Perniciosus Sand Flies Research Articles

Are phlebotomine sand flies (Diptera: Psychodidae) really attracted to UV-light sources?

Phlebotomine sand flies are the sole confirmed vector of leishmaniosis, a group of endemic and re-emerging diseases in southern European countries such as Spain. Light traps are the major surveillance method currently being employed. Nevertheless, color light attraction by sand flies remains mostly unknown. Following prior research, the effect of UV-LED light sources, and its synergic effect with different color lights has been evaluated employing a modified Flebocollect (FC) light trap model. Results suggest that female Phlebotomus perniciosus sand flies are more attracted to a FC trap lured with the combination of blue and UV LED light sources than commercial CDC (Center for Disease Control and prevention) traps (Bonferroni post-hoc test; p < 0.08; blue/UV mean = 0.50; CDC mean = 0.13), while the combination of red and UV modified-traps excel in capturing Sergentomyia minuta sand flies (Bonferroni post-hoc test; p < 0.04; blue/UV mean = 1.19; CDC mean = 0.66). However, based on our prior studies, incorporating UV light sources into sand fly traps does not seem to enhance their attractiveness, as it has not resulted in higher capture rates. These findings contribute to understanding how sand flies’ vision and light color detection is. Further research is recommended to standardize trap construction procedures and explore variations in different endemic regions according to different sand fly species.

Examining the Relationship of Clinical and Laboratory Parameters With Infectiousness to Phlebotomus perniciosus and Its Potential Infectivity in Dogs With Overt Clinical Leishmaniasis.

Infected dogs are considered the main domestic animal reservoirs for Leishmania infantum parasite. Infectiousness to competent phlebotomine vectors has been associated with many factors, the main being the severity of the disease exhibited by infected dogs. This study examines the relationship between different clinical parameters and the infectiousness to colonized Phlebotomus perniciosus sand flies having a blood meal on dogs. Data obtained in the present study come from an untreated group of Leishmania sick dogs submitted to xenodiagnosis for the evaluation of a spot on insecticide solution. Seventeen dogs were diagnosed as affected by leishmaniasis through clinical examination, immunofluorescence antibody test (IFAT) serology, and loop-mediated isothermal amplification (LAMP). The disease severity (clinical score) was staged by using a numeric value derived from eight clinical and parasitological parameters. Xenodiagnosis was performed on caged dogs exposed for 1.5 h to sand-fly bites. The following parameters related to sand flies were examined: blood feeding (% of blood engorged females), promastigote detection (% of promastigote-positive sand flies), promastigote burden, and the promastigote stage maturation (potential transmissibility rate). Statistical relationship between the clinical score and entomological parameters was investigated, as well as the possible correlation between each clinical and laboratory parameters and sand fly infection/infectivity. The severity of clinical score may influence the blood feeding by, and the probability of promastigote detection in, sand flies; skin lesions seem to be the main factor that influences the rate of blood feeding. Promastigote burden is related to IFAT titer, skin lesions, and clinical score. All entomological parameters are strongly related among them. This study confirms that both P. perniciosus infection and infectivity are influenced by a dog's clinical condition.

Leishmania infantum in Tigers and Sand Flies from a Leishmaniasis-Endemic Area, Southern Italy.

We detected Leishmania infantum infection in 45% of tigers and 5.3% of sand flies tested at a zoo in southern Italy in 2019. These infections in tigers and the abundance of Phlebotomus perniciosus sand flies represent a potential risk to other animals and humans living in or visiting the zoo.

Impact of clinically acquired miltefosine resistance by Leishmania infantum on mouse and sand fly infection

ObjectivesThis study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. MethodsExperimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. ResultsThe fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. ConclusionThe natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.

Assessment of the insecticidal activity of oral afoxolaner against Phlebotomus perniciosus in dogs.

Twelve healthy dogs were included in this laboratory efficacy study. Six dogs were randomly allocated based on body weight to an untreated control group and six to an afoxolaner (NexGard®) treated group. In the treatment group, afoxolaner was administered orally on Day 0 in accordance with label instructions. On Days 1, 14 and 28, each dog was exposed to 60 unfed female and 10 male Phlebotomus perniciosus sandflies for 1 h. At the end of each exposure period, sandflies were counted and assessed for viability and feeding status. There was no statistical difference in mortality (0.0–5.4%), nor in feeding proportion (61.6–78%) between the control and the treated groups at all 1-h post-exposure assessments. After collection, live fed and unfed sandflies were kept for viability assessments at 48 and 72 h post-exposure. In the untreated control group, the average percentages of live, fed, female sandflies after exposure, on Days 1, 14 and 28, ranged from 51% to 74% at 48 h and from 46% to 57% at 72 h, demonstrating model robustness over the 28 days of the study. Significantly fewer live fed sandflies were recorded for the afoxolaner treated group (p < 0.01). The insecticidal efficacy was 100%, 95.9% and 75.2% at 48 h post Days 1, 14 and 28 exposures, respectively, and 100%, 100% and 86.3% at 72 h post Days 1, 14, and 28 exposures, respectively. A single administration of oral afoxolaner (NexGard®) to dogs significantly killed P. perniciosus sandflies 48 and 72 h after blood feeding for 1 month.

Rabbit trypanosome detection in Phlebotomus perniciosus sand flies from the leishmaniasis outbreak in Madrid, Spain

Phlebotomine sand flies are known vectors of several pathogens. In Spain, Phlebotomus perniciosus and Phlebotomus ariasi are the proven vectors of Leishmania infantum. Since 2010 a human leishmaniasis outbreak has been notified in Madrid region, central Spain. Studies have shown that P. perniciosus is the only vector confirmed in the focus area and that rabbits and hares are the wild reservoirs incriminated in the cycle of the parasite in the outbreak. Trypanosoma nabiasi is a trypanosomatid found in wild rabbits and its presence has been reported in wild rabbits from southern Spain. Moreover, co-infection with L. infantum was found in some of these animals. However, in Madrid region, there is no information about the transmission of this trypanosome in rabbits. Hence, in this study we investigate if T. nabiasi could be circulating in the aforementioned leishmaniasis focus. Wild P. perniciosus female sand flies were captured in the affected area and analyzed using molecular methods. T. nabiasi DNA was detected in 20 out of 155 female sand flies fed on rabbits by amplification and subsequent sequencing of ITS1 and SSU rRNA fragments. Therefore, we describe for the first time the presence of T. nabiasi and its co-infection with L. infantum in P. perniciosus female sand flies. More investigation is needed in order to elucidate the role of P. perniciosus in the transmission of T. nabiasi among rabbits and its potential consequences.

Evaluation of the anti-feeding and insecticidal effects of a topically administered combination of imidacloprid and permethrin (Advantix\xae) against Phlebotomus (Larroussius) perniciosus (Newstead, 1911) in dogs following monthly administration

BackgroundTwo laboratory experiments (Studies 1 and 2) were conducted to confirm the efficacy of an imidacloprid and permethrin combination (Advantix® Spot-on, Bayer) to repel and kill Phlebotomus (Larroussius) perniciosus sand flies when applied once a month topically to dogs.MethodsBoth studies compared dogs treated with a combination containing 100 mg/ml imidacloprid + 500 mg/ml permethrin (Advantix® Spot-on, Bayer) to placebo treated dogs. The treatments were applied topically on Day -28 (Study 2) and Day 0 (Studies 1 and 2). Sand fly exposures with 80 unfed females were performed before the first treatment for allocation purposes and post-treatment on study days (SDs) 1, 7, 14, 21 and 28 (following first or second monthly treatment for Studies 1 and 2, respectively). After 60 min, sand flies were assessed for mortality and engorgement status.ResultsRepellent evaluation (anti-feeding effect) on all days post-infestation showed efficacies that ranged between 88.1–99.3% during the first month and 92.2–98.9% during the second. Analyses of the comparison of fed sand fly counts for each treatment group resulted in a highly significant reduction (P < 0.0001) at all post-infestation time points for those dogs treated with Advantix®. A significant (P < 0.0001 for all time points) insecticidal effect was equally demonstrated. No treatment related adverse events were observed during the study.ConclusionsIn the present studies Advantix® Spot-on demonstrated to be safe and to provide excellent four-week sand fly (P. perniciosus) repellency of ≥88.1% and ≥92.2% after a first and second monthly treatment, respectively. A significant insecticidal effect was also observed.

Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR

Since 2010 a human leishmaniasis outbreak has been notified in southwestern Madrid region that still remains active. Entomological surveys have been carried out in the affected area in order to obtain information about species diversity, distribution, and density of sand flies. Moreover, molecular identification of blood meal preferences of sand flies and molecular detection of Leishmania infantum has been performed. In this work, we optimized a real time PCR assay in order to determine parasite loads in unfed and blood-fed Phlebotomus perniciosus female sand flies caught in the focus area. Results showed elevated parasite loads in nearly 70% of the studied positive sand flies. Furthermore, significantly higher parasite loads were observed in females without blood in their guts. In conclusion, high L. infantum loads found in P. perniciosus sand flies from the Madrid focus support the exceptional characteristics of this outbreak.

Genetic variability of the S segment of Toscana virus

Toscana virus (TOSV) was originally isolated in 1971 from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy). Since its first isolation, several studies have been conducted in Italy and other Mediterranean countries in order to identify its possible animal reservoirs, spread of infection and genetic variability. Phylogenetic analysis conducted on TOSV genome demonstrated the co-circulation of two major lineages in the Mediterranean areas, TOSV A and TOSV B. This study reports the results of the genetic analysis of 32 viral strains isolated in Italy in the last 30 years from patients hospitalized with neurological disease, from sandflies and from the brain of a bat. The genetic diversity of TOSV was investigated by determining the sequences of the whole S segment. Phylogenetic analysis showed that TOSV A lineage represents the lineage circulating in Italy. Moreover, the current variability of lineage A is similar to that of lineage B.

First detection of Toscana virus in Corsica, France

Toscana virus (TOSV) was detected for the first time from Phlebotomus perniciosus sandflies in Corsica, a French Mediterranean island. Genetic analysis showed that Corsican TOSV belongs to lineage A, together with Italian, Tunisian, Turkish and other French strains. The demonstration of TOSV in Corsica indicates that autochthonous and tourist populations are at risk of infection. Hence, physicians must consider TOSV as a possible cause of aseptic meningitis and unidentified febrile illness during the warm season.

Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

BackgroundLeishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes.ResultsA total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations.ConclusionsThe up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-849) contains supplementary material, which is available to authorized users.

Toscana Virus Isolated from Sandflies, Tunisia

Toscana Virus Isolated from Sandflies, Tunisia

Possibility of Leishmaniasis Transmission in Jura, France

To the Editor: The report of a human cutaneous leishmaniasis case acquired in Clairvaux-les-lacs (1) led us to carry out an investigation with the veterinary clinics in Jura Department, France. Clairvaux-les-lacs is a lakeside resort located in Jura, one of the areas in France with the coldest average temperatures, and is clearly located outside the usual leishmaniasis-endemic area. At least 31 cases of canine leishmaniasis were diagnosed by veterinary clinics in Jura during 2007–2011. Because these dogs were native of or traveled in the leishmaniasis-endemic area along the Mediterranean Sea, all veterinarians considered the infections as acquired outside Jura. Although phlebotomine sand flies have not been reported in Jura to date, Phlebotomus perniciosus sand flies, proven vectors of leishmaniasis, have been found in 2 areas neighboring Jura: Cote-d'Or and Saone-et-Loire (2,3). We have also recently caught P. mascittii sand flies, a species with an unknown vectorial competence, in the Swiss region of Jura, Alsace, Champagne-Ardennes, and Belgium. Therefore, the presence of sand flies in Jura, particularly in wet and milder microclimatic areas (as Clairvaux-les-lacs), is likely, and canine infections could have been acquired locally. A recent model predicted that new at-risk areas are mostly located in western France along the Atlantic coast (4). In accordance with this model, we report new foci of autochthonous canine leishmaniasis in Deux Sevres, Loire-Atlantique, and Loiret. Canine leishmaniasis cases contracted in the Rhine Valley in Germany (5) and the canine cases in Jura argue for a northeastern spread of the disease-endemic area along the Rhone-Rhine axis and mild microclimatic niches. Entomologic and serologic surveys will be carried out in summer 2012 in Jura to look for evidence of possible indigenous transmission of leishmaniasis. These data should supplement the current model of northern spread of leishmaniasis-endemic areas.

Massilia Virus, A Novel Phlebovirus (Bunyaviridae) Isolated from Sandflies in the Mediterranean

A new virus was isolated from three independent pools of Phlebotomus perniciosus sandflies (Diptera; Psychodidae) trapped in two regions of southeastern France, located 90 miles apart. Microscopic, antigenic and genetic analyses indicate that this novel virus belongs to the genus Phlebovirus in the family Bunyaviridae. The new virus is designated Massilia virus since the first isolate was obtained from sandflies collected in the suburban area of Marseille. The complete genome sequence was determined and used to compare the genetic and phylogenetic relationships of Massilia virus with other phleboviruses. Genetic and antigenic properties were employed to address whether or not Massilia virus should be considered a new species within the genus, or a member of a previously recognized species. Cerebrospinal fluid specimens, collected from local patients with central nervous system infections during the previous four-year period were tested for the presence of Massilia virus RNA, but gave negative results. In conclusion, Massilia virus is proposed as a member of the Sandfly fever Naples virus complex; its public health importance has yet to be determined.

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that ∼90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.

RNA genome stability of Toscana virus during serial transovarial transmission in the sandfly Phlebotomus perniciosus

We have carried out a T1 ribonuclease fingerprinting analysis of the RNA genomes of Toscana virus isolates from successive generations of an experimentally virus-infected laboratory colony of Phlebotomus perniciosus sandflies. This analysis detected no virus RNA genome changes during transovarial transmission of the virus over 12 sandfly generations (a period of almost 2 years). These results demonstrate that although RNA viruses can exhibit high rates of mutational change under a variety of conditions, Toscana virus RNA genomes can be maintained in a stable manner during repeated transovarial virus transmission in the natural insect host. The implications of these results for insect RNA virus evolution are discussed.