Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates.

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.