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Objective--To estimate the association between ceftiofur use and the isolation of Escherichia coli with reduced ceftriaxone susceptibility from fecal samples of dairy cow populations. Animals--1,266 dairy cows on 18 farms in Ohio. Procedures--Individual fecal samples from all cows in the study herds were tested for Escherichia coli with reduced ceftriaxone susceptibility. Herd antimicrobial use policy and antimicrobial treatment records were also obtained. Plasmid DNA from these isolates was tested for the presence of the AmpC β-lactamase gene (blaCMY-2). Minimum inhibitory concentrations to a standard panel of 16 antimicrobial drugs were determined by use of a broth microdilution system. Results--Herds for which ceftiofur use was reported were more likely to have cows from which reduced-susceptibility E coli was isolated than herds that did not report ceftiofur use (odds ratio, 25.0). However, at the individual cow level, no association was found between recent ceftiofur treatment and isolation of reduced-susceptibility E coli (adjusted odds ratio, 1.01). No observed linear relationship was found between the percentage of cows from which E coli with reduced ceftriaxone susceptibility was isolated and the percentage of cows in the herd recently treat ed with ceftiofur. Conclusions and Clinical Relevance--Our observation of a herd-level but not an individual cow-level association between ceftiofur use and isolation of E coli with reduced ceftriaxone susceptibility from fecal samples suggests that interventions to reduce the spread of antimicrobial resistance genes in agricultural animals will be most effective at the herd level. [ABSTRACT FROM AUTHOR]