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

Chapter 7: Lyophilisation of Theileria parva stabilates 97
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Katete
Stabilates were reconstituted by adding 0.8 ml of sterile water to each vial. After centrifugations
and separations of supernates, they were distributed in 50 µl aliquots in microtitration-plate wells,
40 wells per cryopreservative type. An equal volume of PBMC suspension containing 3*10 5 cells
was then added to each well and the plates incubated for 10 days.
For control, cryopreserved (un-lyophilised) stabilate was assessed in the same in vitro sessions.
7.2.4.2. In vivo assessment
Lyophilised stabilate CA was appraised by injecting 20 ml of reconstituted material into a Friesian
heifer 10 ml subcutaneously below the right ln. parotideus superficialis and 10 ml intravenously
into the jugular vein.
7.2.5. Vital staining
Vital staining was performed using Ethidium monoazide (EMA, 3-amino-8-azido-5-ethyl-6-phenylphenanthridium
bromide: C 21 H 18 N 5 Br) (Sigma-Aldrich ® , Germany). EMA penetrates damaged cell
membranes (dead cells) to covalently bind with genomic DNA and prevents such bound DNA from
being amplified by PCR. Thus, presence of a PCR product means the membranes are intact and
therefore from viable cells (Nogva et al., 2003; Rudi et al., 2005). Samples of lyophilised and nonlyophilised
stabilates (CA) were EMA stained to check viability by PCR.
Briefly, EMA was added to reconstituted and purified stabilate at a final concentration of 20 µg/ml
in 1.5 ml microtubes. The mixtures were placed on ice and photo-activated by placing the tubes for
30 min under laboratory fluorescent lighting. The tubes were then centrifuged at 14,000 g for one
minute, the pellet washed once in 400 µl PBS buffer and DNA extraction performed by the method
of Boom et al. (1990; 1999). DNA extracts were then subjected to PCR and products resolved by
electrophoresis through a 2% (w/v) Agarose gel.
7.3. Results
7.3.1. In vitro and in vivo infectivity assessment
Cultures of PBMC incubated with various T. parva lyophilised sporozoite materials were negative.
The steer that had been injected with lyophilised stabilate did not show any clinical or
parasitological reactions. PBMC incubated with control stabilates (cryopreserved) became infected
and demonstrated schizonts.