Infectivity of Theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing: evaluation by in vitro titration

Theileria parva sporozoite stabilates are used for immunizing cattle against East Coast fever and in in vitro sporozoite neutralization assays. In this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. Pools of stabilates prepared using Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI 1640), foetal calf serum (FCS) and phosphate-buffered saline (PBS) were compared. All were supplemented with bovine serum albumin except the FCS. RPMI 1640 was as effective as MEM in maintaining sporozoite infectivity while the infectivity in PBS and FCS reached only 59% and 67%, respectively. In a second experiment, a stabiiate based on MEM was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. Refrozen stabilate gave an average sporozoite infectivity loss of 35% per cycle. The results indicate that RPMI can be used as a cheaper freezing medium for T. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35% loss of infectivity.