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Simple Summary  Leptospirosis is a ubiquitous distributed infectious disease present in wild and domestic animals that can be transmitted to humans. This study aimed to estimate the burden of the bacteria in dairy cows from southern Chile and identify the factors associated with the herd-level status. We studied 147 herds and 4876 lactating cows from the area, and an infected herd was defined when at least one serologically positive reactor to MAT was detected. An epidemiological survey was applied to the herd’s owner. The estimated overall individual prevalence was 5.3% (95% CI 2.9–7.7), the overall herd-level prevalence was 42.2% (95% CI 34.2–50.2), and there was variation in both between different herd sizes.  L. borgpetersenii  serovars Hardjo and Tarassovi and  L. interrogans  serovar Pomona were the more frequent serovars in  Leptospira  non-vaccinated herds. Attenuated  Leptospira  vaccine usage was assessed as a factor that decreases the risk of a farm being infected (OR = 0.04; 95% CI = 0.02–0.11), and variables that increase that risk were using bulls for mating (OR = 3.43; (95% CI = 1.1–10.1) and continuous calving distribution (OR = 3.4; 95% CI = 1.3–8.8). The results from this study will contribute to unravelling the infection burden in the main dairy area of the country and designing control strategies.    Abstract  Leptospirosis is a ubiquitous distributed zoonotic infectious disease present in wild and domestic animals. This study aimed to estimate within-herd and herd-level seroprevalence against pathogenic  Leptospira  spp. in dairy cows from southern Chile and identify risk factors associated with the herd-level status. We used a multi-stage strategy combined with a stratified sample strategy for randomly sampling 147 herds and 4876 lactating cows. We considered as infected a herd with at least one positive reactor to MAT. In addition, an epidemiological survey was applied to the herd’s owners and a logistic regression (LR) model was constructed to analyze it. The overall within-herd prevalence was 5.9% (95% CI 4.9–6.8), the overall herd-level prevalence was 42.2% (95% CI 9.2–47.9), and there was variation in both between different herd sizes.  L.   borgpetersenii  serovars Hardjo and Tarassovi and  L. interrogans  serovar Pomona were the more frequent serovars in non-vaccinated herds. A factor that decreases the risk of a farm being infected was  Leptospira  vaccine usage (OR = 0.04; 95% CI = 0.02–0.11), and variables that increase risk were using bulls for mating (OR = 3.43; 95% CI = 1.1–10.1) and continuous calving distribution (OR = 3.4; 95% CI = 1.3–8.8). The study’s results will contribute to unravelling the infection burden in the main dairy area of the country and designing control strategies.

Pathogenic Leptospira spp. Seroprevalence and Herd-Level Risk Factors Associated with Chilean Dairy Cattle