Validation of a minimally-invasive method for sampling epithelial-associated microorganisms on the rumen wall

Authors: HENNIGER, MADISON - University Of Tennessee; ROWAN, TROY - University Of Tennessee; BEEVER, JONATHAN - University Of Tennessee; MULON, PIERRE-YVES - University Of Tennessee; SMITH, JOE - University Of Tennessee; VOY, BRYNN - University Of Tennessee; Wells, James - Jim; Kuehn, Larry; MYER, PHILLIP - University Of Tennessee

Submitted to: Frontiers in Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2023
Publication Date: 10/13/2023
Citation: Henniger, M.T., Rowan, T.N., Beever, J.E., Mulon, P., Smith, J.S., Voy, B.H., Wells, J.E., Kuehn, L.A., Myer, P.R. 2023. Validation of a minimally-invasive method for sampling epithelial-associated microorganisms on the rumen wall. Frontiers in Animal Science. 4. Article 1270550. https://doi.org/10.3389/fanim.2023.1270550.
DOI: https://doi.org/10.3389/fanim.2023.1270550

Interpretive Summary: The rumen is a compartment of the ruminant stomach and is a reservoir for a variety of bacteria. Bacteria in the rumen can attach to the tissue of the wall of the rumen, and these bacteria can include possibly pathogenic bacteria and can impact animal performance and well-being. The bacteria in the fluid of the rumen have been studied extensively but because the rumen wall cannot be easily sampled, the bacteria attached to the wall of the rumen is poorly understood. A novel surgical method was developed that is minimally invasive, and this method was utilized to determine differences in bacteria in the fluid and attached to the wall of the rumen. The wall of the rumen did harbor bacteria different than those found in the fluid. The use of this novel technique to sample the wall of the rumen will provide a minimally invasive method to identify invasive and pathogenic bacteria associated with the tissue of the rumen wall.

Technical Abstract: The rumen microbiome provides approximately 70% of the required energy for the host by converting low-quality feedstuffs into usable energy for ruminants. The energy produced by the microorganisms is subsequently absorbed through the rumen epithelium and used towards growth and energy maintenance. There is evidence that ruminal epimural microbes directly interact with the rumen epithelium, acting as an intermediary communicator between the rumen liquid fraction and the host. Epimural microbiota have been demonstrated to be distinct from the ruminal liquid microbiome and perform unique roles within the rumen environment. However, methods to sample epimural communities from the rumen wall are limited and typically invasive, requiring animal fistulation or harvesting. To characterize the epimural communities present on the rumen wall, a novel and minimally-invasive surgical method was developed to swab the epithelium of the ventral sac of the rumen. The objective of this study was to validate this sampling method by comparing epimural and liquid fraction bacterial communities. During a 70-day feeding trial, Angus steers (n = 45) were sampled on day 35 using the novel surgery method and tubed on day 70 to sample rumen liquid content. Genomic DNA was used to generate amplicon libraries of the V4 region of the 16S rRNA gene. There were no differences between alpha diversity indices when comparing rumen versus epimural bacterial communities (P > 0.05). The Bray-Curtis dissimilarity was used to ordinate ASV counts, and then tested for differences between rumen and epimural communities using a PERMANOVA with 999 permutations (P < 0.05). Differential abundances of bacterial communities were tested using ANCOM-BC and MaAsLin2, where significance was determined by Q < 0.05 and overlap between both analysis methods. Within the 91 taxa that differed in abundance, 451 ASVs were found to be different between sample types (Q < 0.05). Unique ASVs associated with Prevotella, Succinivibrio, family-level Eubacterium, and family-level Succinivibrio were in greater abundance for the rumen epithelial-associated bacterial communities (Q < 0.05). The results demonstrate that the novel method of sampling from the rumen wall can capture differences between epimural and ruminal fluid bacterial communities, thus facilitating studies investigating the interactions between epimural bacteria with the host.