The main objectives of this study were to identify the sand fly vector and Leishmania species responsible for the recent outbreaks of CL in El Hanchane locality, a semi rural area in central Morocco where the first cases of CL appeared in 2006. Two molecular techniques were used for this purpose, ITS1 PCR-RFLP and nested PCR. Of the 7 samples of CL patients, 4 were successfully amplified and characterized by PCR-RFLP. The three remaining specimens were possibly either inhibited, or failed to amplify because of the low amount of DNA in stained slides. Several studies have exploited the amplification of the ITS-1 region for identifying Leishmania spp. in bone marrow aspirates or stained smears [13, 17–19]. The major advantage of the ITS1-PCR is that species identification can be achieved by simple digestion of the PCR products. Thus, all clinically important species can be distinguished by their RFLP patterns. However, previous reports have shown that ITS1 PCR-RFLP is not sensitive enough for diagnostic purposes [20, 21]. Using nested PCR, the 7 patients were all positive, confirming the better sensitivity of this technique .
In this survey, a total of 643 sand flies were collected indoors from July to September 2011 in an emerging focus of CL. The sandfly populations were mainly represented by P. sergenti (76.67%) and P. longicuspis (11.51%). Three unengorged female P. sergenti were infected by Leishmania, similar to L. tropica as demonstrated first by their ITS1PCR-RFLP profiles and then confirmed by the sequence identity of ITS-rDNA gene. In Morocco, L. tropica was first isolated from P. sergenti over three decades ago . Since then and despite the large number of foci of CL due to L. tropica in Morocco, the vectors have never been identified with certainty because most epidemiological studies have focused on the identification of the parasite isolated from humans. P.sergenti is suggested as a vector based only on the circumstantial evidence of its ecology and its high frequency in an endemic area.
Leishmania infection of sand flies has classically been examined by dissecting freshly caught individual sand flies. This method allows the culture of Leishmania strains found in the sand fly gut; but it is time-consuming, needs dissecting expertise and a large number of specimens, since the Leishmania infection rate in sand flies is usually very low even in the highly endemic areas . In recent years, molecular techniques have been used increasingly to identify Leishmania infection both in experimentally infected and field captured phlebotomine sand flies, as a sensitive and effective tool useful in epidemiological studies to facilitate strategic planning for the control of human leishmaniasis [24–26]. The increasing application of molecular techniques in this field has considerably reduced the time needed to obtain results; but such methods are underused in Morocco, despite the existence of CL foci in large areas and the lack of information for its vectors. This is the first report that L. tropica DNA was found in naturally infected P. sergenti from a CL focus of this country. The high density of P.sergenti indoors (i.e., anthropophilic) and their infection with L. tropica among unengorged females, suggest that this species plays a major role as the principal, if not the only vector in this locality.
P. sergenti is the confirmed vector of L. tropica throughout North Africa, Middle East and Central Asia [27–31]. However, P. chabaudi and the closely related species P. riouxi were also suspected as vectors based on their abundance in Tunisian and Algerian foci of CL due to L. tropica[32, 33]. In Israel, in addition to P. sergenti, P. arabicus has been involved in the transmission of L. tropica[34, 35]. None of these sand fly species were found in the present CL focus, where P. sergenti represents 76.67% of the total number of sand flies collected. On the other hand, the screening for Leishmania infection of the other sand fly species, separated into 5 monospecific pools, showed no PCR positives (data no shown), suggesting that P. sergenti is the only vector in this locality.
L. tropica is classically considered to be anthroponotic, however, in the present emerging focus, an anthroponotic transmission is very improbable, since the number of CL cases is too small, and their geographical distribution is too sparse and sporadic to consider humans as an adequate reservoir. Zoonotic outbreaks have been reported in many countries. Rock hyraxes were incriminated as reservoir hosts of L. tropica in Kenya and Namibia [36, 37], in Israel L. tropica was isolated from rock hyrax (Procavia capensis): the strains were identical to those obtained from humans and sand flies in the same focus [38, 39]. In southeast Tunisia gundi (Ctenodactylus gundi) was supported as a reservoir host of L. tropica[40, 41], whereas in Algeria human CL caused by L. killicki is considered as a zoonotic disease with P. sergenti sand flies acting as vectors and gundi rodents (Massoutiera mzabi) as reservoirs . In Morocco L. tropica has also been isolated from dogs in Azilal, a southern CL focus , but the reported infections in dogs were strictly cutaneous, the evolution duration of lesions was too short to consider dogs as the reservoir of the parasite.
L. tropica is recognized as a very heterogeneous species of Leishmania and intraspecific heterogeneity has been readily demonstrated by many investigators [43–45]. Microsatellite analysis revealed that two genetically very distinct populations of L. tropica co-exist within the same focus in Morocco: population ‘Morocco A’ is related to population ‘Asia’, whereas population ‘Morocco B’ is genetically closer to the other African populations .
In terms of isoenzyme profile variability, Morocco is characterized by the largest number of zymodemes ever described in L. tropica, with 8 zymodemes detected from human CL, dogs and the sandflies, e.g. P. sergenti[4, 5, 47]. In the present study, the DNA sequences of L. tropica isolated from P. sergenti showed a great heterogeneity, that was also observed in a southern Moroccan focus where L.tropica isolates from P. sergenti showed a wider range of zymodemes than isolates collected from the vertebrate hosts (dogs and humans) in the same region : 74 L. tropica strains isolated from P. sergenti females were typed and corresponded to four zymodemes (MON-102: one strain; MON-107: 56 strains, MON-122: two strains; and MON-123: 15 strains). Only the first two zymodemes were also identified in humans and their frequencies were different in humans, dogs and the vector . The zymodeme heterogeneity of strains isolated from vectors was also described with L. infantum isolates from P. perniciosus compared to vertebrate hosts in the same Spanish region . Our results reinforce the observation that L. tropica is polymorphic; especially among those detected in P. sergenti compared to human samples.