Leishmania infantum among dogs in Senegal (Senghor et al. 2011) and Leishmania major DNA was detected in Sergentomyia darlingi in a cutaneous leishmaniasis

online | memorias.ioc.fiocruz.br Leishmaniases are parasitic diseases caused by protozoans of the genus Leishmania. These parasites, which infect various wild and domestic mammals, are transmitted by the bite of phlebotomine sandflies. Species of the genus Sergentomyia are widely distributed throughout the Old World and are known to feed on reptiles as well as other vertebrates, including humans (Bates 2007, Berdjane-Brouk et al. 2012). Although these sandflies are considered vectors of lizard Sauroleishmania, it has been recently suggested that certain species of the genus Sergentomyia are involved in the transmission of Leishmania infantum among dogs in Senegal (Senghor et al. 2011) and Leishmania major DNA was detected in Sergentomyia darlingi in a cutaneous leishmaniasis (CL) focus in Mali (Berdjane-Brouk et al. 2012). Earlier, Mukherjee et al. (1997) detected Leishmania donovani DNA in Sergentomyia spp in India. In Portugal, 213 parasite strains have been isolated from sandflies and human and canine leishmaniasis cases have been identified as L. infantum, with Phlebotomus perniciosus and Phlebotomus ariasi as the proven vectors (Campino et al. 2006). However, L. major/L. infantum hybrids have also been isolated from four autochthonous human leishmaniasis cases, two of which were described elsewhere (Ravel et al. 2006). The identification of these multiple hybrids led to the hypothesis that L. major circulates in the country, most likely in infected sandflies. The risk of introduction of new Leishmania species in Portugal from travellers or immigrants from North Africa and the Indian subcontinent is a real concern, especially in the southern part of the country (Algarve region). Portuguese military missions in the Middle East pose an additional introduction risk. An entomological surveillance study of phlebotomine species was performed during the last five years. During Leishmania transmission season, sandflies were captured by CDC miniature light traps and identified using entomological keys. Subsequently, kinetoplastid DNA-polymerase chain reaction (PCR) using primers Uni21 and Lmj4 (Anders et al. 2002) and nuclear ribosomal internal transcribed spacer (ITS)-1 PCR analyses were used to screen female sandflies for Leishmania infection (Maia et al. 2009). After ITS1-PCR, HaeIII digestion was performed on the positive PCR products to differentiate Leishmania species (Schonian et al. 2003). L. major DNA was detected in one Sergentomyia minuta specimen (sample sm3), which was collected in a rural area of the municipality of Albufeira, Algarve region. Furthermore, a characteristic L. major PCR product of 650 base pairs (bp) was obtained using the Uni21 and Lmj4 kinetoplastid primers as determined by agarose gel electrophoresis. After PCR amplification and HaeIII digestion of the product, restriction fragments characteristic of L. major (203 bp and 132 bp) were observed. The amplified fragment obtained by ITS1-PCR was sequenced, aligned and compared with ITS-1 Leishmania sequences that were available in the GenBank database. A neighbour-joining tree (Figure) was constructed using SplitsTree4 (Huson & Bryant 2006). The L. major sequence from sample sm3 had a high level of identity with L. major sequences from strains recovered in the Middle East and Northern Africa (Egypt, accession FJ460456.1, Iran, accessions FN677357.1, AY550178.1, AY260965.1, Israel, accessions EU326229.1, DQ300195.1 and Tunisia, accession FN677342.1). The foci of CL caused by L. major occur in 14 countries of the World Health Organization -Eastern Mediterranean Region (EMR), extending from Morocco to doi: Financial support: EU/FEDER (PTDC/CVT/112371/2009) (FCT/ MMCTES), EU (FP7-261504 EDENext) catalogued by the EDENext Steering Committee as EDENext093. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. CM (SFRH/BPD/44082/2008) and SC (SFRH/BPD/44450/2008) are FCT/MCTES fellows. + Corresponding author: [email protected] Received 31 July 2012 Accepted 4 April 2013 The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal