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

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Chapter 9: General Discussion 123 ______________________________________________________________________________________________ 9.5. Sporozoite Neutralisation Assays The technique described in this work is appropriate for screening sera for sporozoite neutralising antibodies for two reasons. Firstly, it seems not to pick up non-specific antibodies except anti-p67. This is illustrated by the positive PIM-ELISA and SELISA on some samples that were negative on Sporozoite Neutralisation Assay. Secondly, it simulates in vivo conditions more closely by introducing sporozoites into a mixture of antibody and target cells instead of pre-incubating sporozoites with antibody first. Further, the results confirm that unless an animal is repeatedly challenged, the humoral response with respect to p67 is undetectable and that PIM antibodies do not play a major role in in vitro neutralisation of sporozoites. It is also shown that expression of the p67 in the schizont stage is very low as the assay was unable to pick it up despite repeated challenge of the experimental animal with massive numbers of infected lymphoblasts. This low expression has been observed by other workers (Musoke, personal communication 2006). Adjuvanting the dead sporozoites could have probably elicited a higher antibody titre. However, since efficacy of vaccination regimens was not the object of the study, this was not tried. This refined assay may also be used to assess effects of agents directed against the schizont stage e.g. chemotherapeutics. However, caution should be exercised as the effects may not be directed at the parasite per se but at the target host cell. Pre-evaluation of effect on target cells may therefore be necessary. The assay may equally be employed in elucidating the possible interference of maternal antibodies in calf immunisation (Marcotty et al., 2002). If neutralisation of sporozoites by sera from calves can be demonstrated, implications are that calves suckled during periods of heavy ECF challenge may not be immunised as the sporozoite numbers presented to lymphocytes may be reduced severely. 9.6. Conclusions and recommendations The in vitro titration has been optimised it takes 48 hours less and excludes difficulties associated with use of microtubes and shaker placed inside CO 2 incubators. Reading of resultant cultures can be done by PCR further cutting down on time spent processing cyto-centrifuge smears. Stabilates extracted by homogenisers are not inferior to manually extracted ones. It is recommended that, for bulk stabilates, homogenisers are used whereas manual extraction can be employed for smaller batches.
124 Chapter 9: General Discussion ______________________________________________________________________________________________ RPMI is at least as good as MEM in supporting sporozoite infectivity in stabilates. As such it can be used in vaccine production centres where importation of MEM is restrictive. Sucrose has potential to be a more user friendly cryopreservative. It exhibits comparatively better infectivities in vitro and should be investigated further possibly by directly determining amounts of live sporozoites on recovery from cryopreservation using reverse transcription-polymerase chain reaction or by in vivo comparison. Refrozen stabilates lose 35% of initial infectivity. It is recommended that in procedures were refreezing is necessary e.g. polyvalent vaccine production, this factor be considered in determining immunising doses. Attempts of stabilate refreezing in field immunisation should not be made as this would result in poor quality vaccine. Snap freezing in present stabilate formulation and packaging is not a feasible alternative to slow freezing. However, results indicate potential to improve the technique and it is therefore recommended that further studies be undertaken. Snap freezing or indeed vitrification would reduce on equipment needed during stabilate production further simplifying the protocol. Short term storage of stabilates on ice does not result in significant infectivity loss rates. This is true irrespective of parasite stock (Chitongo or Katete) or cryopreservative (glycerol or sucrose). With this observation, it is recommended to deliver the Chitongo stabilate on ice during immunisation in the Southern Province of Zambia after an in vivo test. In contrast, trials to store in a domestic freezer failed to yield sufficient sporozoite viability upon thawing. This method is not recommended. Attempts to repeat a documented occasion of T. parva lyophilisation failed. It may be worthwhile to test other lyoprotectants and combinations of such agents. The Sporozoite Neutralisation Assay was refined and analysis of data made more precise by use of ED ratios with respective confidence intervals. The use of binary observations makes it easier to interpret than counting proportion of infected lymphoblasts as practised before. PCR reading of microplates further simplifies the procedure. It was observed that this assay is quite specific for anti-p67 antibodies. The challenge of relating in vitro results to in vivo implications remains. Not until we can predict the proportions of protected animals from in vitro results can there be any meaningful use for in vitro titrations as regards determination of ECF immunising doses. This calls for a concerted research effort from all institutions, both private and public, that are involved in the control of this
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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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Chapter 4: Suspension media and mul
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Chapter 5 - Snap freezing of Theile
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Curriculum Vitae
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List of Abbreviations 183 _________
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