Potential distributions of phlebotomine sandfly vectors of human visceral leishmaniasis caused by Leishmania infantum in Morocco.

Zoonotic visceral leishmaniasis is a common vector-borne systemic disease caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). In Morocco the situation is complex: many sandfly species have been collected in areas in which the disease is endemic, but only Phlebotomus ariasi, Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) have been confirmed to have vectorial roles. The objective of the present study was to ascertain the potential distribution of L. infantum and its vectors in Morocco, using ecological niche modelling. Vector records were obtained from field collections of the Laboratory team and from previously published entomological observations. Epidemiological data for L. infantum modelling were obtained from Moroccan Ministry of Health reports. The jackknife test indicated that the bioclimatic variables with the greatest influence on model development for all species were annual precipitation and precipitation in the driest quarter of the year. MaxEnt model representations for sandfly species that act as vectors of L. infantum showed the widespread geographic distribution of these species in Morocco, specifically in northern and central Morocco, where foci of visceral leishmaniasis are found. The ecological niche modelling points out areas in which the probability of occurrence of these species is higher. This information should be considered as a starting point for further research to fully elucidate the ecology and epidemiology of these species, as well as of the pathogens they transmit.

Molecular identification of Leishmania tropica and L. infantum isolated from cutaneous human leishmaniasis samples in central Morocco.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis (CL) in Marrakesh-Safi region located in the central-south part of Morocco is a public health problem. This study assessed the efficiency of a microscopic examination method in establishing the diagnosis of CL and PCR for the characterization and identification of the circulating Leishmania strains in different CL foci of the study area. METHODS: A total of 297 smears obtained from cutaneous lesions of suspected patients with CL were stained with May-Grünwald Giemsa (MGG) for microscopic examination. For each positive smear, genomic DNA was extracted and PCR-analysed, targeting the small subunit ribosomal ribonucleic acid (ssu rRNA) gene to detect Leishmania DNA. Then, the internal transcribed spacer 1 (ITS1) was amplified and sequenced in order to identify the Leishmania species. The sensitivity and specificity of the conventional microscopy with ssu rRNA gene were compared by Leishmania nested PCR (LnPCR) and ITS1 gene by ITS-PCR. RESULTS: A total of 257 smears were positive in the microscopic examination, i.e. the detection rate of amastigotes by optical microscopy was 86.53% (257/297). The LnPCR was found to have a specificity and a sensitivity of 100%, each. Interestingly, the sequencing results showed that 99.61% (256/257) of the isolates had Leishmania tropica and 0.39% (1/257) had L. infantum infection. INTERPRETATION & CONCLUSION: Though, classical microscopic examination is useful and economical, it is not sensitive enough, especially in endemic regions where several Leishmania species coexist. In such situations, PCR constitutes a complementary method for the identification of the causal species. The results indicate that both the L. tropica (dominant) and L. infantum are the causative agents of CL in the Marrakesh-Safi region. The rate of CL infection is high in Imintanout, and Chichaoua provinces. Hence, early diagnosis and prompt treatment of CL patients is necessary to prevent its extension to neighboring localities.

Asymptomatic carriers of Leishmania infantum in patients infected with human immunodeficiency virus (HIV) in Morocco.

In Morocco, visceral leishmaniasis (VL) is a parasitic disease caused by the flagellated protozoan parasite Leishmania infantum. L. infantum is transmitted by the bite of female phlebotomine sandflies, and its main reservoir hosts are domestic dogs. Asymptomatic infection with L. infantum is more frequent than clinically apparent disease. In HIV-infected patients, the risk of clinical VL is increased due to immunosuppression that may reactivate latent infections. However, coinfected subjects do not necessarily develop VL and may remain as asymptomatic carriers depending on their immune status. The present study investigates the asymptomatic carriers of L. infantum in HIV-infected patients in central Morocco, where human cases of visceral leishmaniasis by L. infantum have been reported. A total of 200 HIV-infected patients attending the Infectious Diseases Unit of the Ibn Zohar Hospital of Marrakech participated in the study. Parasitological and serological blood analyses included a direct microscopic examination (DME), culture in Novy-McNeal-Nicolle (NNN) medium, and serology by indirect immunofluorescence (IFI). We found prevalence rates of 5% (10/200) by IFI, 3% (6/200) by DME, and 2.5% (5/200) by culture. The parasite was identified as L. infantum by PCR from positive cultures.

Phlebotomine sandflies (Diptera: Psychodidae) of southern Morocco: results of entomological surveys along the Marrakech-Ouarzazat and Marrakech-Azilal roads.

Since the 1970s, Azilal and Ouarzazat have been the main foci for human cutaneous leishmaniasis (CL) in Morocco. The sandflies along the main roads linking these two foci to Marrakech city, which is considered to be an area at risk of CL, were recently surveyed. Among the 872 sandflies collected, in June 2005, on the Marrakech-Ouarzazat road, Sergentomyia fallax was the most common species (36.1%), followed by Phlebotomus sergenti (21.1%), P. papatasi (14.2%), S. minuta (11.7%), P. longicuspis (5.5%), P. alexandri (5.4%), P. perniciosus (4.1%), P. ariasi (0.9%), S. africana (0.6%) and S. dreyfussi (0.3%). On the Marrakech-Azilal road, however, S. minuta was by far the most prevalent species (63.5% of the 1983 sandflies that were collected in August 2006), followed by S. fallax (12.9%), P. perniciosus (12.4%), P. sergenti (4.0%), P. longicuspis (3.0%), P. papatasi (2.8%), S. dreyfussi (1.1%) and P. alexandri (0.2%). The distribution of potential vectors along the two transects, according to altitude and bioclimate, was explored.

Habitat preferences of phlebotomine sand flies (Diptera: Psychodidae) in southwestern Morocco.

Data from a 4-yr study of phlebotomine sand flies in 44 stations in southwestern Morocco are presented. Correspondence analysis is used to describe the habitat preferences of nine sand fly species on a meso-scale (station) and a micro-scale (biotope). This work, based on highly diversified data from various stations, comprising several biotopes sampled during different seasons, provides new information on the ecology of sand flies in southwestern Morocco. It highlights many natural and artificial factors that affect the abundance and distribution of sand fly populations. The impact of climate, urbanization, proximity of humans and domestic animals, organic matter in the soil, shelter, and vegetation type is discussed.

Phlebotomine sandflies (Diptera: Psychodidae) of the genus Sergentomyia in Marrakech region, Morocco.

We reported the results of an entomological investigation in Marrakech area, in the aim to study the present Sergentomyia species composition. One hundred thirty seven sandflies were collected by sticky papers and they comprised three sub-genera: Parrotomyia (43.1%), Sergentomyia (36.5%), and Grassomyia (20.4%). Four species were identified; Sergentomyia (Parrotomyia) africana Newstead (43.1%) followed by S. (Grassomyia) dreyfussi Parrot, S. (Sergentomyia) fallax Parrot, and S. (S.) minuta Rondani accounted for 20.4%, 19.7%, and 16.8%, respectively. Ecological study subdivides these species into rural species (S. africana and S. dreyfussi) and ubiquitous species (S. minuta and S. fallax) which were collected in both urban and rural areas. Enzymatic analysis identified three monomorphic loci (alphaGPDH, ICD, and ME) and six polymorphic loci (PGI, HK, FUM, MDH2, 6PGD, and ACO) in the four species. At FUM and ACO loci, some alleles appeared to be fixed in each species. Morphological (counts of cibarial teeth) and isoenzymatic analysis of wild populations of S. minuta parroti from Morocco and of S. minuta minuta from continental Europe (France, Spain, and Portugal) was carried out. Morphological results showed significant differences between France and Portugal populations and south Spain populations. In contrast, there was no significant difference between northern and southern Moroccan populations. Genetic variability showed a separation between northern and southern European populations and S. minuta from Andalusia clustered with Moroccan populations.

Risk mapping of human HIV-Leishmaniasis co-infection in Morocco.

BACKGROUND: HIV/AIDS is frequently associated with opportunistic diseases such as leishmaniasis. Hence, the co-infection HIV-Leishmania spp. is the result of the geographical overlap between leishmaniasis and HIV/AIDS cases. To the best of our knowledge, this is the first report describing the spatial distribution of HIV-Leishmaniasis co-infection in Morocco where both infections are endemic. METHODS: In the current study, we discuss the HIV-Leishmania spp. co-infection vulnerability in Morocco by using the cartography tools. Thus, epidemiological data of both infections (Leishmaniasis and HIV/AIDS) in different administrative regions of Morocco were collected and co-registered for Digital maps making. RESULTS & CONCLUSION: The results showed a high risk of HIV-Leishmania infantum co-infection in northern and central regions in Morocco. These results should be taken into account for efficient control strategies and epidemiological surveillance of HIV -Leishmania spp. co-infection in Morocco.

Morphological and isoenzymatic differentiation of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) in Southern Morocco.

Morphological and enzymatic characterization of Phlebotomus perniciosus and Phlebotomus longicuspis in Morocco is reported. Twenty-nine localities in central and southern of Morocco were sampled and compared with three localities from the Rif (northern Morocco). For morphological study, sand flies were collected by sticky-paper traps. For males, the morphology of the copulatory valves (aedeagi) was examined and the number of coxite hairs was recorded. For isoenzyme analyses, specimens were collected in CDC light traps and immediately conserved at -80 degrees C. P. perniciosus samples from the south of Morocco, up to 150 km from Marrakech, showed single-pointed aedeagi curved at their apices, indistinguishable from the atypical morph of P. perniciosus, previously described in northern Morocco. Twelve enzyme systems were tested and the qualitative analysis of zymogram profiles revealed eight polymorphic loci (glucosephosphate isomerase (GPI), phosphoglucomutase (PGM), hexokinase (HK), fumarate hydratase (FUM), malate dehydrogenase 1 (MDH1), malate dehydrogenase 2 (MDH2), 6 phosphogluconate dehydrogenase (6PGD) and aconitase (ACO)). Enzyme loci showed fixed alleles diagnostic for P. perniciosus (aconitase) and P. longicuspis (aconitase and hexokinase).

Isoenzymatic characterization of Phlebotomus papatasi (Diptera: Psychodidae) of the Marrakech area, Morocco.

This study reports the genetic characterization of urban and rural populations of Phlebotomus (Phlebotomus) papatasi Scopoli (Diptera: Psychodidae) in Marrakech, Morocco. Using isoenzymatic analysis, four Moroccan populations were compared with other Mediterranean basin populations from Spain, Cyprus, and Syria. Morphological anomalies were noted in the male genitalia of 5.3% of the specimens collected from Marrakech area. Qualitative analysis of zymogram profiles revealed nine polymorphic enzymes (HK, PGM, PGI, 6PGD, MDH1, MDH2, ICD2, FUM, and ACO) and three monomorphic enzymes (ME, ICD1, and alphaGPDH). Genetic distances clearly separated the populations of western Mediterranean countries (Morocco and Spain) from eastern countries (Syria and Cyprus), but they could not be used to differentiate between urban and rural populations in Marrakech area.

Altitudinal structuring of sand flies (Diptera: Psychodidae) in the High-Atlas mountains (Morocco) and its relation to the risk of leishmaniasis transmission.

This paper presents the results of entomological surveys on phlebotomine sand flies (Diptera: Psychodidae) in the Haouz of Marrakech and High-Atlas mountains (Morocco). Sand flies were captured with sticky traps from 25 stations with altitudes ranging between 400 and 1400 m. A total of 2742 specimens belonging to nine phlebotomine species was collected, Phlebotomus (Larroussius) perniciosus Newstead being the predominant species. There was a remarkable difference in the diversity of the sand fly fauna among the altitudes. Two associations of sand fly faunas were determined, the first one in lower altitude and the second one in higher altitude. The significance of the predominant species at any altitude range was discussed in terms of the risk of transmission of leishmaniasis.

Nocturnal activity of phlebotomine sandflies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in Chichaoua, Morocco.

The nocturnal activity of phlebotomine sandflies (Diptera: Psychodidae) was studied "at an epidemic focus" on human cutaneous leishmaniasis due to Leishmania tropica Wright in Chichaoua province, in Morocco. Sandflies were collected using light and sticky-paper traps changed at 2-h intervals, inside and around houses, in August and October 2004. Overall, 633 sandflies, belonging to six species of Phlebotomus and three of Sergentomyia, were collected. Sandfly activity was nocturnal and higher at twilight. Several activity patterns were observed according to the species. Phlebotomus (Paraphlebotomus) sergenti Parrot, 1917, the suspected vector of L. tropica in this focus, was caught during each collection performed from 1900 to 0500 hours, the numbers of species caught peaked at 1900-2100 hours. There were seasonal variations of the nocturnal activity, which could be related to the variations in temperature and relative humidity.

Seasonal fluctuations of phlebotomine sand fly populations (Diptera: Psychodidae) in the urban area of Marrakech, Morocco.

Phlebotomine sand flies (Diptera: Psychodidae) were collected continuously, using sticky traps, during 1 year from October 2002 to September 2003, in an urban area of Marrakech city (Morocco). A total of 3277 specimens were collected belonging to five species. Phlebotomus (Phlebotomus) papatasi (54.6%) is the predominant species followed by Sergentomyia (Sergentomyia) minuta (20%), S. (S.) fallax (11.3%), P. (Paraphlebotomus) sergenti (10.3%) and P. (Larroussius) longicuspis (3.8%). Data analyses showed a mono-modal annual pattern for P. sergenti and a bi-modal one for the other species. P. papatasi, the proven vector of Leishmania major in Morocco, was active throughout the year. This species did not diapause in this region. P. papatasi population peaked in June and November, which relating to the periods of risk in this area. Its preferred temperature ranged between 32 and 36 degrees C but no significant correlation was found between its density and the temperature. Considering the high density and long activity period of P. papatasi, the area of Marrakech should be regarded as a potential focus for L. major. This suggests the need for a continuously surveillance to prevent risk of zoonotic cutaneous leishmaniasis.

Entomological investigations in Chichaoua: an emerging epidemic focus of cutaneous leishmaniasis in Morocco.

Cutaneous leishmaniasis due to Leishmania tropica Wright seems to be an emerging disease in Chichaoua, a province located in southwestern Morocco. In this study, sand flies (Diptera: Psychodidae) were collected from 12 stations. Sticky traps were placed in domestic, peridomestic, and sylvatic sites. In total, 3,787 specimens consisting of 10 species (seven Phlebotomus and three Sergentomiya) were identified. Phlebotomus perniciosus Newstead, the predominant species, was abundant, especially in mountainous areas. Phlebotomus sergenti Parrot (12%) was found in all studied villages where it was associated with domestic and peridomestic habitats. On the basis of its abundance, distribution, and notable anthropophily, P. sergenti, a proven vector of L. tropica elsewhere, is considered the cutaneous leishmaniasis vector in this emerging focus.

Spatial relations among environmental factors and phlebotomine sand fly populations (Diptera: Psychodidae) in central and southern Morocco.

Phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) are of considerable public health importance because of their ability to transmit several human parasites, mainly as vectors of Leishmania spp. Over the past decade, the epidemiological situation of cutaneous leishmaniasis (CL) has significantly increased with its geographic expansion to previously free areas and the emergence of overlapping foci of cutaneous leishmaniasis and visceral leishmaniasis (VL) in several provinces of Morocco. A total of 15,313 specimens was collected during this entomological survey. The genera Phlebotomus (57.38%) and Sergentomyia (42.62%) were identified. Sergentomyia minuta (22.01%) was the most prevalent species, followed by S. fallax (18.21%), Phlebotomus perniciosus (14.35%), P. papatasi (14.06%), P. sergenti (12.85%), P. longicuspis (10.74%), P. ariasi (2.68%), S. dreyfussi (1.53%), P. alexandri (1.31%), P. bergeroti (1.14%), S. christophersi (0.62%), S. africana (0.25%), P. chabaudi (0.14%), P. chadlii (0.05%), and P. kazeruni (0.04%). We aimed to determine current distribution of leishmaniases vectors, their ecological characteristics, and the significance of the predominant species at any bioclimate stage, altitude range, and soil texture in terms of the risk of leishmaniasis transmission.

Bacterial flora as indicated by PCR-temperature gradient gel electrophoresis (TGGE) of 16S rDNA gene fragments from isolated guts of phlebotomine sand flies (Diptera: Psychodidae).

In this study, we tested the capacity of Temperature Gradient Gel Electrophoresis (TGGE)-based fingerprinting of 16S rDNA PCR fragments to assess bacterial composition in a single isolated sand fly gut. Bacterial content was studied in different life stages of a laboratory-reared colony of Phlebotomus duboscqi and in a wild-caught Phlebotomus papatasi population. Our study demonstrates that a major reorganization in the gut bacterial community occurs during metamorphosis of sand flies. Chloroflexi spp. was dominant in the guts of pre-imaginal stages, although Microbacterium spp. and another as yet unidentified bacteria were detected in the gut of the adult specimen. Interestingly, Microbacterium spp. was also found in all the adult guts of both species. We demonstrate that the analysis of bacterial diversity in an individualized sand fly gut is possible with fingerprinting of 16S rDNA. The use of such methodology, in conjunction with other culture-based methods, will be of great help in investigating the behavior of the Leishmania-bacterial community in an ecological context.

Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae).

Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps.

Faunistic study of the sandflies (Diptera: Psychodidae) in an emerging focus of cutaneous leishmaniasis in Al Haouz province, Morocco.

The Moroccan province of Al Haouz is an emerging focus of human cutaneous leishmaniasis caused by Leishmania tropica. In an entomological investigation of three communes in the province (Ourika, Stti Fatma and Rhmate), 3440 sandflies were collected on sticky traps in 2005-2007. Of the six Phlebotomus species caught, Phlebotomus papatasi (13.3% of the total collection), P. sergenti (12.8%) and P. perniciosus (12.8%) were predominant, followed by P. longicuspis (7.9%), P. ariasi (2.6%) and P. alexandri (1.1%). All the other sandflies were Sergentomyia fallax (24.2%), S. minuta (22.0%) or S. dreyfussi (3.4%). The distribution of these species among the many biotopes, at varying altitudes (667-1772 m), is discussed. When the variability of the P. sergenti populations in Ourika and Rhmate and those at two sites in northern Morocco (Taounate and Chefchaouene) was investigated by iso-enzyme analysis, seven polymorphic loci (PGI, PGM, HK, MDH1, ME, 6PGD and ACO) were identified. None of the investigated populations of P. sergenti was in Hardy-Weinberg equilibrium for all the polymorphic loci. The results indicate that the taxon known as Phlebotomus sergenti in Morocco does not have the genetic characteristics of a single species.

Entomological and epidemiological study of a new focus of cutaneous leishmaniasis in Morocco.

Cutaneous leishmaniasis (CL) due to Leishmania tropica Wright has become an increasingly important problem in Al-Haouz province, Morocco. The objective of this paper was to undertake a retrospective analysis of CL between 2000 and 2006 and to study the sandfly fauna for the first time in this focus. The consultation of bulletins, registers and monthly and annual reports published by local and national medical services permitted a global synthesis of CL in Al-Haouz between 2000 and 2006. Sandflies were collected using sticky-paper traps in five selected stations in two communes, Ourika and Sti Fadma, the most affected by CL in Al-Haouz. Overall, 2,648 sandflies, belonging to five species of Phlebotomus and three of Sergentomyia, were collected. Phlebotomus sergenti Parrot (14.5%) was found in all studied stations where it was associated with domestic and peridomestic habitats. Based on this first report on ecology of sandflies and epidemiology of CL, Al-Haouz seems to be a typical focus of L. tropica and P. sergenti like the other Moroccan foci. Two communes, Sti Fadma and Ourika, constituted an important CL micro-focus in this region. Considering their economical importance (trade and tourism), they contribute probably to the dissemination of Leishmania parasite in bordering regions.

Phlebotomine sandflies (Diptera: Psychodidae) of Marrakech city, Morocco.

The sandflies at 17 sites within or near the Moroccan city of Marrakech were surveyed between 2002 and 2006. Overall 2310 specimens were collected and five species were identified. Phlebotomus (Phlebotomus) papatasi was the most prevalent species (47.4%), followed by Sergentomyia (Sergentomyia) minuta (16.6%), S. (S.) fallax (16.1%), P. (Paraphlebotomus) sergenti (12.8%) and P. (Larroussius) longicuspis (7.1%). Phlebotomus sergenti appeared to be the most endophilic species. When the collection sites were categorized into five types of habitat, only P. papatasi and S. minuta were found to be common in every habitat type. Urbanization seemed to have a significant effect on the numbers and species of sandfly. In the year after the demolition and redevelopment of the Akioud neighbourhood, there were far fewer sandflies and species of sandfly than observed before the redevelopment. Two years later, however, the sandfly population was found to be much larger than that seen before the neighbourhood was demolished.

Improvement of Cupressus atlantica Gaussen growth by inoculation with native arbuscular mycorrhizal fungi.

AIMS: The study aimed to determine whether inoculation with native arbuscular mycorrhizal (AM) fungi could improve survival and growth of seedlings in degraded soils of Morocco. METHODS AND RESULTS: Soil samples were collected from the rhizosphere of Cupressus atlantica trees in the N'Fis valley (Haut Atlas, Morocco). AM spores were extracted from the soil, identified and this mixture of native AM fungi was propagated on maize for 12 weeks on a sterilized soil to enrich the fungal inoculum. Then C. atlantica seedlings were inoculated with and without (control) mycorrhizal maize roots, cultured in glasshouse conditions and further, transplanted into the field. The experiment was a randomized block design with one factor and three replication blocks. The results showed that a high AM fungal diversity was associated with C. atlantica; native AM fungi inoculation was very effective on the growth of C. atlantica seedlings in glasshouse conditions and this plant growth stimulation was maintained for 1 year after outplanting. CONCLUSIONS: Inoculation of C. atlantica with AM fungi increased growth and survival in greenhouse and field. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that use of native species of AM fungi may accelerate reforestation of degraded soils. Further studies have to be performed to determine the persistence of these mycorrhizae for a longer period of plantation and to measure the effects of this microbial inoculation on soil biofunctioning.