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.

Departament de Medicina i Cirurgia Animals Facultat de Veterinària Universitat Autònoma de Barcelona 08193 Bellaterra Spain

Department of Parasitology Faculty of Science Charles University 12800 Prague Czech Republic

Secció de Parasitologia Departament de Biologia Salut i Medi Ambient Facultat de Farmàcia i Ciències de l'Alimentació Universitat de Barcelona 08028 Barcelona Spain

Zobrazit více v PubMed

Akhoundi M., Kuhls K., Cannet A., Votýpka J., Marty P., Delaunay P., Sereno D. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Neglected Trop. Dis. 2016;10:e0004770. doi: 10.1371/journal.pntd.0004349. PubMed DOI PMC

Jariyapan N., Bates M.D., Bates P.A. Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3′ untranslated region of the heat shock protein 70 (type I) gene. PLoS Neglected Trop. Dis. 2021;15:e0009982. doi: 10.1371/journal.pntd.0009982. PubMed DOI PMC

Dantas-Torres F. Canine leishmaniasis in the Americas: Etiology, distribution, and clinical and zoonotic importance. Parasit. Vectors. 2024;17:198. doi: 10.1186/s13071-024-06282-w. PubMed DOI PMC

Alten B., Maia C., Afonso M.O., Campino L., Jiménez M., González E., Molina R., Bañuls A.L., Prudhomme J., Vergnes B., et al. Seasonal dynamics of Phlebotomine sand fly species proven vectors of mediterranean leishmaniasis caused by Leishmania infantum. PLoS Neglected Trop. Dis. 2016;10:e0004458. doi: 10.1371/journal.pntd.0004458. PubMed DOI PMC

Gálvez R., Montoya A., Cruz I., Fernández C., Martín O., Checa R., Chicharro C., Migueláñez S., Marino V., Miró G. Latest trends in Leishmania infantum infection in dogs in Spain, Part I: Mapped seroprevalence and sand fly distributions. Parasit. Vectors. 2020;13:204. doi: 10.1186/s13071-020-04081-7. PubMed DOI PMC

Díaz-Sáez V., Corpas-López V., Merino-Espinosa G., Morillas-Mancilla M.J., Abattouy N., Martín-Sánchez J. Seasonal dynamics of phlebotomine sand flies and autochthonous transmission of Leishmania infantum in high-altitude ecosystems in southern Spain. Acta Trop. 2021;213:105749. doi: 10.1016/j.actatropica.2020.105749. PubMed DOI

Lestinova T., Rohousova I., Sima M., de Oliveira C.I., Volf P. Insights into the sand fly saliva: Blood-feeding and immune interactions between sand flies, hosts, and Leishmania. PLoS Neglected Trop. Dis. 2017;11:e0005600. doi: 10.1371/journal.pntd.0005600. PubMed DOI PMC

Rohoušová I., Volf P. Sand fly saliva: Effects on host immune response and Leishmania transmission. Folia Parasitol. 2006;53:161–171. doi: 10.14411/fp.2006.022. PubMed DOI

Morales-Yuste M., Martín-Sánchez J., Corpas-Lopez V. Canine leishmaniasis: Update on epidemiology, diagnosis, treatment, and prevention. Vet. Sci. 2022;9:387. doi: 10.3390/vetsci9080387. PubMed DOI PMC

Velez R., Ballart C., Domenech E., Abras A., Fernández-Arévalo A., Gómez S.A., Tebar S., Muñoz C., Cairó J., Gállego M. Seroprevalence of canine Leishmania infantum infection in the Mediterranean region and identification of risk factors: The example of North-Eastern and Pyrenean areas of Spain. Prev. Vet. Med. 2019;162:67–75. doi: 10.1016/j.prevetmed.2018.10.015. PubMed DOI

Cosma C., Maia C., Khan N., Infantino M., Del Riccio M. Leishmaniasis in humans and animals: A One Health approach for surveillance, prevention and control in a changing world. Trop. Med. Infect. Dis. 2024;9:258. doi: 10.3390/tropicalmed9110258. PubMed DOI PMC

de Vasconcelos T.C.B., Furtado M.C., Belo V.S., Morgado F.N., Figueiredo F.B. Canine susceptibility to visceral leishmaniasis: A systematic review upon genetic aspects, considering breed factors and immunological concepts. Infect. Genet. Evol. 2019;74:103293. doi: 10.1016/j.meegid.2017.10.005. PubMed DOI

Álvarez L., Marín-García P.J., Llobat L. Serum levels and genetic variations of cytokines in two canine breeds (Ibizan hound and boxer) in the Mediterranean region, in terms of Leishmania infantum infection. Comp. Immunol. Microbiol. Infect. Dis. 2022;90–91:101908. doi: 10.1016/j.cimid.2022.101908. PubMed DOI

Álvarez L., Marín-García P.J., Llobat L. Immunological and genomic characterization of Ibizan Hound dogs in an endemic Leishmania infantum region. Parasit. Vectors. 2022;15:445. doi: 10.1186/s13071-022-05504-3. PubMed DOI PMC

Burnham A.C., Ordeix L., Alcover M.M., Martínez-Orellana P., Montserrat-Sangrà S., Willen L., Spitzova T., Volf P., Solano-Gallego L. Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain. Parasit. Vectors. 2020;13:129. doi: 10.1186/s13071-020-3992-8. PubMed DOI PMC

Toepp A.J., Petersen C.A. The balancing act: Immunology of leishmaniosis. Res. Vet. Sci. 2020;130:19–25. doi: 10.1016/j.rvsc.2020.02.004. PubMed DOI PMC

Álvarez L., Marín-García P.J., Llobat L. Genetic haplotypes associated with immune response to Leishmania infantum infection in dogs. Vet. Res. Commun. 2023;47:1675–1685. doi: 10.1007/s11259-023-10123-z. PubMed DOI

Kostalova T., Lestinova T., Maia C., Sumova P., Vlkova M., Willen L., Polanska N., Fiorentino E., Scalone A., Oliva G., et al. The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis. Med. Vet. Entomol. 2017;31:88–93. doi: 10.1111/mve.12192. PubMed DOI

Maia C., Cristóvão J., Pereira A., Kostalova T., Lestinova T., Sumova P., Volf P., Campino L. Monitoring Leishmania infection and exposure to Phlebotomus perniciosus using minimal and non-invasive canine samples. Parasit. Vectors. 2020;13:119. doi: 10.1186/s13071-020-3993-7. PubMed DOI PMC

Ordeix L. Ph.D. Thesis. Universitat Autònoma de Barcelona; Barcelona, Spain: 2018. The Spectrum of Cutaneous Manifestations in Canine Leishmaniosis: Insights into Diagnosis and Immune Responses.

Solano-Gallego L., Montserrat-Sangrà S., Ordeix L., Martínez-Orellana P. Leishmania infantum-specific production of IFN-γ and IL-10 in stimulated blood from dogs with clinical leishmaniosis. Parasit. Vectors. 2016;9:317. doi: 10.1186/s13071-016-1598-y. PubMed DOI PMC

Solano-Gallego L., Di Filippo L., Ordeix L., Planellas M., Roura X., Altet L., Martínez-Orellana P., Montserrat S. Early reduction of Leishmania infantum-specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease. Parasit. Vectors. 2016;9:235. doi: 10.1186/s13071-016-1519-0. PubMed DOI PMC

Riera C., Valladares J.E., Gállego M., Aisa M.J., Castillejo S., Fisa R., Ribas N., Carrió J., Alberola J., Arboix M. Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate. Vet. Parasitol. 1999;84:33–47. doi: 10.1016/S0304-4017(99)00084-9. PubMed DOI

Willen L., Lestinova T., Kalousková B., Sumova P., Spitzova T., Velez R., Domenech E., Vaněk O., Gállego M., Mertens P., et al. Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis. PLoS Neglected Trop. Dis. 2019;13:e0007832. doi: 10.1371/journal.pntd.0007832. PubMed DOI PMC

Aoki V., Abdeladhim M., Li N., Cecilio P., Prisayanh P., Diaz L.A., Valenzuela J.G. Some good and some bad: Sand fly salivary proteins in the control of leishmaniasis and in autoimmunity. Front. Cell Infect. Microbiol. 2022;25:839932. doi: 10.3389/fcimb.2022.839932. PubMed DOI PMC

Oliveira F., Giorgobiani E., Guimarães-Costa A.B., Abdeladhim M., Oristian J., Tskhvaradze L., Tsertsvadze N., Zakalashvili M., Valenzuela J.G., Kamhawi S. Immunity to vector saliva is compromised by short sand fly seasons in endemic regions with temperate climates. Sci Rep. 2020;10:7990. doi: 10.1038/s41598-020-64820-9. PubMed DOI PMC

Collin N., Gomes R., Teixeira C., Cheng L., Laughinghouse A., Ward J.M., Elnaiem D.E., Fischer L., Valenzuela J.G., Kamhawi S. Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania. PLoS Pathog. 2009;5:e1000441. doi: 10.1371/journal.ppat.1000441. PubMed DOI PMC

Kammoun-Rebai W., Bahi-Jaber N., Naouar I., Toumi A., Ben Salah A., Louzir H., Meddeb-Garnaoui A. Human cellular and humoral immune responses to Phlebotomus papatasi salivary gland antigens in endemic areas differing in prevalence of Leishmania major infection. PLoS Neglected Trop. Dis. 2017;11:e0005905. doi: 10.1371/journal.pntd.0005905. PubMed DOI PMC

Velez R., Spitzova T., Domenech E., Willen L., Cairó J., Volf P., Gállego M. Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum. Parasit. Vectors. 2018;11:545. doi: 10.1186/s13071-018-3123-y. PubMed DOI PMC

Quinnell R.J., Soremekun S., Bates P.A., Rogers M.E., Garcez L.M., Courtenay O. Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum. Parasit. Vectors. 2018;11:7. doi: 10.1186/s13071-017-2587-5. PubMed DOI PMC

Chapman L.A.C., Morgan A.L.K., Adams E.R., Bern C., Medley G.F., Hollingsworth T.D. Age trends in asymptomatic and symptomatic Leishmania donovani infection in the Indian subcontinent: A review and analysis of data from diagnostic and epidemiological studies. PLoS Neglected Trop. Dis. 2018;12:e0006803. doi: 10.1371/journal.pntd.0006803. PubMed DOI PMC

Carstens-Kass J., Paulini K., Lypaczewski P., Matlashewski G. A review of the leishmanin skin test: A neglected test for a neglected disease. PLoS Neglected Trop. Dis. 2021;15:e0009531. doi: 10.1371/journal.pntd.0009531. PubMed DOI PMC

Bekele F., Belay T., Zeynudin A., Hailu A. Visceral leishmaniasis in selected communities of Hamar and Banna-Tsamai districts in Lower Omo Valley, South West Ethiopia: Sero-epidemological and Leishmanin Skin Test Surveys. PLoS ONE. 2018;13:e0197430. doi: 10.1371/journal.pone.0197430. PubMed DOI PMC

Checa R., Sánchez-Vizcaíno F., Miró G., Pinchbeck G., Jones H., Noble P.J., Radford A.D. Updating the epidemiology of canine leishmaniosis in the United Kingdom through the use of electronic health data. Vet. Parasitol. 2025;333:110350. doi: 10.1016/j.vetpar.2024.110350. PubMed DOI

Almeida M., Maia C., Cristóvão J.M., Morgado C., Barbosa I., Ibars R.F., Campino L., Gonçalves L., Cortes S. Seroprevalence and risk factors associated with Leishmania infection in dogs from Portugal. Microorganisms. 2022;10:2262. doi: 10.3390/microorganisms10112262. PubMed DOI PMC

Lopes R., Garcês A., Silva A., Brilhante-Simões P., Martins Â., Duarte E.L., Coelho A.C., Cardoso L. Distribution of and relationships between epidemiological and clinicopathological parameters in canine leishmaniosis: A retrospective study of 15 years (2009–2023) Pathogens. 2024;13:635. doi: 10.3390/pathogens13080635. PubMed DOI PMC

Andrade B.B., Teixeira C.R. Biomarkers for exposure to sand flies bites as tools to aid control of leishmaniasis. Front. Immunol. 2012;3:121. doi: 10.3389/fimmu.2012.00121. PubMed DOI PMC