Summary :The present report describes the first case of equine leishmaniasis in Portugal. Leishmania infection was detected in one animal, which presented an ulcerated skin lesion. Diagnosis was based on serology by CIE, and parasite DNA detection by real-time PCR using a probe specific for L. infantum. This finding requests further leishmaniasis equine surveys in order to clarify the role of the horse as reservoir host in european endemic areas.
Résumé
MATERIAL AND RESULTS
The present study was conducted within the framework of a canine epidemiological survey in the endemic Metropolitan Region of Lisbon. A serological screening was carried out in 13 horses living in a farm from the referred endemic area, where three dogs were previously diagnosed with leishmaniasis. The horses were born in Portugal and had never travelled abroad. Serological samples were analysed by counterimmunoelectrophoresis (CIE). The CIE was carried out according to the procedure of Campino et al. (1995), using cultured promastigotes of L. infantum MON-1 as antigen. Sera samples were used undiluted and all the reactions with at least one precipitation arc were considered positive. DNA was extracted from skin biopsy samples (PCR template preparation kit, Roche, Germany) and analysed by real-time TaqMan ' PCR for Leishmania DNA detection (Rolão et al., 2004). The PCR primers (forward, 5'-GGTTAGCCGATGGTGGTCTT-3', reverse, 5'-GCTATATCATATGTCCAAGCACTTACCT-3') and the TaqMan ® internal probe (5'-ACCACCTAAGGTCAACCC-3') (Applied Biosystems, Foster City, CA, USA) were designed from a kDNA minicircle sequence of a mediterrean region isolate of L. infantum (Genebank A/N AF169140). Briefly, 2 µl of each DNA sample were added to a reaction mix consisting of et al., 1984) and Spain (Fisa et al., 1999), and black rats in Italy (Gradoni et al., 1983) and Spain (Morillas-Márquez et al., 1985), which have been considered wild reservoirs. Cats in Portugal (Durão et al., 1994), Spain (Hervas et al., 1999), France (Ozon et al., 1998) and Italy (Poli et al., 2002;Pennisi et al., 2004), and a wolf in Portugal (Rebelo, personal communication) have also been found parasitized and generally considered as accidental hosts. Horses infected with Leishmania have been detected in South and Central America (Aguilar et al., 1984;Oliveira-Neto et al., 1988;Ramos-Vara et al. 1996), Germany (Koehler et al., 2002) and Spain (Solano-Gállego et al., 2003). In this report, we describe the first autochthonous case of equine infection due to L. infantum in Portugal. a final volume of 20 µl. Optimal conditions for PCR amplification were: 95º C for 10 min and 40 cycles consisting of 95º C for 15 sec and 60º C for 60 sec. The PCR reactions were performed in the ABI PRISM ' 5700 System (Perkin-Elmer, Applied Biosystems). One of the horses presented a single irregular ulcerative skin lesion of 2.5 × 1 cm in size in the right metatarsus, which evolved from a small erosion within two months. Analysis by CIE (Fig. 1) revealed the presence of anti-Leishmania antibodies in the horse se...
