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

Chapter 8: SNA and refinement of titration protocol 107
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8.2.5.2. Dead sporozoite inoculation
Animal number 6146 was inoculated with 20 ml of a lyophilised T. parva (Katete AV) stabilate.
Lyophilisation of this stabilate is reported in chapter 7. The dose was divided into two with 10ml
being injected subcutaneously above the left parotid gland (ln. parotideus superficialis) and the
other 10 ml on the contra-lateral lymph node. The animal was inoculated with similar amounts two
weeks later. Serum and plasma were collected before inoculation, at week four, week five and week
six post inoculation and stored at -20°C.
8.2.5.3. Schizont inoculation
A culture of schizont-infected cells was established with PBMC collected from a Friesian heifer
number 6148. Briefly, 2 ml of the stabilate were thawed and purified by centrifugation (Marcotty et
al., 2004). 200µl Aliquots were transferred into six 1.5 ml micro-tubes (Sarstedt, Numbrect) to
which 500µl of PBMC at 6*10 6 /ml were added. Tubes were incubated for 20 min in a thermoshaker
at 37°C (1000 rpm) after which the cells were spun down, pellets re-suspended in 2 ml fresh culture
medium and transferred to each well of a 12 well culture plate. Medium was changed after 12 days
and some cells were transferred to Petri dishes.
At weeks one, three and four after collection and infection of PBMC, cultures were centrifuged and
re-suspended in fresh culture medium at a concentration of 100,000 cells/ml and 1 ml of this
inoculated at each instance subcutaneously below the right parotid lymph node (ln. parotideus
superficialis) of animal number 6148. Serum and plasma were collected before inoculation, at
weeks three, four and five post first inoculation and stored at -20°C.
8.2.6. Sporozoite Neutralization Assay
The three sets of sera from animals 131, 6146 and 6148 were assessed in a Sporozoite
Neutralization Assay as described in Appendix VII. Since the percentage inhibition was the unit of
interest, sera dilutions were kept constant (1/25) and stabilate diluted serially as in in vitro titrations.
P67-hyperimmune serum and plain FCS were used as positive and negative controls, respectively.
The p67 serum had been obtained from animals inoculated with a recombinant p67 antigen
(GFP:p67deltaSS) constructed by Kaba et al. (2004) and kindly provided by Intervet ® . Two
samples were assayed from each animal i.e. pre inoculation and post inoculation serum. The post
inoculation serum was that which was positive in the SELISA. All six sera plus positive (anti-p67
serum) and negative (heat inactivated FCS) controls were assayed in triplicate rows in each of four
sessions. A sucrose T. parva stabilate was used as source for sporozoites.