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

Summary 169
______________________________________________________________________________________________
might have occurred as stabilates were raised to this temperature resulting in freeze-injury of the
sporozoites. Due to the high infectivity loss, this method of storage is not recommended.
Chapter seven is an account of stabilate lyophilisation attempts aimed at repeating and improving a
documented protocol. Lyophilisation is potentially a cheaper method than cryopreservation for
stabilate storage. Authors of the documented attempt noted that although cattle inoculated with the
product developed patent infections, less than 1% of sporozoites had survived the freeze-drying
procedure. The rationale was that if it was repeatable, optimization of the procedure would raise the
surviving proportion of sporozoites. Katete and Chitongo T. parva stabilates cryopreserved with
sucrose or trehalose were tested. In addition, whole infected ticks, tick salivary glands and top
layers of stabilate debris left after centrifugation at production were lyophilised. No sporozoite
viability was evident in the lyophilised materials despite various modifications of the protocol and
parasite presentations. Possible sources of failure are discussed.
In chapter eight, refinements to the Sporozoite Neutralization Assay are outlined and the improved
protocol is used to assay various sera. Presently, SNA analyses are based on counting infected cells
which is laborious and prone to high variability. The idea of scoring wells positive or negative in
plates is proposed. It is more user-friendly and presents binary data that are easily analysed by
logistic regression. The chapter also argues for the less cumbersome use of microtitration plates
instead of microtubes for stabilate in vitro titrations. By shaking plates for a minute and keeping the
target cells in the refrigerator before infection instead of 37ºC, the system as a whole was still as
efficient as the original protocol. In addition, a quicker way of reading the culture plates by PCR
was explored. The in vitro protocol was shortened by 48 hours by skipping the step of activating
Peripheral Blood Mononuclear Cells with Con-A.
Sera collected at weekly intervals from cattle inoculated with schizonts and sporozoites were
assayed in parallel with a positive (anti-p67) and negative (FCS) control. The tested sera were also
subjected to PIM-ELISA, p67-ELISA and a SELISA using infected lymphoblasts as antigen. No
significant sporozoite inhibition was evident from all three sera. Albeit, the assay demonstrated a
clear distinction between the controls. Serum from the live sporozoite inoculation was positive on
all three serology tests and that from the schizont inoculation was positive both on SELISA and
PIM-ELISA. The lack of sporozoite neutralisation with serum from the animal receiving repeated
schizont inoculation lends support to the documented observation of low p67 molecule expression
in the schizont stage. This result also indicates inefficiency of sporozoite neutralisation by anti-PIM
antibodies in vitro and that SNA is specific for anti-p67 antibodies.