across columns. Then 50 µl of PBMC were added to 50 µl of sporozoite suspension in each well. The plates were incubated for 1 h at 37°C in a CO 2 incubator (5% in air), centrifuged (210 g for 10 min) and the excess medium was decanted. Fresh culture medium (150 µl per well) was added to the plates which were then re-incubated for 10 days under the same conditions. On day 10, cyto-centrifuged samples were prepared and stained with Giemsa’s stain. Wells positive or negative for schizonts were scored 1 or 0 respectively. A total of 6 sessions were set up as shown in Table 2.
50 Chapter 3: Theileria parva extraction methods ______________________________________________________________________________________________ Table 2: Titration sessions for comparison of extraction methods. M-manual, H-homogeniser Stabilate Session M1 M2 H1 H2 1 48 48 2 71 72 3 72 72 4 93 91 5 88 95 6 48 48 TOTALS 212 208 215 211 3.2.4. Statistical analysis For the comparison of extraction methods, the binary results were analysed using a random effect logistic regression in Stata ® (Stata/SE 8.0 for Windows Statistical Software. Stata Corporation, Texas). The proportion of positive wells was the response variable and the logarithm of the stabilate concentration and the method of extraction were used as explanatory variables. The test session was taken as a random effect given the importance of the clustering effect within test sessions (Marcotty et al., 2004). The model can be written as follows: ⎛ π ⎞ log ⎜ ⎟ = α + β ln( t . e.) × ln( t. e.) + β ⎝1− π ⎠ method × method + ν + ε where π is the proportion of positive wells, α a constant, ln(t.e.) the natural logarithm of the sporozoite dose in tick-equivalents, β ln(t.e.) the coefficient of the stabilate dose, method the method used (0 for homogenizer and 1 for hand-extraction), β method the coefficient of the method, n the cross-sectional random effect of the session and e the residual error. The coefficients of the model were used to estimate ED 50 , the dose that is effective in 50% of the cases, for the two methods: ln ( ED ) 50 α + β method × method = − β ln( t. e.) A comparison of the sporozoite yield by the multiplicative model of the ratio ED hand /ED machine was then made by non-linear combinations of estimators: ln β ⎛ − = − method ED ⇒ hand = ⎜ β − method machine exp β ln( t. e.) EDmachine ⎝ β ln( t. e.) ( ED ) ln( ED ) hand ⎞ ⎟ ⎠
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 44 and 45: Chapter 2: Objectives 43 __________
Page 46: Chapter 3 -Comparison of two Theile
Page 49: 48 Chapter 3: Theileria parva extra
Page 53 and 54: 52 Chapter 3: Theileria parva extra
Page 55 and 56: 54 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
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 106 and 107:
Page 108 and 109:
Page 110 and 111:
Page 112 and 113:
Page 114 and 115:
Page 116:
Page 120 and 121:
Chapter 9: General Discussion 119 _
Page 122 and 123:
Page 124 and 125:
Page 126:
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?