Culled beef cows (cows from cow-calf operations that have reached the end of productive life span) and culled dairy cows amount to approximately 18% of the cattle harvested in the U.S. annually, but data on antimicrobial resistance (AMR) levels in U.S. cull cattle is extremely limited. To address this data gap, colon contents were obtained from 180 culled beef cows (conventional beef), 179 culled dairy cows (conventional dairy), and 176 culled organic dairy cows produced without using antimicrobials (organic dairy). Also, 181 conventional beef, 173 conventional dairy, and 180 organic dairy carcasses were sponge sampled. These samples were obtained on 6 days (3 each at two beef harvest and processing establishments). At one establishment 30 samples of beef manufacturing trimmings (trim) from conventional cows and 30 samples of trim from organic dairy cows were acquired. All 1,129 samples were cultured for Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella, and 3GCrSalmonella.Metagenomic DNA was isolated from 535 colon content samples and quantitative PCR was performed to assess the abundances of the following 10 antimicrobial resistance genes: aac(6’)-Ie-aph(2”)-Ia, aadA1, blaCMY-2,blaCTX-M,blaKPC-2, erm(B),mecA, tet(A),tet(B), and tet(M). For colon contents only TETrE. coli (P < 0.01), 3GCrE. coli (P < 0.01), and erm(B) (P = 0.03) levels were higher in conventional than organic cows. Sampling day also significantly (P < 0.01) affected each of these levels. Production system did not affect the levels of any measured antimicrobial resistance on carcasses or trim. The human health impact of the few statistically significant AMR differences are undefinable due to the lack of standards for normal, background, safe, or basal levels. Study results provide key heretofore unavailable data that may inform quantitative microbial risk assessments to address these gaps.

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