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

Chapter 1: Quantitation of Theileria parva sporozoites: Review of literature 35
______________________________________________________________________________________________
against East Coast fever and for T. parva research. Quantitation of infectivity is critical for
immunisation and research purposes in general, and in particular, when the effects of different
preparation procedures, cryopreservatives (Cunningham et al., 1973b; Kimbita et al., 2001),
composition of media and storage conditions (Musisi et al., 1996b; Marcotty et al., 2001; Kimbita
et al., 2004) are being explored in the development of improved storage and cryopreservation
methods. Another important use for stabilates is in sporozoite neutralisation assays that are used to
quantitate the degree of inhibition of sporozoite infectivity exerted by various immune sera in
vitro (Musoke et al., 1982).
It is critical to have a measure of the infectivity of newly produced T. parva stabilates in order to
determine immunizing doses and also to make comparisons between stabilates originating from
different tick batches. Quantitating the potency of a stabilate based on the number of sporozoites
per dose is difficult. Apart from the strain-related variability in virulence, the technical difficulties
in assessing the number of viable sporozoites are considerable. Two quantitations are usually done
during the course of stabilate production: one on the infected tick batches and the other on the
resulting stabilate. The first one is mainly to exclude poorly infected batches from production.
Assuming that an infected acinus contains 100,000 sporozoites (Fawcett et al., 1982a), the first
step in quantitation involves screening tick batches to determine the average number of infected
acini per tick (abundance). Tick batches from the field or laboratory will have varying degrees of
infection depending on a number of factors that include piroplasm parasitemia, host susceptibility
to ticks, tick stock, susceptibility of tick stock to particular T. parva parasite stock, duration of
infection in ticks, ambient and or moulting temperatures (Young et al., 1984; Ochanda et al.,
1988; Young et al., 1996). Quantitation is done by histological examination of stained salivary
glands for presence of sporozoites (Büscher and Otim, 1986).
To determine the efficacy of a stabilate for use in immunisation, a second quantitation is made by
inoculating groups of cattle with a selection of doses of stabilate. This is necessitated because
irrespective of the tick infection level, the recommended OIE standard is 10 ticks/ml (OIE, 2005).
Secondly, because of the many factors influencing infection in ticks, stabilates of a stock of
T. parva from different tick batches are likely to have varying degrees of infectivity. In addition,
there may not be a direct correlation between tick infection and concentration of infective
sporozoites in the stabilate (Marcotty et al., 2004).