Phlebotomus langeroni Nitzulescu (Diptera, Psychodidae) a new vector for Leishmania infantum in Europe.

Burrows of the wild rabbit, Oryctolagus cuniculus, a lagomorph that has been recently suggested as a Leishmania infantum reservoir, constitute an unspoilt biotope in phlebotomine studies in Europe. We hypothesize that Phlebotomus langeroni, a proven vector of L. infantum in North Africa, is associated with rabbits and may have been overlooked in Europe. Sandfly captures were carried out with CDC light traps in an L. infantum endemic area of southern Spain with a high density of lagomorphs and a large numbers of burrows. The stable, permanent, and highly abundant presence of P. langeroni was assessed. After morphological identification, this sandfly species was characterized by comparing it with P. perniciosus and other P. langeroni populations from North Africa through molecular techniques. P. langeroni had not been found in southern Spain to date, despite being a highly investigated area, except for this particular biotope. Its activity period turned out to begin in mid-July, ending in late October, accounting for a maximum activity during this month. This study shows that P. langeroni is associated with the existence of rabbit burrows and has been overlooked in Europe. L. infantum DNA was found in almost half of the female specimens (47.6%) captured inside a biotope where wild rabbits are infected as well.

Ruling out nosocomial transmission of Cryptosporidium in a renal transplantation unit: case report.

BACKGROUND: Cryptosporidium spp. is a ubiquitous parasite affecting humans as well as domestic and wild vertebrates, causing diarrhea in both immunocompetent and immunocompromised hosts worldwide. Its transmission occurs primarily by the fecal-oral route. In humans, C. parvum and C. hominis are the most prevalent species, whereas immunocompetent and immunocompromised individuals can also be infected by other zoonotic species. Renal transplant patients are prone to develop cryptosporidiosis, which can induce severe and life-threatening diarrhea. CASE PRESENTATION: We report here a series of nearly concomitant cases of acute symptomatic cryptosporidiosis in three renal transplant patients attending the Strasbourg University Hospital Nephrology Unit. The clinical presentation was persistent diarrhea and acute renal failure. The diagnosis was confirmed by microscopic stool examination using a modified Ziehl-Neelsen staining method and species identification by molecular tools. All patients were treated with nitazoxanide and recovered from diarrhea after 14 days of therapy. CONCLUSION: Genotypic species identification was not consistent with an epidemic context, thus underlining the need for genotyping to monitor at risk patients.

Mitochondrial DNA Intraspecific Variability in Sergentomyia minuta (Diptera: Psychodidae).

Recently, there has been growing interest in analysis of the geographical variation between populations of different Phlebotomus spp. and American sand flies by comparing the sequences of various genes. However, little is known about the genetic structure of the genus Sergentomyia França & Parrot. No study has been carried out on Sergentomyia minuta Rondani. Most authors recognize this as a species with a high degree of morphological polymorphism, and some suspect that there are two subspecies: Se. minuta minuta Rondani in Europe, having about 40 horizontal cibarial teeth (sticks aligned along a straight line in the cibarial cavity), and Se. minuta parroti Adler & Theodor in North Africa, having about 70 cibarial teeth. Here we analyzed phylogeographic patterns using cytochrome b (Cytb) and cytochrome C oxidase I mtDNA for 29 populations from 10 countries: Algeria, Cyprus, France (continental and Corsica), Greece (continental and Crete), Malta, Montenegro, Morocco, Portugal (continental and Atlantic Savage Islands), Spain, and Tunisia. We analyzed intra- and interpopulation patterns of genetic diversity. Our results from Bayesian inference showed a complex genetic structure of Se. minuta with four haplogroups including many different haplotypes. One haplogroup includes all the specimens from North Africa. A second haplogroup includes a few specimens from the south of France, Spain, and one from Portugal. The third includes many specimens from southern France, all the specimens from Corsica, one from Spain, and all specimen from Portugal except one. A fourth branch includes specimens from the Balkans, Malta, Crete, Cyprus, and curiously some from the Atlantic Savage Islands; settlement of the latter population remains unexplained. However, our results suggest that the settlement of the Mediterranean basin could have occurred at the same time for Se. minuta and both Phlebotomus perniciosus Newstead and Phlebotomus ariasi Tonnoir. The spatial distribution of haplotypes was congruent with phylogenetic findings.

A study of a population of Nyssomyia trapidoi (Diptera: Psychodidae) caught on the Pacific coast of Ecuador.

BACKGROUND: Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species. METHODS: Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed. RESULTS: Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA. CONCLUSION: Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.

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.

Multiple genetic divergences and population expansions of a Mediterranean sandfly, Phlebotomus ariasi, in Europe during the Pleistocene glacial cycles.

Phlebotomus ariasi is one of the two sandflies transmitting the causative agent of zoonotic leishmaniasis, Leishmania infantum, in France and Iberia, and provides a rare case study of the postglacial re-colonization of France by a Mediterranean species. Four DNA sequences were analysed-mitochondrial cytochrome b (cyt b), nuclear elongation factor-1α (EF-1α) and two anonymous nuclear loci-for 14-15 French populations and single populations from northeast Spain, northwest Spain, Portugal and Morocco. The presence of cryptic sibling species was not revealed by phylogenetic analyses and testing for reproductive isolation between sympatric populations defined by the two most divergent cyt b haplogroups. No locus was shown to be under positive directional or balancing selection and, therefore, molecular variation was explained demographically. Each nuclear locus showed shallow isolation by distance from Portugal to the French Pyrenees, but for both cyt b and EF-1α there was then a step change to the upland Massif Central, where leading-edge populations showed low diversity at all loci. Multiple genetic divergences and population expansions were detected by analyses of cyt b and dated to the Pleistocene. Endemicity of one cyt b sub-lineage suggested the presence of a refuge north of the Pyrenees during the last glacial period. Monopolization of the Massif Central by genetically differentiated populations of P. ariasi might possibly hinder the northwards spread of leishmaniasis.

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.

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.

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.

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.

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).

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.

Infection by Leishmania infantum in cats: epidemiological study in Spain.

More than 40 cases of feline leishmaniasis have been reported in the scientific literature. The influence of some immunodepressive conditions of viral origin, such as leukemia and feline immunodeficiency, are still unknown. The purpose of this study was to assess the prevalence of Leishmania infection in cats and possible relations with these viral infections. Markers of Leishmania infection were searched in 183 cats from Southern Spain by IFAT, PCR, Giemsa stain and culture, with a follow-up of positive cats. Seropositivity was 60.0% (Ab titer > or =10) and 28.3% of animals presented Ab titers > or =40. Around 25.7% of the cats studied were parasitemic and some of them remained positive for months. Combining both data, 70.6% of the feline population was, or could be, infected. We observed a negative association between seropositivity to Leishmania and infection by FeLV. Hence, production of antibodies against the parasite appears to be compromised in cats with leukemia, which have a prevalence of 36% in our study. In contrast, we found no association with feline immunodeficiency. The results makes us doubt the value of conventional serological methods to detect active Leishmania infection in cats.

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.

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.

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.

Distribution of phlebotomine sandflies, of the subgenus Larroussius, in Morocco.

Adult sandflies (Diptera: Psychodidae) of the genus Phlebotomus and of the subgenus Larroussius were collected with sticky traps from thirty-four localities in central and south-western Morocco. Four Larroussius species were present in this area, Phlebotomus perniciosus Newstead, being the most widespread and abundant species particularly in the mountainous area, was sympatric with the closely related species Phlebotomus longicuspis Nitzulescu. Only one male of P. perniciosus displayed the bifid copulatory valves characteristic of the species. All specimens showed copulatory valves curved at their apex. The number of coxite hairs is a taxonomical tool that differentiates this morph of P. perniciosus from P. longicuspis. Phlebotomus ariasi Tonnoir was collected from six mountainous stations. Phlebotomus mariae Rioux et al. was scarce; only five males were collected and were often associated with P. ariasi.

Postglacial dispersal of Phlebotomus perniciosus into France.

Phlebotomus perniciosus was identified morphologically in samples from France and northeast Spain, and individuals were then characterized at three polymorphic isoenzyme loci (by isoelectrofocusing) and at the mitochondrial DNA locus (by comparative DNA sequence analysis of a fragment of the Cytochrome b gene). The four polymorphic loci gave conflicting patterns of population relationships, which can be explained by hypothesizing different amounts of gene introgression at each locus when two distinctive lineages met in southern France or northeast Spain after isolation in southern Italy and Spain during the Pleistocene Ice Ages. P. perniciosus is an important vector of leishmania infantum and so these population differentiation studies are relevant for predicting the emergence and spread of leishmaniasis in relation to environmental changes, including climate.

Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif.

The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type-locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis-like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.

Variation of esterase electromorphs associated with bloodmeal digestion in phlebotomine sand flies (Diptera: Psychodidae).

Esterases are often used to measure genetic variation, yet they may be influenced by external factors. Isoelectrofocusing was used to investigate the effects of bloodmeal on the esterase variation in wild populations of Phlebotomus (Larroussius) ariasi Tonnoir and P. (L.) perniciosus Newstead and in a colonized population of P. (Phlebotomus) duboscqi Neveu-Lemaire. Modified electrophoretic profiles were demonstrated to be associated with blood digestion. The bloodmeal-induced esterase electromorphs were only found in the abdomen homogenates of the sand flies. These changes in esterase phenotype were probably due to a proteolysis phenomenon and disappeared at the end of bloodmeal digestion. The effect was eliminated by adding trypsin inhibitors.