Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinic... more Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinical form of leishmaniasis in the Americas. Migonemyia migonei is a widely distributed phlebotomine sand fly species in Brazil and has been implicated as a vector for L. (V.) braziliensis. In the present study, we investigated the effects of salivary gland homogenates (SGH) of Mg. migonei on the course of L. (V.) braziliensis infection in BALB/c mice. Mice were separated into four groups (six mice per group): CRTL (uninfected mice); SGH (mice inoculated with Mg. migonei SGH); SGH+LEISH (mice inoculated with Mg. migonei SGH plus L. (V.) braziliensis promastigotes); LEISH (mice inoculated with L. (V.) braziliensis promastigotes). Mice were followed up for 8 weeks and the cellular immune response was evaluated by flow cytometry at the end of the experiment. Analysis of cytokine production by splenic cells stimulated with 0.5 SGH, 0.25 SGH of Mg. migonei or L. (V.) braziliensis soluble antigen stimulation (LSA) demonstrated that upon stimulation with SGH 0.25, the production of IL-17A and TNF was not sustained in the SGH group, with decreasing levels of these cytokines after 5 days compared to 3 days of incubation. Analyzing the production of cytokines after LSA stimulation, we observed lower levels of IL-17A in the SGH group after 5 days compared to 3 days. The same was observed for IFN-γ in the SGH group. Yet, the levels of TNF were significantly higher in the LEISH group after 5 days compared to 3 days. Among SGH+LEISH and LEISH mice, three animals in each group developed skin lesions on the tail, the mean lesion size was significantly higher in the LEISH group. Our study suggests that Mg. migonei SGH may modulate BALB/c immune response, as reflected by the low production or early decrease of pro-inflammatory cytokines in splenic cell cultures following stimulation with L. (V.) braziliensis antigen. Our data also suggest that Mg. migonei saliva may reduce the lesion size in BALB/c mice, but further research with a larger sample size is needed to confirm this hypothesis.
Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in th... more Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH-and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.
Frontiers in Cellular and Infection Microbiology, Aug 24, 2020
Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect v... more Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect vector. It transmits the unicellular parasite Leishmania infantum that multiplies intracellularly in macrophages causing life-threatening visceral diseases. Leishmania establishment in the vertebrate host is substantially influenced by immunomodulatory properties of vector saliva that are obligatorily co-injected into the feeding site. The repertoire of P. perniciosus salivary molecules has already been revealed and, subsequently, several salivary proteins have been expressed. However, their immunogenic properties have never been studied. In our study, we tested three P. perniciosus recombinant salivary proteins-an apyrase rSP01 and yellow-related proteins rSP03 and rSP03B-and showed their anti-inflammatory nature on the murine bone-marrow derived macrophages. Even in the presence of pro-inflammatory stimuli (IFN-γ and bacterial lipopolysaccharide, LPS), all three recombinant proteins inhibited nitric oxide production. Moreover, rSP03 seems to have a very strong anti-inflammatory effect since it enhanced arginase activity, increased the production of IL-10, and inhibited the production of TNF-α even in macrophages stimulated with IFN-γ and LPS. These results suggest that P. perniciosus apyrase and yellow-related proteins may serve as enhancing factors in sand fly saliva, facilitating the development of Leishmania infection along with their anti-haemostatic properties. Additionally, rSP03 and rSP03B did not elicit the delayed-type hypersensitivity response in mice pre-exposed to P. perniciosus bites (measured as visible skin reaction). The results of our study may help to understand the potential function of recombinant's native counterparts and their role in Leishmania transmission and establishment within the host.
Background: During blood feeding, sand flies inoculate salivary proteins that interact with the h... more Background: During blood feeding, sand flies inoculate salivary proteins that interact with the host haemostatic system. The blocking of biogenic amines such as serotonin and histamine helps to limit vasodilatation and clot formation, and thus enables the insect to finish the blood-feeding process. In sand flies, an amine-binding ability is known only for the yellow-related proteins of Phlebotomus and Lutzomyia vectors, but not yet for members of the genus Sergentomyia. The ability of Phlebotomus argentipes and Sergentomyia schwetzi recombinant yellow-related salivary proteins to bind histamine and serotonin was measured by microscale thermophoresis. Both sand fly species were also fed through a chicken-skin membrane on blood mixed with histamine or serotonin in order to check the effects of biogenic amines on sand fly fitness. Additionally, fecundity and mortality were compared in two groups of P. argentipes females fed on repeatedly-bitten and naive hamsters, respectively. Results: The P. argentipes recombinant yellow-related protein PagSP04 showed high binding affinity to serotonin and low affinity to histamine. No binding activity was detected for two yellow-related proteins of S. schwetzi. Elevated concentrations of serotonin significantly reduced the amount of eggs laid by P. argentipes when compared to the control. The fecundity of S. schwetzi and the mortality of both sand fly species were not impaired after the experimental membrane feeding. Additionally, there were no differences in oviposition or mortality between P. argentipes females fed on immunized or naive hamsters. Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or antisaliva antibodies without any influence on their fitness. The serotonin binding by salivary yellow-related proteins may play an important role in Phlebotomus species feeding on mammalian hosts, but not in S. schwetzi, which is adapted to reptiles.
<p>The heatmap shows chromosomal copy number for all 36 chromosomes across the 12 CUK strai... more <p>The heatmap shows chromosomal copy number for all 36 chromosomes across the 12 CUK strains, based on normalized chromosome read-depth. Copy number varies from disomic (light yellow) to pentasomic (dark-red).</p
Antibodies against Phlebotomus perniciosus sand fly salivary gland homogenate (SGH) and recombina... more Antibodies against Phlebotomus perniciosus sand fly salivary gland homogenate (SGH) and recombinant protein rSP03B, sand fly-borne Toscana virus (TOSV), Sand Fly Fever Sicilian virus (SFSV) and Leishmania, as well as DNA of the latter parasite, were investigated in 670 blood samples from 575 human donors in Murcia Region, southeast Spain, in 2017 and 2018. The estimated SGH and rSP03B seroprevalences were 69% and 88%, respectively, although correlation between test results was relatively low (ρ = 0.39). Similarly, TOSV, SFSV and Leishmania seroprevalences were 26%, 0% and 1%, respectively, and Leishmania PCR prevalence was 2%. Prevalences were significantly greater in 2017, overdispersed and not spatially related to each other although both were positively associated with SGH but not to rSP03B antibody optical densities, questioning the value of the latter as a diagnostic marker for these infections in humans. This article is protected by copyright. All rights reserved.
The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a majo... more The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly. We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 9...
The review is devoted to the gut lectins/hemagglutinins of the following representatives of impor... more The review is devoted to the gut lectins/hemagglutinins of the following representatives of important disease vectors : ticks, kissing-bugs, mosquitoes, sandflies and tsetse flies. The paper surveys the recent knowledge on these carbohydrate binding factors with respect to their structural and functional properties, and their significance for pathogen/parasite transmission by the blood-sucking arthropods. Recent results suggest that in most vectors the gut lectin activities are blood-meal enhanced, might participate in blood-meal processing and digestion and could serve as antibacterial and antiparasitic agents.
<b>Copyright information:</b>Taken from "High degree of conservancy among secret... more <b>Copyright information:</b>Taken from "High degree of conservancy among secreted salivary gland proteins from two geographically distant sandflies populations (Mali and Kenya)"BMC Genomics 2006;7():226-226.Published online 4 Sep 2006PMCID:PMC1574310.
Sandfly females, while feeding on the host, excrete urine to concentrate proteins of the bloodmea... more Sandfly females, while feeding on the host, excrete urine to concentrate proteins of the bloodmeal and restore weight and water balance. This process, analogous to prediuresis in mosquitoes, was ob served in 100% of Phlebotomus papatasi (Scopoli) and 85% of P. duboscqi Neveu-Lemaire females studied. Individual females, however, differed in duration of prediuresis and in the number of ejected urine droplets. In both species the prediuresis generally started 1-2 min after the commencement of feed ing and the variation in urine production was positively correlated with the length of feeding. The first one or two droplets were opaque whitish while the remaining ones were clear. Erythrocytes were found sporadically in first droplets of some females. Representative prediuresis in P. duboscqi included 27 drop lets, i.e., about 325 nl urine in total, ejected during 8 min of feeding. The study revealed prediuresis in P. papatasi and P. duboscqi as a regular physiological process which may have consequences in transmis sion of infective diseases.
<p>The non MON-1 strains from Turkey as well as the Cyprian canine isolate clone 1 analysed... more <p>The non MON-1 strains from Turkey as well as the Cyprian canine isolate clone 1 analysed herein are presented in bold.</p><p>VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; PKDL, post Kala Azar dermal leishmaniasis; CanL, canine leishmaniasis.</p><p>*Only one of the three MON-37 clones (cl.1) isolated from the parent CD44 strain was further analyzed;</p><p>**Hybrid strain; CY, Cyprus; ET, Ethiopia; GR, Greece; IN, India; KE, Kenya; LK, Sri Lanka; PT, Portugal; SD, Sudan; SP, Spain; TR, Turkey; n.d., not defined.</p
BackgroundSand flies are vectors ofLeishmaniaspp., the causative agents of leishmaniasis in verte... more BackgroundSand flies are vectors ofLeishmaniaspp., the causative agents of leishmaniasis in vertebrates, including man. The sand fly saliva contains powerful pharmacologically active substances that prevent hemostasis and enhanceLeishmaniaspp. infections. On the other hand, salivary proteins can protect vaccinated mice challenged with parasites. Therefore, sand fly salivary proteins are relevant for the epidemiology of leishmaniasis and can be a potential target for a vaccine against leishmaniasis. Despite this, studies on sand fly salivary glands (SGs) are limited.MethodsThe present study analyzes, in detail, the morphology, anatomy and ultrastructure of the SGs of sand fly vectors of the generaLutzomyiaandPhlebotomus. We used histology, transmission and scanning electron microscopy and lectin labeling associated with confocal laser microscopy.ResultsThe SGs have conserved and distinct morphological aspects according to the distinct sand fly species. Each SG has a single rounded lo...
Background: Phlebotomine sand ies are the principal vectors of Leishmania spp. (Kinetoplastida: T... more Background: Phlebotomine sand ies are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). Sand y ndings in Central Europe are scarce and in Austria, to date only Phlebotomus mascittii has been recorded. In 2018 and 2019, entomological surveys were conducted in Austria with the aim to further clarify sand y distribution and species composition. Results: In 2019, a Ph. simici specimen was trapped in Austria for the rst time. Analyses of two commonly used marker genes, coxI and cytb, revealed high sequence identity with Ph. simici specimens from North Macedonia and Greece. Phylogenetic analyses showed high intraspeci c distances within Ph. simici, thereby dividing this species into three lineages, from Europe, Turkey and Israel, respectively. Low interspeci c distances between Ph. simici, Ph. brevis and a yet unidenti ed Adlerius sp. from Turkey and Armenia highlights that molecular identi cation can be challenging within the Adlerius complex, even when applying standard marker genes. Conclusion: This study provides the rst nding of Ph. simici in Austria and the northernmost record so far. Moreover, it reveals valuable insights into the phylogenetic relationships of species within the Adlerius subgenus. Ph. simici is a suspected vector of Leishmania infantum and therefore of medical and veterinary importance. Potential sand y expansion in Central Europe due to climatic change and the increasing import of Leishmania-infected dogs from endemic areas, urge the need for further studies on sand y distribution in Austria and Central Europe in general.
Background Identification of blood sources of hematophagous arthropods is crucial for understandi... more Background Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA-and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. Conclusions/Significance PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool
<b>Copyright information:</b>Taken from "High degree of conservancy among secret... more <b>Copyright information:</b>Taken from "High degree of conservancy among secreted salivary gland proteins from two geographically distant sandflies populations (Mali and Kenya)"BMC Genomics 2006;7():226-226.Published online 4 Sep 2006PMCID:PMC1574310. (B) Sequence alignment of the orthologues PduK06 and PduM35; (C) PduK04 and PduM10. Black-shaded amino acids represent identical amino acids, grey-shaded amino acids represent conserved amino acids, and * at the top of the amino acids denotes potential T cell epitopes as searched by using the TEPITOPE software.
We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Bo... more We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19610 4 promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.
Differentiation between distinct stages is fundamental for the life cycle of intracellular protoz... more Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.
Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects ... more Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand y. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the y. Methods In this work we wanted to identify y systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand ies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Results Of these, only L. major is able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not speci c to success or failure of a speci c infection and were not different from each other. They were also indistinguishable from non-contaminated blood. Conclusions This implies that sand ies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinic... more Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinical form of leishmaniasis in the Americas. Migonemyia migonei is a widely distributed phlebotomine sand fly species in Brazil and has been implicated as a vector for L. (V.) braziliensis. In the present study, we investigated the effects of salivary gland homogenates (SGH) of Mg. migonei on the course of L. (V.) braziliensis infection in BALB/c mice. Mice were separated into four groups (six mice per group): CRTL (uninfected mice); SGH (mice inoculated with Mg. migonei SGH); SGH+LEISH (mice inoculated with Mg. migonei SGH plus L. (V.) braziliensis promastigotes); LEISH (mice inoculated with L. (V.) braziliensis promastigotes). Mice were followed up for 8 weeks and the cellular immune response was evaluated by flow cytometry at the end of the experiment. Analysis of cytokine production by splenic cells stimulated with 0.5 SGH, 0.25 SGH of Mg. migonei or L. (V.) braziliensis soluble antigen stimulation (LSA) demonstrated that upon stimulation with SGH 0.25, the production of IL-17A and TNF was not sustained in the SGH group, with decreasing levels of these cytokines after 5 days compared to 3 days of incubation. Analyzing the production of cytokines after LSA stimulation, we observed lower levels of IL-17A in the SGH group after 5 days compared to 3 days. The same was observed for IFN-γ in the SGH group. Yet, the levels of TNF were significantly higher in the LEISH group after 5 days compared to 3 days. Among SGH+LEISH and LEISH mice, three animals in each group developed skin lesions on the tail, the mean lesion size was significantly higher in the LEISH group. Our study suggests that Mg. migonei SGH may modulate BALB/c immune response, as reflected by the low production or early decrease of pro-inflammatory cytokines in splenic cell cultures following stimulation with L. (V.) braziliensis antigen. Our data also suggest that Mg. migonei saliva may reduce the lesion size in BALB/c mice, but further research with a larger sample size is needed to confirm this hypothesis.
Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in th... more Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH-and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.
Frontiers in Cellular and Infection Microbiology, Aug 24, 2020
Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect v... more Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect vector. It transmits the unicellular parasite Leishmania infantum that multiplies intracellularly in macrophages causing life-threatening visceral diseases. Leishmania establishment in the vertebrate host is substantially influenced by immunomodulatory properties of vector saliva that are obligatorily co-injected into the feeding site. The repertoire of P. perniciosus salivary molecules has already been revealed and, subsequently, several salivary proteins have been expressed. However, their immunogenic properties have never been studied. In our study, we tested three P. perniciosus recombinant salivary proteins-an apyrase rSP01 and yellow-related proteins rSP03 and rSP03B-and showed their anti-inflammatory nature on the murine bone-marrow derived macrophages. Even in the presence of pro-inflammatory stimuli (IFN-γ and bacterial lipopolysaccharide, LPS), all three recombinant proteins inhibited nitric oxide production. Moreover, rSP03 seems to have a very strong anti-inflammatory effect since it enhanced arginase activity, increased the production of IL-10, and inhibited the production of TNF-α even in macrophages stimulated with IFN-γ and LPS. These results suggest that P. perniciosus apyrase and yellow-related proteins may serve as enhancing factors in sand fly saliva, facilitating the development of Leishmania infection along with their anti-haemostatic properties. Additionally, rSP03 and rSP03B did not elicit the delayed-type hypersensitivity response in mice pre-exposed to P. perniciosus bites (measured as visible skin reaction). The results of our study may help to understand the potential function of recombinant's native counterparts and their role in Leishmania transmission and establishment within the host.
Background: During blood feeding, sand flies inoculate salivary proteins that interact with the h... more Background: During blood feeding, sand flies inoculate salivary proteins that interact with the host haemostatic system. The blocking of biogenic amines such as serotonin and histamine helps to limit vasodilatation and clot formation, and thus enables the insect to finish the blood-feeding process. In sand flies, an amine-binding ability is known only for the yellow-related proteins of Phlebotomus and Lutzomyia vectors, but not yet for members of the genus Sergentomyia. The ability of Phlebotomus argentipes and Sergentomyia schwetzi recombinant yellow-related salivary proteins to bind histamine and serotonin was measured by microscale thermophoresis. Both sand fly species were also fed through a chicken-skin membrane on blood mixed with histamine or serotonin in order to check the effects of biogenic amines on sand fly fitness. Additionally, fecundity and mortality were compared in two groups of P. argentipes females fed on repeatedly-bitten and naive hamsters, respectively. Results: The P. argentipes recombinant yellow-related protein PagSP04 showed high binding affinity to serotonin and low affinity to histamine. No binding activity was detected for two yellow-related proteins of S. schwetzi. Elevated concentrations of serotonin significantly reduced the amount of eggs laid by P. argentipes when compared to the control. The fecundity of S. schwetzi and the mortality of both sand fly species were not impaired after the experimental membrane feeding. Additionally, there were no differences in oviposition or mortality between P. argentipes females fed on immunized or naive hamsters. Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or antisaliva antibodies without any influence on their fitness. The serotonin binding by salivary yellow-related proteins may play an important role in Phlebotomus species feeding on mammalian hosts, but not in S. schwetzi, which is adapted to reptiles.
<p>The heatmap shows chromosomal copy number for all 36 chromosomes across the 12 CUK strai... more <p>The heatmap shows chromosomal copy number for all 36 chromosomes across the 12 CUK strains, based on normalized chromosome read-depth. Copy number varies from disomic (light yellow) to pentasomic (dark-red).</p
Antibodies against Phlebotomus perniciosus sand fly salivary gland homogenate (SGH) and recombina... more Antibodies against Phlebotomus perniciosus sand fly salivary gland homogenate (SGH) and recombinant protein rSP03B, sand fly-borne Toscana virus (TOSV), Sand Fly Fever Sicilian virus (SFSV) and Leishmania, as well as DNA of the latter parasite, were investigated in 670 blood samples from 575 human donors in Murcia Region, southeast Spain, in 2017 and 2018. The estimated SGH and rSP03B seroprevalences were 69% and 88%, respectively, although correlation between test results was relatively low (ρ = 0.39). Similarly, TOSV, SFSV and Leishmania seroprevalences were 26%, 0% and 1%, respectively, and Leishmania PCR prevalence was 2%. Prevalences were significantly greater in 2017, overdispersed and not spatially related to each other although both were positively associated with SGH but not to rSP03B antibody optical densities, questioning the value of the latter as a diagnostic marker for these infections in humans. This article is protected by copyright. All rights reserved.
The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a majo... more The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly. We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 9...
The review is devoted to the gut lectins/hemagglutinins of the following representatives of impor... more The review is devoted to the gut lectins/hemagglutinins of the following representatives of important disease vectors : ticks, kissing-bugs, mosquitoes, sandflies and tsetse flies. The paper surveys the recent knowledge on these carbohydrate binding factors with respect to their structural and functional properties, and their significance for pathogen/parasite transmission by the blood-sucking arthropods. Recent results suggest that in most vectors the gut lectin activities are blood-meal enhanced, might participate in blood-meal processing and digestion and could serve as antibacterial and antiparasitic agents.
<b>Copyright information:</b>Taken from "High degree of conservancy among secret... more <b>Copyright information:</b>Taken from "High degree of conservancy among secreted salivary gland proteins from two geographically distant sandflies populations (Mali and Kenya)"BMC Genomics 2006;7():226-226.Published online 4 Sep 2006PMCID:PMC1574310.
Sandfly females, while feeding on the host, excrete urine to concentrate proteins of the bloodmea... more Sandfly females, while feeding on the host, excrete urine to concentrate proteins of the bloodmeal and restore weight and water balance. This process, analogous to prediuresis in mosquitoes, was ob served in 100% of Phlebotomus papatasi (Scopoli) and 85% of P. duboscqi Neveu-Lemaire females studied. Individual females, however, differed in duration of prediuresis and in the number of ejected urine droplets. In both species the prediuresis generally started 1-2 min after the commencement of feed ing and the variation in urine production was positively correlated with the length of feeding. The first one or two droplets were opaque whitish while the remaining ones were clear. Erythrocytes were found sporadically in first droplets of some females. Representative prediuresis in P. duboscqi included 27 drop lets, i.e., about 325 nl urine in total, ejected during 8 min of feeding. The study revealed prediuresis in P. papatasi and P. duboscqi as a regular physiological process which may have consequences in transmis sion of infective diseases.
<p>The non MON-1 strains from Turkey as well as the Cyprian canine isolate clone 1 analysed... more <p>The non MON-1 strains from Turkey as well as the Cyprian canine isolate clone 1 analysed herein are presented in bold.</p><p>VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; PKDL, post Kala Azar dermal leishmaniasis; CanL, canine leishmaniasis.</p><p>*Only one of the three MON-37 clones (cl.1) isolated from the parent CD44 strain was further analyzed;</p><p>**Hybrid strain; CY, Cyprus; ET, Ethiopia; GR, Greece; IN, India; KE, Kenya; LK, Sri Lanka; PT, Portugal; SD, Sudan; SP, Spain; TR, Turkey; n.d., not defined.</p
BackgroundSand flies are vectors ofLeishmaniaspp., the causative agents of leishmaniasis in verte... more BackgroundSand flies are vectors ofLeishmaniaspp., the causative agents of leishmaniasis in vertebrates, including man. The sand fly saliva contains powerful pharmacologically active substances that prevent hemostasis and enhanceLeishmaniaspp. infections. On the other hand, salivary proteins can protect vaccinated mice challenged with parasites. Therefore, sand fly salivary proteins are relevant for the epidemiology of leishmaniasis and can be a potential target for a vaccine against leishmaniasis. Despite this, studies on sand fly salivary glands (SGs) are limited.MethodsThe present study analyzes, in detail, the morphology, anatomy and ultrastructure of the SGs of sand fly vectors of the generaLutzomyiaandPhlebotomus. We used histology, transmission and scanning electron microscopy and lectin labeling associated with confocal laser microscopy.ResultsThe SGs have conserved and distinct morphological aspects according to the distinct sand fly species. Each SG has a single rounded lo...
Background: Phlebotomine sand ies are the principal vectors of Leishmania spp. (Kinetoplastida: T... more Background: Phlebotomine sand ies are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). Sand y ndings in Central Europe are scarce and in Austria, to date only Phlebotomus mascittii has been recorded. In 2018 and 2019, entomological surveys were conducted in Austria with the aim to further clarify sand y distribution and species composition. Results: In 2019, a Ph. simici specimen was trapped in Austria for the rst time. Analyses of two commonly used marker genes, coxI and cytb, revealed high sequence identity with Ph. simici specimens from North Macedonia and Greece. Phylogenetic analyses showed high intraspeci c distances within Ph. simici, thereby dividing this species into three lineages, from Europe, Turkey and Israel, respectively. Low interspeci c distances between Ph. simici, Ph. brevis and a yet unidenti ed Adlerius sp. from Turkey and Armenia highlights that molecular identi cation can be challenging within the Adlerius complex, even when applying standard marker genes. Conclusion: This study provides the rst nding of Ph. simici in Austria and the northernmost record so far. Moreover, it reveals valuable insights into the phylogenetic relationships of species within the Adlerius subgenus. Ph. simici is a suspected vector of Leishmania infantum and therefore of medical and veterinary importance. Potential sand y expansion in Central Europe due to climatic change and the increasing import of Leishmania-infected dogs from endemic areas, urge the need for further studies on sand y distribution in Austria and Central Europe in general.
Background Identification of blood sources of hematophagous arthropods is crucial for understandi... more Background Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA-and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. Conclusions/Significance PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool
<b>Copyright information:</b>Taken from "High degree of conservancy among secret... more <b>Copyright information:</b>Taken from "High degree of conservancy among secreted salivary gland proteins from two geographically distant sandflies populations (Mali and Kenya)"BMC Genomics 2006;7():226-226.Published online 4 Sep 2006PMCID:PMC1574310. (B) Sequence alignment of the orthologues PduK06 and PduM35; (C) PduK04 and PduM10. Black-shaded amino acids represent identical amino acids, grey-shaded amino acids represent conserved amino acids, and * at the top of the amino acids denotes potential T cell epitopes as searched by using the TEPITOPE software.
We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Bo... more We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19610 4 promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.
Differentiation between distinct stages is fundamental for the life cycle of intracellular protoz... more Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.
Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects ... more Background Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that effects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand y. Novel strategies for disease control, require a better understanding of the key step for transmission namely, the establishment of infection inside the y. Methods In this work we wanted to identify y systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand ies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, Leishmania donovani and Herpetomonas muscarum: a parasite not transmitted to humans. Results Of these, only L. major is able to successfully establish an infection in P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not speci c to success or failure of a speci c infection and were not different from each other. They were also indistinguishable from non-contaminated blood. Conclusions This implies that sand ies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
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