BACKGROUND: Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE: Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

• MeSH
• antigeny genetika   imunologie   MeSH
• hmotnostní spektrometrie MeSH
• hospodářská zvířata * MeSH
• imunoblotting MeSH
• kousnutí a bodnutí hmyzem diagnóza   MeSH
• kozy MeSH
• ovce MeSH
• protilátky krev   MeSH
• psi MeSH
• Psychodidae genetika   imunologie   MeSH
• rekombinantní proteiny genetika   imunologie   MeSH
• slinné proteiny a peptidy genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.

• MeSH
• antigeny imunologie   MeSH
• cytokiny imunologie   MeSH
• druhová specificita MeSH
• Leishmania major imunologie   MeSH
• leishmanióza imunologie   MeSH
• myši MeSH
• Phlebotomus imunologie   parazitologie   MeSH
• protilátky protozoální imunologie   MeSH
• slinné proteiny a peptidy imunologie   MeSH
• slinné žlázy MeSH
• zkřížená ochrana imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. METHODS AND FINDINGS: In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. CONCLUSIONS: In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host-sand fly-parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

• MeSH
• Leishmania imunologie   MeSH
• Psychodidae parazitologie   fyziologie   MeSH
• slinné proteiny a peptidy imunologie   metabolismus   MeSH
• sliny imunologie   parazitologie   MeSH
• stravovací zvyklosti * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.

• MeSH
• fylogeneze MeSH
• glykosylace MeSH
• hmyzí proteiny chemie   metabolismus   MeSH
• konformace proteinů MeSH
• ligandy MeSH
• molekulární modely MeSH
• Psychodidae * MeSH
• sekvence aminokyselin MeSH
• sliny metabolismus   MeSH
• statická elektřina MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. METHODOLOGY/PRINCIPAL FINDINGS: The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. CONCLUSIONS/SIGNIFICANCE: Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.

• MeSH
• ELISA MeSH
• hmyzí proteiny diagnostické užití   genetika   MeSH
• imunoblotting MeSH
• kousnutí a bodnutí hmyzem diagnóza   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• Phlebotomus imunologie   MeSH
• protilátky krev   MeSH
• psi MeSH
• rekombinantní proteiny diagnostické užití   genetika   MeSH
• sekvenční analýza DNA MeSH
• slinné proteiny a peptidy diagnostické užití   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins. CONCLUSIONS: Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.

• MeSH
• ELISA MeSH
• hmotnostní spektrometrie MeSH
• hmyzí proteiny imunologie   MeSH
• hodnocení rizik MeSH
• imunoglobulin G krev   MeSH
• kousnutí a bodnutí hmyzem komplikace   MeSH
• Leishmania infantum izolace a purifikace   MeSH
• leishmanióza přenos   veterinární   MeSH
• nemoci psů prevence a kontrola   přenos   MeSH
• Phlebotomus imunologie   MeSH
• psi MeSH
• slinné proteiny a peptidy imunologie   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH
• Německo MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• hmyz - vektory MeSH
• imunizace MeSH
• infekce přenášené vektorem MeSH
• interakce hostitele a parazita imunologie   MeSH
• Leishmania patogenita   MeSH
• leishmanióza imunologie   prevence a kontrola   terapie   MeSH
• Phlebotomus chemie   imunologie   MeSH
• Psychodidae chemie   imunologie   MeSH
• sliny chemie   imunologie   MeSH

• MeSH
• antiparazitární látky * klasifikace   škodlivé účinky   zásobování a distribuce   MeSH
• internet MeSH
• komplementární terapie MeSH
• lidé MeSH
• nelegální distribuce léčiv na předpis * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• zprávy MeSH

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. METHODOLOGY/PRINCIPAL FINDINGS: Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. CONCLUSIONS: These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

• MeSH
• ELISA MeSH
• endemické nemoci veterinární   MeSH
• leishmanióza imunologie   veterinární   MeSH
• longitudinální studie MeSH
• nemoci psů imunologie   MeSH
• Phlebotomus imunologie   MeSH
• polymerázová řetězová reakce veterinární   MeSH
• protilátky imunologie   MeSH
• psi MeSH
• rekombinantní proteiny imunologie   MeSH
• rizikové faktory MeSH
• slinné proteiny a peptidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH

Pooled salivary gland samples are frequently used to ensure the sufficient amount of material for the experiments; however, this could mask an individual variability. Thus, we compared salivary protein profiles in seven colonies of three Phlebotomus species: Phlebotomus sergenti, Phlebotomus perniciosus, and Phlebotomus papatasi. Surprisingly, the individual profiles differed significantly between the colonies as well as between individuals. The highest variability was observed in proteins with molecular masses of 42-46 kDa corresponding to the yellow-related proteins. The phenogram constructed from salivary gland profiles revealed the existence of two main groups in P. sergenti, corresponding well with the geographical origin. The F1 progeny obtained from cross-mating studies between P. sergenti colonies of different geographical origin formed a distinct subgroup within the parental groups. In P. papatasi, several groups of protein profiles were observed with no relationship to the geographical origin. The biological role of salivary proteins variability is discussed.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fenotyp MeSH
• křížení genetické MeSH
• molekulová hmotnost MeSH
• Phlebotomus chemie   MeSH
• proteom analýza   MeSH
• shluková analýza MeSH
• slinné proteiny a peptidy analýza   chemie   MeSH
• slinné žlázy chemie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH