Given the seroprevalence found in this study, the role these animals might play in the leishmaniosis cycle should not be underestimated, especially as the cats live close to humans in an endemic area. Future studies should focus on determining the urban cycle of infection in apparently healthy stray cats in the city of Zaragoza. of the different techniques. Methods The prevalence of infection was studied in stray cats captured in urban and peri-urban areas of Zaragoza. Blood was collected from each animal for serology and molecular analysis. Three serological methods, namely the immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were used to detect antibodies and a real-time PCR (qPCR) assay was used to detect DNA. The results were analyzed by Fishers exact test and Cohens kappa statistic (infection positivity by IFAT, 2.8% (5/179) by ELISA and 14.5% (26/179) by WB. DNA was detected by qPCR in 5.6% (10/179) of the cats. Sixteen cats (8.9%) tested positive by only one serological technique and four tested positive by all three serological methods used. AMG 837 sodium salt The overall rate of infected cats (calculated as the number of cats seropositive and/or qPCR positive) was 15.6%, and only two cats tested positive by all the diagnostic methods. A significant association was found between male cats and a positive qPCR result. Comparison of the techniques revealed a fair agreement in seropositivity between blood qPCR and IFAT (ELISA and IFAT seropositivity were statistically associated with FIV status by the chi-square test. Conclusions The results obtained in this study, using serological tests and qPCR, indicate the existence of asymptomatic infection in apparently healthy stray cats in the city of Zaragoza, an endemic area in Spain. that is endemic in 88 countries of southern Europe, Africa, Asia and South and Central America. More than 350 million people are estimated to be at risk of the disease , with dogs being the main reservoir for infection. Based on seroprevalence studies and environmental variables, AMG 837 sodium salt 23.2% of dogs in Italy, Spain, France and Portugal are estimated to be infected . Although infection in dogs is well documented, there is a need for more information on its prevalence in cats in the same areas. Various phlebotomine species are implicated in the transmission of in Europe, of which only two are found in Spain: and . Female spp. feed on a variety of vertebrate reservoirs, including humans, livestock, dogs, wild rabbits, hares, rodents and cats , with variable impacts on the epidemiology of leishmaniosis. Colonies of stray cats could play AMG 837 sodium salt a role in the maintenance of the life-cycle in an urban environment, where cats are naturally exposed to active vectors. Free-roaming cats in European cities are a potential source of zoonotic diseases. In the case of . The most common clinical signs of feline leishmaniosis (FeL) include peripheral lymphadenomegaly, cutaneous and mucocutaneous lesions (e.g. nodular and/or ulcerative dermatitis), generalized weakness, weight loss, anorexia and ocular and oral lesions. Some of the most frequent clinicopathological abnormalities seen in FeL are non-regenerative anemia, hyperproteinemia with hyperglobulinemia and hypoalbuminemia and proteinuria TSPAN10 . To carry out the complex diagnosis of FeL, serological and molecular techniques are commonly employed in clinical and epidemiological studies . Serological studies have included the immunofluorescent antibody test (IFAT) , the direct agglutination test (DAT) , the enzyme-linked immunosorbent assay (ELISA)  and western blot (WB) . In contrast with serology techniques, molecular diagnostic techniques are not restricted to bodily fluids, but can also be applied to bone marrow, the lymph node, spleen and skin tissues. Nevertheless, blood is the most commonly used sample for molecular testing in epidemiological surveys. In an epidemiological survey carried out in the area of Barcelona (Spain), molecular test results indicated that subclinical infections outnumber clinical infections in cat populations in endemic regions . As serological methods may not be sufficiently sensitive to bring subclinical infections to light, a combination of at least two positive results by molecular and serological techniques is recommended for a more accurate estimation of infection . Several studies have provided seroprevalence and molecular data in endemic southern European countries, particularly Spain , France , Portugal,  and Italy , but considerable gaps in regional data remain to be filled. Given the absence of epidemiological studies on feline infection in the city of Zaragoza, an endemic region of Spain, the aims of the present study were: (1) to investigate the prevalence of infection in stray cats in Zaragoza using serology and quantitative (qPCR) assays; and (2) to evaluate the screening results of apparently healthy stray cats living in an endemic region by comparing serological and qPCR test data. Methods Study areas, cats and sampling The study was carried out in the city of Zaragoza (413858.8948?N, 05315.7632?W, the Aragon region of Spain) on cats.