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

Abstract

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 stabilate 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.

Infection and treatment method (ITM) vaccine against East Coast fever: reducing the number of doses per straw for use in smallholder dairy herds by thawing, diluting and refreezing already packaged vaccine

“…In order to assess the infectivity of the diluted stabilate two groups of six cattle were inoculated with 1 ml of the stabilate, diluted either 1:20 or 1:14. The dilution of 1:20 represents a dose equivalent to the recommended field dose of ILRI 0804 (1:100) assuming no loss of viability, whereas the 1:14 dilution assumes there was about a 33–35% loss of infective sporozoites during repackaging [6]. The cattle were inoculated without simultaneous administration of oxytetracycline, in line with previous procedures [13].…”

“…It does, however, rely on there being minimal loss of sporozoite viability or of key antigenic components of the vaccine during the repackaging process. In a previous study, Mbao et al [6] showed that sporozoites from a single stabilate showed 33–35% loss of in vitro infectivity following thawing and refreezing. However, no dilution step or in vivo analysis was reported.…”

Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system

“…To demonstrate the infectivity of secreted T. parva sporozoites for bovine PBMC, we performed serial dilutions of secreted sporozoites per previous in vitro studies that used parasites from ground infected ticks [ 33 , 34 ]. To reduce potential immature parasite forms, T. parva sporozoites were collected from the in vitro tick feeding system on day 5 post-tick attachment.…”