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

40 Chapter 1: Quantitation of Theileria parva sporozoites: Review of literature
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1.3.4. Applications of in vitro quantitation
1.3.4.1. Stabilate production and storage parameters
The detrimental effects of various handling and storage processes on the infectivity of stabilates
have been an area of much interest. These have been investigated by evaluating the alteration in
infectivity of sporozoites for lymphocytes in vitro. Kimbita et al. (2004) used in vitro systems to
quantitate effects of holding temperature and compared various media as candidates for stabilate
production. They found L-15 medium stabilates to be superior in conserving infectivity over
MEM, Optimem, Iscoves modified MEM and RPMI 1640. They also evaluated infectivities of
sporozoites harvested from ticks at various stages of maturation (Kimbita and Silayo, 1997) and
the effects of various cryoprotectants (Kimbita et al., 2001). Marcotty et al. (2004) investigated
the effect of high speed centrifugation on sporozoites. These studies helped in identifying some of
the processes and reagents that influence sporozoite survival during stabilate production and that
are critical for optimization of production parameters.
1.3.4.2. Sporozoite Neutralisation Assays
Based on the evidence that serum from theileriosis recovered calves neutralises sporozoite
infectivity in vitro (Gray and Brown, 1981), techniques have been developed to quantitate titres of
antibodies against native and recombinant sporozoite antigenic proteins (Musoke et al., 1984;
Musoke et al., 1992; Kaba et al., 2004). Briefly, the techniques involve a short incubation of
sporozoite suspensions with the test sera prior to introduction of PBMC. This is then followed by
a single or serial sampling for schizont scoring. Another application is the study of cross reactivity
of inter-species sporozoite antigens as used by Knight et al. (1996).
However, these techniques may give antibodies more time to bind to epitopes than actually
happens in vivo in which the parasite has only a few minutes before being internalised (Shaw et
al., 1991). A better approximation would be to introduce the sporozoites into a mixture of cells
and antiserum thus simulating the competition that ensues in vivo. Sporozoite Neutralisation
Assays will become important tools in the current efforts to develop and assess efficacy of
potential sub-unit vaccines against ECF that elicit a humoral response against sporozoites. It is
therefore important to explore and implement assay systems that are standardised, reproducible,
robust and easy to evaluate.