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

Chapter 7: Lyophilisation of Theileria parva stabilates 93
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"Now faith is being sure of what we hope for and certain of what we do not see"
Hebrews 11:1
7. CHAPTER 7
7.1. Introduction
East Coast fever (ECF), a disease of cattle caused by Theileria parva is of major economic concern
due to direct losses incurred through its control and mortality (Mukhebi et al., 1992). Among the
control measures targeted at East Coast fever is immunisation by the "Infection and Treatment"
(I & T) method. However, the method has a few constraints. Since the immunity generated is strain
specific, it calls for isolation and judicial use of different T. parva stocks for specific localities.
Secondly, it relies on a costly cold chain both for storage and delivery. T. parva stabilates have to
be kept in liquid nitrogen to preserve parasite viability. Simplification of this cold chain by
assessing viability of stored stabilates on ice (Musisi et al., 1996b; Marcotty et al., 2001) and
attempts to freeze-dry the stabilate (Marcotty et al., 2003) has been explored. Delivery of stabilate
on ice is currently the method of choice in the eastern province of Zambia.
Successful resuscitation of freeze-dried sporozoites was demonstrated by Marcotty et al. (2003).
However, studies to repeat this success or optimize it were not conclusive. As this is a potentially
less expensive strategy for storage and delivery of immunising stabilates, we conducted further
T. parva stabilates lyophilisation experiments using two lyoprotectants and several lyophilisation
protocols.
7.2. Materials and Methods
7.2.1. Parasite material and cryoprotectants
Two stocks of T. parva, Katete and Chitongo, were used. The Chitongo and Katete stocks were
isolated from Namwala and Katete districts of the southern and eastern provinces of Zambia,
respectively in the early 1980's, (Berkvens et al.,1988; Geysen et al., 1999). Parasite materials were
suspended in RPMI 1640 (25 mM HEPES, Life Technologies # 52400-025) medium supplemented
with BSA (Acros organics # 240401000 and #240400100) at 3.5% (w/v) and antibiotics. Two
lyoprotectants were used: Sucrose (Sigma #S1888-500g) or Trehalose (Sigma # T-0167) at molar
concentrations of 0.3 M and 0.1 M, respectively.