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

96 Chapter 7: Lyophilisation of Theileria parva stabilates
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Table 11. Lyophilisation protocol for L9. P c – Chamber pressure
Shelf temp
(°C) P c (hPa) Time
Prefreezing -40 1 h
Put vials in lyophiliser
Hold -40 7 h
Drying -35 0.08 30 min
-25 0.08 15 min
-25 0.08 10 h
-20 0.05 15 min
-20 0.05 2 h
Close vials and stop the
machine
Table 12. Lyophilisation protocol for L10. P c – Chamber pressure
Shelf temp
(°C) P c (hPa) Time
Prefreezing -40 1.5 h
Put vials in lyophiliser
Drying -35 0.04 30 min
-25 0.03 19.5 h
-25 0.08 10 h
Close vials and stop the
machine
7.2.4. Assessment of infectivity
7.2.4.1. In vitro assessment
Chitongo
For controls, un-lyophilised stabilate from the same batches was used. Stabilates CA and CA-sed
were assessed in separate sessions. For each stabilate, 20 microtitre-plate wells were set up. About
3*10 5 PBMC were incubated with 50 µl of stabilate in each well.
Whole ticks and tick glands were first reconstituted by adding sterile water equal to weight
difference recorded after drying and gently mixing for half a minute. The tick materials were left to
equilibrate for about 15 min before being homogenized using an Ultra-turrax ® tissue grinder for 5
min followed by centrifugation at 400 g for10 min. Recovered supernatant was transferred in 200 µl
aliquots to twelve 1.5 ml microtubes to which 500 µl PBMC suspension were added. The tubes
were incubated for 40 min in an Eppendorf Thermomixer Compact ® (Eppendorf, Hamburg) at
37°C and 1000 rpm after which they were centrifuged and the pellets re-suspended in 1 ml aliquots
of culture medium in 12-well plates. They were incubated at 37°C in a CO 2 incubator for 10 days.
The cultures were examined at the end of incubation and scored for presence of schizonts.