Inbred BALB/c mice, 6~8 week-old females, were used throughout the experiments. These mice were obtained from the animal breeding facility of Pasteur Institute of Iran.

Leishmania tropica strain MHOM/AF/88/KK27 is a cutaneous L. tropica isolated from Afghanistan, and was initially described by Dr. R. Killick-Kendrick (Lira et al., 1998). It was a gift from Dr. D. Sacks (Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institute of Health, Bethesda, Maryland, USA). The L. major strain MRHO/IR/75/ER is an isolate from Iran, and was a gift from Dr. M. Mohebali (School of Public Health, Tehran University of Medical Sciences, Tehran, Iran). The parasites were grown at 23-24℃ in 50-ml disposable centrifuge tubes containing 4 ml of culture medium. The culture medium consisted of 2 ml NNN and 2 ml RPMI-1640 medium (Sigma Chemical Co., St. Louis, Missouri, USA). The NNN medium consisted of 12% rabbit blood (with sodium citrate as an anticoagulant), 1.35% glucose (Sigma Chemical Co.), 1.4% agar (Merck, Darmstadt, Germany), and 0.6% (w/v) NaCl (Arasto Pharmaceutical Chemicals Co., Saveh, Iran). Penicillin (100 IU/ml) and streptomycin (100 µg/ml) were used as antibiotics. The parasite growth was determined by daily counting of viable parasites in culture. The promastigotes were considered stationary, if the number of viable parasites was approximately the same on 2 consecutive days. The stationary phase of promastigote growth reached 7-9 days after the beginning of promastigote cultures from starting concentration of 10⁶ promastigotes per milliliter.

Mice were infected subcutaneously with 10⁶ live stationary phase promastigotes in the right hind footpad. Disease evolution was monitored by measurement of the thickness of footpad at weekly intervals. The thickness was determined by a dial-gauge caliper (Mitutoyu, Kawasaki, Kanagawa, Japan). The footpad thickness increase due to the infection was defined as the difference between the thickness of the infected footpad and the thickness of the uninfected contralateral footpad.

The parasite load assays were carried out as previously described (Sacks and Melby, 1998). Footpad tissues, draining lymph nodes, or spleens were homogenized in 400, 1000, and 1,000 µl of RPMI 1640, respectively. One hundred microliters from each homogenate were diluted by 2-fold serial dilutions in consecutive wells.

Groups of 5-10 BALB/c mice were infected with 10⁶ stationary phase promastigotes of either L. major or L. tropica. The infection was done by injecting into the right hind footpad of mice. The disease evolution was monitored by determination of the footpad thickness at weekly intervals. Parasite burdens were determined in the footpads, draining lymph nodes, and spleens at 4 intervals: day 10, and mo 1, 4, and 6 post-infection (PI). The weight of the spleen and lymph node were recorded at intervals mentioned above. The study was approved by Ethical Committee of Pasteur Institute of Iran.

Footpad thicknesses were compared between different experimental groups by the Student's t-test. Parasite loads in each group were calculated by the geometric means of reciprocal titers of individual tissue samples in that group. Geometric mean titers were compared by the Student's t-test. The weights of spleens and lymph nodes were compared between different groups by nonparametric Mann-Whitney U test. P-values less than 0.05 were considered significant.

Lesion development in footpads was followed up for 6 mo after infection. The results are shown in Fig. 1. The footpad thickness increased progressively until the end of the experiment in L. major-infected mice (Fig. 1). However, the footpad thickness in L. tropica-infected mice increased only up to 3 mo PI and regressed thereafter. This pattern was the same as reported previously (Lira et al., 1998; Mahmoudzadeh-Niknam, 2004).

The parasite loads in L. major and L. tropica-infected mice are shown in Fig. 2. The parasite load of L. major-infected footpads increased up to 1 mo after infection (P ≤ 0.001) and remained high until the end of the experiment, i.e., 6 mo after the injection. This pattern was quite different in L. tropica-infected footpads: the parasite load increased up to 1 mo, decreased from mo 1 to mo 4 (P ≤ 0.05) and remained low from mo 4 to 6 PI. The parasite load of lymph nodes in L. tropica-infected mice increased up to mo 1 (P ≤ 0.001), remained the same from mo 1 to mo 4 (P ≤ 0.21), and from mo 4 to mo 6 (P ≤ 0.11). The parasite load of lymph nodes in L. major-infected mice increased from day 10 to mo 1 (P ≤ 0.001), remained the same from mo 1 to mo 4 (P ≤ 0.9), and increased from mo 4 to mo 6 (P ≤ 0.001). The parasite load of the spleen in L. major-infected mice increased from day 10 to mo 1 (P ≤ 0.003), remained the same from mo 1 to mo 4 (P ≤ 0.5), and increased from mo 4 to mo 6 (P ≤ 0.001). The parasite load of the spleen in L. tropica-infected mice increased from day 10 to mo 1 (P ≤ 0.001), from mo 1 to mo 4 (P ≤ 0.001), and decreased from mo 4 to mo 6 (P ≤ 0.001). For the purpose of having a preliminary evaluation of the long-term outcome of L. tropica infection in BALB/c mice, parasite loads of the footpad, lymph node, and spleen were determined in 4 mice at mo 11 after L. tropica infection. Results showed that the parasite loads at mo 11 PI were not significantly different from those at mo 6 PI (data not shown).

Parasite loads of L. tropica and L. major in different tissues of infected BALB/c mice are compared in Fig. 2. In the footpad, the parasite load of L. tropica was higher than that of L. major at day 10 (P ≤ 0.002) and day 30 (P ≤ 0.01) after infection. However, the parasite load of L. major became higher than the parasite load of L. tropica at mo 4 and mo 6 after the infection (P ≤ 0.009 for mo 6). In lymph nodes, the parasite load of L. tropica was statistically higher than the parasite load of L. major at day 10, mo 1, and mo 4 after the infection (P ≤ 0.001 for all intervals). In the spleen, the L. tropica parasite load was higher than the L. major parasite load at mo 1 (P ≤ 0.01) and mo 4 (P ≤ 0.001) after the infection. However, the L. major parasite load in the spleen became higher than the L. tropica parasite load 6 mo after the infection (P ≤ 0.01).

The spleen and lymph node weights in L. major and L. tropica-infected BALB/c mice are shown in Fig 3. In L. major-infected mice, the weight of the spleen and lymph node increased progressively throughout the experiment. The weight increase in L. major-infected spleen was statistically significant at mo 1 (P ≤ 0.01) and mo 6 (P ≤ 0.005) after the infection. The weight increase of the L. major-infected lymph node was statistically significant at mo 1(P ≤ 0.004), mo 4 (P ≤ 0.001), and mo 6 (P ≤ 0.04) after the infection. The general trend of the increase was in accordance with the increase in parasite loads in the same organ at the corresponding time. In L. tropica-infected mice, the weight of the spleen increased slightly until mo 1, and remained steady until mo 4, after then diminished. It was noteworthy that these slight differences were not statistically significant. The weight of the lymph node in L. tropica-infected mice increased progressively and significantly between day 10 and mo 1 (P ≤ 0.002), and from mo 1 to mo 4 (P ≤ 0.001). The weight reduced slightly, but insignificantly by mo 6. Comparison of the spleen weights between L. tropica and L. major-infected mice showed that the spleen weight was significantly greater in L. major-infected spleen in comparison to the L. tropica-infected spleen at mo 6 PI. The weight of spleens at other intervals or the lymph node weights throughout the experiment were not statistically different between L. tropica and L. major-infected mice.