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Background: The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods. Methods: Blood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing. Results: Of the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5–46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9–58.9) livestock. The identified Leishmania spp. were L. infantum and L. major. Conclusions: The findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection. [ABSTRACT FROM AUTHOR]