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

122 Chapter 9: General Discussion
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procedure than two step slow freezing and has potential for even higher yields of viable sporozoites
at resuscitation.
9.4. Storage conditions
The rate of stabilate infectivity loss upon storage on ice is minimal. This may allow delivery on this
medium during immunisations. The loss rates were similar for glycerol and sucrose protected
stabilates. In addition, we have shown that loss dynamics for Katete are similar to those of the
Chitongo stock and can therefore speculate that ice storage can work for the southern province of
Zambia where Chitongo is used. Caution should however be taken to include an in vivo assessment
before implementation of the technique. Our attempt to store stabilates at -20°C resulted in high
sporozoite infectivity loss. This method of short term storage therefore seems not to have a practical
field application.
Some disparities between glycerol and sucrose stabilates extracted from single tick batches were
observed. Sucrose seemed to give consistently higher infectivities compared to glycerol, except in
the case of the storage at -20°C where sucrose stabilates deteriorated faster than glycerol stabilates.
Though there is strong evidence that sucrose stabilates had relatively higher infectivity, this may be
a result of an interaction between the cryopreservatives and the target cells. It was further observed
that some wells of higher concentration of glycerol stabilates gave false negatives (chapter 8). For
these reasons, it is recommended that the comparative effects of these cryopreservatives on PBMC
be assessed in vitro. Sucrose stabilates are at least as infective as glycerol stabilates in vitro and
should therefore be assessed in vivo with the view of replacing glycerol which requires drop-wise
addition during vaccine production.
Resuscitation of lyophilised T. parva sporozoites was not achieved in this work. Although survival
of lyophilisation is commonly limited to prokaryotic cells, this was once demonstrated for T. parva
(Marcotty et al., 2003). Since liquid nitrogen storage is very expensive and the I & T method is the
only practical technique for immunising cattle at present, efforts at identifying cheaper storage
methods should be continued. The various attempts made are a basis for trying other protocols and
lyoprotectants. In this light, attempts at lyophilisation using cocktails of various lyoprotectants
should be made, as long as the reagents do not make the vaccine too expensive for cattle keepers.