Background and Objectives: Liver abscesses (LAs) present a prevalent and costly challenge in the North American beef industry. While 16S rRNA amplicon sequencing has been employed to characterize the LA microbiome, it has limitations in detecting low-abundance taxa, classifying taxa beyond the genus level, and providing functional information. Shotgun metagenomics can help overcome these limitations, enabling both functional profiling and species-level classification to guide future target-enriched (TE) bait design. Consequently, this study leverages shotgun metagenomics to characterize the microbial species prevalent in LAs, compare functional profiles among LA types for the first time, and inform the ongoing design of TE tools.

Methods: Liver abscess samples were collected from 24 cattle as part of a previous study. In that study, 16S rRNA sequencing was utilized to classify three types of LA communities: high Fusobacterium, high Bacteroides, and high diversity. Here, we selected 8 samples from each LA type (n=24) for shotgun metagenomic library preparation and sequencing on an Illumina NovaSeq 6000 instrument using paired-end chemistry at the North Texas Genome Center. Raw sequence reads were trimmed and quality-filtered using trimmomatic, and host DNA was identified and removed by aligning the trimmed reads to the Bos taurus genome using BWA. The remaining non-host reads were classified taxonomically using Kraken2 and the ‘core_nt’ database. Functional profiling was performed using HUMAnN3.

Results: Across all LAs, the three most abundant species were Fusobacterium necrophorum (41.64% RA ± 6.88), Bacteroides haparinolyticus (17.70% RA ± 3.35), and Trueperella pyogenes (15.39% RA ± 5.63). However, each of the three previously defined LA types, or ‘flavors’, displayed a distinct species-level taxonomic profile. High Fusobacterium LAs were overwhelmingly dominated by Fusobacterium necrophorum (81.73% RA ± 12.07), followed by Trueperella pyogenes (4.14% RA ± 4.11). High Bacteroides LAs primarily consisted of Bacteroides haparinolyticus (33.18% RA ± 3.57) and Fusobacterium necrophorum (37.31% RA ± 4.98), with Bacteroides zoogleoformans (6.00% RA ± 0.65) being considerably less abundant. In high diversity LAs, the most abundant species included Actinomycetaceae bacterium MB13-C1-2 (29.90% RA ± 11.40), Lellottia amnigena (17.32% RA ± 4.84), and/or Cellvibrio sp. PSBB006 (16.60% RA ± 3.85). Preliminary results from ongoing functional profiling suggest potential differences in gene family composition among LA types.

Conclusion: We have demonstrated that LAs are highly polymicrobial, with significant differences in community composition among abscesses. By enhancing our understanding of the pathogens linked to LA formation and their functional genes, this research underscores potential disease pathogenesis, and the challenges related to LA treatment and prevention. These findings will also guide the development of TE-based tools aimed at tracing LA-associated taxa to their likely origins throughout the bovine gastrointestinal tract.