Compounds in sand fly saliva elicit specific immune responses that may play a role in the establishment of canine Leishmania infection. Although canine antibodies to anti-sand fly saliva antigens have been extensively studied, little is known about cellular immune responses against Phlebotomus perniciosus salivary proteins. This study aimed to explore humoral and T-cell-mediated immunity against P. perniciosus salivary proteins in dogs (n = 85) from Mallorca (Spain), a leishmaniosis-endemic area, and find correlations with demographic (age, sex, and breed) and parasite-specific immunological parameters. Anti-sand fly saliva IgG was examined using a P. perniciosus whole salivary gland homogenate (SGH) ELISA and recombinant salivary protein rSP03B ELISA. Interferon gamma (IFN-γ) release whole blood assays with L. infantum soluble antigen (LSA), SGH, and rSP03B were also performed. Positive correlations were found between IgG levels in the SGH and rSP03B tests and between concentrations of SGH IFN-γ and rSP03B IFN-γ. While concentrations of SGH IFN-γ and rSP03B IFN-γ were low and produced only by a minority of dogs (less than 20%), high levels and frequencies of LSA IFN-γ as well as anti-saliva IgG for SGH and rSP03B were detected in a majority of dogs (61% and 75%, respectively). LSA IFN-γ levels were positively correlated with age and Leishmania-specific antibodies. In conclusion, dogs from a leishmaniosis-endemic area presented high humoral immunity against P. perniciosus salivary proteins, but their cellular immunity to these proteins was low and less frequent.

• Keywords
• Leishmania infantum, anti-saliva antibodies, canine, recombinant salivary proteins, specific P. perniciosus saliva IFN-γ,
• MeSH
• Immunity, Cellular * MeSH
• Endemic Diseases MeSH
• Insect Proteins * immunology   MeSH
• Immunity, Humoral * MeSH
• Immunoglobulin G blood   immunology   MeSH
• Interferon-gamma MeSH
• Leishmaniasis * immunology   veterinary   epidemiology   MeSH
• Dog Diseases * immunology   parasitology   epidemiology   MeSH
• Phlebotomus * immunology   MeSH
• Dogs MeSH
• Salivary Proteins and Peptides * immunology   MeSH
• T-Lymphocytes * immunology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain epidemiology   MeSH
• Names of Substances
• Insect Proteins * MeSH
• Immunoglobulin G MeSH
• Interferon-gamma MeSH
• Salivary Proteins and Peptides * MeSH

Phlebotomus perniciosus is a major vector of Leishmania infantum in the Mediterranean. While the seroprevalence of leishmaniosis in Spanish dogs and cats has been studied, data on the exposure of cats to P. perniciosus bites under natural conditions without repellents is limited. Stray cats could serve as sentinels for L. infantum and P. perniciosus exposure. This study analyzed sera from 204 apparently healthy stray cats, collected from January 2021 to January 2022, for antibodies against P. perniciosus saliva and L. infantum parasites. Anti-sand fly antibodies were detected in 40.69% of cats using an ELISA with the recombinant salivary protein SP03B of P. perniciosus. Seroprevalence of L. infantum infection was 23.52% by Western blot and 27.41% by ELISA, with an overall seroprevalence of 40.69% (95% CI 34.18-47.54%). This is the first assessment of antibody response to P. perniciosus saliva and L. infantum in naturally exposed stray cats in Spain. Further research is needed to examine the salivary antigens recognized by cats and to explore the relationship between P. perniciosus exposure and L. infantum infection severity in cats.

• Keywords
• Cat, ELISA, Leishmania infantum, Phlebotomus perniciosus, serology, western blotting,
• MeSH
• Enzyme-Linked Immunosorbent Assay veterinary   MeSH
• Insect Vectors parasitology   MeSH
• Cats MeSH
• Leishmania infantum * immunology   MeSH
• Leishmaniasis, Visceral * veterinary   epidemiology   immunology   MeSH
• Cat Diseases * epidemiology   parasitology   immunology   MeSH
• Phlebotomus * parasitology   immunology   MeSH
• Antibodies, Protozoan * blood   MeSH
• Seroepidemiologic Studies MeSH
• Animals MeSH
• Check Tag
• Cats MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain epidemiology   MeSH
• Names of Substances
• Antibodies, Protozoan * MeSH

Sand flies (Diptera, Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis, as well as phleboviruses. In the Balkans, the endemicity and spreading of sand fly-borne diseases are evident, particularly in the Republic of Kosovo, a country with a predominantly humid continental climate. To date, understanding the drivers behind the spatial structure and diversity patterns of sand fly communities in humid continental regions remains limited. Therefore, elucidating the geographical and ecological factors contributing to the presence of potential vector species in the country is crucial. We aimed to enhance our understanding of factors influencing sand fly occurrence in cool and wet wintering humid continental areas, which could serve as a model for other countries with similar climatic conditions. Therefore, we assessed the currently known sand fly fauna through detailed environmental analyses, including Voronoi tessellation patterns, entropy calculations, Principal Coordinate and Component Analyses, Hierarchical Clustering, Random Trees, and climatic suitability patterns. Notable differences in the ecological tolerance of the species were detected, and the most important climatic features limiting sand fly presence were wind speed and temperature seasonality. Sand flies were observed to prefer topographical environments with little roughness, and the modelled climatic suitability values indicated that, dominantly, the western plain regions of Kosovo harbour the most diverse sand fly fauna; and are the most threatened by sand fly-borne diseases. Phlebotomus neglectus and P. perfiliewi, both confirmed vectors for L. infantum and phleboviruses, were identified as two main species with vast distribution in Kosovo. Contrary to this, most other present species are relatively sparse and restricted to temperate rather than humid continental regions. Our findings reveal a diverse potential sand fly fauna in Kosovo, indicating the need for tailored strategies to address varying risks across the country's western and eastern regions in relation to leishmaniasis control amidst changing environmental conditions.

• Keywords
• Balkan, Environmental analysis, Leishmania, Machine learning, Phlebovirus, Sand fly, Spatial patterns,
• Publication type
• Journal Article MeSH

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

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.

• Keywords
• Density, Distribution, Environment, Leishmania, Phlebotomus, Residential,
• MeSH
• Insect Vectors MeSH
• Leishmania infantum * MeSH
• Leishmaniasis * epidemiology   veterinary   MeSH
• Phlebotomus * MeSH
• Dogs MeSH
• Psychodidae * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain MeSH

BACKGROUND: Sand flies are principal vectors of the protozoan parasites Leishmania spp. and are widely distributed in all warmer regions of the world, including the Mediterranean parts of Europe. In Central European countries, the sand fly fauna is still under investigation. Phlebotomus mascittii, a suspected but unproven vector of Leishmania infantum, is regarded as the most widely distributed species in Europe. However, many aspects of its biology and ecology remain poorly known. The aim of this study was to provide new data on the biology and ecology of Ph. mascittii in Austria to better understand its current distribution and potential dispersal. METHODS: Sand flies were collected by CDC light traps at four localities in Austria for 11 (2018) and 15 weeks (2019) during the active sand fly season. Climatic parameters (temperature, relative humidity, barometric pressure and wind speed) were retrospectively obtained for the trapping periods. Sand flies were identified by a combined approach (morphology, DNA barcoding, MALDI-TOF protein profiling), and blood meals of engorged females were analysed by DNA sequencing and MALDI-TOF mass spectrometry. RESULTS: In total, 450 individuals of Ph. mascittii were caught. Activity was observed to start at the beginning of June and end at the end of August with peaks in mid-July at three locations and early August at one location. Increased activity was associated with relatively high temperatures and humidity. Also, more individuals were caught on nights with low barometric pressure. Analysis of five identified blood meals revealed chicken (Gallus gallus) and equine (Equus spp.) hosts. Sand fly abundance was generally associated with availability of hosts. CONCLUSION: This study reports unexpectedly high numbers of Ph. mascittii at selected Austrian localities and provides the first detailed analysis of its ecology to date. Temperature and humidity were shown to be good predictors for sand fly activity. Blood meal analyses support the assumption that Ph. mascittii feeds on mammals as well as birds. The study significantly contributes to understanding the ecology of this sand fly species in Central Europe and facilitates prospective entomological surveys.

• Keywords
• Blood meal, Central Europe, Climate, MALDI-TOF, Phlebotomine sand fly,
• MeSH
• Ecology * MeSH
• Insect Vectors * parasitology   MeSH
• Horses MeSH
• Chickens MeSH
• Leishmania infantum MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae MeSH
• Retrospective Studies MeSH
• Seasons * MeSH
• Sequence Analysis, DNA MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH
• Austria MeSH

BACKGROUND: The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. METHODS: All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. RESULTS: Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. CONCLUSIONS: The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

• Keywords
• Crete, Greece, Phlebotominae, Phlebotomus (Adlerius) creticus n. sp., Sand fly fauna,
• MeSH
• Species Specificity MeSH
• Insect Vectors physiology   MeSH
• Leishmaniasis, Cutaneous transmission   MeSH
• Leishmaniasis, Visceral transmission   MeSH
• Phlebotomus anatomy & histology   classification   physiology   MeSH
• Psychodidae anatomy & histology   classification   parasitology   MeSH
• Seasons MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Greece MeSH

BACKGROUND: In endemic areas of zoonotic leishmaniosis caused by L. infantum, early detection of Leishmania infection in dogs is essential to control the dissemination of the parasite to humans. The aim of this study was to evaluate the serological and/or molecular diagnostic performance of minimally and non-invasive samples (conjunctiva cells (CS) and peripheral blood (PB)) for monitoring Leishmania infection/exposure to Phlebotomus perniciosus salivary antigens in dogs at the beginning and the end of sand fly seasonal activity (May and October, respectively) and to assess associated risks factors. METHODS: A total of 208 sheltered dogs from endemic areas of leishmaniosis were screened. Leishmania DNA detection in PB on filter paper and CS was performed by nested-PCR (nPCR), while the detection of anti-Leishmania antibodies was performed using IFAT and ELISA. The exposure to P. perniciosus salivary antigens (SGH, rSP01 and rSP03B + rSP01) was measured by ELISA. RESULTS: Ninety-seven (46.6%) and 116 (55.8%) of the 208 dogs were positive to Leishmania antibodies or DNA by at least one test at the beginning and end of the sand fly season, respectively. IFAT and ELISA presented a substantial agreement in the serodiagnosis of leishmaniosis. Discrepant PB nPCR results were obtained between sampling points. Leishmania DNA was detected in CS of 72 dogs at the end of the phlebotomine season. The presence of antibodies to the parasite measured by ELISA was significantly higher in dogs presenting clinical signs compatible with leishmaniosis at both sampling points. Phlebotomus perniciosus salivary antibodies were detected in 179 (86.1%) and 198 (95.2%) of the screened dogs at the beginning and end of the phlebotomine season, respectively. CONCLUSIONS: The association between ELISA positivity and clinical signs suggests its usefulness to confirm a clinical suspicion. CS nPCR seems to be an effective and non-invasive method for assessing early exposure to the parasite. PB nPCR should not be used as the sole diagnostic tool to monitor Leishmania infection. The correlation between the levels of antibodies to P. perniciosus saliva and Leishmania antibodies suggests the use of a humoral response to sand fly salivary antigens as biomarkers of L. infantum infection.

• Keywords
• Blood, Conjunctival cells, Dog, Exposure, L. infantum, Phlebotomus pernicious, Saliva,
• MeSH
• Antigens, Protozoan immunology   MeSH
• Endemic Diseases prevention & control   MeSH
• Insect Vectors parasitology   MeSH
• Insect Proteins immunology   MeSH
• Immunoglobulin G blood   MeSH
• Conjunctiva cytology   parasitology   MeSH
• Insect Bites and Stings MeSH
• Leishmania infantum isolation & purification   MeSH
• Leishmaniasis blood   immunology   veterinary   MeSH
• Dog Diseases parasitology   prevention & control   transmission   MeSH
• Phlebotomus parasitology   MeSH
• Antibodies, Protozoan blood   MeSH
• Protozoan Proteins immunology   MeSH
• Dogs MeSH
• Risk Factors MeSH
• Serologic Tests MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Protozoan MeSH
• Insect Proteins MeSH
• Immunoglobulin G MeSH
• Antibodies, Protozoan MeSH
• Protozoan Proteins MeSH
• Salivary Proteins and Peptides MeSH

BACKGROUND: Canine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response. METHODS: This study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods. RESULTS: Results indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT. CONCLUSIONS: To our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds.

• Keywords
• Anti-sand fly saliva antibodies, Canine leishmaniosis, Ibizan hounds, Leishmania infantum, Papular dermatitis, Phlebotomus perniciosus, rSP03B,
• MeSH
• Breeding MeSH
• Endemic Diseases MeSH
• Insect Proteins immunology   MeSH
• Immunoglobulin G immunology   MeSH
• Leishmaniasis immunology   veterinary   MeSH
• Disease Susceptibility MeSH
• Dog Diseases immunology   parasitology   MeSH
• Phlebotomus immunology   MeSH
• Dogs MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Saliva immunology   MeSH
• Zoonoses parasitology   transmission   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Spain MeSH
• Names of Substances
• Insect Proteins MeSH
• Immunoglobulin G MeSH
• Salivary Proteins and Peptides 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