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

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Chapter 5: Snap freezing of T. parva stabilates 71 ______________________________________________________________________________________________ 5.2.3. In vitro titrations Titrations of the stabilates were as described by Marcotty et al. (2004) and modified as described earlier (chapter 3). Briefly, stabilates were thawed rapidly in a water bath at 37°C for 5 min. They were then diluted serially eight times (2-fold) and mixed with bovine Peripheral Blood Mononuclear Cells (PBMC) in 96-well flat-bottom microtitration plates (Nunc ® , Roskilde, Denmark). The plates were incubated for 10 days at 37°C in 5% CO 2 in air. After incubation, cytocentrifuged samples of each well were made on microscopy slides. The cells were stained with Giemsa’s stain and examined for the presence of schizonts. 5.2.4. Statistical analysis Data were analyzed in a generalised linear latent and mixed model in Stata ® 9 (StataCorp., Stata Statistical Software: Release 9. College Station, TX: StataCorp LP). The proportion of positive wells was the response variable (binomial) and explanatory variables were the natural logarithm of the stabilate concentration expressed in tick equivalents (ln t.e.), the cryoprotectant and the freezing method. The two sessions during which stabilates were titrated were entered as random effects. To compare the individual infectivities of the stabilates, ratios of their effective doses (ED) [ ED control EDx ] where x = a stabilate undergoing a particular freezing method, were calculated by non-linear combination of estimators [nlcom] in Stata ® 9 (chapter 3). The ED was taken as a stabilate concentration giving a particular proportion of positive wells e.g. ED 50 was the dose at which 50% of the wells were positive. The [nlcom] process also fits 95% confidence intervals of the estimates. The ratios quantified the sporozoite infectivities with respect to the infectivity of the control stabilate (sucrose stabilate frozen by programmed freezing). 5.3. Results Numbers of wells read for sucrose, trehalose and glycerol were 189, 191 and 152, respectively. Sucrose stabilate undergoing the snap-freezing retained 27% of infectivity compared to the same stabilate in the control programmed freezing procedure. There was no significant difference in infectivity between sucrose stabilates from programmed freezing and those from 2-step slow freezing (P=0.88). Table 6 summarizes the results of the titrations and the titration curves are illustrated in Figure 11.
72 Chapter 5: Snap freezing of Theileria parva stabilates ______________________________________________________________________________________________ Table 6: Theileria parva sporozoite infectivity, expressed as Effective Dose (ED) ratios, after 2- step slow freezing, snap-freezing and programmed freezing (control) in glycerol, sucrose and trehalose Method CPA ED ratio Lower CL Upper CL Glycerol 0.09 0.04 0.22 2-step Sucrose 1.1 0.67 1.85 Trehalose 1.05 0.53 2.09 Glycerol 0.02 0.007 0.05 Snap freezing Sucrose 0.24 0.13 0.43 Trehalose 0.22 0.11 0.47 Glycerol 0.08 0.04 0.17 Control Trehalose 0.94 0.58 1.53 This table derives from the model described in the Materials and Methods and presented in figure 11. CPA = Cryoprotectant agent; ED ratio = EDsucrose-control/EDx where x = stabilate undergoing a particular method of freezing; CL = 95% Confidence Limit. On comparing the methods, there was no difference in the efficiency of the two-step slow freezing from the control (P=0.68). The snap-freezing was significantly less efficient (P
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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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APPENDICES
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SUMMARY
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Curriculum Vitae
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