Inbred female BALB/c mice, 6-8 week old, 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 type 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 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.

Mice were infected intradermally by defined doses of live stationary phase promastigotes in the ear dermis for a primary infection and in the hind footpad for a secondary infection. The disease evolution was monitored by measurement of thickness of ear or footpad at weekly intervals. The thickness was deterMice were infected intradermally by defined doses of live stationary phase promastigotes in the ear dermis for a primary infection and in the hind footpad for a secondary infection. The disease evolution was monitored by measurement of thickness of ear or footpad at weekly intervals. The thickness was determined by a dial-gauge caliper (Mitutoyu, Kawasaki, Kanagawa, Japan).

DTH responses to the secondary infection were determined by measuring footpad thickness at 18-24, 48, and 72 hr post-secondary infection. DTH values were calculated as the thickness of parasite-injected footpad minus thickness of uninjected contralateral footpad.

BALB/c mice, in groups of 5-10 mice, were infected in the ear by L. major or L. tropica as a primary infection, and disease evolution was monitored by determination of the ear thickness. A secondary infection was done by injection of L. major or L. tropica in the hind footpad, 2-4 mo after the primary infection. DTH responses to the secondary infection were determined by determination of footpad thickness at defined times after the secondary challenge. Each experiment was repeated at least once, and data from one representative experiment is shown.

Footpads or ear thicknesses were compared between different experimental groups by Student's t-test. P-values, equal to or less than 0.05, were considered significant.

BALB/c mice were infected in the ear with 10³, 10⁴, 10⁵ stationary phase L. major promastigotes. Ear thickness was determined up to 20 weeks after infection. Results show that higher doses of L. major results in earlier appearance of the lesion and higher ear thickness (Fig. 1). The ear thickness differences were statistically significant between all 3 groups at week 8 and 10 after infection (P < 0.008 and P < 0.05, respectively). Injection of 10³ dose of L. major was able to establish infection as evidenced by increase of ear thickness. Our data shows that, although infection by all doses finally results in lesion development, the development of lesion was significantly delayed in lower dose infections. In other words, when we compared the state of disease exacerbation in mice infected with different doses of parasites at a definite time after a primary infection (for example at the time of a secondary infection which was 2 mo after the primary infection), the disease was already exacerbated in mice infected by 10⁵ parasites, while lesion did not even appear in mice infected by 10³ parasites (see below). It means that mice received a secondary L. major infection at different states of disease exacerbation originated from different doses of primary infection.

BALB/c mice were infected by injection with 10⁵ stationary phase L. major promastigotes intradermally into the ear dermis. One million stationary phase L. major promastigotes were injected subcutaneously into hind footpad 2-3 mo after a primary infection, and DTH responses were determined up to 3 days and footpad thickness determined for up to 20 weeks. The results are shown in Figs. 2 and 3. It was shown that primary L. major infection can; (1) exacerbate secondary L. major infection in BALB/c mice in a statistically significant manner (P < 0.001 in all compared intervals), and (2) induce higher DTH responses at 24 hr post-injection (P < 0.0001). So, DTH is associated with more exacerbation of a secondary L. major infection in BALB/c mice in our experimental system.

Experiments were carried out for study on the effect of the dose of primary L. major primary infection on exacerbation of the secondary L. major infection as well as on DTH responses to this secondary infection. Primary infections were done by 10³, 10⁴, or 10⁵ doses of stationary L. major in ear dermis. The secondary infection was done by 10⁶ stationary L. major promastigotes in footpad, 2-3 mo after the primary infection. Disease evolution and DTH response of these experiments are shown in Figs. 4 and 5. Footpad thickness increased more rapidly in mice infected with higher doses of primary L. major infection (P < 0.05 between all 3 groups at week 12 after secondary infection). DTH responses at 24 hr after the secondary infection were also higher in mice receiving higher doses of L. major as the primary infection (P < 0.05 between mice receiving different doses of the primary infection). Based on these results, and for excluding the effect of infecting dose, we decided to compare the same infectious dose of L. major and L. tropica for their DTH induction capability in BALB/c mice.

Mice were infected with 10⁵ stationary phase promastigotes of L. major or L. tropica into ear dermis as the primary infection. Mice received 10⁶ stationary phase promastigotes of L. major or L. tropica as the secondary infection into hind footpad about 3-4 mo after the primary infection. DTH responses against the secondary infection were determined at 18-24, 48, and 72 hr post-secondary infection. As shown in Fig. 6, the DTH response against L. major was statistically higher in L. major-infected mice in comparison to L. tropica-infected mice (P < 0.0001). Similar statistically important differences were observed between the DTH response against L. tropica secondary infection in mice that received L. major or L. tropica as the primary infection (P < 0.0001).