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

Chapter 5: Snap freezing of T. parva stabilates 69
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"Success is to be measured not so much by the position that one has reached in life... as by
the obstacles which he has overcome while trying to succeed" - Booker T. Washington
5. CAP 5
5.1. Introduction
East Coast fever (ECF), is a lymphoproliferative disease of cattle that is often fatal and causes high
morbidity. It is caused by Theileria parva, an obligate intra-cellular Apicomplexan protozoon that is
transmitted by the three-host ticks Rhipicephalus appendiculatus and Rhipicephalus zambeziensis.
The disease causes major economic losses in the livestock industries of eastern, central and
southern Africa (Young et al., 1988). One of the control options is immunisation of susceptible
young cattle stock by the Infection-and-Treatment (I & T) method (Radley et al., 1975a) using
cryopreserved stabilates of live sporozoites of the parasite. Immunisation is achieved by inoculation
of a dose of stabilate with concurrent injection of a long acting tetracycline. The technique is
applied widely in areas where the disease is endemic (Berkvens et al.,1988; Uilenberg, 1999;
Marcotty et al., 2001; Fandamu et al., 2006).
Currently, freezing of triturated tick stabilates at the end of production is a two-step slow freezing
procedure; first cooling in ultra freezers to -80°C and then transferred into liquid nitrogen (-196°C)
(OIE, 2005). The method was adapted from low temperature storage of trypanosomes as described
by Cunningham et al. (1963), cited by Cunningham et al. (1973a). In this work, we tested the effect
of skipping the first step and directly plunging the vials into liquid nitrogen. The rationale was to
approximate vitrification, a process that prevents intra-cellular crystallisation of water during
cooling (James, 2004). Vitrification is widely used in cryopreservation of some helminth species
and insect embryos, and has the potential to be adapted for parasitic protozoa with the advantage of
achieving higher survival rates at recovery from cryo-storage (James, 2004). On the other hand, fast
freezing is expected to be detrimental to the cells when vitrification is not achieved as the exchange
of water and cryoprotectants through the cytoplasmic membrane might not be completed before the
crystallisation of the water (Whittingham, 1980). Snap freezing would have the advantage of
requiring less equipment and speed up stabilate production.