Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
(N/A)
Non Technical Summary
The chronic inflammatory effects of subacute acidosis are the greatest impediment to achieving optimal beef productivity in modern well-managed beef production systems. While ruminal health has received much attention, it is increasingly evident that the post-ruminal gastrointestinal tract (GIT) plays a significant role in the progression ofdisease.These ill effects may be compoundedby restrictions placed on certain dietary antibiotics. As alternatives to antibiotics are sought, there is a surprising lack of knowledge about how they interact with commensalbacteria and pathogens to alter intestinal function and integrity. Microbially modified bile acids are a compelling mechanistic target for enhanced gut barrier function. To address this gap, thespecific objectivesof this proposal are to feed tylosin + monensin to grain-finished feedlot cattle and: 1) characterize changes in the ruminal, intestinal, and cecal microbiome, 2) identify antibiotic-responsive metabolites in digesta 3) determine host response to microbial signals, and 4) integrate metagenome-metabolome-transcriptome data for a comprehensive model of the system. Ourcentral hypothesis, is thatantimicrobial drugs improve host intestinalfunction and integrityby inhibiting bile salt hydrolyzing bacterial species in the lower GIT, resulting in less microbial modification of bile acids and a primary conjugated bile acid profile that dampens physiological inflammation. In turn, gut barrier function will improve and the catabolic cost of maintaining an immune response will decrease. By using multiple-omics techniques, our aim is to reveal elements of the system to target for development of alternatives that improve efficiency, health, and welfare of feedlot cattle managedunder various conditions.
Animal Health Component
10%
Research Effort Categories
Basic
70%
Applied
10%
Developmental
20%
Goals / Objectives
Increasing the proportion of grain in ruminant diets is a widespread practice intended to maximize the conversion of feed to lean tissue growth. However, increased dietary fermentation rate can result in different degrees of rumen acidosis (acute vs. subacute) that causes chronic gastrointestinal tract (GIT) inflammation. This inflammation leads to liver abscesses and heightened immune activation, which have significant catabolic costs that antagonize efficient beef production. While ruminal health has received much attention, it is increasingly evident that the impact of high grain diets on other sections of the GIT substantially contribute to the overall pathophysiology of the disease (Sanz Fernandez et al., 2020). Indeed, the portal vein collects blood from the entire GIT and recent evidence suggests that a considerable number of bacterial endotoxins and liver abscess microbes arise from the post-ruminal GIT (Khafipour et al., 2009;Jennings et al., 2021; Pinnell et al., 2022). Subtherapeutic antimicrobials are routinely fed long-term to grain-fed cattle to ameliorate digestive disturbances and other epidemiological linked disorders, such as bovine respiratory disease (BRD) and foot rot. Auxiliary benefits, including improvements in ruminal energetic efficiency, feed intake, and average daily gain are well established. However, with new restrictions in place regarding use of dietary antibiotics, producers are losing critical production tools. Currently available alternatives to antimicrobial drugs, such as probiotics, prebiotics, organic acids and essential oils lack sufficient efficacy to inhibit disease or dampen inflammation (Schoonmaker, 2015). Thus, new and improved technologies are needed to ensure optimal cattle growth and efficiency. A more mechanistic understanding of how antimicrobial drugs work in post-ruminal regions of the GIT would enlighten mechanisms amenable to manipulation and will facilitate allied industry development of new strategies that target these changes.The established theory for antimicrobial drug inhibition of liver abscesses is a suppression of ruminal pathogen growth which limits their ability to translocate across the ruminal wall following acidosis episodes (Russell, 1987; Nagaraja et al., 1999). Emerging evidence in ruminants confirms that the intestinal microbiome has a marked effect on gut inflammation and immunity (Malmuthuge et al., 2013, 2019; Liang et al., 2016) which could plausibly be the underlying mode of action for antimicrobial drug inhibition of liver abscesses in feedlot cattle (Pinnell et al., 2022). Our preliminary data suggests that the most common antimicrobial drug intervention (tylosin + monensin) increased jejunal villus height:crypt depth ratio in cattle fed 93% concentrate diets, suggesting an improvement in gut health.Much attention has focused on microbiome analysis of ruminant GIT contents in recent years (Thomas et al., 2017; Ogunade et al., 2018), but many of these studies have failed to address how these microbes modulate host intestinal function and integrity. Therefore, a critical gap remains in our knowledge about how antimicrobial drugs, commensal bacteria, and pathogen pressure alter intestinal function and integrity.Research in poultry and swine suggests that tylosin and monensin improve host GIT barrier function by decreasing intestinal bacterial populations responsible for microbial hydrolysis of primary bile acids into secondary bile acids (Lin, 2014; Ipharragurre et al., 2018). This is significant because primary bile acids have antimicrobial and anti-inflammatory properties and play a major role in gut health and peripheral metabolism (de Diego-Cabero et al., 2015; Ridlon et al., 2014). The current understanding of how antimicrobial drugs affect ruminant bile acid metabolism is highly limited. Therefore, ouroverarching goalis todefine changes in the ruminant GIT microbiome and metabolome after antimicrobial drug administration and ascertain the host response. Thespecific objectivesof this proposal are to feed FDA label-defined doses of tylosin + monensin, to grain-finished cattle and:Characterize changes in the ruminal, intestinal, and cecal microbiomeIdentify antibiotic-responsive microbial metabolites in the rumen, intestine, and cecumDetermine host response to microbial signalsIntegrate metagenome-metabolome-transciptome and physiological data for a comprehensive model of the system.
Project Methods
Seventy-two crossbred steers (approx. 450 kg), never fed antimicrobials, will be sourced from the Purdue University herd and allotted to 36 penssuch that body weight and breed composition are similar among treatments. Cattle will be fed the same antimicrobial-free, high forage, 56% roughage diet (DM basis) for 42 days prior to initiation of the study. All steers will be immediately transitioned from the high forage diet to a high grain, 7% roughage diet with treatments applied: 1) control - no dietary antibiotic, and 2) tylosin + monensin - 75 mg of tylosin and 200 mg of monensin fed per head daily. All diets will be formulated to meet or exceed NASEM (2016) requirements for protein, vitamins, and minerals. Diets will be fed for approximately 126 days, and steers will be allowedad libitumaccess to feed and water.Because therapeutic antimicrobials may impact the GIT microbiome, any treated cattle will be removed from the study. Typical treatment rates at the Purdue ASREC beef unit are 1-2%, thus an extra 4 animals (2 pens) per treatment will fed treatment diets.Steers will be serial slaughtered prior to antibiotic treatment administration on day 0, after 7, 21, and 63 days on feed, and at a finished BW ofapproximately 615 kg at 126 days. Four animals per treatment (8 animals total) will be slaughtered on day 0, and eight animals per treatment (16 total) will be slaughtered at each time point starting at day seven.At slaughter, digestive contents and tissue will be collected from therumen, jejunum, and cecumwithin 45 minutes of exsanguinationaccording toLindholm-Perry (2016). Digestasamples designated for microbiome analysis andmetabolite and endotoxin analysis will be immediately snap frozen in liquid nitrogen and storedat -20oC until analysis.Approximately 10 mL of blood will be collected from the jugular vein at slaughter. Serum will be harvested and frozen at -20°C until inflammatory marker analysis.For histological analysis , tissues will be rinsed with phosphate buffered saline and fixed in 10% formalin. Tissuesfor transcriptome analysiswill be flash frozen in liquid nitrogen, then stored at -80°C until RNA extraction and transcriptome analysis.Objective 1. Characterize changes in the ruminal, intestinal, and cecal microbiomeIn order to determine the composition of the total bacterial community, we will conduct 16S rRNA gene amplicon sequencing of total extracted DNA as described previously (Kozich et al., 2013). In order to determine bacterial numbers, the entire community from each sample will be quantified with qPCR. Total bacterial DNA will be extracted from intestinal digesta samples using the MagAttract Power Microbiome DNA/RNA Kit (Qiagen, Valencia, CA, USA) per manufacturer's instructions. Extracted DNA will be quantified using a Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA). PCR will be carried out in a single step with dual-indexed primers targeting the V4 region of the 16S rRNA gene as listed in Kozich et al. (2013). The products of all samples will be purified and normalized with the SequalPrep Normalization Kit (Invitrogen, Carlsbad, CA, USA). Alpha and beta diversity will be assessed with a variety of metrics and tested statistically with Kruskal-Wallis and PERMANOVA tests, respectively. Bacterial taxa with differential abundance according to treatment group will be determined with DESeq2 (Love et al., 2014) and linear discriminant analysis (LDA) effect size (LEfSe) (Segata et al., 2011).Objective 2. Identify antibiotic-responsive microbial metabolites in the rumen, intestine, and cecumLipidome profiling:Lipids will be extracted from 200 uL of digesta samples using theBligh & Dyer (1959)method. Mass spectrometry data will be acquired by flow-injection (no chromatographic separation) from 10 μL of diluted lipid extract stock solution delivered using a micro-autosampler (G1377A) to the ESI source of an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). For LC-MS/MS quantification,isotopically-labeled internal standards will be spiked in the samples andinjected intothe sameAgilent 6460 Triple Quad LC/MS System (Agilent Technologies, Santa Clara, CA).Quantification of LPS and LTA:Samples will be collected from animals slaughtered on day 0, 7, 21, 63 and 126 using sterile techniques to avoid environmental microbial contamination. Lipopolysaccharide will be analyzed with a PyroGene endotoxin detection assay (Lonza, Walkersville, MD).Lipoteichoic acid will be analyzed using an anti-LTA antibody based enzyme linked immunosorbent assay(Aviva Systems Biology, San Diego, CA) and absorbance will be read at 450 nm using a plate reader.Objective 3. Determine host responses to microbial signalsDetermine serum concentration of inflammatory markers:We will use procedures adapted from a commercially available, bovine specificenzyme-linked immunosorbent assays (ELISA) kitvalidatedin our laboratories (Briggs et al., 2020).Serum concentrations of lipopolysaccharide binding protein (LSBio, LS-F7412, Seattle, WA) will be analyzed at 450 nm on a Spark 10M plate reader (Tecan Life Sciences, Männedorf, Zürich, Switzerland).Determine GIT characteristics and histology:Intestinallengths will be determined by looping the intestine across a stationary board, fitted with plastic pegs at 1-m increments, without tension to minimize stretching.For histological analysis, fixed GIT samples will be sectioned and Alcian blue periodic acid-Schiff stained.Imageswill be obtainedfrom 5 non-overlapping fields at 100x magnification. All morphological measurements will bemadeusing ImageJ (National Institutes of Health, Bethesda, MD). For villus height and crypt depth measurements, at least 1 villus per image will be measured representing at least 5 villi/crypts. Goblet cell area will be quantified in each villus.Next generation sequencing of jejunal transcriptome:Samples will be pulverized under liquid nitrogen and total RNA will be extracted using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA, USA).Samples will be submitted to the Purdue University Genomics Core for library preparation using the TruSeq Stranded Total RNA Library Prep Kit for measuring messenger RNA molecules.Library will be sequenced on an Illumina NextSeq or NovaSeq platformto generate at least 50-60 million 2x150bppaired end reads.For biological interpretation of data,differentially expressed genes will be used for GO and pathway enrichment analysis using clusterProfiler (Yu et al., 2012),DAVID (Huang et al. 2007; Huang et al. 2009)and GSEA analysis using GenePattern (Reich et al, 2006; Subramanian et al, 2005) to identify significant pathways and gene sets that are antibiotic-responsive.Objective 4. Integrate metagenome-metabolome-transciptome and physiological data for a comprehensive model of the systemCorrelation analysis will be performed between metabolites and microbial species. Pairs with correlation coefficients above 0.8 or below -0.8 will be extracted to visualize in Cytoscape (Shannon et al., 2003). For a particular metabolite of interest , the top correlated microbial species will be extracted for further investigation. We will also identify pairs of metabolites and genes with high correlation coefficient. We will then use locally installed PaintOmics3 (Hernandez et al., 2018) to map metabolites and genes to KEGG database to perform pathway analysis. For pathway analysis, PaintOmics3 will first perform single and joint pathway enrichment analysis to identify significant KEGG pathways. Then PaintOmics3 will create pathway networks based on metabolomic and transcriptomic data and pathways sharing similar trends will be clustered so thatpathways with the same pattern of change can be grouped and, if also connected by edges, their molecular relationships revealed.