Original Article

Molecular Identification and Intra-species Variations among Leishmania infantum Isolated from Human and Canine Visceral Leishmaniasis in Iran


Background: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been re-ported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary.

Methods: The investigation was conducted from 2014 to 2015 in the northwest and south of Iran, where VL is endemic (7 provinces). Blood samples of patients and infected dogs were collected and sera separated for serologic examinations (DAT, rK39). Spleen or bone marrow samples from infected dogs were also collected to confirm the infection. DNAs of 70 samples amplified by targeting a partial sequence of ITS (18S rRNA–ITS1–5.8S rRNA–ITS2) gene. All the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the TaqI enzyme.

Results: The cause of all 70 VL cases, were L. infantum, so, the dominant specie is L. infantum. The sequencing results of all VL cases and RFLP analysis corroborate each other. Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify and construct the phylogenetic relationship of Iranian isolates. In addition, detection and differentiation of Leishmania spp. DNA was confirmed by amplification of variable area of the minicircle kDNA (conserved sequence blocks (CSB)).

Conclusion: Low divergence and high likelihood were seen among L. infantum isolates of human and dogs from Iran with a very slight divergence was seen between isolates from northwest and south of Iran, thus grouped in a unique clad. No correlation was observed between intraspecies divergence and geographic distribution of the isolates.

World Health Orzation (WHO). 2000. Leishmaniasis and Leishmania/HIV coinfection, WHO/CDC/CSR/ISR.

Desjeux, P., 2004. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. 27:305–318.

Alvar, J., Vélez, I.D., Bern, C., Herrero, M., Desjeux, P., et al., 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7, e35671.

Alborzi, A., Rasouli, M., Shamsizadeh, A., 2006. Leishmania tropica-isolated patient with visceral leishmaniasis in southern Iran. Am. J. Trop. Med. Hyg. 74, 306– 307.

Mohebali, M., 2012. Epidemiological status of visceral leishmaniasis in Iran: experiences and review of literature. J. Clin. Exp. Pathol. S3 (2161-0681).

Badirzadeh, A., Mohebali, M., Ghasemian, M., Amini, H., Zarei, Z., Akhoundi, B., Hajjaran, H., Emdadi, D., Molaei, S., Kusha, A., 2013. Cutaneous and post kala-azar dermal leishmaniasis caused by Leishmania infantum in endemic areas of visceral leishmaniasis, Northwestern Iran 2002–2011: a case series. Pathog. Glob. Health 107, 194–197.

Mohebali, M., Hajjaran, H., Hamzavi, Y., Mobedi, I., Arshi, S., Zarei, Z., Akhoundi, B., Naeini, K.M., Avizeh, R., Fakhar, M., 2005. Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet. Parasitol. 129, 243– 251.

Mohebali, M., Edrissian, G.h., Nadim, A., Hajjaran, H., Akhoundi, B., Hooshmand, B., et al., 2006. Application of Direct Agglutination Test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iranian J Parasitol. 1(1):15-25.

Leite, R.S., Ferreira, S.A., Ituassu, L.T., Melo, M.N., Andrade, A.S.R., 2010. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples. Vet. Parasitol. 170: 201–6.

Van Der Auwera, G., Maes, l., Doncker, S.D., Rvel, C., Cnops, L., Esbroeck, M.V., Clerinx, J., Dujardin, J.C., 2013. Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics. www.eurosurveillance.org.

Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J., Bastien, P., 2002. Comparison of six methods using peripheral blood for detection of canine visceral leishmaniasis. J Clinic Microbiol. 40(1): 210-215.

Nasereddin, A., Ereqat, S., Azmi, K., Baneth, G., Jaffe, C.L., Abdeen, Z., 2006. Serological survey with PCR validation for Canine Visceral Leishmaniasis in northern Palestine. J. Parasitol. 92 (1), 178–183.

Mohammadiha, A., Haghighi, A., Mohebali, M., Mahdian, R., Abadi, A.R., et al. 2013a. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol. 192: 83–90.

Mohammadiha, A., Mohebali, M., Haghighi, A., Mahdian, R., Abadi, A.R., et al., 2013b. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol. 133: 89–94.

Mahmoudzadeh-Niknam, H., Ajdary, S., Riazi-Rad, F., Mirzadegan, E., Rezaeian, A., Khaze, V., Djadid, N.D., Alimohammadian, M.H., 2012. Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran. Trop Med Int Health. 17 (11) 1335–1344.

Hajjaran, H., Mohebali, M., Teimouri, A., Oshaghi, M.A., Mirjalali, H., Kazemi-Rad, E., et al. 2014. Identification and Phylogenetic Relationship of Iranian Strains of Various Leishmania Species Isolated from Cutaneous and Visceral Cases of Leishmaniasis Based on N-Acetyl Glucosamine-1-phosphate Transferase Gene. Infect Genet Evol. 26: 203– 212.

Mirzaei A, Schweynoch C, Rouhani S, Parvizi P, Schonian G. Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leishmaniasis in Iran. Trans R Soc Trop Med Hyg. 2014; 108:502–12. doi: 10.1093/trstmh/tru085.

World Health Orzation (WHO). 1991. Basic laboratory Methods in Medical Parasitology. First ed. WHO, Geneva.

Edrissian, G.H., Hajjaran, H., Mohebali, M., Soleimanzaeh, G., Bokaei, S., 1996. Application and evaluation of direct agglutination test in serodiagnosis of visceral leishmaniasis in man and canine reservoirs in Iran. Iranian J. Med. Sci. 21, 119–124.

Aransay, A.M., Scoulica, E., Tselentis, Y., 2000. Detection and identification of Leishmania DNA within naturally infected sandflies by semi-nested PCR on minicircle kinetoplast DNA. Applied Environ Microbiol. 66: 1933-1938.

Brewster, S., Aslett, M., Barker, D.C., 1998. Kinetoplast DNA minicircle database. Parasitol Today. 14: 437-438.

Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics. 23, 2947–2948.

Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution. 30, 2725–2729.

Shirzadi, M.R. 2008. Epidemiological information of cutaneous and visceral leishmaniasis in I.R. Iran. In: Report of the constructive meeting on cutaneous leishmaniasis. WHO. Geneva. 7–8.

Hosseininejad, M., Mohebali, M., Hosseini, F., Karimi, S., Sharifzad, S., Akhoundi, B., 2011. Seroprevalence of canine visceral leishmaniasis in asymptomatic dogs in Iran. Iranian Journal of Veterinary Research, Shiraz University. 13 (1) 38.

Malmasi, A., Janitabar, S., Mohebali, M., Akhoundi, B., Maazi, N., Aramoon, M., Khorrami, N., Seifi, H.A., 2014. Seroepidemiologic Survey of Canine Visceral Leishmaniasis in Tehran and Alborz Provinces of Iran. J Arthropod-Borne Dis. 8 (2): 132–138.

Gani, Z.H., Hassan, M.K., Jassim, A.M., 2010. Sero-epidemiological study of visceral leishmaniasis in Basrah, Southern Iraq. The Journal of The Pakistan Medical Association 60: 464-469.

Fakhar, M., Motazedian, M.H., Hatam, G.R., Asgari, Q., Kalantari, M., Mohebali, M., 2008. Asymptomatic human carriers of Leishmania infantum: possible reservoirs for Mediterranean visceral leishmaniasis in southern Iran. Ann. Trop. Med. Parasitol. 102, 1–7.

Mirzaei, A., Rouhani, S., Taherkhani, H., Farahmand, M., Kazemi, B., Hedayati, M., Baghaei, A., Davari, B., Parvizi, P., 2011. Isolation and detection of Leishmania species among naturally infected Rhombomys opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkmen Sahara, North East of Iran. Exp. Parasitol. 129, 375–380.

Mirzaei, A., Rouhani, S., Kazerooni, P.A., et al., 2013. Molecular detection and identification of Leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in Fars province, south of Iran. Iranian J Parasitol. 8 (2) 280-288.

Hajjaran, H., Vasigheh, F., Mohebali, M., Rezaei, S., Mamishi, S., Charedar, S., 2011. “Direct diagnosis of Leishmania species on serosity materials punctured from cutaneous leishmaniasis patients using PCR-RFLP”. Journal of Clinical Laboratory Analysis. 25 (1):20–24.

Parvizi, P., Ready, P.D., 2008. Nested PCRs of nuclear ITS-rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 13: 1159–71.

Rassi, Y., Javadian, E., Nadim, A., Zahraii, A., Vatandoost, H., Motazedian, M.H., Azizi, K., Mohebali, M., 2005. Phlebotomus (Larroussius) kandelakii the principal and proven vector of visceral leishmaniasis in north-west of Iran. Pak J Biol Sci. 8(12): 1802-1806.

Nadim, a., Javadian, e., Mohebali, m., Zamen Momeni, a., 2009. “Leishmania parasites and Leishmaniases,” in Epidemiology of Leishmaniasis in Iran, Tehran University Press, Tehran, (Persian).

Waki, K., Dutta, S., Ray, D., Kolli, B.K., Akman, L., Kawazu, S.-I., Lin, C.-P., Chang, K.-P., 2007. Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of transendoplasmic reticulum N-acetylglucosamine-1-phosphate transferase. Eukaryot. Cell 6, 198–210.

Hajjaran, H., Mohebali, M., Mamishi, S., Vasigheh, F., Oshaghi, M.A., Naddaf, S.R., Teimouri, A., Edrissian, G.H., Zarei, Z., 2013. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed. Res. Int. Article ID 789326, 7 pages.

IssueVol 13 No 4 (2018) QRcode
SectionOriginal Article(s)
Phylogeny Leishmania infantum PCR-RFLP Sequencing Iran

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
DALIMI A, MOHAMMADIHA A, MOHEBALI M, MIRZAEI A, MAHMOUDI M. Molecular Identification and Intra-species Variations among Leishmania infantum Isolated from Human and Canine Visceral Leishmaniasis in Iran. Iran J Parasitol. 2018;13(4):567-576.