The most frequently used diagnostic methods were compared in a longitudinal survey with Leishmania infantum-infected asymptomatic dogs from an area of Italy where leishmaniasis is endemic. In February and March 2005, 845 asymptomatic dogs were tested by an immunofluorescence antibody test (IFAT), a dipstick assay (DS), and an enzyme-linked immunosorbent assay (ELISA) for L. infantum and by IFAT for Ehrlichia canis. Dogs seronegative for L. infantum were further parasitologically evaluated by microscopic examination of lymph node tissues and PCR of skin samples. A total of 204 animals both serologically and parasitologically negative for L. infantum at the first sampling were enrolled in the trial and were further examined for canine leishmaniasis (CanL) and canine monocytic ehrlichiosis in November 2005 (i.e., the end of the first sandfly season) and March 2006 and 2007 (1- and 2-year follow-ups, respectively). At the initial screening, the overall rates of L. infantum seroprevalence were 9.5% by IFAT, 17.1% by ELISA, and 9.8% by DS and the overall rate of E. canis seroprevalence was 15%. The rates of concordance between the results of IFAT and DS were almost equal, whereas the rate of concordance between the results of IFAT and DS and those of the ELISA was lower. The results of the annual incidence of Leishmania infection were variable, depending on the test employed, with the highest values registered for PCR (i.e., 5.7% and 11.4% at the 1- and 2-year follow-ups, respectively), followed by ELISA, IFAT, and DS. Over the 2 years of observation, 55 animals (i.e., 26.9%) became positive for L. infantum by one or more diagnostic tests at different follow-up times, with 12.7% showing clinical signs related to CanL, while the remaining 87.3% were asymptomatic. A diagnostic scheme for assessment of the L. infantum infection status in asymptomatic dogs is suggested
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