Molecular detection of Leishmania infantum and Leishmania tropica in rodent species from endemic cutaneous leishmaniasis areas in Morocco

Background Leishmaniasis remains a major public health problem in African nations, including Morocco, where little is known about the vertebrate reservoirs involved in the causal parasites’ transmission cycles. The present study investigates the role of rodent species as potential reservoirs of Leishmania spp. in central Morocco, where both L. tropica and L. infantum have been reported. Methods Rodents were caught from 22 sites in central Morocco, by using Sherman metal traps, and identified morphologically. For each specimen, genomic DNA was extracted from different tissues using the Speed Tools DNA extraction Kit. Then, samples were PCR-analyzed, targeting the SSU rRNA gene to detect Leishmania spp. DNA, followed by amplification of the internal transcribed spacer 1 (ITS1) and its sequencing to identify the species. Results A total of 197 rodents belonging to ten species were captured and identified: Rattus rattus (40.61%), Mus musculus (25.38%), Apodemus sylvaticus (8.63%), Mus spretus (7.11%), Meriones shawi (5.58%), Rattus norvegicus (4.57%), Meriones libycus (3.05%), Mastomys erythroleucus (2.03%), Gerbillus campestris (2.03%) and Lemniscomys barbarus (1.01%). Molecular analysis revealed the presence of Leishmania species in 18 specimens: six R. rattus (out of 80 captured; 7.5%), 11 M. musculus (out of 50 captured; 22%), and one R. norvegicus (out of 9 captured; 11.11%). Conclusions To the best of our knowledge, L. infantum and L. tropica were identified in rodent species for the first time in Morocco. These findings suggest that rodent species may be involved in L. infantum and L. tropica transmission cycles in this country but that further studies are needed to confirm their role as reservoirs of Leishmania species in Morocco. Electronic supplementary material The online version of this article (10.1186/s13071-017-2398-8) contains supplementary material, which is available to authorized users.

The detection of infection in wild and domestic animals is the first step in identifying the different host reservoirs of Leishmania spp. Host incrimination depends on the accumulation of evidence based on five criteria [21,22]: (i) geographical and temporal overlapping of vectors and hosts distributions; (ii) survival of the reservoir host long enough to permit transmission; (iii) higher infection prevalence; (iv) presence and frequency of parasites in the skin or the blood to be infective for the vector; and (v) detection of the same Leishmania species in human cases and the reservoir host.
In the present work, molecular methods were used to detect Leishmania infection in wild-caught rodents from central Morocco, from where both cutaneous and visceral leishmaniasis has been reported in humans [17,23].

Study area
This study was conducted in four regions of central Morocco: Al Haouz, Chichaoua, Essaouira and Marrakesh. A total of 22 sites ( Fig. 1) with altitudes between 318 and 2579 m were sampled. Across the study area, the climate is arid to semi-arid on the plain (up to 450 m in Marrakesh) and humid in the Atlas Mountains (right up to 4167 m on Toubkal Mountain) and on the coast (up to 50 m in Essaouira).

Rodent collection
The presence of rodents was surmised from cut plant stems and fresh droppings. Animals were captured using metal Sherman baited traps placed at the entrances of active burrows. For catching many rodent species, different baits were used in the same trap: bread with olive oil, tomatoes, potatoes and dates. The traps were set in the afternoon and recovered early the next morning. Forty traps were placed twice monthly (every 15 days) at each site between June 2014 and May 2015.

Treatment of captured rodents and DNA extraction
In the laboratory, caught rodents were anaesthetized with ether for the purposes of species identification and collecting data on weight, length (body and tail) and skin lesions (according to European decree NOR: AGRG1238767A 2013). Species identifications were made according to morphological characteristics [24]. Samples of liver, spleen, bone marrow and skin (ear lobe and, when available, skin lesions) (Fig. 2) were then harvested for parasite detection.25-30 mg of each tissue was removed with the aid of single-use forceps, scissors and scalpel blades, placed in sterile tubes, and stored at -20°C for DNA extraction.
Genomic DNA was extracted using the Speed Tools DNA extraction Kit (Biotools, Madrid, Spain) following the manufacturer's recommendations, and was eluted in a final volume of 200 μl of PCR-grade water. The extracts were stored at 4°C until PCR analysis.
Detection of Leishmania spp. DNA The extracted DNA was screened for Leishmania spp. by the LnPCR amplifying a fragment from the small subunit ribosomal ribonucleic acid gene SSU rRNA [25,26]. The first amplification step ( Table 1) was performed using R221 (specific for order Kinetoplastida) and R332 primers (specific for the genera Leishmania and Crithidia) [25]. The PCR product was then tested in a subsequent amplification step with R233 and R333 primers of the genus Leishmania, according to the protocol developed by Van Eys et al. [25] and adapted and modified by Cruz et al. [27].
A reamplification reaction was then performed in a 25 μl final volume, involving 10 μl of a 1/40 dilution of the first PCR amplicons as a template was added to a PCR mixture ( Table 1). The components, total reaction volume and programs for the implementation of the LnPCR are detailed in Table 1. The amplification products were resolved on a 1.5% agarose gel, stained with Gel Red Nucleic Acid stain (Biotium, Fremont, California, USA), and visualized under UV light. Samples yielding a PCR product of 603 bp (Additional file 1: Figure S1) and 358 bp (Additional file 1: Figure S2), respectively, by first and second amplification reaction were deemed positive for Leishmania spp. DNA.
Negative controls without DNA were employed in all assays. DNA from the reference Leishmania infantum strain MHOM/FR/78/LEM75 was used as a positive control (Additional file 1: Figures S1-S3).

Identification of Leishmania species
Samples shown to be positive by LnPCR were further analyzed to identify the Leishmania species by nested amplification of the ribosomal internal transcribed spacer 1 (ITS1-PCR).
The first PCR reaction was carried out using the primers LITSR/L5.8S as described by El Tai et al. [28] and Schönian et al. [29]. Amplification reactions were performed in volumes of 50 μl was added to a PCR mixture described in Table 1. For the second reaction, we used the primers SAC and VAN2 [30]. A reamplification reaction was then performed in a 25 μl final volume, involving 10 μl of a 1/40 dilution of the first PCR amplicons as a template was added to a PCR mixture described in Table 1.
Negative and positive controls were also included in this assay. All reactive were synthesized commercially (Biotools, B&M Labs, S.A., Madrid, Spain). All PCR products were visualized on 1.5% agarose gel as above (Additional file 1: Figure S3). Samples providing a 300-350 bp (Additional file 1: Figure S2) and 280-330 bp (Additional file 1: Figure S3), respectively, were deemed positive. The ITS1-PCR products were excised from the agarose gels and purified using the QIA quick extraction kit (Qiagen). They were then sequenced which allows to correctly identify all species of the genus Leishmania of the Old World [31], using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit v3.1 and an automated ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). These sequences were edited using BioEdit Sequence Alignment Editor Software v. 7.0.9.0 [32] and compared with others held in the GenBank database using BLAST software.
Ten animals showed ear lobe skin lesions (Fig. 2), of which 50% were confirmed positive for L. infantum.  Three of these positive animals showed, in addition, splenomegaly and hepatomegaly (compatible with visceral leishmaniasis).

Discussion
The identification of the natural hosts of Leishmania species is essential to understand the epidemiology of the disease. To our knowledge, there have been no data published since 1982 on natural Leishmania infection in rodent species in Morocco [19].
In the present study, L. infantum DNA was detected in six R. rattus, nine M. musculus (both anthropophilic species) and one R. norvegicus, while L. tropica DNA was detected in two M. musculus. This is the first time that L. infantum and L. tropica infections have been detected in rodent species in Morocco. In the wider Mediterranean region, L. infantum (which causes visceral and cutaneous zoonotic leishmaniasis) has been identified in naturally infected wild rodents including R. rattus, R. norvegicus and M. musculus [8,10,12,15,16,[33][34][35]. In Morocco, dogs make up the main host reservoir for L. infantum [17,36]. However, on the Island of Montecristo (Italy), where no dogs are present, up to 15.5% of R. rattus individuals have been reported infected with L. infantum [37], maintaining a sylvatic transmission cycle.
Leishmania tropica-induced cutaneous leishmaniasis is commonly considered an anthroponotic disease that does not involve an animal reservoir [38], but zoonotic transmission has been demonstrated in Jordan, Palestine and Israel [39,40].
In north Africa, several rodent species have been implicated in the transmission of L. tropica [39][40][41][42]:   Ctenodactylus gundi has been proposed as a reservoir host in Tunisia [42,43], while in Algeria, human cutaneous leishmaniasis caused by Leishmania killicki (a variant of L. tropica) is considered a zoonotic form with P. sergenti as a vector and Massoutiera mzabi as a reservoir [39]. However, in Morocco L. tropicais generally considered an anthroponotic species, despite its identification in dogs on many occasions [44][45][46]. In the present study, the regions of Chichaoua, Al Haouz and Essaouira were known to be endemic foci of L. tropica-induced cutaneous leishmaniasis for humans [47][48][49], while, L. infantum is reported responsible for sporadic human cases of visceral leishmaniasis in these areas [50,51]. In the same study area, Boussaa et al. [23] reported L. infantum-induced leishmaniasis in dogs, which returned very high seroprevalence results (81.8 and 87.8% as determined by ELISA and Western blotting, respectively).
In addition, the vector sand flies Phlebotomus perniciosus and P. sergenti have been found engorged with rodent blood in southern Portugal [52] but also in central Morocco [53] where the sand fly composition is well established [54]. Svobodovà et al. [55] reported the transmission of L. tropica to mice by the bite of Phlebotomus sergenti, a species widespread in our study area [54].
The richness of rodent species (n = 10) across the present study area is favoured by ecological factors. Prevalence and abundance of rodent species areknown to be associated with abundant vegetation [56]. In arid and semi-arid regions, such in the study area, many authors suggested trophic cascade including precipitations, vegetation and rodent density 1 year later [57,58].

Conclusion
To the best of our knowledge, molecular detection of L. infantum DNA in M. musculus, R. norvegicus and R. rattusand of L. tropica DNA in M. musculus is reported here for the first time in Morocco. These results suggest the possible involvement of rodent species in L. tropica and L. infantum cycles. The present findings should be taken into consideration when developing programs to combat this disease in Morocco.

Additional file
Additional file 1: Figure S1. Profile of agarose (1.5%) gel for diagnostic of Leishmania spp. in DNA extracted from tissue from rodent species, using LnPCR for to amplify part of the SSU rRNA gene for diagnosis. Lane MM: 100 bp molecular marker (DNA ladder); Lanes 1, 12: negative controls (no DNA PCR); Lane 13: positive control (MHOM/FR/78/LEM75); Lanes 2-11: DNA extracted of tissue from rodent species. LnPCR for first amplification was carried out using R221/R332 primers. Figure S2. Profile of agarose (1.5%) gel for diagnostic of Leishmania spp. in DNA extracted from tissue from rodent species, using LnPCR for to amplify part of the SSU rRNA gene for diagnosis. Lane MM: 100 bp molecular marker (DNA ladder); Lanes 1, 12, 14: negative controls (no DNA PCR); Lanes 13, 15: positive controls (MHOM/FR/78/LEM75); Lanes 2-11: DNA extracted of tissue from rodent species. LnPCR for second amplification was carried out using R233/R333 primers. Figure S3. Profile of agarose (1.5%) gel for characterization of Leishmania species in DNA extracted from tissue from rodent species, using ITS1-PCR for to amplify the ribosomal internal transcribed spacer 1 (ITS1) region. Authors' contributions ME, SB and AB conceived the project, and were involved in capturing and rodent identification, conducting literature searches and analysis of the results, and participated in the writing of the manuscript. ME, CC, JN, EC, OS and JM were involved in the molecular diagnostics (DNA extraction and PCR) of Leishmania. All authors read and approved the final manuscript.
Ethics approval and consent to participate All animals were treated according to European decree NOR: AGRG1238767A (1st February 2013) regarding the ethical evaluation and authorization of projects involving animals for experimental procedures. All efforts were made to minimize the suffering of the animals used.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

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