127. Integrating sequencing, qPCR, and culture from matched liver abscesses and gut epithelial tissues in feedlot cattle reveals community-wide and Fusobacterium-specific population dynamics.

Abstract

Liver abscesses (LAs) pose a significant challenge in the North American beef industry, resulting in millions of dollars of economic losses annually. Traditionally, LA pathogenesis is attributed to the translocation of Fusobacterium necrophorum from the rumen to the liver. However, recent studies indicate that LAs are polymicrobial and suggest that the hindgut may also serve as a microbial source. To further our understanding of LA etiology and the gut-liver axis, we integrated 16S rRNA gene sequencing, quantitative PCR (qPCR), and culture method to leverage the complementary strengths of each method and characterize matched ruminal epithelial (RE) and colonic epithelial (CE) tissues and LA samples from feedlot cattle. Matched LA, RE, and CE were collected from 96 tylosin-naïve cattle. Purulent abscess material and epithelial tissues were homogenized in sterile PBS and cultured anaerobically for F. necrophorum and F. varium. Genomic DNA was extracted using the MagMax-96 DNA Multi-sample kit. Quantitative PCR targeting leukotxin gene promoter regions (lktA-n and lktA-f) was used to detect and quantify F. necrophorum subsp. necrophorum (Fnn) and subsp. funduliforme (Fnf), and qPCR targeting the hdgA gene was used to detect and quantify F. varium. Microbial community composition was characterized using 16S rRNA gene sequencing targeting the V3-V4 region on an Illumina NovaSeq6000 instrument. Sequence reads were processed in QIIME2 using DADA2 to generate amplicon sequence variants (ASVs), and taxonomy was assigned using a SILVA138.2 classifier. Diversity metrics, community composition, and statistical analyses were conducted in RStudio using phyloseq. Microbial community composition varied significantly between LAs, RE, and CE (PERMANOVA, p < 0.001), with tissue type accounting for 46.1% of the observed variation. LAs exhibited significantly lower richness and diversity compared to both gut sites (Pairwise Wilcoxon Rank-sum Test; p < 0.001), suggesting strong selective pressure within the abscess environment. Partitioning around medoids (PAM) clustering identified three distinct LA community types characterized by high relative abundances (RAs) of Fusobacterium, Bacteroidota, and Pseudomonadota, respectively. Integrating sequencing, qPCR, and culture data revealed distinct F. necrophorum subspecies prevalence across LA types. Overall, Fnn was the most prevalent subspecies, but the prevalence of both Fnn and Fnf differed significantly across LA types (Fisher’s Exact Test; p = 0.0032 and p = 0.0001). Fusobacterium-dominated LAs had 24-fold higher odds of Fnn detection compared to LAs with high RAs of Bacteroidota (Firth Logistic Regression; p = 0.0016). Conversely, LAs with high RAs of Bacteroidota had 7-fold and 6-fold higher odds of Fnf detection compared to LAs with high RAs of Fusobacterium or Pseudomonadota (Firth Logistic Regression; p < 0.001 and p = 0.006). These findings demonstrate that LA microbial communities partition into distinct types with differing subspecies-level F. necrophorum populations, highlighting the value of integrating culture, qPCR, and sequencing approaches to resolve pathogen dynamics beyond genus-level classification.