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

36 Chapter 1: Quantitation of Theileria parva sporozoites: Review of literature
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1.3.1. Direct quantitation
1.3.1.1. Histological
Light microscopy allows for the quantitation of infected acini in dissected salivary glands of ticks
(Büscher and Otim, 1986). Briefly, the procedure involves dissecting the dorsum off partially
engorged ticks to expose the viscera. The salivary glands are then separated from other organs,
excised and placed in a drop of normal saline on a microscope slide. The acini are spread out with
a teasing needle to obtain a monolayer after which the slide is air dried and fixed in pure
methanol. Staining is commonly done with Methyl Green Pyronine (Walker et al., 1979) or
Feulgen's reaction as described by Blewett and Branagan (1973). Infected acini stain a grainy pink
with Feulgen's reaction. The parameters of importance arising from counting of infected acini are:
abundance, which is the mean number of infected acini per tick, or intensity, which is mean
number of infected acini per infected tick or prevalence, which is the proportion of infected ticks
in the batch.
Determining the number of sporozoites in ticks involves estimating the number of sporozoites per
infected acinus. Jarrett et al. (1969) estimated that one infected salivary gland would contain about
5 x 10 4 sporozoites and, therefore, an infected tick would contain 10 5 sporozoites. This was an
underestimation as Fawcett et al. (1982a) found about 10 5 sporozoites per acinus using low power
electron microscopy. Some workers have used this latter estimate e.g. Musoke et al. (1992) when
they reported concentrations of 5 x 10 4 sporozoites in SNA's. Rocchi et al. (2006) put the number
at 10 4 sporozoites per acinus but did not explain how they arrived at this estimate.
1.3.1.2. Molecular
Because of the potentially high sensitivity of the Polymerase Chain Reaction (PCR), molecular
techniques have been used to detect and quantitate T. parva sporoblasts and sporozoites in ticks
(Chen et al., 1991; Watt et al., 1997). Watt et al. (1997) used a direct visual grading (0 to 5) of the
brightness of PCR bands on a 1.6 % agarose gel to quantitate infection in ticks and compare these
with histological techniques. However, this is quite a subjective method and although sufficient
for their objectives, may not be suited for stand-alone routine quantitation. Moreover, it may be
unsuitable for quantitating infectivity of stabilates because it does not discriminate between
sporoblasts and sporozoites.
1.3.1.3. Flow cytometry
Goddeeris et al. (1991) and Yagi et al. (2000) used flow cytometry to quantitate purified T. parva
schizonts and Theileria sergenti piroplasms, respectively. This technique is highly precise with