Wildlife as a Sentinel for Pathogen Introduction in Nonendemic Areas: First Detection of Leishmania tropica in Wildlife in Spain

Leishmaniasis is a chronic global arthropod-borne zoonotic disease produced by several species of Leishmania with cutaneous, mucocutaneous, and visceral clinical manifestations. In Spain, only Leishmania infantum has been reported so far, although other species of Leishmania, such as L. tropica and L. major, are present in surrounding countries. The aim of this work is to analyze the occurrence of Leishmania spp. infection in European wildcats (Felis silvestris) as sentinels, including their genotypic characterization. Necropsies of 18 road-killed wildcats were conducted. Samples of ear skin and spleen were taken for DNA isolation and PCR of the highly sensitive SSU-rDNA target. Subsequent PCR tests were performed using more specific targets for the determination of Leishmania species: hsp70 and ITS1. Positive samples were sequenced, and phylogenetic trees were constructed. Seven wildcats were found positive for Leishmania spp. Based on the hsp70 and ITS1 sequences, an animal was found to be infected only with L. tropica in ear skin samples, while two cats were found to be infected with L. infantum in both the ear skin and the spleen. In one animal, a clear sequence of L. infantum ITS1 and a sequence of L. tropica hsp70 were obtained from the ear skin. Since hsp70 and ITS1 sequencing was not possible in three cats, the species of Leishmania infecting them was not determined. This is the first report of autochthonous infection with L. tropica in the Iberian Peninsula. Health care professionals, including physicians, dermatologists, and veterinarians, must be aware of this for a correct diagnosis, treatment, and management of possible coinfections.