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Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco.


Phlebotomine sandflies (Diptera: Psychodidae) are small-sized blood-sucking insects feeding on a wide range of hosts, and potentially transmit pathogens to man and other animals. These are vectors of bacteria (e.g. Bartonella bacilliformis), viruses (e.g. Phlebovirus and Vesiculovirus), and protozoa (e.g. Leishmania spp) (1-4). Among > 800 phlebotomine sandfly species estimated to exist, only 98 species of Phlebotomus and Lutzomyia genera are proven or suspected vectors of human leishmaniasis (5). This parasitic disease is caused by the unicellular Leishmania parasites which affect 12 million of people worldwide (6).

Leishmania tropica a causative agent of cutaneous leishmaniasis (CL) is transmitted from person-to-person through Phlebotomus (Paraphlebotomus) sergenti in urban settings (5). It was originally described from Algeria, although the species has a broad range of distribution which covers vast areas of southern mediterranean (Morocco, Algeria and Tunisia), northern mediterranean, Saudi Arabia, Afghanistan, Pakistan and northern parts of India. Such a broad distribution suggests a notable intraspecific variability and since the distribution of the vector is broader than the distribution of the transmitted parasite (7), it may play an important role in the epidemiology of the disease. Phlebotomus sergenti has been confirmed as the vector of CL caused by L. tropica in Morocco (8), Algeria (9) and Tunisia (10).

Sandflies are also vectors of many viruses belonging to three different genera: (i) the Phlebovirus (family Bunyaviridae) including sandfly fever Sicilian virus (SFSV) (1), sandfly fever Naples virus (SFNV) (1), and Toscana virus (TOSV) (1); (ii) the Vesiculovirus (11-12); and (iii) the Orbivirus (13). SFNV and SFSV lead to short-term febrile illness, whereas TOSV virus can cause central nervous system (CNS) infections such as meningitis, encephalitis, meningoencephalitis and peripheral neurologic symptoms. TOSV has been isolated from the P. perniciosus (14), P. perfiliewi (15), and TOSV RNA was detected in Sergentomyia minuta (16).

Several species of Leishmania and phleboviruses have a common Phlebotomus spp vector. For example, P. ariasi is a vector of L. infantum (17) and SFSV (18), P. longicuspis is a potential vector of L. infantum (19) which have been found to be infected by SFNV and SFSV (20) and P. perniciosus can transmit both L. infantum (21) and TOSV (5). The first robust evidence for the existence of an epidemiological relationship between Leishmania and phleboviruses, which has been assumed for a long time, was described by Bichaud et al (22). The main objectives of this study were to inventory the Phlebotomine sandfly species in an emerging focus of CL due to L. tropica, in the El Hanchane district in the center of Morocco, and to detect Leishmania DNA and phlebovirus RNA in sandflies using the internal transcribed spacer 1 (ITS1) gene system and a generic nested RT-PCR, respectively.


Study sites and collection of sandflies

Sandfly sampling was carried out from July to September 2011 (during the period of sandfly peak prevalence), in the district of El Hanchane (31[degrees] 31' 11" N and 9[degrees] 26' 02" W) which is 30 km near the town of Essaouira in the centre of Morocco. Population size was 5349 and this semi-rural locality was selected based on the emergence of CL cases. The sandflies were collected using CDC light-traps placed in or near human dwellings or horse stables. The traps were set in the late afternoon and sandflies were collected from the traps the following morning. All the sandflies were identified morphologically by dissecting genital organs according to morphological keys modified by Boussaa (23).

For detection of phlebovirus RNA, sandflies were pooled with a maximum of 30 individuals per pool, based on trapping origin, species and sex, and were placed in 1.5 ml tubes and stored at -80[degrees]C. Phlebotomus sergenti females were tested individually for infection by Leishmania species, a part of nucleic acid (NA) extracted was pooled for the detection of phlebovirus RNA.

Extraction of NA from sandflies

RNA extraction for 33 pools of sandflies was performed using the "Mini kit MACHEREY-NAGEL Nucleospin RNA II" (Duren, Germany) according to the manufacturer's instructions. In 273 females of P. sergenti total nucleic acid was extracted via Biorobot EZ1, by Qiagen kit according to the instructions of the manufacturer.

Detection of Phlebovirus and Leishmania infection

Phlebovirus: The RNA was transcribed into cDNA with the "Transcriptor First Strand cDNA Synthesis Kit, Roche" (Mannheim, Germany), PCRs were performed as previously reported (24). To avoid the risks of contamination, positive controls were not included in the experiment. The PCR product was cloned and sequenced in both directions.

Leishmania typing: The ribosomal internal transcribed spacer 1 (ITS1) was subjected to PCR, using the LITSR and L.5.8S primers (25). Amplification reaction was performed in a volume of 50 [micro]l. The reaction was performed in a thermocycler (S1000[TM] Thermal Cycler, Bio-Rad) with the following steps: initial denaturation at 94[degrees]C for 2 min followed by 32 cycles each, consisting of denaturation at 94[degrees]C for 20 s, annealing at 53[degrees]C for 30 s and extension at 72[degrees]C for 1 min and in the end post-extension phase at 72[degrees]C for 6 min. PCR products were analyzed using a 1.5% agarose gel together with a 100 bp ladder marker, and were visualized by UV-light transillumination.

For Leishmania species discrimination, ITS1-PCR products were assigned to RFLP analysis. The PCR products were digested with the restriction endonuclease HaeIII (fermentase-lithvani) for 2 h at 37[degrees]C. The produced fragments were separated by electrophoresis on 2% agarose gel and compared with those of WHO reference strains of L. major (MHOM/SU/73/5ASKH), L. tropica (MHOM/SU/74/K27) and L. infantum (MHOM/TN/80/ IPT1). Contamination was monitored by negative controls during processes of NA extraction and the PCR amplifications. Genomic NA was extracted in a room where the amplified DNAs were never processed and the PCRs were done in a separate room.


Sandflies collected

All the sandfly samples trapped during this study were identified individually to the species level. A total of 643 were collected (374 females and 269 males). Phelobotomus sergenti was the most abundant species (76.67%), followed by P. longicuspis (11.51%), S. minuta (3.89%), P. alexandri (3.42%), S. antennata (2.18%), and P. perniciosus (1.24%), respectively. The remaining species were P. langeroni (0.62%), P. kazeruni (0.31%), and P. bergeroti (0.16%) (Table 1).

Toscana virus infections identified in sandflies

Out of 33 pools, only one pool of male P. sergenti contained phlebovirus RNA. This 201-nucleotide sequence (excluding primers) is identical at the nucleotide level with the sequence of Toscana virus detected in Morocco (TOSV_Morocco_SLP1_JN832571) handled 2 yr before. The phylogenetic analysis indicated that this sequence was most closely (100% bootstrap support) related to the Toscana virus France strain (GenBank Accession No. FJ153281).



Groupings were determined by a neighbour-joining method with MEGA v.5.05, and the robustness of the groups were tested using 500 bootstrap pseudo-replicates (Fig. 1).

Leishmania infections identified in sandflies

A total of 10 females of P. sergenti were found positive for L. tropica (3.66%) showing a band of approximately 320 bp. The RFLP profiles for 10 infected P. sergenti consisted of two bands (185 and 57/53 bp) were identical to that of the L. tropica (Fig. 2). In another context, this result is confirmed by sequencing of ITS-5, 8rRNA gene which allowed the identification of six different sequences of L. tropica (Data not shown).


Infections by TOSV and Leishmania represent an important public health problem in the countries where these pathogens circulate. TOSV has a tropism for the central nervous system and is a major cause of meningitis and encephalitis in parts of the mediterranean basin. In humans, infection with Leishmania spp ranges from asymptomatic form to severe visceral involvement.

Phlebotomus sergenti, the most abundant species in this survey is related to the semi-arid bioclimatic belt where the vegetation is characterized by Thuja (Tetraclinis articulata), Junipers (Juniperus spp) and to a lesser degree, to the arid belt, by Argan (Argania spinosa) and common Jujube (Ziziphus lotus) (26). Indeed, this type of vegetation is characteristic of El Hanchane area especially the Tetraclinis articulata and Argania spinosa.

In Morocco, L. tropica had been isolated from P. sergenti for the first time over three decades ago (27). Since then, P. sergenti was suggested as a vector in several CL foci based only on epidemiological and entomological findings, including the abundance and endophily of this species. This is the first report of detection of L. tropica DNA within P. sergenti naturally infected in an emerging CL focus in the country.

Leishmania infection of sandfly has classically been examined by dissecting fresh individual sandflies under a microscope. This method needs dissecting expertise and a large number of specimens, since the Leishmania infection rate in sandflies is usually very low (0.01-1%) even in the highly endemic areas (28). In recent years, molecular techniques based on PCR (29-30) are used to identify Leishmania infection and could be a useful tool in epidemiological studies and strategic planning for the control of human leishmaniasis (5,31-32). The infection rate is an essential parameter for estimating the vectorial capacity of the sandfly. In the present study, the rate of P. sergenti females naturally infected by L. tropica was ~3.66%. This value is lower than the rates recently described in Almadinah Al-munawarah province where 31% of P. sergenti females were found positive against kDNA of L. tropica (33).

One pool of P. sergenti males was infected by TOSV; to our knowledge, this is the first time that TOSV has been detected in phlebotomine sandflies other than P. perniciosus, P. perfiliewi and S. minuta. Since, male sandflies are non-haematophagous, virus detection has been interpreted as evidence of transovarial transmission of this phlebovirus under natural conditions. In Morocco, TOSV has been previously detected in male P. perniciosus in Sefrou province (34).

Since, TOSV was previously detected from sandflies in our laboratory, the question of contamination should be discussed. First, we do not grow the virus in cell culture and never use positive controls for RT-PCR screening. Second, previous detection of TOSV RNA and RTPCR was done more than two years earliar to this finding: meanwhile, a total of 22 RT-PCR experiments were conducted without finding any positive results. Third, sequencing of this RT-PCR product was performed not in the laboratory, but in Marseille where the material was shipped. For these reasons, it was believed that the finding of TOSV RNA in these sandflies is valid.

We demonstrated that L. tropica and TOSV could infect the common arthropod vector (P. sergenti). It has been also reported that both TOSV and L. infantum are transmitted by P. perniciosus. Indeed, Bichaud et al (22) confirmed the existence of an epidemiological relation ship between L. infantum and TOSV infections and serological analyses were based on the IgG detection for both Leishmania and TOSV. Another example of this association between Karimabad virus infection and CL was reported in Iran (35).


In conclusion, our data showed that P. sergenti is a potential vector of L. tropica and TOSV. Therefore, more epidemiological investigations are proposed to elucidate the role of P. sergenti in the ecology of TOSV and further serological studies are required to estimate the population risk-factors of infections by TOSV and L. tropica in this focus, because persons exposed to Leishmania parasite infections are at greater risk of being infected with TOSV.


This work was supported by EMRO-COMSTECH Grant RAB&GH 10-11/09 and Action Concertees Inter Pasteuriennes ACIP-A-08-2007. We would like to thank the team of the Department of Parasitology, Direction d'Epidemiologie et de Lutte Contre les Maladies, Moroccan Ministry of Health. The authors are also very grateful to doctors and nurses of the El Hanchane Health Center.


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Nargys Es-Sette [1-2], Malika Ajaoud [1], Laurence Bichaud [3], Salsabil Hamdi [1], Fouad Mellouki [2], Remi N. Charrel [3] & Meryem Lemrani [1]

[1] Laboratoire de parasitologie et de maladies vectorielles, Institut Pasteur du Maroc, Casablanca; [2] Departement de Biologie, Faculte des sciences et techniques, Mohammadia, Morocco; [3] Aix Marseille Univ, IRD French Institute of Research for Development, EHESP French School of Public Health, UMR_D 190 "Emergence des Pathologies Virales", Marseille, France

Correspondence to: Dr Meryem Lemrani, Institut Pasteur du Maroc, Laboratoire de parasitologie et de maladies vectorielles, 1 Place Louis Pasteur, 20360, Casablanca, Morocco.


Received: 24 April 2013

Accepted in revised form: 4 March 2014
Table 1. Species and gender of sandflies processed for
phleboviruses and Leishmania

Species             Sandflies       Pools

                 Male   Female   Male   Female

P. sergenti      220     273       9       10
P. longicuspis    22      52       1        2
P. alexandri      0       22       0       1
P. perniciosus    4        4       1       1
P. kazeruni       0        2       0       1
P. langeroni      4        0       1       1
P. bergeroti      0        1       0       1
S. minuta         6       19       1       1
S. antennata      13       1       1       1

Sub total        269      374     14      19

Total                 643              33
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Author:Es-Sette, Nargys; Ajaoud, Malika; Bichaud, Laurence; Hamdi, Salsabil; Mellouki, Fouad; Charrel, Remi
Publication:Journal of Vector Borne Diseases
Article Type:Report
Geographic Code:6MORO
Date:Jun 1, 2014
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