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

104 Chapter 8: SNA and refinement of titration protocol
______________________________________________________________________________________________
SNA based on the observation that immune serum interferes with sporozoite infectivity in vitro as
described by Gray and Brown (1981).
Typically, SNA's are carried out by incubating sporozoites with test sera (about 30 min) after which
PBMC are introduced. In this system, antibodies have more time to interact with sporozoites before
they contact and enter lymphocytes. This method is not very representative of in vivo events in
which sporozoites are inoculated into a complex environment containing antibody and PBMC.
Moreover, sporozoites invade target cells in a very short time (Shaw et al., 1991) and therefore,
indications are that the period of exposure to antibodies is relatively shorter in vivo than the present
SNA prescribes. Therefore, it was desirable to attempt to approximate the natural process as closely
as possible by introducing sporozoites into a culture of PBMC's containing test sera.
The modifications were aimed at optimising the protocol for use in quantitating T. parva. The
modifications not only address the maintenance of the viability of cells, but also provide for more
efficient management of the culture system and reduced variability. Full descriptions of the existing
procedures are given elsewhere (Musoke et al., 1984; Musoke et al., 1992; Marcotty et al., 2004)
and a comparison of the original and refined protocols are presented in Appendices I and II. The
stages that needed improvement were identified as: a) storage of PBMC, b) discontinuation of
PBMC stimulation with Con-A, c) replacement of microtubes, which are cumbersome and limit the
number of repetitions that can be done per session, with 96-well plates for incubation, d) counting
of positive wells rather than cells, e) incubation of antibody with PBMC and f) PCR reading of
cultures instead of microscopy.
8.2. Materials and methods
8.2.1. PBMC storage
Collection and isolation of PBMC were as described in chapter 3. Cells were stained with
carboxyfluoroscein succinimidyl ester (CFSE) at a pre-titrated concentration of 0.56µl per 1*10 7
PBMC (Appendix V). The cell suspension was split into two 15 ml lots in 50 ml culture flasks, one
flask was stored overnight in a refrigerator (4°C). The other was stored overnight at 37°C in an
incubator (5% CO 2 in air) with the cap loosened.
The following day, samples from the two flasks were analysed by flow cytometry (FACScan ® ,
Becton Dickinson). Data were analysed in WinMDI ® version 2.8 based on 10,000 events acquired
without any gating. Mean CFSE fluorescence in log units were obtained and dot plots of forward