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. 2013 Jul 26:6:217.
doi: 10.1186/1756-3305-6-217.

Detection and molecular typing of Leishmania tropica from Phlebotomus sergenti and lesions of cutaneous leishmaniasis in an emerging focus of Morocco

Malika Ajaoud  1 , Nargys Es-sette, Salsabil Hamdi, Abderahmane Laamrani El-Idrissi, Myriam Riyad, Meryem Lemrani
Affiliations

Detection and molecular typing of Leishmania tropica from Phlebotomus sergenti and lesions of cutaneous leishmaniasis in an emerging focus of Morocco

Malika Ajaoud et al. Parasit Vectors. .

Abstract

Background: Cutaneous leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. In Morocco, anthroponotic cutaneous leishmaniasis due to Leishmania tropica is considered as a public health problem, but its epidemiology has not been fully elucidated. The main objective of this study was to detect Leishmania infection in the vector, Phlebotomus sergenti and in human skin samples, in the El Hanchane locality, an emerging focus of cutaneous leishmaniasis in central Morocco.

Methods: A total of 643 sand flies were collected using CDC miniature light traps and identified morphologically. Leishmania species were characterized by ITS1 PCR-RFLP and ITS1-5.8S rRNA gene nested-PCR of samples from 123 females of Phlebotomus sergenti and 7 cutaneous leishmaniasis patients.

Results: The sand flies collected consisted of 9 species, 7 of which belonged to the genus Phlebotomus and two to the genus Sergentomyia. Phlebotomus sergenti was the most predominant (76.67%).By ITS1 PCR-RFLP Leishmania tropica was found in three Phlebotomus sergenti females and four patients (4/7). Using nested PCR Leishmania tropica was identified in the same three Phlebotomus sergenti females and all the 7 patients. The sequencing of the nested PCR products recognized 7 haplotypes, of which 6 have never been described.

Conclusions: This is the first molecular detection and identification of Leishmania tropica in human skin samples and Phlebotomus sergenti in support of its vector status in El Hanchane. The finding of seven Leishmania tropica haplotypes underscores heterogeneity of this species at a high level in Morocco.

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Figures

Figure 1
Figure 1
Map of Morocco showing El Hanchane locality: the study area.
Figure 2
Figure 2
Sensitivity analysis of nested PCR and ITS1-PCR.L. tropica DNA was diluted 10-fold starting from 103 pg and tested for amplification. The L. tropica nested PCR amplify the 400 bp product (A), and ITS1-PCR amplify about 320 bp product (B). N: Negative control. 100 bp weight molecular (WM) (Bioline).
Figure 3
Figure 3
Nested-PCR of ITS1-5.8S rDNA gene of Leishmania DNA from human samples and P. sergenti sand fly. Lines 1–7: human samples; line 8–10: P. sergenti females. Reference strains: L. infantum (MHOM/TN/80/IPT1), L. major (MHOM/SU/73/5ASKH) and L. tropica (MHOM/SU/74/K27); N: Negative control; 1 kb weight molecular (WM) (Invitrogen).
Figure 4
Figure 4
ITS1-5.8S rDNA gene sequences alignment from patients and from P. sergenti with Tunisian haplotype. Using GENtle software (v.1.9.4). Identities are denoted by points. Gaps are denoted by dashes.
Figure 5
Figure 5
Neighbor-Joining tree showing the relationships of the haplotypes of the ITS1-5.8S rDNA gene sequences of Leishmania tropica infecting Phlebotomus sergenti and patients, using MEGA 5 software, and it relates ITS1-5.8S rDNA gene haplotypes in GenBank. The tree shown is based on the Kimura 2-parameter model of nucleotide substitution. Bootstrap values are based on 1,000 replicates. L. aethiopica was used as outgroup. Black triangles indicate haplotypes of L. tropica from patients and black disks haplotypes of L. tropica from P. sergenti.
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