Leishmania major Inoculum Preparation

The reference strain of Leishmania major (MRHO/IR/75/ER) and two bacterial species of Enterobacter cloacae and Bacillus subtilis were selected for inoculum preparation. At fifth day of culture, stationary phase promastigotes of L. major were harvested from RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco Invitrogen, Carlsbad, CA, USA) and 100 μg/ml of penicillin-streptomycin (Biowest, USA) incubated at 25 ± 1°C. To count parasites, the cultures were centrifuged at 5,000 ×g at room temperature for 10 min and then re-suspended in 0.5% formalin following three washes with PBS. The reason for choosing these two bacterial species was their isolation from the resting, feeding and breeding environments of P. papatasi and their significant effects on the development of Leishmania (Maleki-Ravasan, in press ; Maleki-Ravasan et al., 2015 (link)). Both bacteria were grown at 37°C on a Brain Heart Infusion (BHI) agar medium plate overnight. The single-grown colonies were then subcultured in BHI broth at 100 rpm at 37°C overnight. Bacterial cells were adjusted to 1.5×10⁸ CFU/ml (optical density at 600 nm, ∼0.25) according to Kaplan et al., 2012 (link) protocol. The stock solution was serially diluted up to 1:100 and 1:10,000 to obtain 1.5×10⁶ and 1.5×10³ CFU/ml of each bacterial cell for high and low doses groups, respectively.