From Abattoir to Animal Model: 16S rRNA gene Microbial Profiling of Beef Cattle Lymph Nodes vs. Digesta and Functional Microbiome Changes in C3H/HeJ Mice on High-Fat Casein Diets ± Ammonium-Hydroxide

This literature review synthesizes current evidence indicating that foodborne pathogens continue to pose a significant public health and economic burden in the United States, with recurrent multistate outbreaks frequently implicating beef, particularly ground beef, as a primary vehicle of illness. Within the meat system, peripheral lymph nodes (LNs) embedded in adipose, or muscle can evade surface decontamination, introducing Salmonella and Shiga toxin–producing Escherichia coli (STEC) and other entering taxa into trim, thereby amplifying downstream risk during commingling and grinding. Additionally, a brief review was included summarizing culture-independent approaches that have revealed lymph nodes as reservoirs of enteric pathogenic microbes in mammals. These approaches, including 16S rRNA gene amplicon, metagenomics, and meta-transcriptomics, have become impactful in food safety and microbiology. According to microbiome studies worldwide, the diverse, metabolically active LN microbiota in cattle and swine are frequently enriched with Proteobacteria, including Enterobacterales taxa. Additionally, plant- and feedlot-level studies have shown marked variability by season, geography, and management, with serovars such as Montevideo and Anatum being common in positive nodes. Furthermore, spoilage bacteria and foodborne and airborne pathogens, from a farm-to-fork risk framework perspective, involve upstream exposures (pen surfaces, water troughs, dust/aerosols, wildlife) and lymphatic trafficking, which shape contamination that is not fully mitigated by hide or carcass interventions. Consistent with this systems view, regulatory controls (GMP/GHP, HACCP), verification testing, and targeted pre-harvest strategies (e.g., ration formulation, direct-fed microbials, sanitation and drainage, weather-adaptive practices) are emphasized to reduce LN-mediated hazards. This literature review also discusses a link to an animal model of diet-microbiome-host interactions using C3H/HeJ mice fed high-fat beef diets with or without ammonium-hydroxide supplementation. In this study, long-term feeding regimens were used to profile taxonomic and functional microbial shifts under acidogenic Western-diet conditions, providing a translational lens for obesity-relevant mechanisms in which high-fat feeding, microbiome composition, and microbial metabolites interface with metabolic outcomes. In the second study, since fecal contamination of hides is considered a primary source of enteric pathogens, as contamination from the gut can also occur via bacterial translocation through peripheral LNs, which can be included in trim used in ground beef, becoming a significant additional reservoir of Salmonella and STECs, creating a difficult-to-control route for pathogen entry. This study aims to profile and compare bacterial communities in beef lymph nodes with themselves versus fecal/cecal digesta using 16S rRNA gene sequencing. From eight carcasses, we collected 38 LNs mandibular (MLS), subiliac (SNL), popliteal (PLN), inferior (IILN), superior-iliac (SILN), fecal (BF), and cecal content (CC). DNA was extracted, and the V3–V4 16S rRNA region sequenced. Reads were processed with DADA2 (ASVs; SILVA taxonomy). Alpha diversity (Observed, Chao1, Shannon, Simpson) and beta diversity (Bray–Curtis; PCoA) were tested using standard ecological statistics, and hierarchical clustering applied Pearson correlations across the groups. Results: SILN exhibited the greatest within-sample diversity (Observed/Chao1/Shannon; p≤0.0008), whereas PLN formed a tight, compositionally distinct cluster versus other LNs in PCoA (Axis1+2=46.2% variance; p<0.05). Firmicutes and Proteobacteria dominated phylum-level profiles; Proteobacteria were highest in MLS/SILN (43–44%) and lowest in SNL (<15%). At the order level, Staphylococcales, Lactobacillales, and Enterobacterales predominated, with Staphylococcales reaching 82% in PLN. Genus-level cores included Enterococcus, Staphylococcus, and Escherichia/Shigella. A Legionella-assigned ASV appeared (low abundance) in PLN, while the digesta presented another biological composition by Ruminococcaceae UCG-005, Bacteroides, and Romboutsia. Correlation heatmaps segregated two ecological clusters: (i) MLS+PLN and (ii) SNL+IILN+SILN (r= 0.93, within cluster), indicating shared exposures/functions among the latter group. Conclusions: The presence of Escherichia/Shigella, Enterococcus, Staphylococcus, and Legionella genera and the robust correlation of the bacterial relative abundance in the peripheral. LNs and digesta suggest possible bacterial translocation and the presence of potentially pathogenic bacteria from the Gastrointestinal Tract (GIT), underscoring the importance of risk assessments in slaughterhouses to prevent the spread of spoilage bacteria, foodborne, airborne, and zoonotic pathogens. Additionally, this study confirms the necessity of regulatory agencies for microbial beef carcass inspection based on peripheral LNs. It assures that this decision can contribute to minimal microbial contamination by refining hypotheses on translocation and possible trim-mediated contamination pathways. The third study found that acidogenic Western-style diets disrupt gut bacteria and promote obesity-related diseases. In a recent precursor study of HFD-induced obesity in mice, beef supplemented with ammonium hydroxide exhibited a protective effect against microbiome dysfunction associated with HFD (or diet-induced obesity), indicating the sustainability of beneficial resident taxa. Correspondingly, the functional potential reflected a range of positive cellular response processes. For this study, we investigated whether long-term feeding of alkalinized dietary protein (ammonium hydroxide enhancement, AHE) modulates microbiome structure/function under high-fat conditions, and whether these responses are sex-dimorphic. Methods: C3H/HeJ mice (n=32; equal sex distribution) received one of four diets from 6 to 18 months: control casein (CC), AHE-casein (CCN), high-fat casein (HFC), or AHE high-fat casein (HFCN). Diet pellets differed primarily in pH (CCN = 8.17, CC = 7.29; HFCN = 8.02, HFC = 7.09). Body mass and survival were tracked; fecal samples were collected at 16 months, sequenced, and underwent shotgun metagenomics (assembly, binning, taxonomic profiling, functional analyses, and GO enrichment). Results: Diet and sex jointly shaped host metrics, as AHE diets taxonomically showed an abundance of Verrucomicrobiota phyla predominating in most cohorts, notably Akkermansia muciniphila, whereas Bacillota was predominant only in CC-males. Within Pseudomonadota, Christensenella, along with other beneficial taxa associated with beneficial health outcomes were identified. These included: Lactococcus lactis, Lactococcus cremoris, Pediococcus acidilactici, and the families Lachnospiraceae/Oscillospiraceae. In parallel, ostensibly advantageous enriched functions associated with AHE that enhanced electron transport, B-vitamin cofactor pathways, and mucosal/redox support were all expressed in a manner dependent upon sex and diet. The differences are consistent with altered mucin interaction and energy/defense pathways, aligning with the hypothesis that protein alkalinization, under high-fat load, reprograms community structure and functional potential. Conclusions: In the long term, pH-directed protein chemistry is a tractable lever for gut ecology during high-fat feeding, enriching and promoting the balance of beneficial Gram-positive/Gram-negative taxa. These taxonomic and functional shifts provide a mechanistic bridge between dietary acid load, microbiome remodeling, and obesity phenotypes, motivating sex-aware, diet-chemistry interventions in translational models.