IDENTIFYING MICROBES OF POTENTIAL IMPORTANCE TO THE NUTRITION, HEALTH AND PERFORMANCE OF RUMINANT LIVESTOCK.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012536
• Project Start Date: Jul 1, 2017
• Project End Date: Jun 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717

Performing Department
Animal & Range Sciences

Non Technical Summary
The factors affecting nutrition, health and performance of livestock animals overlap and collectively underpin the economic performance of all livestock operations. Each of these important factors is significantly impacted by microbes occupying the different regions of the animal's gastrointestinal tract. Combining modern molecular approaches to assess composition, functional potential, and small molecule metabolite dynamics with longitudinally collected measures of health and productivity, we will identify microbial species, who produce metabolites, have the potential to produce metabolites, or who themselves correlate with health and production traits for isolation and characterization. These microbes will be assessed for use in novel non-antibiotic, consumer- acceptable direct microbial interventions that optimize neonatal health and productivity and improve the quality of their products. The long-term objectives of this project being to increase the profitability and sustainability of Montana's and the United States' livestock agricultural industries.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
308	3310	1040	25%
308	3610	1040	25%
308	7410	1040	25%
308	4099	1100	25%

Knowledge Area
308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3610 - Sheep, live animal; 4099 - Microorganisms, general/other; 3310 - Beef cattle, live animal; 7410 - General technology;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

agriculture

microbiome

metabolome

neonatal health

immune function

productivity

food security

sustainability

profitability

Goals / Objectives
The long-term goals of this project are to develop designer microbial communities that can be administered directly or maternally as polymicrobial pro-biotics, and favored ecologically through time and route of application, and with the appropriate addition of pre-biotic supplements provided combinatorially (synbiotics). These synbiotics would be designed based on basic science-driven research (informed) to reduce neonatal mortality, increase neonatal health and provide optimally productive livestock animals. The following specific objectives are designed to build upon our current progress toward our long-term objective by enabling functionally-informed hypotheses to be generated and tested:Determine the metagenomic changes associated with the early successional development of GIT microbiota of ruminant livestock.Determine the metabolomic changes associated with the early successional development of GIT microbiota in ruminant livestock.Using a variety of host-systems as models of host-gene-metabolome relationships develop and refine methods to better explore and understand the relationships to host diet, health, and productivityIdentify 1-3 metabolites, genes, and/or microbes that show strong correlative relationships with animal health and/or performance and isolate microbes that produce, express, or are these for characterization

Project Methods
Samples have already been collected in a 2.5 year animal trial carried out in the first phase of this project and spanning 1 month prior to ewe breeding to parturition, to lambs reaching 1 year of age when they were turned back out to the MSU flock. Lambs were additionally sampled at 2 years of age after spending a year with the flock. Samples collected included oral, and rectal swabs, stomach tube collections of pre-rumen and eventually rumen digesta (at birth the rumen is not considered functional being smaller than the abomasum, typically representing ~29% of the total GIT and increases to ~55% following weaning(J.B. Russell and Rychlik, 2001)), and placental and gut fluids obtained from five fetuses (opportunistically collected through late gestational ewe mortality resulting from pregnancy toxemia). Each sample type was collected from more than 24 lambs at each time point from immediately at birth, and then at 1 d, 3 d, 7 d, 2 w, 4 w, 3 m, 6 m, 1 y and 2y of age. Blood sera was also collected via jugular puncture at each time point. Lamb weights were collected every 12 - 14 d to assess birth to weaning weights and growth rates. Morbidities and the details of any treatments were noted. Following weaning, lambs were dry-lotted to assess their growth efficiency and maximum average daily gains (ADG), total body weight, and residual feed intakes for 3 months.Samples will be selected for metagenomic (Objective 1) and metabolomic (Objective 2) interrogation to ensure adequate analytical power to assess longitudinal changes associated with the successional development of the rumen and hind-GIT microbiota while nesting variables including animal health (with a specific focus on scours), performance (based on RFI), and immunoglobin titres.Metagenomic libraries will be produced from DNA extracts using Nextera XT DNA sample preparation kits (Illumina) and sequenced using paired-end 2x150nt sequencing protocols on either an Illumina MiSeq (currently available in the Yeoman lab) or HiSeq. Metagenomic data will be quality trimmed (

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Researchers in the scientific fields of animal sciences and microbiology/microbiome via publications and presentations and students and stakeholders via presentations and opportune discussion Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have been recruited and are presently developing cattle ileum and cecal organoids models and isolating methylcaconitine-degrading bacteria from wild rumen samples, respectively. Additionally, three undergraduate students have been working to learn anaerobic microbiological techniques, organoid and primary cell culture techniques, and general laboratory techniques. Finally, one postdoctoral fellow has been training in addvanced bioinformatic andd biostatistical techniques relatedd to the objectives of this proposal. How have the results been disseminated to communities of interest?Results have been disseminated through 4 new publications described in this report, through online and in-personteaching, and in oral presentations and interactions with stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Several manuscripts are awaiting submission due to various factors, including collaborative and regulatory approval.Grant applications are being developed to further support these research efforts, we presently have bothprivate foundation and federal support. We arerecruiting 1-2 graduate students to advance our work to assess the ability of microbial strains to stimulate and modulate immune function in vitro using bovine small intestinal cell lines and organoids. Additionally, we are working to isolate and identify microbes capable of degrading the major larkspur alkaloid toxin, methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle for testing in animal trials.

Impacts
What was accomplished under these goals? 1. 16S rRNA-basedd methods have been used to assess the microbial changes associated with early succession of both the bovine and ovine gastrointestinal tract. These findings have been published and are subject to additional publications in preparation. 2. GC-MS-based metabolomic data have been developed for the successional development of the ovinegut and is being prepared for publication. 3.Methods have and are continuing to be developed through analyses of a variety of systems, includding bovine, ovis, primate, human, insect, and even soil microbiomes. Major emphases are on the integration of multiple -omics datasets and efforts have resulted in several manuscripts that have been and are in line for publication focussed onunderstand microbial ecology and the microbiomesinfluence on immunological parameters and health.These cross-species analyses continue to provide new insights aiding our interpretation and understanding of more directly ag-related systems. 4.We have identified 56microbial taxa that are being tested for their influences on calf immune development and functional response to endotoxin andd pathogenic challenge. Additionally,we are isolating microbe(s) that are present in the ruminal content of various wild species that facilitate the degradation ofmethyllycaconitine, the primary alkaloid toxin in Larkspur.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled FD. 2020. Soil bacterial communities of wheat vary across the growing season and among dryland farming systems. Geoderma. 358: 113989
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sanglard LP, Schmitz-Esser S, Gray KA, Linhares DCL, Yeoman CJ, Dekkers JCM, Niederwerder MC, Ser�o NVL. 2020. Vaginal microbiota diverges in sows with low and high reproductive performance after porcine reproductive and respiratory syndrome vaccination. Scientific Reports. 10: 3046 DOI: 10.1038/s41598-020-59955-8
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Borgogna JC, Shardell MD, Brotman RM, Yeoman CJ, Ghanem KG, Kadriu H, Ulanov AV, Gaydos CA, Hardick J, Robinson C, Ravel J, Bavoil PM, Tuddenham S. 2020. Vaginal metabolomics profiles: comparing uninfected, C. trachomatis mono- and C. trachomatis/M. genitalium co-infected women. Scientific Reports. 10: 3420 DOI: 10.1038/s41598-020-60179-z
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled F. 2020. Dryland cropping systems, weed communities, and disease status modulate the effect of climate conditions on wheat soil bacterial communities. mSphere. 5:e00340-20 DOI: 10.1128/mSphere.00340-20
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Yeoman CJ, Lachman MM, Ishaq S, Olivo S, Swartz J, Herrygers M, Berardinelli JG. 2020. Development of Climactic Oral and Rectal Microbiomes Corresponds to Peak Immunoglobin Titers in Lambs. Conference of Research Workers in Animal Diseases (CRWAD). Virtual Conference. Dec 5 - 8.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ, Betiku OC, Gaylord TG, Ishaq SL, Sealey WM. 2020. A METAGENOMIC EXPLORATION OF THE FUNCTIONAL POTENTIAL OF GUT MICROBES TOWARD THE NUTRITIVE CAPABILITY OF RAINBOW TROUT (ONCORHYNCHUS MYKISS). World Aquaculture Society Meeting. Honolulu, HI. Feb 9.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ. 2020. Microbes & Agriculture: From Wheat Pests to Livestock. Crop & Pest Management School. Bozeman, MT. Jan 8.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ. 2020. An important microbial community lives within all of us. Provosts Distinguished Lecture Series. Bozeman, MT. Feb 11.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Results have primarily been disseminated to the scientific field via publications, to students through in-class teaching and associated practical activities, and to our departmental stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four students for a graduate-level nutrition class were involved in a study comparing the gut microbiology and productivity of sheep fed alfalfa that was either fed as hay or a reduced particle size pellet resulting in a publication and new insight into the influence of the physical structure of feed on the gut microbiota. A graduate student has also been recruited and is bioprospecting wild rumen samples for toxin degrading activities. How have the results been disseminated to communities of interest?Results have been disseminated through 10 publications described in this report, through in-class teaching, and associated practical activities (resulting in a publication) and in engagements with stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Several manuscripts are presently being prepared along with grant applications. Having recently obtained some private foundation support we are recruiting students to advance our work to assess the ability of microbial strains to stimulate and modulate immune function in vitro using bovine small intestinal cell lines. Our preliminary work looking at these host:microbe interactions has been used to develop a foundational grant that we have not yet heard back on due to the recent move of NIFA to KS but hope to get a positive review on to help accelerate our progress. Additionally, we are working to identify and isolate microbes capable of detoxifing the major larkspur toxin, methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle.

Impacts
What was accomplished under these goals? 1. Taxonomic data has been developed for the successional development of bovine and ovis gut microbiota, however, full metagenomic sequencing has not been completed. 2. Metabolomic data have been developed for the successional development of the ovis gut, is analyzed and is being prepared for publication. 3. Methods have and are continuing to be developed through analyses of bovine, ovis, primate, human, insect, and even soil microbiomes. Each of these have and are being analyzed to understand ecology, including the influence of diet, health, and productivity. These cross-species analyses continue to provide new insights aiding our interpretation and understanding of more directly ag-related systems. 4. Metabolites have been identified that correspond to diminished health, but none have robustly stood out as being associated with health from our current studies. We have also identified 8 microbial species that are being tested for their influences on immune development and function and are presently searching for microbe(s) that are facilitating a methyllycaconitine- (primary toxin in Larkspur) degrading activity that we have variably detected in wild rumen samples.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ishaq SL, Lachman MM, Wenner BA, Baeza A, Butler M, Gates E, Olivo S, Geddes JB, Hatfield P, Yeoman CJ. 2019. Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed. PLoS One 14: e0215797. DOI: 10.1371/journal.pone.0215797
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yeoman CJ, Brutscher LM, Esen �, ?bao?lu F, Fowler C, Eren AM, Wanner K, Weaver DK. 2019. Genome-resolved insights into a novel Spiroplasma symbiont of the Wheat Stem Sawfly (Cephus cinctus). PeerJ 7:e7548 DOI: 10.7717/peerj.7548
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled FD. 2019. Soil bacterial communities of wheat vary across the growing season and among dryland farming systems. Geoderma. In press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Sanglard LP, Schmitz-Esser S, Gray KA, Linhares DCL, Yeoman CJ, Dekkers JCM, Niederwerder MC, Ser�o NVL. 2019. Investigating the relationship between vaginal microbiota and host-genetics and their impact on immune response and farrowing traits in commercial gilts. Journal of Animal Breeding and Genetics. In press. DOI: 10.1111/jbg.12456
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lei B, Flores AR, Yeoman CJ, Liu M. 2019. Complete genome sequence of hypervirulent Streptococcus pyogenes emm3 strain 1838. Microbiology Resource Announcements. 8: e01494  18 DOI: 10.1128/MRA.01494-18
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Miles MP, Wilson S, Yeoman CJ. 2019. Physical activity and inflammation phenotype conversion. Journal of Clinical Exercise Physiology. 8: 64-73
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Pafco B, Sharma AK, Petrzelkova KJ, Vlckova K, Todd A, Yeoman CJ, Wilson BA, Stumpf R, White BA, Nelson KE, Leigh SR, Gomez A. 2019. Gut microbiome composition of wild western lowland gorillas is associated with age and sex factors. American Journal of Physical Anthropology. 9: 1202. DOI: 10.3389/fmicb.2018.01202.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gomez A, Sharma A, Mallott E, Petrzelkova K, Robinson CJ, Yeoman CJ, Carbonero F, Pafco B, Rothman J, Ulanov A, Vlckova K, Amato KR, Schnorr S, Dominy N, Modry D, Todd A, Torralba M, Nelson KE, Burns M, Blekhman R, Remis M, Stumpf RM, Wilson BA, Gaskins HR, Garber P, White BA, Leigh SR. 2019. Plasticity in the human gut microbiome defies evolutionary constraints. mSphere 4: e00271-19 DOI: 10.1128/mSphere.00271-19
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Borgogna JC, Shardell MD, Santori EK, Nelson TM, Rath JM, Glover ED, Ravel J, Gravitt P, Yeoman CJ, Brotman RM. 2019. The vaginal metabolome and microbiota of cervical HPV-positive and HPV-negative women: a cross-sectional analysis. British Journal of Obstetrics & Gynecology. In press. DOI: 10.1111/1471-0528.15981
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ishaq SL, Page CM, Yeoman CJ, Murphy TW, Van Emon ML, Stewart WC. 2019. Zinc-amino-acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not. Journal of Animal Sciences. 97: 687  697. DOI: 10.1093/jas/sky456.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The research performed to date are primarily of value to the research community, specifically those in the fields of microbiology, microbial ecology, and animal nutrition, health, and production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students (Kathryn Parea & Dusti East) have undertaken research internships with the lab and been trained in bioinformatic and biostatistical techniques of 16S rRNA gene and metabolomic data, respectively. One project resulted in a manuscript (Perea et al. 2017) and the other is currently being prepared as part of a larger publication. One MS student (Medora Lachman) is in continued training on an integrated project involving animal handling and husbandry, molecular techniques, including microbiome and metagenomic sample collection and processing, and bioinformatics. An additional graduate student (Deanna DeSonn) and undergraduate student (Eden Manuel) will be joining the lab during the next reporting period on research extending from the current work. How have the results been disseminated to communities of interest?Results have been disseminated through the publications described in this report, in the W4177 annual meeting, and in two invited talks: • Invited speaker Animal Microbiome Congress (Kansas City, KS) • Plenary speaker Midwestern Section of the American Society for Animal Sciences (Omaha, NE) What do you plan to do during the next reporting period to accomplish the goals?In addition to completing analysis, and writing and submitting manuscripts developed from data elaborated during the current funding period, we will advance objective 4 of research objectives by testing our newly acquired microbial strains for their ability to stimulate immune function in vitro using bovine small intestinal cell lines in collaboration with Dr. Mark Jutila here at Montana State U. We hope this provide compelling evidence to support a competitive foundational grant to rapidly accelrate our progress. Additionally, we have recieved private foundation funding to look at microbial detoxification of the major larkspur toxin methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle.

Impacts
What was accomplished under these goals? Objective 1: 16S rRNA gene sequence profiles have been obtained from both the lumen and mucosa of ten gastrointestinal tract regions (including, the pharynx, rumen, reticulum, omasum, abomasum, duodenum,proximal, medial, and distal jejunum, ileum, and colon of neonatal calves at 1, 3, 7, and 21 days post parturition. These samples were compared to each other and to 16S rRNA gene profiles collected from the dam's colostrum, outer udder skin, and vaginal scrapings collected at calving. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Bacteroidetes was greater in the proximal GIT, while Firmicutes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal. These results were published in Nature's Scientific reports (Yeoman et al. 2018. Sci. Rep. 8: 3197) and highlighted in various news articles, including the Bighorn county Newspaper (https://www.bighorncountynews.com/content/msu-faculty-publishes-breakthrough-study-cattle-microbes), on the Northern Radio News network (http://www.northernbroadcasting.com/our-brands/northern-news-network/), and online (http://www.montana.edu/news/17826/msu-faculty-publishes-breakthrough-animal-health-discovery-in-nature-s-scientific-reports). An additional study looking at oral, ruminal, and rectal samples collected from lambs over a 1 year period and compared to environmental and maternal profiles has also been completed and is currently being analyzed and prepared for publication. Objective 2: 99 Metabolomic samples collected from lambs from pre-parturition (i.e. amniotic fluid, fetal rumen, abomasum, and meconium), peri-parturition (amniotic fluid), and up to 360 days post-parturition (rumen and fecal) have been profiled. A subset of these samples have been analyzed and presented (East et al. 2017) and are currently being prepared for a manuscript. Objective 3: Several studies on systems ranging from Soil, Rainbow trout, Gorillas, and humans have been undertaken with methods being refined for the integrated analyses of omics data (including metabolomics and metagenomics), and the understanding of factors such as dietary (including supplementation), environmental, and infection impact on microbial ecosystem compostion and function. Six manuscripts have resulted from this objective in the most recent reporting period, with another two presently in review. Objective 4: Eight microbes have been identified from molecular studies that show strong correlative relationships with animal health and/or performance and have been sourced from collaborative relationships with the multi-national rumen microbial census group. These strains were collected as part of their Hungate 1000 objective. Metabolomic data continues to be analyzed and mined for metabolites of potential importance. No efforts have been made in this reporting period to identify genes related to animal health or performance.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Perea K, Perz K, Olivo SK, Williams A, Lachman M, Ishaq SL, Thomson J, Yeoman CJ. 2017. Feed efficiency phenotypes in lambs involve changes in ruminal, colonic, and small intestine-located microbiota. Journal of Animal Sciences. 95(6):2585-2592. doi: 10.2527/jas.2016.1222
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ishaq SL, Yeoman CJ, Whitney TR. 2017. Ground redberry juniper and urea in supplements fed to Rambouillet ewe lambs. Part 2: Ewe lamb rumen microbial communities. Journal of Animal Sciences. 95: 4587. DOI: 10.2527/jas2017.1731
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yeoman CJ, Ishaq SL, Bichi E, Olivo SK, Lowe JL, Aldridge BM. 2018. Biogeographical differences in the influence of maternal microbial sources on the early successional development of the bovine neonatal gastrointestinal tract. Scientific Reports. 8: 3197. DOI:10.1038/s41598-018-21440-8
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ishaq SL, Page CM, Yeoman CJ, Murphy TW, Van Emon ML, Stewart WC. 2019. Zinc-amino-acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not. Journal of Animal Sciences. In Press

Progress 07/01/17 to 09/30/17

Outputs
Target Audience:To date, the progress has been limited to the preparation of a grant application and re-analysis of existing data. Therefore we have not yet disseminated our findings to our target audiences beyond the person's reviewing our grant. However, papers are already in preparation which point to potential target microbes and metabolites. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate student (Dusti East) has been involved in the analysis of the metabolomic data How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are currently analyzing data and drafting several manuscripts describing longitudinally collected microbiome and metabolome samples and data of cattle and sheep from prior to birth through until up to 1 year of age. Metagenomic analyses will be conducted in the next period if funding permits. We will continue to identify potential metabolites and microbes for strong correlative relationships to health and productivity and further seek to secure funding for their isolation and characterization.

Impacts
What was accomplished under these goals? 1. Metabolomic data has been obtained and is currently being analyzed (Goal 2) 2. A microbial taxa has been identified as of great interest, having strong correlative relationships to animal health and productivity (Goal 4). A Seed grant application has been submitted to the 2017 USDA-NIFA AFRI Animal Health & Production & Animal Products Foundational call to pursue research into this organsim.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Yeoman CJ, Ishaq SL, Bichi E, Olivo S, Lowe J, Aldridge BM. Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract. In Review Scientific Reports

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Researchers in the scientific fields of animal sciences and microbiology/microbiome via publications and presentations and students and stakeholders via presentations and opportune discussion Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have been recruited and are presently developing cattle ileum and cecal organoids models and isolating methylcaconitine-degrading bacteria from wild rumen samples, respectively. Additionally, three undergraduate students have been working to learn anaerobic microbiological techniques, organoid and primary cell culture techniques, and general laboratory techniques. Finally, one postdoctoral fellow has been training in addvanced bioinformatic andd biostatistical techniques relatedd to the objectives of this proposal. How have the results been disseminated to communities of interest?Results have been disseminated through 4 new publications described in this report, through online and in-personteaching, and in oral presentations and interactions with stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Several manuscripts are awaiting submission due to various factors, including collaborative and regulatory approval.Grant applications are being developed to further support these research efforts, we presently have bothprivate foundation and federal support. We arerecruiting 1-2 graduate students to advance our work to assess the ability of microbial strains to stimulate and modulate immune function in vitro using bovine small intestinal cell lines and organoids. Additionally, we are working to isolate and identify microbes capable of degrading the major larkspur alkaloid toxin, methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle for testing in animal trials.

Impacts
What was accomplished under these goals? 1. 16S rRNA-basedd methods have been used to assess the microbial changes associated with early succession of both the bovine and ovine gastrointestinal tract. These findings have been published and are subject to additional publications in preparation. 2. GC-MS-based metabolomic data have been developed for the successional development of the ovinegut and is being prepared for publication. 3.Methods have and are continuing to be developed through analyses of a variety of systems, includding bovine, ovis, primate, human, insect, and even soil microbiomes. Major emphases are on the integration of multiple -omics datasets and efforts have resulted in several manuscripts that have been and are in line for publication focussed onunderstand microbial ecology and the microbiomesinfluence on immunological parameters and health.These cross-species analyses continue to provide new insights aiding our interpretation and understanding of more directly ag-related systems. 4.We have identified 56microbial taxa that are being tested for their influences on calf immune development and functional response to endotoxin andd pathogenic challenge. Additionally,we are isolating microbe(s) that are present in the ruminal content of various wild species that facilitate the degradation ofmethyllycaconitine, the primary alkaloid toxin in Larkspur.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled FD. 2020. Soil bacterial communities of wheat vary across the growing season and among dryland farming systems. Geoderma. 358: 113989
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sanglard LP, Schmitz-Esser S, Gray KA, Linhares DCL, Yeoman CJ, Dekkers JCM, Niederwerder MC, Ser�o NVL. 2020. Vaginal microbiota diverges in sows with low and high reproductive performance after porcine reproductive and respiratory syndrome vaccination. Scientific Reports. 10: 3046 DOI: 10.1038/s41598-020-59955-8
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Borgogna JC, Shardell MD, Brotman RM, Yeoman CJ, Ghanem KG, Kadriu H, Ulanov AV, Gaydos CA, Hardick J, Robinson C, Ravel J, Bavoil PM, Tuddenham S. 2020. Vaginal metabolomics profiles: comparing uninfected, C. trachomatis mono- and C. trachomatis/M. genitalium co-infected women. Scientific Reports. 10: 3420 DOI: 10.1038/s41598-020-60179-z
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled F. 2020. Dryland cropping systems, weed communities, and disease status modulate the effect of climate conditions on wheat soil bacterial communities. mSphere. 5:e00340-20 DOI: 10.1128/mSphere.00340-20
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Yeoman CJ, Lachman MM, Ishaq S, Olivo S, Swartz J, Herrygers M, Berardinelli JG. 2020. Development of Climactic Oral and Rectal Microbiomes Corresponds to Peak Immunoglobin Titers in Lambs. Conference of Research Workers in Animal Diseases (CRWAD). Virtual Conference. Dec 5 - 8.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ, Betiku OC, Gaylord TG, Ishaq SL, Sealey WM. 2020. A METAGENOMIC EXPLORATION OF THE FUNCTIONAL POTENTIAL OF GUT MICROBES TOWARD THE NUTRITIVE CAPABILITY OF RAINBOW TROUT (ONCORHYNCHUS MYKISS). World Aquaculture Society Meeting. Honolulu, HI. Feb 9.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ. 2020. Microbes & Agriculture: From Wheat Pests to Livestock. Crop & Pest Management School. Bozeman, MT. Jan 8.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yeoman CJ. 2020. An important microbial community lives within all of us. Provosts Distinguished Lecture Series. Bozeman, MT. Feb 11.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Results have primarily been disseminated to the scientific field via publications, to students through in-class teaching and associated practical activities, and to our departmental stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four students for a graduate-level nutrition class were involved in a study comparing the gut microbiology and productivity of sheep fed alfalfa that was either fed as hay or a reduced particle size pellet resulting in a publication and new insight into the influence of the physical structure of feed on the gut microbiota. A graduate student has also been recruited and is bioprospecting wild rumen samples for toxin degrading activities. How have the results been disseminated to communities of interest?Results have been disseminated through 10 publications described in this report, through in-class teaching, and associated practical activities (resulting in a publication) and in engagements with stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Several manuscripts are presently being prepared along with grant applications. Having recently obtained some private foundation support we are recruiting students to advance our work to assess the ability of microbial strains to stimulate and modulate immune function in vitro using bovine small intestinal cell lines. Our preliminary work looking at these host:microbe interactions has been used to develop a foundational grant that we have not yet heard back on due to the recent move of NIFA to KS but hope to get a positive review on to help accelerate our progress. Additionally, we are working to identify and isolate microbes capable of detoxifing the major larkspur toxin, methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle.

Impacts
What was accomplished under these goals? 1. Taxonomic data has been developed for the successional development of bovine and ovis gut microbiota, however, full metagenomic sequencing has not been completed. 2. Metabolomic data have been developed for the successional development of the ovis gut, is analyzed and is being prepared for publication. 3. Methods have and are continuing to be developed through analyses of bovine, ovis, primate, human, insect, and even soil microbiomes. Each of these have and are being analyzed to understand ecology, including the influence of diet, health, and productivity. These cross-species analyses continue to provide new insights aiding our interpretation and understanding of more directly ag-related systems. 4. Metabolites have been identified that correspond to diminished health, but none have robustly stood out as being associated with health from our current studies. We have also identified 8 microbial species that are being tested for their influences on immune development and function and are presently searching for microbe(s) that are facilitating a methyllycaconitine- (primary toxin in Larkspur) degrading activity that we have variably detected in wild rumen samples.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ishaq SL, Lachman MM, Wenner BA, Baeza A, Butler M, Gates E, Olivo S, Geddes JB, Hatfield P, Yeoman CJ. 2019. Pelleted-hay alfalfa feed increases sheep wether weight gain and rumen bacterial richness over loose-hay alfalfa feed. PLoS One 14: e0215797. DOI: 10.1371/journal.pone.0215797
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yeoman CJ, Brutscher LM, Esen �, ?bao?lu F, Fowler C, Eren AM, Wanner K, Weaver DK. 2019. Genome-resolved insights into a novel Spiroplasma symbiont of the Wheat Stem Sawfly (Cephus cinctus). PeerJ 7:e7548 DOI: 10.7717/peerj.7548
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Ishaq SL, Seipel T, Yeoman CJ, Menalled FD. 2019. Soil bacterial communities of wheat vary across the growing season and among dryland farming systems. Geoderma. In press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Sanglard LP, Schmitz-Esser S, Gray KA, Linhares DCL, Yeoman CJ, Dekkers JCM, Niederwerder MC, Ser�o NVL. 2019. Investigating the relationship between vaginal microbiota and host-genetics and their impact on immune response and farrowing traits in commercial gilts. Journal of Animal Breeding and Genetics. In press. DOI: 10.1111/jbg.12456
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lei B, Flores AR, Yeoman CJ, Liu M. 2019. Complete genome sequence of hypervirulent Streptococcus pyogenes emm3 strain 1838. Microbiology Resource Announcements. 8: e01494  18 DOI: 10.1128/MRA.01494-18
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Miles MP, Wilson S, Yeoman CJ. 2019. Physical activity and inflammation phenotype conversion. Journal of Clinical Exercise Physiology. 8: 64-73
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Pafco B, Sharma AK, Petrzelkova KJ, Vlckova K, Todd A, Yeoman CJ, Wilson BA, Stumpf R, White BA, Nelson KE, Leigh SR, Gomez A. 2019. Gut microbiome composition of wild western lowland gorillas is associated with age and sex factors. American Journal of Physical Anthropology. 9: 1202. DOI: 10.3389/fmicb.2018.01202.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gomez A, Sharma A, Mallott E, Petrzelkova K, Robinson CJ, Yeoman CJ, Carbonero F, Pafco B, Rothman J, Ulanov A, Vlckova K, Amato KR, Schnorr S, Dominy N, Modry D, Todd A, Torralba M, Nelson KE, Burns M, Blekhman R, Remis M, Stumpf RM, Wilson BA, Gaskins HR, Garber P, White BA, Leigh SR. 2019. Plasticity in the human gut microbiome defies evolutionary constraints. mSphere 4: e00271-19 DOI: 10.1128/mSphere.00271-19
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Borgogna JC, Shardell MD, Santori EK, Nelson TM, Rath JM, Glover ED, Ravel J, Gravitt P, Yeoman CJ, Brotman RM. 2019. The vaginal metabolome and microbiota of cervical HPV-positive and HPV-negative women: a cross-sectional analysis. British Journal of Obstetrics & Gynecology. In press. DOI: 10.1111/1471-0528.15981
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ishaq SL, Page CM, Yeoman CJ, Murphy TW, Van Emon ML, Stewart WC. 2019. Zinc-amino-acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not. Journal of Animal Sciences. 97: 687  697. DOI: 10.1093/jas/sky456.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The research performed to date are primarily of value to the research community, specifically those in the fields of microbiology, microbial ecology, and animal nutrition, health, and production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students (Kathryn Parea & Dusti East) have undertaken research internships with the lab and been trained in bioinformatic and biostatistical techniques of 16S rRNA gene and metabolomic data, respectively. One project resulted in a manuscript (Perea et al. 2017) and the other is currently being prepared as part of a larger publication. One MS student (Medora Lachman) is in continued training on an integrated project involving animal handling and husbandry, molecular techniques, including microbiome and metagenomic sample collection and processing, and bioinformatics. An additional graduate student (Deanna DeSonn) and undergraduate student (Eden Manuel) will be joining the lab during the next reporting period on research extending from the current work. How have the results been disseminated to communities of interest?Results have been disseminated through the publications described in this report, in the W4177 annual meeting, and in two invited talks: • Invited speaker Animal Microbiome Congress (Kansas City, KS) • Plenary speaker Midwestern Section of the American Society for Animal Sciences (Omaha, NE) What do you plan to do during the next reporting period to accomplish the goals?In addition to completing analysis, and writing and submitting manuscripts developed from data elaborated during the current funding period, we will advance objective 4 of research objectives by testing our newly acquired microbial strains for their ability to stimulate immune function in vitro using bovine small intestinal cell lines in collaboration with Dr. Mark Jutila here at Montana State U. We hope this provide compelling evidence to support a competitive foundational grant to rapidly accelrate our progress. Additionally, we have recieved private foundation funding to look at microbial detoxification of the major larkspur toxin methyllycaconitine, which is responsible for numerous cases of morbidity and mortiality among range cattle.

Impacts
What was accomplished under these goals? Objective 1: 16S rRNA gene sequence profiles have been obtained from both the lumen and mucosa of ten gastrointestinal tract regions (including, the pharynx, rumen, reticulum, omasum, abomasum, duodenum,proximal, medial, and distal jejunum, ileum, and colon of neonatal calves at 1, 3, 7, and 21 days post parturition. These samples were compared to each other and to 16S rRNA gene profiles collected from the dam's colostrum, outer udder skin, and vaginal scrapings collected at calving. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Bacteroidetes was greater in the proximal GIT, while Firmicutes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal. These results were published in Nature's Scientific reports (Yeoman et al. 2018. Sci. Rep. 8: 3197) and highlighted in various news articles, including the Bighorn county Newspaper (https://www.bighorncountynews.com/content/msu-faculty-publishes-breakthrough-study-cattle-microbes), on the Northern Radio News network (http://www.northernbroadcasting.com/our-brands/northern-news-network/), and online (http://www.montana.edu/news/17826/msu-faculty-publishes-breakthrough-animal-health-discovery-in-nature-s-scientific-reports). An additional study looking at oral, ruminal, and rectal samples collected from lambs over a 1 year period and compared to environmental and maternal profiles has also been completed and is currently being analyzed and prepared for publication. Objective 2: 99 Metabolomic samples collected from lambs from pre-parturition (i.e. amniotic fluid, fetal rumen, abomasum, and meconium), peri-parturition (amniotic fluid), and up to 360 days post-parturition (rumen and fecal) have been profiled. A subset of these samples have been analyzed and presented (East et al. 2017) and are currently being prepared for a manuscript. Objective 3: Several studies on systems ranging from Soil, Rainbow trout, Gorillas, and humans have been undertaken with methods being refined for the integrated analyses of omics data (including metabolomics and metagenomics), and the understanding of factors such as dietary (including supplementation), environmental, and infection impact on microbial ecosystem compostion and function. Six manuscripts have resulted from this objective in the most recent reporting period, with another two presently in review. Objective 4: Eight microbes have been identified from molecular studies that show strong correlative relationships with animal health and/or performance and have been sourced from collaborative relationships with the multi-national rumen microbial census group. These strains were collected as part of their Hungate 1000 objective. Metabolomic data continues to be analyzed and mined for metabolites of potential importance. No efforts have been made in this reporting period to identify genes related to animal health or performance.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Perea K, Perz K, Olivo SK, Williams A, Lachman M, Ishaq SL, Thomson J, Yeoman CJ. 2017. Feed efficiency phenotypes in lambs involve changes in ruminal, colonic, and small intestine-located microbiota. Journal of Animal Sciences. 95(6):2585-2592. doi: 10.2527/jas.2016.1222
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ishaq SL, Yeoman CJ, Whitney TR. 2017. Ground redberry juniper and urea in supplements fed to Rambouillet ewe lambs. Part 2: Ewe lamb rumen microbial communities. Journal of Animal Sciences. 95: 4587. DOI: 10.2527/jas2017.1731
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yeoman CJ, Ishaq SL, Bichi E, Olivo SK, Lowe JL, Aldridge BM. 2018. Biogeographical differences in the influence of maternal microbial sources on the early successional development of the bovine neonatal gastrointestinal tract. Scientific Reports. 8: 3197. DOI:10.1038/s41598-018-21440-8
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ishaq SL, Page CM, Yeoman CJ, Murphy TW, Van Emon ML, Stewart WC. 2019. Zinc-amino-acid supplementation alters yearling ram rumen bacterial communities but zinc sulfate supplementation does not. Journal of Animal Sciences. In Press

Progress 07/01/17 to 09/30/17

Outputs
Target Audience:To date, the progress has been limited to the preparation of a grant application and re-analysis of existing data. Therefore we have not yet disseminated our findings to our target audiences beyond the person's reviewing our grant. However, papers are already in preparation which point to potential target microbes and metabolites. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate student (Dusti East) has been involved in the analysis of the metabolomic data How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are currently analyzing data and drafting several manuscripts describing longitudinally collected microbiome and metabolome samples and data of cattle and sheep from prior to birth through until up to 1 year of age. Metagenomic analyses will be conducted in the next period if funding permits. We will continue to identify potential metabolites and microbes for strong correlative relationships to health and productivity and further seek to secure funding for their isolation and characterization.

Impacts
What was accomplished under these goals? 1. Metabolomic data has been obtained and is currently being analyzed (Goal 2) 2. A microbial taxa has been identified as of great interest, having strong correlative relationships to animal health and productivity (Goal 4). A Seed grant application has been submitted to the 2017 USDA-NIFA AFRI Animal Health & Production & Animal Products Foundational call to pursue research into this organsim.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Yeoman CJ, Ishaq SL, Bichi E, Olivo S, Lowe J, Aldridge BM. Biogeographical Differences in the Influence of Maternal Microbial Sources on the Early Successional Development of the Bovine Neonatal Gastrointestinal tract. In Review Scientific Reports