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Background Relapsing fever borreliosis is an infectious disease caused by bacteria of the genus Borrelia, inflicting recurrent episodes of fever and spirochetemia in humans. Borrelia persica, the causative agent of relapsing fever in Israel, is prevalent over a broad geographic area that extends from India to Egypt. It is transmitted by the soft tick Ornithodoros tholozani and causes disease in humans as well as domestic cats and dogs. The goal of this study was to survey domestic dogs and cats in Israel for infection with B. persica. Methods Blood, sera and demographic and clinical data were collected from dogs and cats brought for veterinary care in central Israel. PCR followed by DNA sequencing was used to detect B. persica DNA in blood samples, and an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies reactive with B. persica antigens in sera from the same animals. This is the first serological survey of B. persica in dogs and the first survey for antibodies reactive with a relapsing fever Borrelia sp. in cats globally. Results Four of the 208 dogs (1.9%) and three of 103 cats (2.9%) sampled were positive by PCR for B. persica DNA, and 24 dogs (11.5%) and 18 cats (17.5%) were seropositive for B. persica antigen by ELISA. The ratio between PCR-positivity and seropositivity in both the dog and cat populations was 1:6. All four PCR-positive dogs and two of three PCR-positive cats were seronegative, suggesting a probable recent infection. Thrombocytopenia showed significant association with seropositivity in dogs (P = 0.003). In cats, anemia had a significant association with seropositivity (P = 0.0001), and thrombocytopenia was associated with the combined prevalence of seropositivity or PCR-positivity (P = 0.022). Conclusions Borrelia persica infection is more prevalent and widespread in domestic canine and feline populations in Israel than previously thought. Dogs and cats may play a role as reservoirs and sentinels for human infection. Precautions should be taken to prevent transfusion-transmitted infection between blood donor and recipient animals. Graphic Abstract