Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of -hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions. Ketosis or hyperketonemia, a condition in which blood levels of -hydroxybutyrate (BHBA) and its metabolite acetoacetate are elevated, is common in many animal species, resulting from impaired glucose homeostasis. Important examples are diabetes in humans and other animal species, ketosis in dairy cows, pregnancy toxemia in sheep and goats, and decreased energy intake in all animal species. In both humans and farm animals, these conditions are known to be associated with increased risk for infectious diseases (14,16,17,27,28,33,34). Many specific defects in innate and adaptive immune functions mediated by diverse mechanisms were identified under these diverse hyperketonemic conditions (29,33,35,44,45,49). Abnormally high levels of BHBA and acetoacetate in blood and other body fluids is one of these deleterious mechanisms affecting immune functions in many animal species. BHBA was reported to affect human, bovine, and ovine neutrophil function and chemotaxis (7, 10, 20, 21, 36-38, 43, 48). Phagocytosis, microbial killing, and various antimicrobial mechanisms of neutrophils, like reactive oxygen species (ROS) production, were impaired by exposure to BHBA levels similar to those measured under hyperketonemic disease conditions.Mastitis, an inflammatory response of the mammary tissue to invading bacteria, is a worldwide problem leading to multibillion dollar economic losses, and Escherichia coli is a leading cause of acute mastitis in dairy animals. Dairy cows with elevated serum, urine, and milk levels of BHBA or acetoacetate had a significantly higher risk for mastitis (14,31,44). Furthermore, hyperketonemia in experimentally induced E. coli bovine mastitis was associated with an increased severity of disease that was attributed to neutrophil dysfunction (22). The efficacy and s...