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

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Chapter 8: SNA and refinement of titration protocol 105 ______________________________________________________________________________________________ scatter (FSC) versus side scatter (SSC) constructed. FSC measures size of cells while SSC the complexity of intracellular contents. On another occasion, PBMC were collected and portion of the suspension distributed to 96-well microtitration plates (Nunc ® , Roskilde, Denmark) at 40 µl/well. The 96-well plates were incubated overnight at 37°C in an incubator (5% CO 2 in air). The remaining portion was stored in a 50 ml Cellstar ® tube (Greiner Bio-One, Frickenhausen, Germany) in a refrigerator. This set was for demonstrating sporozoite neutralisation. 8.2.2. Exclusion of Con-A A 96-well culture plate of T. parva-infected lymphoblasts was prepared as described in Appendix I.A with the activation step excluded. Another plate was set up with the original protocol (Appendix I.A without alterations) as a control. Cyto-centrifuge smears were made after 10 days of incubation and compared. 8.2.3. Use of 96-wellmicrotitration plates Vs microtubes Two titrations, one with the original (Appendix I.A) and the other with the refined (Appendix I.B) protocols were conducted using glycerol and sucrose stabilates to assess the effect of using plates for dilutions and incubation. The PBMC were collected, isolated and stored as described in Appendix I.B. The titrations were done the following day and cultures scored from cyto-centrifuge smears after 10 days. The refined protocol included a 1 minute shaking of plates after 30 minutes of primary incubation instead of the 1 hour shaking in microtubes for the original protocol. Effects of this reduced period of shaking on the infectivity of stabilates were analysed. 8.2.4. Use of PCR for reading titration results A titration of glycerol, sucrose and trehalose stabilates was set up as described in chapter 5. After removal of samples for cyto-centrifuge smears (100 µl from each well), the plates were treated as described in Appendix VI for DNA extraction and PCR reading. The PCR products were then run though 2 % (w/v) Agarose gel. Each well tested on PCR was scored positive or negative and compared to the corresponding result from Giemsa-stained smears (Figure 19). Analysis of the agreement between the two scoring procedures was done by calculating Cohen's kappa and McNemar's chi-square. Landis & Koch (1977) proposed the following scale to describe the degree of agreement between tests: 0.21-0.40, "Fair"; 0.41-0.60,"Moderate"; 0.61-0.80,
106 Chapter 8: SNA and refinement of titration protocol ______________________________________________________________________________________________ "Substantial"; 0.81-1.00 "Almost perfect". A kappa greater than 0.7 is considered a satisfactory agreement between tests. A B C D Figure 19: Cyto-centrifuge smears of PBMC at day 10 of culture: (A) smear at 20X, (B) positive smear at 400X, (C) Theileria parva schizont infected lymphoblasts at 1000X, (D) a negative smear at 400X. 8.2.5. Inoculations 8.2.5.1. Live sporozoite inoculation Animal number 131 was inoculated with 1ml of a 1/100 T. parva (Katete) stabilate subcutaneously below the parotid gland (ln. parotideus superficialis). At three weeks post inoculation, it was injected with Butalex (Schering-Plough, Uxbridge) at 2.4 mg/kg. Serum and plasma were collected before inoculation and once every week thereafter until week six post inoculation.
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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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SUMMARY
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Samenvatting 173 __________________
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Curriculum Vitae
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List of Abbreviations 183 _________
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