Hares collected in a green park adjacent to the focus (n = 59) showed positive exposure to P. perniciosus bites in comparison to hares from a non-endemic area (Czech Republic, n = 18). A significant positive correlation was found between IgG response to yellow protein rSP03B and salivary gland homogenate (r = 0.902) and between apyrase rSP01B and salivary gland homogenate (r = 0.710). Wild rabbits captured in the study area (n = 21) presented higher anti-saliva antibody levels than negative control sera and their IgG response against recombinant salivary proteins were positively correlated with salivary gland homogenate (rSP03B: r = 0.710; rSP01B: r = 0.666). All sera of dogs from the focus (n = 34) showed higher anti-saliva IgG levels than that of non-exposed dogs. Moreover, dogs protected against sand fly bites through the use of topical insecticides and sleeping indoors showed significantly lower antibody levels than the non-protected ones. Antibody response to all three recombinant salivary proteins tested showed positive correlation with salivary gland extract (rSP03B: r = 0.858; rSP01: r = 0.864; and rSP01B: r = 0.861). Data confirmed the exposure of hares, rabbits and dogs to P. perniciosus bites in the context of an outbreak of human leishmaniosis in Spain, highlighting their involvement in Leishmania transmission by supporting their role as potential reservoirs.
This novel methodology represents a promising tool for further epidemiological studies that would help to design better strategies for the control of leishmaniosis in this area and other foci.