Expression of A2 proteins in amastigotes of Leishmania infantum produced from canine isolates collected in the district of Meshkinshahr, in north-western Iran.

The differentiation of Leishmania parasites from the stage found in the sandfly vectors (the promastigote) to that found in mammalian hosts (the amastigote) is triggered by the rise in ambient temperature encountered during transmission from the vectors (Wiesgigl and Clos, 2001). This warming, to 35–37uC, induces the synthesis of several antigens (Brandau et al., 1995), and there have been several attempts to characterise these and other molecules that are specifically expressed by the amastigote stage (Zhang et al., 1996). Using electrophoresis, it is possible to separate .50 polypeptides, of 12–170 kDa, from extracts of L. infantum promastigotes, including 37 antigenic components recognised by sera from dogs naturally infected with the parasite (Rami et al., 2005). Some stage-specific antigens play a role in the host pathology and/or hostprotection mechanisms encountered in leishmaniasis. The A2 proteins of L. donovani, for example, are common in the amastigote stage and appear to facilitate visceralization of the parasites in the mammalian host (Zhang and Matlashewski, 2001; Ghosh et al., 2002). In L. donovani, these proteins range in size from 42 to 100 kDa, have highly conserved repetitive elements (Carvalho et al., 2002), and are mainly located in the cytoplasm of the amastigotes (Charest and Matlashewski, 1994; Ghedin et al., 1998; Zhang and Matlashewski, 2001). Patients suffering from active visceral leishmaniasis (VL) have anti-A2 antibodies in their sera (Ghedin et al., 1997) and, since they can induce a potent immune response and protection against VL (Brandau et al., 1995; Coelho et al., 2003), A2 antigens might be useful in the development of a vaccine for the prevention of VL (Ghosh et al., 2002). The aim of the present study was to investigate the presence of A2 proteins in amastigotes produced from isolates of L. infantum from dogs in the Meshkinshahr district of Ardebil province, in north– western Iran. Dogs are the principal mammalian hosts of L. infantum, the parasite that causes human VL in the Mediterranean basin (WHO, 1990; Mohebali et al., 2005) and some areas of north–western and southern Iran (Mohebali et al., 2005). Dogs in rural areas of Meshkinshahr were selected at random and, with their owners’ consent, screened for L. infantum. The screening involved the collection of blood from the radial vein (5 ml/dog), the centrifugation of each sample (at 8006g for 5 min) and the testing of each resultant serum sample (after a period of storage at 220uC) for antileishmanial antibodies. Each serum was tested using a commercial rk39 dipstick (Cypress Diagnostics. Langdorp, Belgium) and in a direct agglutination test (DAT) based on L. infantum antigens (a kind gift from the Protozoology Unit at the Tehran University of Medical Sciences), with a cut-off titre for DAT positivity of 1:320 (Mohebali et al., 2004). With the approval of the local health authority, the three dogs found seropositive, out of the 30 screened, were killed by terminal anaesthesia, transferred to the Parasitology Laboratory at Meshkinshahr Health Research Centre, and then carefully Annals of Tropical Medicine & Parasitology, Vol. 102, No. 1, 81–84 (2008)