Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran

Abstract

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

Figure 1

(a) Random amplified polymorphic DNA (RAPD) profiles obtained from Leishmania stocks and isolates with the AB1-O7 primer. Lane 1, L. infantum stock, lanes 2–12, L. infantum isolates. (b) Lane 1, L. tropica stock, lanes 2-3, L. tropica. M: 100 bp size marker (Roche).

Figure 2

Flow chart which outlines the major parts of the study and results. B PCR: blood PCR, BM: bone marrow, S: spleen tissue, L: liver tissue, and DAT: direct agglutination test.

Figure 3

Restriction fragment length polymorphism (RFLP) patterns obtained from Leishmania stocks and patients samples. lane 1, negative control, lane 2, L. tropica, lanes 3, 4, L. major, lanes 5,6, L. infantum. M: 100-bp size marker (Fermentas).

Figure 4

Distribution of Leishmania species isolated from human and animal hosts from some parts of Iran, identified based on ITS1-PCR-RFLP (2004–2012).

Figure 5

Phylogenic tree of the ITS1 region nucleotide sequences of Leishmania isolates recovered from humans and reservoir animals infected with cutaneous and visceral leishmaniasis. The tree was constructed by using the maximum likelihood, Tamura 3-parameter model in MEGA software version 5. The numbers above the branches indicate the percentage of bootstrap. Branches without numbers have frequencies of less than 70%.