Antibodies against Phlebotomus perniciosus sandfly salivary gland homogenate (SGH) and recombinant protein rSP03B, sandfly-borne Toscana virus (TOSV), Sandfly 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.

• Keywords
• Leishmania infantum, anti-saliva antibodies, blood donors, sandflies, sandfly fever sicilian virus, toscana virus,
• MeSH
• Blood Donors MeSH
• Leishmania infantum * MeSH
• Leishmaniasis * parasitology   veterinary   MeSH
• Humans MeSH
• Phlebotomus * parasitology   MeSH
• Antibodies MeSH
• Psychodidae * MeSH
• Recombinant Proteins MeSH
• Sandfly fever Naples virus * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain epidemiology   MeSH
• Names of Substances
• Antibodies MeSH
• Recombinant Proteins MeSH

Phlebotomus argentipes is a predominant vector of Leishmania donovani, the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes, sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti-P. argentipes saliva IgG responses. The anti-rPagSP06 and anti-P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti-P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area.

• Keywords
• Bangladesh, IgG antibodies, Leishmania donovani, Marker of exposure, Phlebotomus argentipes, Salivary glands,
• MeSH
• Insect Proteins * immunology   MeSH
• Bites and Stings epidemiology   MeSH
• Leishmania donovani MeSH
• Humans MeSH
• Phlebotomus * MeSH
• Salivary Proteins and Peptides * immunology   MeSH
• Saliva MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Bangladesh epidemiology   MeSH
• Names of Substances
• Insect Proteins * MeSH
• Salivary Proteins and Peptides * MeSH

BACKGROUND: Sand flies are vectors of Leishmania spp., the causative agents of leishmaniasis in vertebrates, including man. The sand fly saliva contains powerful pharmacologically active substances that prevent hemostasis and enhance Leishmania spp. 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. METHODS: The present study analyzes, in detail, the morphology, anatomy and ultrastructure of the SGs of sand fly vectors of the genera Lutzomyia and Phlebotomus. We used histology, transmission and scanning electron microscopy and lectin labeling associated with confocal laser microscopy. RESULTS: The SGs have conserved and distinct morphological aspects according to the distinct sand fly species. Each SG has a single rounded lobe constituting of c.100-120 secretory cells. The SG secretory cells, according to their ultrastructure and lectin binding, were classified into five different subpopulations, which may differ in secretory pathways. CONCLUSIONS: To the best of our knowledge, these morphological details of sand fly salivary glands are described for the first time. Further studies are necessary to better understand the role of these different cell types and better relate them with the production and secretion of the saliva substances, which has a fundamental role in the interaction of the sand fly vectors with Leishmania.

• Keywords
• Lectin binding, Sand fly vectors, Secretory cell population, Ultrastructure,
• MeSH
• Microscopy, Electron MeSH
• Disease Vectors MeSH
• Mosquito Vectors anatomy & histology   parasitology   ultrastructure   MeSH
• Leishmaniasis transmission   MeSH
• Phlebotomus anatomy & histology   parasitology   ultrastructure   MeSH
• Psychodidae anatomy & histology   parasitology   ultrastructure   MeSH
• Salivary Glands parasitology   ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field.

• MeSH
• Insect Vectors parasitology   physiology   MeSH
• Immunoassay methods   MeSH
• Leishmania infantum physiology   MeSH
• Leishmaniasis blood   diagnosis   parasitology   veterinary   MeSH
• Mice, Inbred BALB C MeSH
• Dog Diseases blood   diagnosis   parasitology   MeSH
• Phlebotomus parasitology   physiology   MeSH
• Dogs MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. CONCLUSIONS/SIGNIFICANCE: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations.

• MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Immunoglobulin G blood   MeSH
• Goats MeSH
• Sheep MeSH
• Peptides immunology   MeSH
• Phlebotomus immunology   MeSH
• Mass Screening methods   MeSH
• Antibodies blood   MeSH
• Dogs MeSH
• Sensitivity and Specificity MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ethiopia MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Peptides MeSH
• Antibodies MeSH
• Salivary Proteins and Peptides MeSH

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

• MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Insect Proteins genetics   immunology   MeSH
• Immunoglobulin G immunology   MeSH
• Insect Bites and Stings immunology   parasitology   MeSH
• Humans MeSH
• Phlebotomus genetics   immunology   physiology   MeSH
• Recombinant Proteins genetics   immunology   MeSH
• Salivary Proteins and Peptides genetics   immunology   MeSH
• Saliva immunology   MeSH
• Antibody Formation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Africa, Eastern MeSH
• Names of Substances
• Insect Proteins MeSH
• Immunoglobulin G MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH

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.

• Keywords
• Canine leishmaniosis, Longitudinal study, Markers of exposure, North-east Spain, Phlebotomus perniciosus, Saliva proteins,
• MeSH
• Endemic Diseases veterinary   MeSH
• Insect Vectors parasitology   MeSH
• Immunity, Humoral MeSH
• Immunoglobulin G analysis   MeSH
• Leishmania infantum isolation & purification   MeSH
• Leishmaniasis blood   parasitology   veterinary   MeSH
• Longitudinal Studies MeSH
• Dog Diseases diagnosis   immunology   parasitology   MeSH
• Phlebotomus immunology   MeSH
• Antibodies, Protozoan blood   MeSH
• Antibodies blood   MeSH
• Dogs immunology   parasitology   MeSH
• Seasons MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Salivary Glands chemistry   parasitology   MeSH
• Saliva immunology   microbiology   parasitology   MeSH
• Animals MeSH
• Check Tag
• Dogs immunology   parasitology   MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain epidemiology   MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Antibodies, Protozoan MeSH
• Antibodies MeSH
• Salivary Proteins and Peptides MeSH

Leishmania spp. are medically important unicellular parasites transmitted by phlebotomine sand flies. The World Health Organization recently highlighted the importance of reliable diagnostic tools for leishmaniasis. Our study of human infection was conducted in two endemic foci of Leishmania tropica in the Galilee region, northern Israel. Elevated anti-Leishmania antibodies were present in the majority (78.6%) of L. tropica-PCR positive individuals. Moreover, the enzyme-linked immunosorbent assay showed high sensitivity, specificity, and negative and positive predictive values (ranging between 73% and 79%), thus fulfilling the basic requirement for future development of a serodiagnostic and screening tool. The anti-sand fly saliva antibodies used as biomarkers of exposure reflected the composition of the local sand fly fauna as well as the abundance of individual species. High levels of antibodies against vector salivary proteins may further indicate frequent exposure to sand flies and consequently a higher probability of Leishmania transmission.

• MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Leishmania tropica * MeSH
• Leishmaniasis, Cutaneous diagnosis   epidemiology   MeSH
• Humans MeSH
• Psychodidae parasitology   MeSH
• Sensitivity and Specificity MeSH
• Seroepidemiologic Studies MeSH
• Serologic Tests methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Israel epidemiology   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 immunology   MeSH
• Psychodidae parasitology   physiology   MeSH
• Salivary Proteins and Peptides immunology   metabolism   MeSH
• Saliva immunology   parasitology   MeSH
• Feeding Behavior * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Salivary Proteins and Peptides MeSH

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
• Antigens genetics   immunology   MeSH
• Mass Spectrometry MeSH
• Animals, Domestic * MeSH
• Immunoblotting MeSH
• Insect Bites and Stings diagnosis   MeSH
• Goats MeSH
• Sheep MeSH
• Antibodies blood   MeSH
• Dogs MeSH
• Psychodidae genetics   immunology   MeSH
• Recombinant Proteins genetics   immunology   MeSH
• Salivary Proteins and Peptides genetics   immunology   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Antibodies MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH