Simple SummaryThere are limited data on Lyme borreliosis, a tick-borne disease caused by the bacteria of theBorrelia burgdorferisensu lato complex, in horses. Seropositivity is not necessarily associated with clinical disease. Data on seropositivity againstBorrelia burgdorferisensu lato andAnaplasma phagocytophilumin German horses are sparse. Therefore, serum samples from horses (n= 123) suspected of having Lyme borreliosis and clinically healthy horses (n= 113) residing at the same stables were tested for specific antibodies againstBorrelia burgdorferisensu lato andAnaplasma phagocytophilum. First, the horses were screened for antibodies againstBorrelia burgdorferisensu lato. Afterward, the horses were screened for antibodies againstBorrelia burgdorferisensu lato andAnaplasma phagocytophilum(SNAP®4Dx Plus®ELISA). The clinical signs of suspect horses included lameness (n= 36), poor performance (n= 19), and apathy (n= 12). Twenty-three percent (n= 26) of suspect horses and 17% (n= 18) of clinically healthy horses were seropositive for having aBorrelia burgdorferisensu lato infection (p= 0.371), showing that the detection of specific antibodies againstBorrelia burgdorferialone is not sufficient for a diagnosis of equine LB.Anaplasma phagocytophilum-seropositivity and seropositivity against both pathogens was 20%/6% in suspect horses and 16%/2% in the clinically healthy population, showing only minor differences (p= 0.108). Unspecific testing for antibodies againstBorrelia burgdorferisensu lato without clinical suspicion of Lyme borreliosis is not recommended since the clinical relevance of seropositivity againstBorrelia burgdorferisensu lato remains unknown.AbstractThere are limited data on Lyme borreliosis (LB), a tick-borne disease caused by theBorrelia burgdorferisensu lato complex, in horses. Seropositivity is not necessarily associated with clinical disease. Data on seropositivity againstBorrelia burgdorferiandAnaplasma phagocytophilumin German horses are sparse. Therefore, serum samples from horses (n= 123) suspected of having Lyme borreliosis and clinically healthy horses (n= 113) from the same stables were tested for specific antibodies againstBorrelia burgdorferisensu lato andAnaplasma phagocytophilum. The samples were screened for antibodies againstBorrelia burgdorferi(ELISA and an IgG line immunoblot assay). Furthermore, the samples were examined for antibodies againstB. burgdorferiandAnaplasma phagocytophilumwith a validated rapid in-house test (SNAP®4Dx Plus®ELISA). The clinical signs of suspect horses included lameness (n= 36), poor performance (n= 19), and apathy (n= 12). Twenty-three percent (n= 26) of suspect horses and 17% (n= 18) of clinically healthy horses were seropositive for having aBorrelia burgdorferisensu lato infection (p= 0.371), showing that the detection of specific antibodies againstB. burgdorferialone is not sufficient for a diagnosis of equine LB.Anaplasma phagocytophilumseropositivity and seropositivity against both pathogens was 20%/6% in suspect horses and 16%/2% in the clinically healthy population, showing only minor differences (p= 0.108). Unspecific testing for antibodies againstB. burgdorferiwithout clinical suspicion of Lyme borreliosis is not recommended since the clinical relevance of seropositivity againstBorrelia burgdorferisensu lato remains to be elucidated.