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

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: Simulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC-MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC-MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase. CONCLUSIONS/SIGNIFICANCE: This study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.

• MeSH
• Child MeSH
• Adult MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Insect Vectors physiology   MeSH
• Immunoglobulin G blood   MeSH
• Immunoglobulin M blood   MeSH
• Insect Bites and Stings epidemiology   immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Onchocerciasis MeSH
• Child, Preschool MeSH
• Aged MeSH
• Simuliidae physiology   MeSH
• Saliva * MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ghana MeSH
• Sudan MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Immunoglobulin M MeSH

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.

• Keywords
• Phlebotomus, apyrase, immunogenicity, macrophage polarization, sand fly saliva, yellow-related proteins,
• MeSH
• Anti-Inflammatory Agents MeSH
• Phenotype MeSH
• Macrophages MeSH
• Mice MeSH
• Phlebotomus * MeSH
• Dogs MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Inflammatory Agents MeSH
• Recombinant Proteins MeSH
• Salivary Proteins and Peptides MeSH

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. METHODS: 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. CONCLUSIONS: Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or anti-saliva 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.

• Keywords
• Anti-saliva antibodies, Histamine, Mortality, Oviposition, Phlebotomus argentipes, Sergentomyia schwetzi, Serotonin, Yellow-related proteins,
• MeSH
• Biogenic Amines * blood   pharmacology   MeSH
• Fertility drug effects   MeSH
• Histamine blood   MeSH
• Insect Proteins chemistry   metabolism   MeSH
• Insect Bites and Stings immunology   MeSH
• Cricetinae MeSH
• Blood metabolism   MeSH
• Evolution, Molecular MeSH
• Mortality MeSH
• Phlebotomus metabolism   MeSH
• Reptiles MeSH
• Antibodies MeSH
• Psychodidae metabolism   MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Mammals MeSH
• Serotonin blood   MeSH
• Salivary Proteins and Peptides * chemistry   metabolism   MeSH
• Saliva immunology   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biogenic Amines * MeSH
• Histamine MeSH
• Insect Proteins MeSH
• Antibodies MeSH
• Recombinant Proteins MeSH
• Serotonin 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