Evaluating the Diagnostic Performance of Long-Read Metagenomic Sequencing Compared to Culture and Antimicrobial Susceptibility Testing for Detection of Bovine Respiratory Bacteria and Indicators of Antimicrobial Resistance

<b>Background/Objectives:</b> Long-read metagenomic sequencing can detect bacteria and antimicrobial resistance genes (ARGs) from bovine respiratory samples, providing an alternative to culture and antimicrobial susceptibility testing (C/S). This study applied Bayesian latent class models (BLCMs) to estimate the sensitivity (Se) and specificity (Sp) of long-read metagenomic sequencing compared to C/S for detecting <i>Mannheimia haemolytica</i>, <i>Pasteurella multocida</i>, and <i>Histophilus somni</i>, as well as associated macrolide and tetracycline resistance potential. <b>Methods:</b> Deep nasopharyngeal swabs were collected from fall-placed feedlot calves at arrival, 13, and 36 days on feed across two years and two metaphylaxis protocols. Samples underwent C/S and long-read metagenomic sequencing. BLCMs were used to estimate Se and Sp for the detection of bacteria and potential for antimicrobial resistance (AMR). <b>Results:</b> Se and Sp for detecting respiratory bacteria by metagenomics were not significantly different than culture, with four exceptions. For the 2020 samples, Se for <i>M. haemolytica</i> was lower than culture, and Sp for <i>H. somni</i> was lower, while in both 2020 and 2021 samples, Se for <i>P. multocida</i> was higher for metagenomics than culture. The estimated Se and Sp of metagenomics for the detection of <i>msrE-mphE</i>, <i>EstT</i>, and <i>tet(H)</i> within bacterial reads were either not significantly different or were lower than AST, with Sp > 95% with one exception. <b>Conclusions:</b> This study provided BLCM-based estimates of clinical Se and Sp of metagenomics and C/S without assuming a gold standard in a large pen research setting. These findings demonstrate the potential of long-read metagenomics to support bovine respiratory disease diagnostics, AMR surveillance, and antimicrobial stewardship in feedlot cattle.