The most frequently used diagnostic methods were compared in a longitudinal survey with and by Panipenem IFAT for were further parasitologically evaluated by microscopic examination of lymph node tissues and PCR of skin samples. ELISA and 9.8% by DS and the overall rate of 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 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 2 years of observation 55 animals (i.e. 26.9%) became positive for 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 infection status in asymptomatic dogs is suggested. Canine leishmaniasis (CanL) due to is transmitted by different species of sandflies and is considered one of the most important canine protozoal diseases of zoonotic concern (2). is widely distributed in many Mediterranean countries; and in Italy stable endemic foci consisting of dogs from the central and southern areas of the country have Panipenem been reported (4 5 30 32 with high percentages (up to 53.1%) of animals being serologically positive (5). Recently infections have pass on throughout the north parts of Italy (19). In central European countries CanL can be a well-known and growing travel-associated disease and the casual focal autochthonous transmitting of continues to be suspected (16). In canines infections could cause serious medical forms or canines may stay asymptomatic for a long period (5 7 17 Many medical top features of CanL (e.g. lethargy pounds reduction anorexia epistaxis lymphadenomegaly and splenomegaly) could be just like those of additional illnesses including canine monocytic ehrlichiosis (CME) (13). Along with CanL CME due to spp Certainly. to receptive pets and human beings (22 24 Therefore the reliable recognition of disease in asymptomatic pets is problematic since both serological and IL18BP antibody parasitological strategies have inherent restrictions (23). Indeed serology may not be a good indicator of infection when it is used in cross-sectional studies due to the various times Panipenem that span between infection and seroconversion (i.e. from 3 months to Panipenem 7 years [1]). Additionally asymptomatic infected animals may remain seronegative as a consequence of their individual immune response (3). Among the direct parasitological tests microscopic examination is a rapid and simple method; but it has a low sensitivity particularly with asymptomatic dogs and Panipenem thus it is not recommended for use for mass screenings in areas of endemicity. Although in vitro culture techniques are Panipenem reliable and sensitive they are prone to microbiological contamination (12) especially if skin samples or samples collected under field conditions are used. Molecular tools that detect DNA in putative dog reservoirs have been developed (33) and they have been shown to be more sensitive than serology and culture techniques (10). Thus although PCRs can be useful for the detection of asymptomatic infected animals (10 27 definitions of the methodologies amplification protocols and gene targets to be used and the tissue type to be tested are a matter of debate among scientists (6 36 As a consequence data currently available in the literature about diagnosis of CanL in asymptomatic animals are controversial and a diagnostic “gold standard” is far from being clearly stated (23). Again no longitudinal studies are available to investigate the serological and parasitological features that appear over the course of the first infection in asymptomatic animals from an area of endemicity for canine vector-borne diseases. Thus it was the aim of the present study to compare the most frequently used diagnostic methods in a longitudinal survey of antibodies and by IFAT for the presence of specific anti-antibodies. Dogs without detectable anti-antibodies were further examined for the presence of amastigote stages of parasites in stained lymph node smears and for DNA in dermal tissue samples by PCR (see below). In March 2005 204 animals (i.e. 102 from each.