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

General Introduction 15
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cryopreserved as stabilates. The presence of several antigenic types in a locality complicates both
the epidemiology and the control of the disease by immunisation.
Before field use, stabilates are titrated to assess their efficacy and to determine the optimal
immunising doses. The principle objective is to calculate a dose that gives maximum protection
with minimum clinical effects. Determining the actual number of viable sporozoites contained in a
stabilate dose is difficult and cumbersome for routine dose determination. The method used is to
inoculate groups of cattle with stabilate at different dilutions and evaluate clinical and
parasitological reactions. While the major demand for quantitation of T. parva stabilates is dose
determination, it is also required in research to compare diverse stocks as well as in improving
vaccine production, storage and distribution. In improving stabilate production protocols,
quantitation is necessary to prove that proposed modifications do not alter significantly the efficacy
of the stabilate. Evaluating these effects in vivo is at great cost and is not precise due to variations in
reactions of individual animals. In addition, it raises animal welfare concerns. It is for these reasons
that less expensive, more reliable and ethically acceptable substitute techniques are being sought for
quantitating sporozoite concentrations and studying immunological mechanisms.
In vitro techniques have been shown to offer a possible solution. They are relatively cheap, ethically
acceptable and give repeatable results. In addition, results are obtained in a fraction of the time that
in vivo procedures take. In this thesis, we explore the optimization of two such techniques, in vitro
titration and Sporozoite Neutralisation Assays (SNA) and use them to quantitate several variations
of stabilates. The work attempts to show that viable sporozoites in stabilates can be quantitated
relative to a control stabilate and SNA's can be adapted to approximate the in vivo conditions
thereby making them more accurate. The statistical analyses required to analyse the two procedures
are also discussed and improvements proposed that take into account random factors which may
result in erroneous interpretation of results if ignored. This is more critical for experiments that
require many repetitions in order to generating adequate sample sizes.
Using these in vitro procedures we first evaluate several critical processes in stabilate production
and storage as they affect the survival of sporozoites and consequently stabilate efficacy. Tick
homogenisation, medium composition and cryoprotection, freezing rates, storage on ice or in a
domestic freezer and lyophilisation are investigated. Secondly, we use the refined Sporozoite
Neutralisation Assay to screen sera obtained from cattle that had been inoculated with various forms
of T. parva parasite material: live sporozoites, dead sporozoites and live schizont-infected