Background Recently, the blacklegged tick (Ixodes scapularis), which is the vector of Borrelia burgdorferi, has undergone a range expansion from the northeastern and mid‐west United States to areas of southeastern Canada, including parts of Ontario. Understanding the seroprevalence of antibodies against B. burgdorferi in horses and risk factors for exposure is important for monitoring and preventing this emerging disease. Study design Cross‐sectional study of 551 horses in southern, central, and eastern Ontario, Canada. Objectives To assess the seroprevalence of B. burgdorferi in horses in Ontario, Canada; to evaluate risk factors associated with seropositivity; and, to compare the performance of two diagnostic tests. Methods Serum samples were obtained from clinically healthy horses in Ontario, Canada, along with completed questionnaires that were used for the risk factor analysis. Sera were tested with a Multiplex ELISA (Animal Health Diagnostic Center, Cornell University) and C6 ELISA (IDEXX SNAP® 4Dx® Plus test, IDEXX Laboratories). Results The seroprevalence of B. burgdorferi on at least one test was 17% (91/551), though only 15 (16%) horses tested positive with both tests. A spatial cluster of cases was detected in Eastern Ontario. The odds of being seropositive for B. burgdorferi on the C6 ELISA were significantly increased when oak trees were present by pastures (OR = 7.3 (1.8‐29.2), P = .005), while the odds were significantly decreased when regular tick checks were performed (OR = 0.1 (0.01‐0.7), P = 0.02). Main limitations Recruitment focused on known areas with blacklegged ticks as well as areas of higher horse density, which may have led to selection bias. Conclusions The expansion of blacklegged tick populations poses an ongoing risk for horses. Assessment of diagnostic testing options and risk factors is important for diagnosis and prevention, and with further investigation this information may be used to propose changes in management.

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The blacklegged tick ( Ixodes scapularis), which transmits Borrelia burgdorferi, the causative agent of Lyme disease, has undergone rapid range expansion in Ontario. In horses, Lyme disease remains an enigmatic disease, with limited understanding of the pathogenesis and many issues pertaining to selection and interpretation of laboratory tests. We evaluated B. burgdorferi seropositivity in naturally exposed horses over a 12-mo period and compared paired samples with 2 common serologic tests. Serum samples were collected in 2017, ~1 y after initial testing, from a cohort of 22 horses that were seropositive in a 2016 seroprevalence study. Samples were tested using a C6 ELISA and a multiplex ELISA targeting outer surface proteins A, C, and F. 1 y after initial testing, 14 of 22 (64%) horses remained seropositive; 7 (32%) were positive on the multiplex ELISA, 2 (9%) on C6 ELISA, and 5 (23%) on both tests. Repeatability was 100% for the C6 ELISA, and 95% for the multiplex ELISA, with no significant difference between paired sample multiplex titer values. Our results indicate strong intra-test reliability, although further investigation is required to determine the clinical significance of serologic testing.