First molecular detection of Leishmania infantum in Sergentomyia minuta (Diptera, Psychodidae) in Alentejo, southern Portugal.

Protozoan parasites, such as Leishmania spp., are the causative agents of many insect-borne infectious diseases with medical and veterinary importance. Leishmaniasis, caused by Leishmania spp., is transmitted by female phlebotomine sand flies. In the Alentejo region of Portugal, located at the north of Algarve, cases of human and canine leishmaniasis caused by Leishmania infantum have been notified. However, no recent studies regarding the sand fly fauna in the region are available. We therefore aimed to explore the phlebotomine sand fly species found in both, Évora and Beja Districts, to gain an insight about the leishmaniasis epidemiology in these areas. After the identification of the insect species, PCR molecular tests were used to assess L. infantum infection rate in the sand fly captured females, together with the analysis of blood meal sources of the insect vectors. One Sergentomyia minuta female was positive for L. infantum infection and another for human blood as a meal source. The occurrence of this phlebotomine species infected with L. infantum may suggest that, in the Mediterranean basin, leishmaniasis epidemiology is changing. Also, if the importance of S. minuta for the zoonotic and anthroponotic cycle of leishmaniasis is later proven, the strategies to control its vector will inevitably to be rethought.

Detection of Leishmania DNA and blood meal sources in phlebotomine sand flies (Diptera: Psychodidae) in western of Spain: Update on distribution and risk factors associated.

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012-2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

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.

Rotation of the external genitalia in male Phlebotomine sand flies (Diptera, Psychodidae) in laboratory conditions and in captured specimens in Algarve, Portugal.

Protozoal parasites are the causative agents of many insect-borne infectious diseases worldwide with impact on human and animal health. Leishmaniasis is caused by Leishmania spp. and transmitted by female Phlebotomine sand flies. In Portugal, two species of Phlebotomus (Larroussius), namely Phlebotomus perniciosus and Phlebotomus ariasi are the proven vectors of Leishmania infantum. Phlebotomine females and males rest and breed in the same sites; and these locations can be predicted according to the male external genitalia maturation. Therefore, this study aimed to determine the timing of complete rotation of the male external genitalia in laboratory conditions and to characterize the external genitalia rotation in field captured males to predict the male and female sand fly breeding and resting sites. This knowledge can be applied in the design and implementation of integrated sand fly control strategies targeting these sites.

Entomological and ecological studies in a new potential zoonotic leishmaniasis focus in Torres Novas municipality, Central Region, Portugal.

In Portugal human and canine leishmaniasis are caused by Leishmania infantum, and Phlebotomus perniciosus and P. ariasi are the proven vectors. Three main foci were identified in eighty's decade: Trás-os-Montes and Alto Douro region, Lisbon region and Algarve region, but according to OnLeish observatory data, canine leishmaniasis cases have been reported from several other regions, for which sand fly species and their infection rates are unknown. This study is the first phlebotomine survey in Torres Novas municipality, Santarém District, Portugal. The main objectives were to identify the phlebotomine species, their bioecological aspects, Leishmania infection rate and the risk factors for the presence of phlebotomine species in the municipality. From June to November, 2010, 275 biotopes were surveyed with CDC light-traps. Captures covered the 17 parishes of the municipality and included domestic, peridomestic and sylvatic biotopes. Specimens were identified morphologically and females were used for molecular detection of Leishmania and bloodmeal identification. Simple and multiple logistic regression analysis were used to identify risk factors for phlebotomine presence. Nonparametric tests were used to compare densities of independent groups. A total of 1262 sand flies were captured and identified, and four species detected: P. perniciosus (73.69%), P. ariasi (8.16%), P. sergenti (6.58%) and Sergentomyia minuta (11.57%). In 71.4% localities at least one L. infantum proven vector species was present. Risk factors were identified as: high average temperatures and low relative humidities, sheltered locations and absence of strong wind, presence of pine trees as dominant vegetation, peridomestic biotopes, particularly sheep pens or proximity of sheep, poultry and house martin nests. L. infantum infection rate was 4% for P. ariasi and 0.48% for the total of Larroussius females. P. perniciosus females exhibited an opportunistic behavior, feeding in a wide variety of vertebrate hosts. The high abundance and distribution of proven vector species, together with a canine leishmaniasis seroprevalence of 7.93% in the District, and the capture of a gravid infective sand fly female, suggests that Torres Novas municipality is a potential zoonotic leishmaniasis focus in the country.

Molecular detection of Leishmania infantum in naturally infected Phlebotomus perniciosus from Algarve region, Portugal.

BACKGROUND & OBJECTIVES: In Portugal, Phlebotomus perniciosus and P. ariasi, (Subgenus Larroussius; Diptera: Psychodidae) are the proven vectors of leishmaniasis caused by Leishmania infantum. The Algarve Region in southern Portugal has been considered an endemic focus of leishmaniasis since 1980s. The main objective of the present study was to validate a molecular approach to detect Leishmania infection in phlebotomines based on DNA extraction from the female sandfly whole body, minus genitalia, followed by PCR for application on epidemiological surveys. METHODS: In Algarve Region, from early May until early November 2006, sandflies were captured by CDC miniature light-traps. kDNA-PCR and ITS1-PCR were used to screen the presence of Leishmania DNA in female sandflies after species identification by entomological keys. RESULTS: A total of 474 sandflies were collected in 108 biotopes. One female of P. perniciosus, the predominant species, was found infected with L. infantum reflecting an overall infection rate of 0.47%. INTERPRETATION & CONCLUSION: PCR associated with morphological characterization of the sandflies will be a powerful epidemiological tool for the determination of the number of phlebotomines infected with Leishmania spp in nature. In addition, the simultaneous occurrence of dogs and P. perniciosus infected with L. infantum shows that Algarve continues to be an endemic focus of canine leishmaniasis. Furthermore, as P. sergenti and P. papatasi which transmit L. tropica and L. major, respectively were present, the future introduction of these two Leishmania species in southern region of Portugal should not be neglected.

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains.

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.

The phlebotomine sandflies of Portugal. XIII--Occurrence of Phlebotomus sergenti Parrot, 1917 in the Arrabida leishmaniasis focus.

In a survey carried out during the summer in 2002 and 2003, in the canine and vulpine leishmaniasis focus of Arrabida, 665 phlebotomine sandflies were caught. 13.83% were P. ariasi, 58.65% P. pemiciosus, 0.45% P. sergenti and 27.07% S. minuta. Despite the low abundance, the finding of the three adults P. sergenti (two males in Aldeia Grande and one female in Quinta da Ramada) confirm the colonization of the Arrabida leishmaniasis focus by this species, which presence had been previously reported but thought accidental. The abundance of P. ariasi observed at this time is significantly different from that in previous years (Pires, 1984 and Fernandes pers. com., 1994). The occurrence of P. sergenti in this region, in association with the decrease in abundance of P. ariasi, may reflect an increased aridness of this region, a consequence of current climate and environmental changes.