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.

Departament de Biologia Sanitat i Medi Ambient Facultat de Farmàcia i Ciències de l'Alimentació Universitat de Barcelona Barcelona Spain

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

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

Servei de Dermatologia Fundació Hospital Clínic Veterinari Universitat Autònoma de Barcelona Bellaterra Spain

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Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE. 2012;7:e35671. doi: 10.1371/journal.pone.0035671. PubMed DOI PMC

Abdeladhim M, Kamhawi S, Valenzuela JG. What’s behind a sand fly bite? The profound effect of sand fly saliva on host hemostasis, inflammation and immunity. Infect Genet Evol. 2014;28:691–703. doi: 10.1016/j.meegid.2014.07.028. PubMed DOI PMC

Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol. 2009;165:1–18. doi: 10.1016/j.vetpar.2009.05.022. PubMed DOI

Solano-Gallego L, Morell P, Arboix M, Alberola J, Ferrer L. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J Clin Microbiol. 2001;39:560–563. doi: 10.1128/JCM.39.2.560-563.2001. PubMed DOI PMC

Lombardo G, Pennisi MG, Lupo T, Chicharro C, Solano-Gallego L. Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome. Parasit Vectors. 2014;7:120. doi: 10.1186/1756-3305-7-120. PubMed DOI PMC

Ordeix L, Solano-Gallego L, Fondevila D, Ferrer L, Fondati A. Papular dermatitis due to Leishmania spp. infection in dogs with parasite-specific cellular immune responses. Vet Dermatol. 2005;16:187–191. doi: 10.1111/j.1365-3164.2005.00454.x. PubMed DOI

Borja LS, de Sousa OMF, da Solcà MS, Bastos LA, Bordoni M, Magalhães JT, et al. Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors. Vet Parasitol. 2016;229:110–117. doi: 10.1016/j.vetpar.2016.10.004. PubMed DOI

Molina R, Amela C, Nieto J, San-Andrés M, González F, Castillo JA, et al. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Trans R Soc Trop Med Hyg. 1994;88:491–493. doi: 10.1016/0035-9203(94)90446-4. PubMed DOI

Baneth G, Yasur-Landau D, Gilad M, Nachum-Biala Y. Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology. Parasit Vectors. 2017;10:113. doi: 10.1186/s13071-017-2050-7. PubMed DOI PMC

Pinelli E, Killick-Kendrick R, Wagenaar J, Bernadina W, del Real G, Ruitenberg J. Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum. Infect Immun. 1994;62:229–235. doi: 10.1128/IAI.62.1.229-235.1994. PubMed DOI PMC

Hosein S, Blake DP, Solano-Gallego L. Insights on adaptive and innate immunity in canine leishmaniosis. Parasitol. 2017;144:95–115. doi: 10.1017/S003118201600055X. PubMed DOI PMC

Carrillo E, Moreno J. Cytokine profiles in canine visceral leishmaniasis. Vet Immunol Immunopathol. 2009;128:67–70. doi: 10.1016/j.vetimm.2008.10.310. PubMed DOI

Maia C, Campino L. Biomarkers associated with Leishmania infantum exposure, infection, and disease in Dogs. Front Cell Infect Microbiol. 2018;8:302. doi: 10.3389/fcimb.2018.00302. PubMed DOI PMC

Moreira MAB, Luvizotto MCR, Garcia JF, Corbett CEP, Laurenti MD. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. Vet Parasitol. 2007;145:245–252. doi: 10.1016/j.vetpar.2006.12.012. PubMed DOI

dos Santos WLC, Jesus EE, Paranhos-Silva M, Pereira AM, Santos JC, Baleeiro CO, et al. Associations among immunological, parasitological and clinical parameters in canine visceral leishmaniasis: emaciation, spleen parasitism, specific antibodies and leishmanin skin test reaction. Vet Immunol Immunopathol. 2008;123:251–259. doi: 10.1016/j.vetimm.2008.02.004. PubMed DOI

Carrillo E, Ahmed S, Goldsmith-Pestana K, Nieto J, Osorio Y, Travi B, et al. Immunogenicity of the P-8 amastigote antigen in the experimental model of canine visceral leishmaniasis. Vaccine. 2007;25:1534–1543. doi: 10.1016/j.vaccine.2006.10.036. PubMed DOI PMC

Solano-Gallego L, Montserrrat-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

Martínez-Orellana P, Marí-Martorell D, Montserrat-Sangrà S, Ordeix L, Baneth G, Solano-Gallego L. Leishmania infantum-specific IFN-γ production in stimulated blood from dogs with clinical leishmaniosis at diagnosis and during treatment. Vet Parasitol. 2017;248:39–47. doi: 10.1016/j.vetpar.2017.10.018. PubMed DOI

Montserrat-Sangrà S, Ordeix L, Martínez-Orellana P, Solano-Gallego L, Montserrat-Sangrà S, Ordeix L, et al. Parasite specific antibody levels, Interferon-γ and TLR2 and TLR4 transcripts in blood from dogs with different clinical stages of leishmaniosis. Vet Sci. 2018;5:31. doi: 10.3390/vetsci5010031. PubMed DOI PMC

Manna L, Reale S, Viola E, Vitale F, Manzillo VF, Michele PL, et al. Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol. 2006;142:271–280. doi: 10.1016/j.vetpar.2006.06.028. PubMed DOI

de Vasconcelos TCB, Furtado MC, Belo VS, Morgado FN, Figueiredo FB. 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

Quinnell RJ, Kennedy LJ, Barnes A, Courtenay O, Dye C, Garcez LM, et al. Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism. Immunogenet. 2003;55:23–28. doi: 10.1007/s00251-003-0545-1. PubMed DOI

Sanchez-Robert E, Altet L, Utzet-Sadurni M, Giger U, Sanchez A, Francino O. Slc11a1 (formerly Nramp1) and susceptibility to canine visceral leishmaniasis. Vet Res. 2008;39:36. doi: 10.1051/vetres:2008013. PubMed DOI PMC

Sanchez-Robert E, Altet L, Sanchez A, Francino O. Polymorphism of Slc11a1 (Nramp1) gene and canine leishmaniasis in a case-control study. J Hered. 2005;96:755–758. doi: 10.1093/jhered/esi111. PubMed DOI

Quilez J, Martínez V, Woolliams JA, Sanchez A, Pong-Wong R, Kennedy LJ, et al. genetic control of canine leishmaniasis: genome-wide association study and genomic selection analysis. PLoS ONE. 2012;7:e35349. doi: 10.1371/journal.pone.0035349. PubMed DOI PMC

Solano-Gallego L, Llull J, Ramos G, Riera C, Arboix M, Alberola J, et al. The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Vet Parasitol. 2000;90:37–45. doi: 10.1016/S0304-4017(00)00223-5. PubMed DOI

Solano-Gallego L, Llull J, Ramis A, Fernández-Bellon H, Rodríguez A, Ferrer L, et al. Longitudinal study of dogs living in an area of Spain highly endemic for leishmaniasis by serologic analysis and the leishmanin skin test. Am J Trop Med Hyg. 2005;72:815–818. doi: 10.4269/ajtmh.2005.72.815. PubMed DOI

Martínez-Orellana P, Quirola-Amores P, Montserrat-Sangrà S, Ordeix L, Llull J, Álvarez-Fernández A, et al. The inflammatory cytokine effect of Pam3CSK4 TLR2 agonist alone or in combination with Leishmania infantum antigen on ex-vivo whole blood from sick and resistant dogs. Parasit Vectors. 2017;10:123. doi: 10.1186/s13071-017-2062-3. PubMed DOI PMC

Vlkova M, Rohousova I, Drahota J, Stanneck D, Kruedewagen EM, Mencke N, et al. Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission. PLoS Negl Trop Dis. 2011;5:e1344. doi: 10.1371/journal.pntd.0001344. PubMed DOI PMC

Hostomska J, Rohousova I, Volfova V, Stanneck D, Mencke N, Volf P. Kinetics of canine antibody response to saliva of the sand fly Lutzomyia longipalpis. Vector Borne Zoonotic Dis. 2008;8:443–450. doi: 10.1089/vbz.2007.0214. PubMed DOI

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

Quinnell RJ, Soremekun S, Bates PA, Rogers ME, Garcez LM, 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

Kostalova T, Lestinova T, Sumova P, Vlkova M, Rohousova I, Berriatua E, et al. Canine antibodies against salivary recombinant proteins of Phlebotomus perniciosus: a longitudinal study in an endemic focus of canine leishmaniasis. PLoS Negl Trop Dis. 2015;9:e0003855. doi: 10.1371/journal.pntd.0003855. PubMed DOI PMC

Kostalova T, Lestinova T, Maia C, Sumova P, Vlkova M, Willen L, 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

Gomes R, Oliveira F. The immune response to sand fly salivary proteins and its influence on Leishmania immunity. Front Immunol. 2012;3:110. doi: 10.3389/fimmu.2012.00110. PubMed DOI PMC

Lima T, Martínez-Sogues L, Montserrat-Sangrà S, Solano-Gallego L, Ordeix L. Diagnostic performance of a qPCR for Leishmania on stained cytological specimens and on filter paper impressions obtained from cutaneous lesions suggestive of canine leishmaniosis. Vet Dermatol. 2019;30:318. PubMed

Solano-Gallego L, Di Filippo L, Ordeix L, Planellas M, Roura X, Altet L, et al. 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

Willen L, Mertens P, Volf P. Evaluation of the rSP03B sero-strip, a newly proposed rapid test for canine exposure to Phlebotomus perniciosus, vector of Leishmania infantum. PLoS Negl Trop Dis. 2018;12:1–14. doi: 10.1371/journal.pntd.0006607. PubMed DOI PMC

López-Ratón M, Rodríguez-Álvarez MX, Suárez CC, Sampedro FG. Optimal cutpoints: an R package for selecting optimal cutpoints in diagnostic tests. J Stat Softw. 2014;61:1–36. doi: 10.18637/jss.v061.i08. DOI

R Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2015. http://www.R-project.org. Accessed 1 Mar 2019.

Youden WJ. Index for rating diagnostic tests. Cancer. 1950;3:32–35. doi: 10.1002/1097-0142(1950)3:1<32::AID-CNCR2820030106>3.0.CO;2-3. PubMed DOI

Greiner M. Two-graph receiver operating characteristic (TG-ROC): a Microsoft-Excel template for the selection of cut-off values in diagnostic tests. J Immunol Methods. 1995;185:145–146. doi: 10.1016/0022-1759(95)00078-O. PubMed DOI

Hosmer DW, Lemeshow S. Applied logistic regression. Hoboken: John Wiley & Sons, Inc.; 2000.

Peng CJ, Lee KL, Ingersoll GM. An introduction to logistic regression analysis and reporting. J Educ Res. 2002;96:3–14. doi: 10.1080/00220670209598786. DOI

Minitab 17 Statistical Software. State College, PA: Minitab, Inc.; 2010. http://www.minitab.com/. Accessed 10 Sep 2018.

Cohen J. A coefficient of agreement for nominal scales. Educ Psychol Meas. 1960;20:37–46. doi: 10.1177/001316446002000104. DOI

Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Canine leishmaniosis—new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 2008;24:324–330. doi: 10.1016/j.pt.2008.04.001. PubMed DOI

Vlkova M, Rohousova I, Hostomska J, Pohankova L, Zidkova L, Drahota J, et al. Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi. PLoS Negl Trop Dis. 2012;6:e1719. doi: 10.1371/journal.pntd.0001719. PubMed DOI PMC

Martín-Martín I, Molina R, Jiménez M. Kinetics of Anti-Phlebotomus perniciosus saliva antibodies in experimentally bitten mice and rabbits. PLoS ONE. 2015;10:e0140722. doi: 10.1371/journal.pone.0140722. PubMed DOI PMC

Martín-Martín I, Molina R, Rohoušová I, Drahota J, Volf P, Jiménez M. High levels of anti-Phlebotomus perniciosus saliva antibodies in different vertebrate hosts from the re-emerging leishmaniosis focus in Madrid. Spain. Vet Parasitol. 2014;202:201–216. doi: 10.1016/j.vetpar.2014.02.053. PubMed DOI

Ockenfels B, Michael E, McDowell MA. Meta-analysis of the effects of insect vector saliva on host immune responses and infection of vector-transmitted pathogens: a focus on leishmaniasis. PLoS Negl Trop Dis. 2014;8:e3197. doi: 10.1371/journal.pntd.0003197. PubMed DOI PMC

Abbehusen MMC, dos Almeida VA, da Solcà MS, da Pereira LS, Costa DJ, Gil-Santana L, et al. Clinical and immunopathological findings during long term follow-up in Leishmania infantum experimentally infected dogs. Sci Rep. 2017;7:15914. doi: 10.1038/s41598-017-15651-8. PubMed DOI PMC

Courtenay O, Macdonald DW, Lainson R, Shaw JJ, Dye C. Epidemiology of canine leishmaniasis: a comparative serological study of dogs and foxes in Amazon Brazil. Parasitology. 1994;109:273–279. doi: 10.1017/S0031182000078306. PubMed DOI

Hepburn N. Cutaneous leishmaniasis: an overview. J Postgrad Med. 2003;49:50. doi: 10.4103/0022-3859.928. PubMed DOI

Velez R, Spitzova T, Domenech E, Willen L, Cairó J, Volf P, et al. 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

Oliva G, Scalone A, Foglia Manzillo V, Gramiccia M, Pagano A, Di Muccio T, et al. Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-PCR techniques in a cohort of naive dogs exposed to three consecutive transmission seasons. J Clin Microbiol. 2006;44:1318–1322. doi: 10.1128/JCM.44.4.1318-1322.2006. PubMed DOI PMC

de Ferreira EC, de Lana M, Carneiro M, Reis AB, Paes DV, da Silva ES, et al. Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations. Vet Parasitol. 2007;146:235–241. doi: 10.1016/j.vetpar.2007.02.015. PubMed DOI

Zanette MF, de Lima VMF, Laurenti MD, Rossi CN, Vides JP, da Vieira RFC, et al. Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs. Rev Soc Bras Med Trop. 2014;47:105–107. doi: 10.1590/0037-8682-1723-2013. PubMed DOI

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

Andrade BB, Teixeira CR. 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

Alcover MM, Ballart C, Martín-Sánchez J, Serra T, Castillejo S, Portús M, et al. Factors influencing the presence of sand flies in Majorca (Balearic Islands, Spain) with special reference to Phlebotomus pernicious, vector of Leishmania infantum. Parasit Vectors. 2014;7:421. doi: 10.1186/1756-3305-7-421. PubMed DOI PMC

Alten B, Maia C, Afonso MO, Campino L, Jiménez M, González E, et al. Seasonal dynamics of Phlebotomine sand fly species proven vectors of Mediterranean leishmaniasis caused by Leishmania infantum. PLoS Negl Trop Dis. 2016;10:e0004458. doi: 10.1371/journal.pntd.0004458. PubMed DOI PMC

Solcà MS, Andrade BB, Abbehusen MMC, Teixeira CR, Khouri R, Valenzuela JG, et al. Circulating biomarkers of immune activation, oxidative stress and inflammation characterize severe canine visceral leishmaniasis. Sci Rep. 2016;6:32619. doi: 10.1038/srep32619. PubMed DOI PMC

Courtenay O, Carson C, Calvo-Bado L, Garcez LM, Quinnell RJ. Heterogeneities in Leishmania infantum infection: using skin parasite burdens to identify highly infectious dogs. PLoS Negl Trop Dis. 2014;8:e2583. doi: 10.1371/journal.pntd.0002583. PubMed DOI PMC

Phlebotomus perniciosus Recombinant Salivary Proteins Polarize Murine Macrophages Toward the Anti-Inflammatory Phenotype