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.

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.

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.

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.

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.

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.

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.

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences.

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.

Isoenzyme characterization of Leishmania from man, dog and sandflies in the Maltese islands.

As a part of a general survey on leishmaniases and sandflies of the Maltese islands, 22 Leishmania stocks were isolated from human visceral (1) and cutaneous (1) cases, dogs (16) and sandflies (4). They were characterized by the analysis of 15 enzymes. The commonest Mediterranean L. infantum zymodeme, MON 1, was found to cause human and canine visceral leishmaniasis; L. infantum MON 78, which has so far been isolated only in Malta, was the agent of human cutaneous leishmaniasis. Both zymodemes were isolated from the same sandfly species, Phlebotomus perniciosus.

Differentiation of females in Sergentomyia sensu stricto (Diptera: Psychodidae) using scanning electron microscopy of pharyngeal armatures.

Scanning electron microscopy of external ornamentation and internal armature of the pharynx was used to identify females of Sergentomyia sensu stricto. Five species from the eastern Mediterranean basin were compared; S. minuta clearly was separated from species of the fallax-group. Within the fallax-group, S. fallax was distinguished readily by its heart-shaped pharynx and the difference in armature between the dorsal and lateral plates.

Electrophoretic and morphological differentiation between two sympatric species of Adlerius: Phlebotomus brevis and Phlebotomus simici (Diptera: Psychodidae).

Phlebotomus simici Theodor and P. brevis Artemiev & Dergacheva are closely related species in the subgenus Adlerius that only may be identified by the male terminalia. Females only may be differentiated by collecting them in copula. Because females of P. simici may be involved in Leishmania transmission, their identity should be verified. Our study separated individual from 2 sympatric populations in Lebanon by means of agarose enzyme electrophoresis. Differential patterns were found at the PGM and HK loci. Results were confirmed by morphological reexamination of female spermathecae and pharyngeal armature.

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.

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

The phlebotomine sandfly Phlebotomus perniciosus Newstead is a vector of Leishmania infantum Nicolle in the western Mediterranean basin. In northern Morocco it is often caught in sympatry with a closely related species, P. longicuspis Nitzulescu. In the locality of Chefchaouene where females of both species were morphologically distinguishable, none of the collected males exhibited forked copulatory valves, which is considered a specific character for P. perniciosus. They 1st were identified as P. longicuspis. Isoelectrofocusing of 7 enzyme systems was used to compare these specimens with the sympatric females. Two groups of males were separated and assigned to each species by their isoenzyme patterns associated with morphological differences in the shape of the aedeagus and in the number of coxite hairs. Based on our results, a reexamination of the distribution of both species is necessary especially in many locations where P. longicuspis has been identified only from males.

[Comparison between Phlebotomus duboscqi Neveu-Lemaire, 1906, and Phlebotomus papatasi (Scopoli, 1786): morphological and isoenzymatic studies]. / Comparaison entre Phlebotomus duboscqi Neveu-Lemaire, 1906 et Phlebotomus papatasi (Scopoli, 1786): études morphologique et isoenzymatique.

Phlebotomus duboscqi et P.papatasi are morphologically closely related. Both species can be vectors of zoonotic cutaneous leishmaniasis in the Old World. In some African areas these two species are sympatric. Until now the females of the two species could not be differentiated. The authors investigated morphological characters and isoenzymatic characterization. Constant differences were observed. Moreover three interbreeding attempts were unsuccessful. These two species seemed to be considered as genuine species.