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.

Department of Microbiology and Molecular Genetics The Kuvin Centre for the Study of Infectious and Tropical Diseases The Hebrew University Hadassah Medical School The Hebrew University of Jerusalem Jerusalem Israel

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

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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. 10.1016/j.meegid.2014.07.028 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. 10.1089/vbz.2007.0214 PubMed DOI

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 10.1371/journal.pntd.0001344 PubMed DOI PMC

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 10.1371/journal.pntd.0001719 PubMed DOI PMC

Martin-Martin I, Molina R, Jimenez M. Kinetics of Anti-Phlebotomus perniciosus Saliva Antibodies in Experimentally Bitten Mice and Rabbits. PLoS ONE. 2015; 10: e0140722 10.1371/journal.pone.0140722 PubMed DOI PMC

Vinhas V, Andrade BB, Paes F, Bomura A, Clarencio J, Miranda JC, et al. Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis. Eur J Immunol. 2007; 37: 3111–3121. PubMed

Clements MF, Gidwani K, Kumar R, Hostomska J, Dinesh DS, Kumar V, et al. Measurement of recent exposure to Phlebotomus argentipes, the vector of Indian visceral leishmaniasis, by using human antibody responses to sand fly saliva. Am J Trop Med Hyg. 2010; 82: 801–807. 10.4269/ajtmh.2010.09-0336 PubMed DOI PMC

Gidwani K, Picado A, Rijal S, Singh SP, Roy L, Volf P, et al. Antibody response to sand fly saliva to evaluate human visceral leishmaniasis vector exposure in India and Nepal: effect of long-lasting insecticidal nets. Trop Med Int Health. 2011; 16: 66–66

Rohousova I, Ozensoy S, Ozbel Y, Volf P. Detection of species-specific antibody response of humans and mice bitten by sand flies. Parasitology. 2005; 130: 493–499. PubMed

de Moura TR, Oliveira F, Novais FO, Miranda JC, Clarêncio J, Follador I, et al. Enhanced Leishmania braziliensis infection following pre-exposure to sandfly saliva. PLoS Negl Trop Dis. 2007; 1: e84 PubMed PMC

Marzouki S, Ben Ahmed M, Boussoffara T, Abdeladhim M, Ben Aleya-Bouafif N, Namane A, et al. Characterization of the antibody response to the saliva of Phlebotomus papatasi in people living in endemic areas of cutaneous leishmaniasis. Am J Trop Med Hyg. 2011; 84: 653–661. 10.4269/ajtmh.2011.10-0598 PubMed DOI PMC

Teixeira C, Gomes R, Collin N, Reynoso D, Jochim R, Oliveira F, et al. Discovery of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi in Latin America. PLoS Negl Trop Dis. 2010; 4: e638 10.1371/journal.pntd.0000638 PubMed DOI PMC

Gomes RB, Mendonça IL, Silva VC, Ruas J, Silva MB, Cruz MS, et al. Antibodies against Lutzomyia longipalpis saliva in the fox Cerdocyon thous and the sylvatic cycle of Leishmania chagasi. Trans R Soc Trop Med Hyg. 2007; 101: 127–133. PubMed

Rohousova I, Talmi-Frank D, Kostalova T, Polanska N, Lestinova T, Kassahun A, et al. Exposure to Leishmania spp. and sand flies in domestic animals in northwestern Ethiopia. Parasit Vectors. 2015; 8: 360 10.1186/s13071-015-0976-1 PubMed DOI PMC

Hostomska J, Volfova V, Mu JB, Garfield M, Rohousova I, Volf P, et al. Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus. BMC Genomics. 2009; 10: 282 10.1186/1471-2164-10-282 PubMed DOI PMC

Martin-Martin I, Molina R, Jimenez M. An insight into the Phlebotomus perniciosus saliva by a proteomic approach. Acta Trop. 2012; 123: 22–30. 10.1016/j.actatropica.2012.03.003 PubMed DOI

Rohousova I, Volfova V, Nova S, Volf P. Individual variability of salivary gland proteins in three Phlebotomus species. Acta Trop. 2012; 122: 80–86. 10.1016/j.actatropica.2011.12.004 PubMed DOI

Souza AP, Andrade BB, Aquino D, Entringer P, Miranda JC, Alcántara R, et al. Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral leishmaniasis endemic areas. PLoS Negl Trop Dis. 2010; 4: e649 10.1371/journal.pntd.0000649 PubMed DOI PMC

Marzouki S, Abdeladhim M, Abdessalem CB, Oliveira F, Ferjani B, Gilmore D, et al. Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans. PLoS Negl Trop Dis. 2012; 6: e1911 10.1371/journal.pntd.0001911 PubMed DOI PMC

Drahota J, Martin-Martin I, Sumova P, Rohousova I, Jimenez M, Molina R, et al. Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector. PLoS Negl Trop Dis. 2014; 8: e2597 10.1371/journal.pntd.0002597 PubMed DOI PMC

Martin-Martin I, Molina R, Rohousova I, Drahota J, Volf P, Jimenez 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: 207–216. 10.1016/j.vetpar.2014.02.045 PubMed DOI

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 10.1371/journal.pntd.0003855 PubMed DOI PMC

Marzouki S, Kammoun-Rebai W, Bettaieb J, Abdeladhim M, Hadj Kacem S, Abdelkader R, et al. Validation of recombinant salivary protein PpSP32 as a suitable marker of human exposure to Phlebotomus papatasi, the vector of Leishmania major in Tunisia. PLoS Negl Trop Dis. 2015; 9: e0003991 10.1371/journal.pntd.0003991 PubMed DOI PMC

Mondragon-Shem K, Al-Salem WS, Kelly-Hope L, Abdeladhim M, Al-Zahrani M, Valenzuela JG, et al. Severity of Old World Cutaneous Leishmaniasis Is Influenced by Previous Exposure to Sandfly Bites in Saudi Arabia. PLoS Negl Trop Dis. 2015; 9: e3449. PubMed PMC

Elnaiem DE. Ecology and control of the sand fly vectors of Leishmania donovani in East Africa, with special emphasis on Phlebotomus orientalis. J Vector Ecol. 2011; 36: S23–S31. 10.1111/j.1948-7134.2011.00109.x PubMed DOI

Elnaiem DA, Ward R, Hassan KH, Miles MA, Frame IA. Infection rates of Leishmania donovani in Phlebotomus orientalis from a focus of visceral leishmaniasis in eastern Sudan. Ann Trop Med Parasitol. 1998; 92: 229–232. PubMed

Gebresilassie A, Kirstein OD, Yared S, Aklilu E, Moncaz A, Tekie H, et al. Species composition of phlebotomine sand flies and bionomics of Phlebotomus orientalis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in Tahtay Adiyabo district, Northern Ethiopia. Parasit Vectors. 2015; 8: 248 10.1186/s13071-015-0849-7 PubMed DOI PMC

Gebre-Michael T, Balkew M, Berhe N, Hailu A, Mekonnen Y. Further studies on the phlebotomine sandflies of the kala-azar endemic lowlands of Humera-Metema (north-west Ethiopia) with observations on their natural blood meal sources. Parasit Vectors. 2010; 3: 6 10.1186/1756-3305-3-6 PubMed DOI PMC

Gebresilassie A, Yared S, Aklilu E, Kirstein OD, Moncaz A, Tekie H, et al. Host choice of Phlebotomus orientalis (Diptera: Psychodidae) in animal baited experiments: a field study in Tahtay Adiyabo district, northern Ethiopia. Parasit Vectors. 2015; 8: 190 10.1186/s13071-015-0807-4 PubMed DOI PMC

Gebresilassie A, Abbasi I, Aklilu E, Yared S, Kirstein OD, Moncaz A, et al. Host-feeding preference of Phlebotomus orientalis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in northern Ethiopia. Parasit Vectors. 2015; 8: 270 10.1186/s13071-015-0883-5 PubMed DOI PMC

Seblova V, Volfova V, Dvorak V, Pruzinova K, Votypka J, Kassahun A, et al. Phlebotomus orientalis Sand Flies from Two Geographically Distant Ethiopian Localities: Biology, Genetic Analyses and Susceptibility to Leishmania donovani. PLoS Negl Trop Dis. 2013; 7: e2187 10.1371/journal.pntd.0002187 PubMed DOI PMC

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011; 36: S1–S9. 10.1111/j.1948-7134.2011.00106.x PubMed DOI

Volf P, Rohousova I. Species-specific antigens in salivary glands of phlebotomine sandflies. Parasitology. 2001; 122: 37–41 PubMed

Vlkova M, Sima M, Rohousova I, Kostalova T, Sumova P, Volfova, et al. Comparative analysis of salivary gland transcriptomes of Phlebotomus orientalis sand flies from endemic and non-endemic foci of visceral leishmaniasis. PLoS Negl Trop Dis. 2014; 8: e2709 10.1371/journal.pntd.0002709 PubMed DOI PMC

Bahia D, Gontijo NF, Leon IR, Perales J, Pereira MH, Oliveira G, et al. Antibodies from dogs with canine visceral leishmaniasis recognise two proteins from the saliva of Lutzomyia longipalpis. Parasitol Res. 2007; 100: 449–454. PubMed

Martin-Martin I, Molina R, Jimenez M. Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach. Acta Trop. 2013; 126: 229–239. 10.1016/j.actatropica.2013.02.008 PubMed DOI

Ribeiro JMC, Arca B. From sialomes to the sialoverse: An insight into the salivary potion of blood feeding insects. Adv In Insect Phys. 2009; 37: 59–118.

Hamasaki R, Kato H, Terayama Y, Iwata H, Valenzuela JG. Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major. J Insect Physiol. 2009. 55: 1044–1049. 10.1016/j.jinsphys.2009.07.010 PubMed DOI PMC

Gomes RB, Brodskyn C, de Oliveira CI, Costa J, Miranda JC, Caldas A, et al. Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. J Infect Dis. 2002; 186: 1530–1534. PubMed

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