Phlebotomine Sand Flies Research Articles

Cutaneous leishmaniasis in protected environmental areas in the Eastern Amazon: the case of São Félix do Xingu, Pará, Brazil

Cutaneous Leishmaniasis is a disease transmitted to men and animals by infected female phlebotomine sandflies and is considered a great environmental and public health problem in the Amazon region. Thus, the study aimed to analyze the spatial distribution of this disease in São Félix do Xingu, in the state of Pará, Brazil, and its relationship with epidemiological and environmental variables, in the period from 2012 to 2016. The data used were from the Information System of the Pará State Secretariat of Health, the National Institute for Space Research and the Brazilian Institute of Geography and Statistics. The statistical and spatial analysis of the variables were done using non-parametric chi-square statistical test, kernel interpolation technique and the Bivariate Global Moran Index. The municipality had 183 confirmed cases, non-homogeneously distributed in 5 administrative districts. The individuals most affected were adult males with brown skin, an elementary level of education and rural area residents. In the case series, a direct relationship was observed between the increase of the number of cases and deforestation in the study area. The spatial analysis showed different types of land use and cover related to case clusters in the municipality. Additionally, there was a presence of cases in protected areas and a great epidemiological silence in indigenous lands. Thus, this disease is a large and complex public health problem in the municipality, related to social and environmental risk factors.

Molecular Detection of Bartonella sp. in Psathyromyia shannoni and Lutzomyia cruciata From Northeastern Mexico

Phlebotomine sand flies are vectors of Leishmania spp., Bartonella bacilliformis, and several arboviruses worldwide. In Mexico, the presence of Bartonella species is associated sporadically with arthropods and little is known on the diversity of insects that could be incriminated with its transmission. The aim of this study was to perform a molecular detection of Bartonella DNA in sand fly species collected in northeastern Mexico. Sand flies were collected at the states of Nuevo Leon and Tamaulipas from June to August 2010, using 16 light traps per night. Sand fly species were morphologically identified, and for Bartonella detection, we amplified ~378 bp of the citrate synthase gene (gltA). DNA sequences were compared in a phylogenetic reconstruction based on maximum likelihood. A total of 532 specimens from seven sand fly species were morphologically identified, where 11 specimens from Tamaulipas tested positive for the presence of a new lineage of Bartonella sp. associated with Psathyromyia shannoni and Lutzomyia cruciata. This work represents the second record of Bartonella-associated with sand flies outside of the endemic area of Carrion’s disease. More studies are necessary to understand their life cycle, transmission dynamics, and their relationship with sand fly species.

A new cluster of rhabdovirus detected in field-caught sand flies (Diptera: Psychodidae: Phlebotominae) collected from southern Thailand

BackgroundThe distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand.MethodsSand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis.ResultsThe results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae family virus presence in Phlebotomus papatasi (3/331, 0.9%). The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies.ConclusionsThere is limited knowledge on the genetic diversity and ecology of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae.Graphical abstract

Leishmania Promastigotes Enhance Neutrophil Recruitment through the Production of CXCL8 by Endothelial Cells

Endothelial cells represent one of the first cell types encountered by Leishmania promastigotes when inoculated into the skin of the human hosts by the bite of phlebotomine sand flies. However, little is known on their role in the early recruitment of phagocytic cells and in the establishment of the infection. Initially, neutrophils, rapidly recruited to the site of promastigotes deposition, phagocytize Leishmania promastigotes, which elude the killing mechanisms of the host cells, survive, and infect other phagocytic cells. Here, we show that Leishmania promastigotes co-incubated with HMEC-1, a microvascular endothelial cell line, exhibited significant morphological changes and loss of infectivity. Moreover, promastigotes of different Leishmania species stimulated the production of CXCL8 by HMEC-1 in a dose- and TLR4-dependent manner. Interestingly, we observed that the conditioned media from Leishmania-stimulated HMEC-1 cells attracted leukocytes, mostly neutrophils, after 2 h of incubation. After 24 h, a higher percentage of monocytes was detected in conditioned media of unstimulated HMEC-1 cells, whereas neutrophils still predominated in conditioned medium from Leishmania-stimulated cells. The same supernatants did not contain CCL5, a chemokine recruiting T cells and monocytes. On the contrary, inhibition of the production of CCL5 induced by TNF-α was seen. These data indicate that the interaction of Leishmania promastigotes with endothelial cells leads to the production of chemokines and the recruitment of neutrophils, which contribute to the establishment of Leishmania infection.

Live attenuated Leishmania major p27 gene knockout as a novel vaccine candidate: A study on safety, protective immunity and efficacy against canine leishmaniasis caused by Leishmania infantum

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27−/−) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27−/− candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27−/− vaccinated group showed no clinical signs after inoculation with Lmp27−/− mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27−/− parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum.

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism.

Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.

On Professor Jean-Antoine Rioux' Phlebotomine sand flies collection (Diptera: Psychodidae)

RésuméIntroductionLe Professeur Jean-Antoine Rioux (1925-2017) a passé une grande partie de sa longue et fructueuse carrière scientifique à explorer et comprendre le fonctionnement de foyers de leishmanioses dans plusieurs régions de Méditerranée, du Proche et du Moyen-Orient et d'Afrique. L’étude des phlébotomes constitua une partie importante de son travail dans les foyers étudiés et un grand nombre de spécimens ont été mis en collection, objet de cette note.Matériel et MéthodeLa collection a été complètement inventoriée. Les phlébotomes y sont classés par espèces et par pays. Chaque phlébotome est monté in toto individuellement sur une lame dans du baume du Canada.RésultatsLa collection est riche de 130 840 spécimens provenant de 10 pays: France (continentale et Corse), Italie, Espagne, Algérie, Tunisie, Maroc, Yémen, Syrie, Soudan et République du Congo. Elle regroupe 26 espèces: Phlebotomus alexandri, Ph. ariasi, Ph. bergeroti, Ph. chabaudi, Ph. chadlii, Ph. kazeruni, Ph. longicuspis, Ph. mascittii, Ph. mongolensis, Ph. orientalis, Ph. papatasi, Ph. perfiliewi, Ph. perniciosus, Ph. riouxi, Ph. sergenti, Sergentomyia africana, Se. antennata, Se. christophersi, Se. clydei, Se. fallax, Se. minuta, Se. schwetzi, Se. silva, Se. taizi, Se. tiberiadis et Grassomyia dreyfussi.DiscussionLa collection des phlébotomes de Jean-Antoine Rioux (1925-2017) a été transférée dans la Plateforme des centres de ressources biologiques de Reims (PF CRBs Reims) où elle est entretenue et inventoriée afin d’être mise à la disposition des personnes désireuse d'y accéder à toutes fins scientifiques. Outre des topotypes de Ph. chabaudi et de Ph. chadlii, elle regroupe de nombreux exemplaires d'Afrique du Nord, essentiellement du Maroc, mais également des exemplaires de Syrie ou du Yémen dont l'intérêt est évident compte tenu du contexte géopolitique actuel.

The Psychodidae (Diptera) of Ja National Park (Amazonas, Brazil) with description of two new species of Psychodinae.

The Ja National Park, located in the Amazon, is the largest National Park of Brazil and still its insect fauna is barely known. Herein we describe two new species of Psychodinae, Alepia iy sp. nov. and Parasetomima timmirima sp. nov., and report 19 other species of Psychodidae, subfamilies Phlebotominae, Psychodinae and Trichomyiinae collected from this Conservation Unit. Micrommatos Quate Brown, represented by M. stephaniae Quate Brown, and Platyplastinx culmosus Quate Brown are recorded for the first time for Brazil. Among the collected phlebotomine sand fly species, three species have previously been implicated in transmission of Leishmania: Nyssomyia anduzei (Rozeboom), Psychodopygus ayrozai (Barretto Coutinho) and Trichophoromyia ubiquitalis (Mangabeira).

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.

Elevated and sustained anti-feeding effect of Scalibor® deltamethrin collar against the sand fly Phlebotomus perniciosus in dogs confirmed for 1 year following treatment.

Dogs are reservoir hosts for Leishmania infantum, a protozoan parasite transmitted by phlebotomine sand flies. The anti‐feeding and fast‐killing efficacy of Scalibor® deltamethrin collars against experimental Phlebotomus perniciosus challenges on dogs was determined over 1 year. Two groups of 8 dogs each were fitted with placebo (control) or deltamethrin collars (treated) on Day 0 and exposed to sand flies approximately every 28 days up to Day 364. After each exposure, anti‐feeding and fast‐killing efficacy rates were determined by comparing blood‐fed or live insects, respectively, in the treated vs. the control group. Blood‐fed and live sand flies were significantly less in treated dogs as compared to control dogs at each assessment. The anti‐feeding efficacy rate exceeded 90% except on Day 337 (89%) but increased again (96%) on Day 364. Fast killing efficacy was <74% over the study when considering all flies. However, this value increased cumulatively to 98% when only blood‐fed flies were compared between groups. Scalibor® collars are highly effective at preventing P. perniciosus blood‐feeding and in fast‐killing flies taking a blood meal for up to 1 year after application. These strong and long‐lasting effects are an important strategic component for L. infantum transmission control.

A clinical study to optimise a sand fly biting protocol for use in a controlled human infection model of cutaneous leishmaniasis (the FLYBITE study)

Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019)

Density assessment and reporting for Phlebotomus perniciosus and other sand fly species in periurban residential estates in Spain

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using “sticky” interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution.

Leishmaniosis in Rodents Caused by Leishmania infantum: A Review of Studies in the Mediterranean Area.

Leishmaniosis infection begins when a phlebotomine sand fly vector inoculates pathogenic protozoan parasites of the genus Leishmania into a mammalian host. In the case of Leishmania infantum, the domestic dog is considered to be the main parasite reservoir, and canine leishmaniosis (CanL) has a high mortality rate in untreated dogs. Hundreds of cases of human leishmaniosis (HL) are reported in the world each year, the incidence in Europe being relatively low. Leishmaniosis control is primarily focused on the dog, combining methods that prevent sand fly bites and boost host resistance to infection. However, these measures are only partially effective and new solutions need to be found. One of the main factors limiting CanL and HL control is the existence of a sylvatic Leishmania transmission cycle that interacts with the domestic cycle maintained by dogs. It is suspected that the main reservoir of infection in wildlife are rodents, whose expansion and rapid population growth worldwide is increasing the risk of human and zoonotic pathogen transfer. The aim of this review is therefore to analyze reports in the literature that may shed light on the potential role of rodents in the leishmaniosis transmission cycle in the Mediterranean area. Following the general methodology recommended for reviews, six databases (Google Scholar, Ovid, PubMed, Science Direct, Scopus and Web of Science) were explored for the period January 1995 to December 2020. The results extracted from 39 publications that met the established inclusion criteria were analyzed. It was found that 23 species of rodents have been studied in nine countries of the Mediterranean basin. Of the 3,643 specimens studied, 302 tested positive for L. infantum infection by serology, microscopy and/or molecular techniques.

Natural Products against Sand Fly Vectors of Leishmaniosis: A Systematic Review.

Leishmaniosis is a vector-borne disease transmitted to animals and humans by the bite of blood-sucking phlebotomine sand flies. These small insects play a crucial role in the diffusion of the disease. To date, the sole strategy recognized for the prevention of leishmaniosis is the use of topical repellent compounds against sand fly bites. Several synthetic insecticides and repellents have been developed; however, the wide and unprejudiced use of these formulations have led to the loss of their effectiveness and the development of resistance phenomena. Moreover, some of these synthetic repellents have severe detrimental effects on the environment and could represent a serious threat to both animal and human health. Recently, an increased interest in the research on alternative approaches to sand fly control has been expressed. In this study, we systematically reviewed the efforts of the scientific community to individuate a phytochemical alternative for the control of sand fly species recognized as vectors of Leishmania spp. Based on literature research using different electronic databases, a total of 527 potentially relevant studies were screened and narrowed down to a final 14 eligible scientific reports. Our analysis suggests that although there is a rapidly growing body of literature dedicated to botanical insecticides and repellents against sand fly vectors of Leishmania spp., much of this literature is limited to in vitro studies conducted in laboratory conditions, and only a few of them investigated the repellency of plant-based products. These studies highlighted that natural compounds display a really short period of action and this significantly limits the use of these products as an alternative to chemical-based repellents.

Behavioural responses of Phlebotomus duboscqi to plant‐derived volatile organic compounds

Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography-mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p-cymene and the benzenoid m-cresol, p-cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose-response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five-component blends, referred to as Blend-f and Blend-m for females and males respectively, were formulated and tested in dose-response assays against 1-octen-3-ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant-derived VOCs can be exploited for sand fly management.

Sandfly Fever Sicilian Virus-Leishmania major co-infection modulates innate inflammatory response favoring myeloid cell infections and skin hyperinflammation.

BackgroundThe leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV).Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis.Methodology/Principal findingsIn vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV’s hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone.Conclusion/SignificanceOverall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.

Parasitological and molecular search for Leishmania natural infection in phlebotomine sand flies in peri-urban and rural sites of an Argentinean area endemic for tegumentary leishmaniasis

Leishmaniases are neglected tropical diseases caused by Leishmania spp. parasites transmitted by the bite of phlebotomine sand flies. In Argentina, the most endemic area of American tegumentary leishmaniasis (ATL) has been Orán department, Province of Salta, where Leishmania (Viannia) braziliensis prevails and Nyssomyia neivai is considered as its vector, although there is no accurate and sufficient information in this regard. The aim of this work was to search for natural infection by Leishmania spp. in sand flies from peri-urban and rural sites with ATL background in Orán department. For this, sand flies were caught at five sites; female sand flies captured with Shannon trap were dissected to microscopically examine their gut contents, while females captured with CDC traps were molecularly analyzed by duplex PCR with two primer pairs to simultaneously amplify kinetoplast DNA (kDNA) and mammalian actin. A total of 1921 females were captured, with Ny. neivai being the most abundant species (89%), followed by Migonemyia migonei (6%) and cortelezzii complex (3%). No natural infection was found in any of them neither by dissection nor by PCR, although the detection limit of kDNA PCR was up to 25 promastigotes. The absence of infected females in peri-urban sites suggest that the transmission did not take place in those environments during the study period. Future searches for natural infection should focus on rural settings to deepen knowledge and elucidate the role of the circulating sand fly species as all have been linked to ATL transmission at other sites.

Phlebotomine sand fly survey in the Republic of Moldova: species composition, distribution and host preferences

BackgroundPhlebotomine sand flies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a first update on their occurrence, species composition and bloodmeal sources after 50 years.MethodsDuring 5 seasons (2013–2017), 58 localities from 20 regions were surveyed for presence of sand flies using CDC light traps and manual aspirators. Species identification was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profiling). In engorged females, host blood was identified by three molecular techniques (RFLP, cytb sequencing and MALDI-TOF peptide mass mapping). Population structure of most abundant species was studied by cox1 haplotyping; phylogenetic analyses of ITS2 and cox1 genetic markers were used to resolve relationships of other detected species.ResultsIn total, 793 sand flies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identified by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfiliewi and Phlebotomus sp. (Adlerius), the first being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only five haplotypes of cox1 detected. Distinct morphological and molecular characters of Phlebotomus sp. (Adlerius) specimens suggest the presence of a yet undescribed species.ConclusionsOur study revealed the presence of stable sand fly populations of three species in Moldova that represent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied.Graphical abstract

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers.

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9-8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3-5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3-2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.