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The severity of experimental Escherichia coli mastitis in relation to in vitro chemotaxis of polymorphonuclear leukocytes was investigated in cows during negative energy balance. The negative energy balance was induced by feed restriction. Cows were classified into two groups, ketonemic and nonketonemic, based on the β-hydroxybutyrate concentration in the peripheral blood at the moment of inoculation. Bacterial growth in the inoculated quarter was used as a parameter to indicate the severity of experimental mastitis. In the nonketonemic cows, experimental mastitis ranged from moderate to severe. Severity of experimental mastitis was negatively related to preinfection chemotactic response of polymorphonuclear leukocytes. In contrast, the course of experimental mastitis in the ketonemic group was relatively severe in all cows, regardless of preinfection chemotactic response.