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

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Chapter 4: Suspension media and multiple freezing of Theileria parva stabilates 65 ______________________________________________________________________________________________ 4.4. Discussion Sporozoite infectivities for stabilates prepared in PBS, RPMI and FCS when compared to MEM were not statistically significantly different. However, the RPMI stabilates were closest in infectivity to MEM stabilates as shown by the equivalence test estimate (minimum 0.63 times). This observation agrees with the findings of Kimbita et al. (2004) when they compared different media including Leibovitz-15, Optimem and Iscove’s MEM. In their study, RPMI and MEM stabilates showed similar infectivity when compared to L-15. Gray & Brown (1981) showed that neat serum could have inhibitory effects on sporozoite infectivity and this could explain why the ED of FCS was lower than that of MEM. However, the effect may not be very pronounced as the confidence interval is wide. FCS is expensive (about €107/l) due to the stringent standards required for international distribution. If it could be produced locally in existing regional laboratories with nationally or regionally accepted quality control standards from known naïve dams, it could be very much cheaper than international grade FCS. Newborn calf serum might also be used (€67.4/l) and would be much cheaper from local sources. The cost of using locally produced reagents would then be lower than commercial media supplemented with BSA (1 litre of RPMI with BSA at 3.5 % costs €72).The PBS stabilates were also less infective than MEM stabilates but still showed a reasonably high infectivity (59 % of that of MEM). It could be that the nutritive value of the defined medium components may not be as critical as their buffering and cryoprotective qualities during storage. If this is the case, PBS supplemented with BSA has the potential to be the cheapest alternative medium (€62/l). Although PBS and FCS were found to maintain a high proportion of the infectivity of the sporozoite dose, their suitability needs further investigation. One refreezing cycle significantly decreased the infectivity of sporozoites in comparison to storage for 1.5 h on ice. The latter should not affect the infectivity as Marcotty et al. (2001) found that keeping a stabilate on ice for up to 6 h did not reduce sporozoite infectivity significantly. Although the actual freezing temperature was not measured in these experiments, 1.5 h was found to be sufficient to freeze the vial contents to -60 o C (Njuguna and Musisi, 1996). This is below the critical temperature range (-20 o C to -50 o C) at which ice crystals form and raise extra-cellular solute concentrations, a process that is detrimental to cell integrity in cryopreservation (Farrant, 1970). Subsequent re-freezing cycles caused similar losses in infectivity.
66 Chapter 4: Suspension media and multiple freezing of Theileria parva stabilates ______________________________________________________________________________________________ Refreezing of stabilates seems to induce considerable loss in infectivity. Attempts to refreeze stabilates left over from field immunisation would result in low quality stabilates that may not be protective upon inoculation. It would be helpful to have this cautionary information in the extension packages for stabilate delivery especially for the private sector involved in ECF immunisations. A re-titration of such stabilate may be necessary to determine appropriate immunising doses. This would be beyond the expertise of the service provider. Moreover, they may not have appropriate equipment to undertake a standard refreezing process. In unavoidable situations e.g. polyvalent ECF vaccine production, it would be advisable that the infectivity loss is considered and immunising doses adjusted accordingly. The process of refreezing may be useful in research work for reducing variability arising from different storage vials. This involves thawing the stabilate, pooling the contents of vials and refreezing for later use. Holding the stabilates for up to 1 h on ice did not reduce the quality or infectivity of stabilates significantly. This is important as the process of preparing stabilates, particularly centrifuging and aliquoting, takes time before the stabilate is ready for freezing. In addition, including “batch” as a random effect not only takes into account the tick batches but the different methods of grinding (Omni-mixer Homogeniser ® , Ultraturax ® and manual) used in these experiments. In conclusion, the study confirms the findings of Kimbita et al. (2004) that RPMI 1640 is as effective as MEM in supporting sporozoite infectivity. Therefore, it is recommended that RPMI 1640 that is properly supplemented can be used as an alternatively cheaper freezing medium in T. parva stabilate production where MEM is either too costly or not available. We also showed that there is an estimated loss in sporozoite infectivity of 35 % when stabilates are refrozen. This loss needs to be adjusted for in both research and field use stabilates.
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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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Chapter 8: SNA and refinement of ti
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APPENDICES
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SUMMARY
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Curriculum Vitae
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List of Abbreviations 183 _________
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