The study was approved by the ethics committee of Pasteur Institute of Iran. The four strains along with the reference strain (RS) of L. major (MRHO/IR/75/ER) as a control, were used for inoculation of BALB/c mice. Fifty thousand stationary

phase promastigotes were inoculated in the right foot pad of BALB/c mice. Parasite was grown in RPMI 1640 media, supplemented with 2 mM L-glutamine, 10% foetal bovine serum, 100 U/mL penicillin and 100 μg streptomycin and then harvested and washed with Phosphate buffered saline (PBS) by centrifugation at 3000 rpm for 30 min. Species of the strains were characterized by isoenzyme electrophoresis and PCR as L. major. Genetic heterogeneity of the four strains was analysed by single-strand conformation polymorphism MK-8669 concentration (SSCP).The internal transcribed spacer 1 (ITS1) was amplified by primer pairs L5.8S (5′- TGATACCACTTATCG-CACTT -3′) and LITSR (5′ – CTGGATCATTTTCCGATG -3′) as described

previously [15]. Amplification reactions were carried out in volumes of 25 μL: 60 ng DNA was mixed with a PCR mixture containing 200 μM dNTPs mix, 1.5 mM MgCl2, 1 U Taq polymerase and 10 pmol of each primer. The amplification of samples was performed at: 95°C for 5 min for initial denaturing followed by 35 cycles consisting of denaturation at 95ºC for AZD3965 ic50 40 s, annealing at 60ºC for 40 s and extension at 72ºC for 1 min. Final extension was followed at 72ºC for 7 min. PCR products were analysed on 1.5% agarose gel, and the bands NADPH-cytochrome-c2 reductase were visualized by ethidium bromide staining [16]. Single-strand conformation polymorphism was performed by denaturing the double-strand DNA products as described (15), with a few modifications. Briefly, 4 μL of PCR products were mixed with 6 μL denaturing buffer (95% formamide, 10 mm NaOH, 0·25% bromophenol blue, 0·25% Xylene) and 4 μL loading buffer (40% Sucrose, 0·25% bromophenol blue,

0·25% Xylene). After heating at 98ºC, the mixture was immediately frozen in liquid nitrogen for 15 min. The samples were loaded then on 5.5% polyacrylamide gel and silver stained. For assessment of cytokine mRNA, 35 mice in each group were inoculated with five strains (175 mice), and cytokine transcripts were analysed in each time point of 3, 16, 40 h, 1 week, 3, 5 and 8 weeks post-infection (five mice for each time point). One group containing five un-infected mice were used as a control. Parasite load was measured by inoculation of four mice in each group with five strains (20 mice in total) and after 8 weeks, parasite burdens were determined in LN of each mouse. Parasite load was estimated at 8 weeks post-infection.