PS5-21. Investigating Rumen Microbiome Dynamics During Immune Challenge in Beef Cattle.

Abstract Vaccine-induced adaptive immunity varies substantially among beef cattle, yet it remains unclear whether immune challenge reshapes the rumen microbiome and whether such remodeling reflects individual differences in immune responses. To address this, we immunized purebred Brahman (BRA; n = 60) and crossbred Angus-Brahman (MAB; n = 74) heifers with keyhole limpet hemocyanin (KLH) on day 0, followed by a booster on day 14. KLH-specific IgG concentrations were quantified by indirect ELISA on days 0, 14, and 28, and cell-mediated immunity was assessed via delayed-type hypersensitivity (DTH) on day 28, calculated as the mean change in skinfold thickness at two KLH injection sites minus the change at a PBS control site, measured 24 hours post-injection. Rumen fluid collected on days 0 and 28 underwent 16S rRNA V4 amplicon sequencing on an Illumina MiSeq platform. Reads were quality-filtered, denoised, and chimera-removed using DADA2, and taxonomy was assigned with SILVA v138.2. Because cohorts were raised on separate farms, they were analyzed independently, with MAB models additionally adjusted for continuous Brahman breed composition. Alpha diversity was compared between timepoints using linear models adjusted for pen and animal age, and beta diversity was compared using PERMANOVA with the same explanatory variables, stratifying permutations by pen. Differential abundance was assessed by modeling the within-animal change in CLR abundance from day 0 to day 28 as the response using Huber M-estimation with pen and animal age as fixed effects. The association between genus-level microbiome change and immune outcomes was assessed using the same estimation framework, with the within-animal delta CLR as the predictor and IgG or DTH response as the outcome. Alpha diversity responses were cohort-dependent, with BRA heifers exhibiting significant increases in richness, Shannon diversity, and inverse Simpson diversity over the 28-day period (all FDR < 0.001), whereas MAB heifers showed a significant decrease in Faith's phylogenetic diversity (FDR < 0.001). Microbial community composition shifted significantly in both cohorts between days 0 and 28, with timepoint explaining 21.7-24.8% of Aitchison distance variance and 12.0-14.8% of weighted UniFrac variance (PERMANOVA, all P = 0.001). Huber M-estimation identified widespread taxonomic shifts over the study period, with 107 of 176 prevalent genera significantly altered in BRA (FDR < 0.05) and 120 of 183 in MAB. Despite this pervasive restructuring, no genus-level change was associated with KLH-specific IgG or DTH response after correction for multiple comparisons in either cohort. Overall, these results demonstrate that broad microbiome restructuring occurred over the 28-day KLH vaccination period at the diversity, compositional, and taxonomic levels in a breed-dependent manner, but the observed changes did not correspond to individual differences in measured adaptive immunity under these conditions. Further studies incorporating higher-resolution microbiome profiling, larger cohorts, and more frequent sampling could help clarify whether and how microbial dynamics are linked to individual adaptive immune responses.