BACKGROUND: Canine leishmaniasis (CanL) is a zoonotic disease, caused by Leishmania infantum and transmitted by Phlebotomus perniciosus in the Mediterranean basin. Previously, an ELISA based on the P. perniciosus salivary protein SP03B was proposed as a valid tool to screen for canine exposure to sand fly bites across regions endemic for CanL. Although this approach is useful in laboratory settings, a practical tool for immediate application in the field is needed. In this study we propose the rSP03B sero-strip, the first immunochromatographic test (ICT) in the field of vector exposure able to rapidly screen dogs living in endemic areas for the presence of P. perniciosus and to aid in the evaluation of vector control programs. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was prepared using the bacterially expressed recombinant protein rSP03B as antigen. For test optimization, pre-immune sera from non-bitten laboratory-bred Beagles were used as negative controls. In order to validate the test, sera from laboratory-bred Beagles experimentally exposed to P. perniciosus bites were used as positive controls. Additionally, all samples were tested by ELISA using whole salivary gland homogenate (SGH) and the rSP03B protein as antigen. An almost perfect degree of agreement was found between the ICT and the SGH-ELISA. Furthermore, the newly proposed rSP03B sero-strip showed a sensitivity of 100% and a specificity of 86.79%. CONCLUSIONS/SIGNIFICANCE: We developed a simple and rapid ICT based on the P. perniciosus rSP03B salivary protein, able to replace the standard ELISA used in previous studies. Our rSP03B sero-strip showed to be highly sensitive and specific in the detection of antibodies (IgG) against P. perniciosus saliva. In the future, this test can be employed during large-scale epidemiological studies of CanL in the Mediterranean area to evaluate the efficacy of vector control programs.

Coris BioConcept Crealys Park Gembloux Belgium

Department of Parasitology Faculty of Science Charles University Prague Czech Republic

See more in PubMed

Gramiccia M, Gradoni L. The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol. 2005;35: 1169–1180. 10.1016/j.ijpara.2005.07.001 PubMed DOI

Desjeux P. The increase in risk factor for leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 2001;95: 239–243. PubMed

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. 10.1016/j.pt.2008.04.001 PubMed DOI

Moreno J, Alvar J. Canine leishmaniasis: Epidemiological risk and the experimental model. Trends Parasitol. 2002;18: 399–405. 10.1016/S1471-4922(02)02347-4 PubMed DOI

Dujardin JC, Campino L, Cañavate C, Dedet JP, Gradoni L, Soteriadou K, et al. Spread of vector-borne diseases and neglect of leishmaniasis, Europe. Emerg Infect Dis. 2008;14: 1013–1018. 10.3201/eid1407.071589 PubMed DOI PMC

Medlock JM, Hansford KM, Van Bortel W, Zeller H, Alten B. A summary of the evidence for the change in European distribution of phlebotomine sand flies (Diptera: Psychodidae) of public health importance. J Vector Ecol. 2014;39: 72–7. 10.1111/j.1948-7134.2014.12072.x PubMed DOI

Maroli M, Rossi L, Baldelli R, Capelli G, Ferroglio E, Genchi C, et al. The northward spread of leishmaniasis in Italy: Evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors. Trop Med Int Heal. 2008;13: 256–264. 10.1111/j.1365-3156.2007.01998.x PubMed DOI

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 10.1371/journal.pone.0035671 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: 1–22. 10.1371/journal.pntd.0004458 PubMed DOI PMC

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: 1–26. 10.1371/journal.pntd.0005600 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: 1–9. 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: 1–9. 10.1371/journal.pntd.0001719 PubMed DOI PMC

Doucoure S, Mouchet F, Cornelie S, Drame PM, D’Ortenzio E, Dehecq JS, et al. Human antibody response to aedes albopictus salivary proteins: A potential biomarker to evaluate the efficacy of vector control in an area of chikungunya and dengue virus transmission. Biomed Res Int. 2014;2014: 8 10.1155/2014/746509 PubMed DOI PMC

Drame PM, Diallo A, Poinsignon A, Boussari O, Dos Santos S, Machault V, et al. Evaluation of the Effectiveness of Malaria Vector Control Measures in Urban Settings of Dakar by a Specific Anopheles Salivary Biomarker. PLoS One. 2013;8: 1–9. 10.1371/journal.pone.0066354 PubMed DOI PMC

Brosseau L, Drame PM, Besnard P, Toto JC, Foumane V, Le Mire J, et al. Human Antibody Response to Anopheles Saliva for Comparing the Efficacy of Three Malaria Vector Control Methods in Balombo, Angola. PLoS One. 2012;7 10.1371/journal.pone.0044189 PubMed DOI PMC

Schwarz A, Sternberg JM, Johnston V, Medrano-Mercado N, Anderson JM, Hume JCC, et al. Antibody responses of domestic animals to salivary antigens of Triatoma infestans as biomarkers for low-level infestation of triatomines. Int J Parasitol. 2009;39: 1021–1029. 10.1016/j.ijpara.2009.01.010 PubMed DOI PMC

Gidwani K, Picado A, Rijal S, Singh SP, Roy L, Volfova V, et al. Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: A cluster-randomized trial. PLoS Negl Trop Dis. 2011;5 10.1371/journal.pntd.0001296 PubMed DOI PMC

Volf P, Tesarová P, Nohýnková E. Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age. Med Vet Entomol. 2000;14: 251–256. PubMed

Andrade BB, Teixeira CR. Biomarkers for exposure to sand flies bites as tools to aid control of leishmaniasis. Front Immunol. 2012;3 10.3389/fimmu.2012.00121 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 chagasiin Latin America. PLoS Negl Trop Dis. 2010;4: 13–15. 10.1371/journal.pntd.0000638 PubMed DOI PMC

Souza AP, Andrade BB, Aquino D, Entringer P, Miranda JC, Alcantara 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: 1–8. 10.1371/journal.pntd.0000649 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. 10.1111/mve.12192 PubMed DOI

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: 1–14. 10.1371/journal.pntd.0003991 PubMed DOI PMC

Anderson JM, Oliveira F, Kamhawi S, Mans BJ, Reynoso D, Seitz AE, et al. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics. 2006;7 10.1186/1471-2164-7-52 PubMed DOI PMC

Xu X, Oliveira F, Chang BW, Collin N, Gomes R, Teixeira C, et al. Structure and function of a “yellow” protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection. J Biol Chem. 2011;286: 32383–32393. 10.1074/jbc.M111.268904 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 10.1371/journal.pntd.0003855 PubMed DOI PMC

Sima M, Ferencova B, Warburg A, Rohousova I, Volf P. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites. PLoS Negl Trop Dis. 2016;10: 1–14. 10.1371/journal.pntd.0004553 PubMed DOI PMC

Osikowicz G, Beggs M, Brookhart P, Caplan D, Ching S, Eck P, et al. One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine. Clin Chem. 1990;36: 1586. PubMed

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

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

R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria: [Internet]. https://www.r-project.org/

Eklund A. Beeswarm: The Bee Swarm Plot, an Alternative to Stripchart. R package version 0.2.1. [Internet]. 2015. http://cran.r-project.org/package=beeswarm

Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag; New York, 2009.

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

Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, et al. Protein production and purification. Nat Methods. 2008;5: 135–146. 10.1038/nmeth.f.202 PubMed DOI PMC

Lestinova T, Vlkova M, Votypka J, Volf P, Rohousova I. Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate. Acta Trop. Elsevier B.V.; 2015;144: 9–18. 10.1016/j.actatropica.2015.01.005 PubMed DOI

Lawyer P, Killick-Kendrick M, Rowland T, Rowton E, Volf P. Laboratory colonization and mass rearing of phlebotomine sand flies (Diptera, Psychodidae). Parasite. 2017;24: 42 10.1051/parasite/2017041 PubMed DOI PMC

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

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

Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa