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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Animal Biosciences & Biotechnology Laboratory Research





- Davies, Cary
- Ramsay, Timothy
- 2023
- Publications
- Food Animal Production (101)

Research Project: Alternatives to Antibiotics: Developing Novel Strategies to Improve Production Efficiency in Swine **Location:
                            Animal Biosciences & Biotechnology Laboratory** 2023 Annual Report **Objectives**
                                

Objective 1. Determine interactions occurring across kingdoms (bacterial, fungal, porcine) at the microbe-lumen interface of the gut.
Sub-objective 1A: Identify quorum sensing molecules used by K. slooffiae.
Sub-objective 1B: Use microbiome datasets to determine interactions occurring between taxa, identify keystone taxa, and identify molecules present in EV that may be mediating these interactions.
Sub-objective 1C. Identify the mechanism of action for the QSM identified in the pig intestinal tract for biological effects on the IPEC-J2 jejunal cell line.
Objective 2. Identify alternatives to antibiotics through bioinformatics-based approaches, including the microbiome, metagenome, and metatranscriptome approaches, and examine their effects on the innate immune response at the gut mucosa.
Sub-objective 2A: Use ML to model microbiome data to identify species that are critical determinants of good and poor growth.
Sub-objective 2B: Identify potential probiotic candidates through in vivo testing of hypotheses generated during network analyses in Objectives 1B and 2A. 
Objective 3. Identify the role of Kazachstania slooffiae, a porcine fungal commensal, in pig performance through in vivo and in vitro studies. 
Sub-objective 3A: IPEC-J2 cell line challenges with live or heat killed K. slooffiae to analyze innate immune response. 
Sub-objective 3B: Dose-response feeding trial of K. slooffiae to post-weaning pigs.
Objective 4. Identify mechanisms of action of antibiotics and of their alternatives in promoting the growth and well-being of swine by examining metagenomes and metatranscriptomes in the swine GI tract.
Sub-objective 4A: Feeding trial in pigs with in-feed antibiotics. 
Sub-objective 4B. Dietary probiotic effects on pre- and post-weaning growth and intestinal physiology.
Sub-objective 4C. Evaluate Clostridium scindens to promote the preweaning growth of pigs.
Sub-objective 4D: Use ML in conjunction with metagenomic data to identify species, genes, and pathways that are critical in microbiome response to low dose antibiotics. **Approach**
                                

This project aims to determine the mechanisms behind antibiotic-induced animal growth and identify potential alternative growth promotants in swine during the weaning transition. Weaning is a critical point in piglet development marked by elevated stress and a predisposition to infections by opportunistic pathogens. These infections result in financial loss to farmers and producers due to increased mortality rates, reduced growth performance, increased feed costs, and veterinary expenses. Previously, in-feed antibiotics were utilized to prevent infections with the added benefit of antibiotic-associated weight gain, but the ban of in-feed antibiotics for agricultural animals presents a new challenge. Identification of alternative interventions and improved production strategies are needed to increase animal growth and disease resilience, but the mechanism behind antibiotic-induced growth remains unknown. We propose to utilize a combinatorial approach of in vitro, in vivo, and bioinformatics-based methods to identify mechanisms behind antibiotic-induced growth performance in piglets and physiological pathways altered by in-feed antibiotics to allow targeted identification of alternatives to antibiotics (ATA). These data will be utilized to optimize machine learning (ML) methods to identify microbiome members and molecules of interest in growth performance. These findings will be used to clarify the mechanism of growth promotion, thus permitting educated targeting of species and physiological pathways as ATA. Further, the microbial interactions in the gut of piglets will be analyzed to determine mechanisms behind cross-kingdom signaling that alter piglet health and growth during the weaning transition. This project will enhance the scientific understanding of the microbial network in the porcine gut and its role in growth promotion. This proposal will also determine physiological pathways altered by in-feed antibiotics to allow targeted identification of ATA in swine during the weaning transition. Farmers and producers will directly benefit from implementing validated alternate production management practices that will directly impact swine growth efficiency. **Progress Report**
                                

This is the report for the first year of project 8042-31440-002-000D. This project, similar to that of the prior cycle, aims to determine ways to reduce the use of antibiotics in farm animals. However, the current project focuses on the identification of mechanisms by which in-feed antibiotics confer positive health benefits in the weanling pig. This mechanistic understanding will facilitate the targeted development of antibiotic alternatives. At the beginning of the year, there were two unfilled positions, but one scientist resigned (microbiologist) in March 2023, therefore there are currently three unfilled positions. A support scientist with extensive experience in microbiology was hired. 

Under the prior project, ARS scientists received an Antimicrobial Resistance (AMR) and Alternatives to Antibiotics (ATA) award from the Office of National Programs to collaborate with scientists at US-Meat Animal Research Center (USMARC) to feed piglets Kazachstania slooffiae during the weaning transition to look for enhanced growth and health. Fecal and gastrointestinal organ samples (726 total) were sequenced using 16S (bacterial) and ITS (fungal) markers (milestone 2a) and analyses to determine the effect of feeding K. slooffiae on piglet growth and gut microbiome composition were completed. No effect of K. slooffiae on piglet growth, intestinal cell morphology, or immune response was observed. However, these data are the first to describe the mucosal micro- and mycobiome along the GI tract in piglets over time, and they also provide a robust dataset to correlate additional microbiome species with piglet growth. Samples were subdivided into “good” and “poor” growers, and a recently hired ARS Headquarters funded postdoc initiated machine learning analyses to identify additional microbial species, genes, and pathways which contribute to increased piglet growth.  

A subset of fecal samples from healthy piglets in the above USMARC project were also analyzed by advanced chemical methods to identify all proteins expressed by gut microbes at the time of weaning and two weeks post-weaning. Changes in protein expression during the shift from a milk to plant-based diet were established, and bacteria which play critical roles in digestion and production of bioactive metabolites such as short chain fatty acids (SCFAs) were identified. Gut microbes contribute to roughly 10% of digestion in humans, with values likely similar in piglets. In addition, secondary metabolites such as SCFAs and others are critical to host health. Thus, understanding gut microbial metabolism is a first step towards designing targeted therapeutics such as probiotics to manipulate gut microbiota and improve host health. Not only is this the first study to examine protein expression in the piglet gut microbiome during the weaning transition, but the development of advanced chemical methods for this work provides a foundation to apply similar methods to complete Subobjective 1c and 4a.

ARS scientists initiated a large scale PacBio metagenomic sequencing project in collaboration with FDA scientists to develop whole genome/metagenome references for microbes in piglet feces. PacBio sequencing provides the longest and highest accuracy reads currently available and will thus provide high quality genomes for many unsequenced organisms in the piglet gut. These references will permit the identification of microbes to the species and strain level and provide a robust gene catalogue for gut microbiome members, thus facilitating the completion of subsequent objectives which rely on bioinformatics approaches to identify mechanisms by which in-feed antibiotics contribute to piglet health (Objectives 2 and 4).

Quorum sensing molecules (QSM) are chemical signal molecules released by microbes whose secretion changes as the microbiome changes in the gut. QSM mediate communicate among microbes and orchestrate population level functions. Farnesol is a QSM that is produced in the intestinal tract by the fungus Candida albicans which is a common microorganism in the human gut and is the best characterized of these QSMs. ARS scientists at Beltsville, Maryland, demonstrated that farnesol inhibited cell proliferation rates and amino acid oxidation with no effect on protein synthesis in the IPEC-J2 pig jejunal cell line, indicating that farnesol specifically alters oxidative metabolism versus overall amino acid utilization by the gut. In addition, farnesol stimulated the secretion of interleukin-8, suggesting farnesol may induce an inflammatory response in the gut, although the secretion of another inflammatory cytokine, interleukin-6, was unaffected. The overall data imply that farnesol produced by the microbiome directly communicates with the cells lining the intestine in the pig to affect gut wall metabolism and immunological responses. Not all QSMs produced by the microbiome function alike. For example, ARS scientists at Beltsville, Maryland, demonstrated that indole (secreted by bacteria in the microbiome) stimulated cell replication in IPEC-J2 pig intestinal cell line but had no effect on protein synthesis or amino acid oxidation. Proliferation of cells derived from the pig gut was extremely sensitive to the stimulatory effect of indole, responding to the lowest concentration tested.

A high pressure liquid chromatography (HPLC) method was developed to detect and quantify the QSMs farnesol and tyrosol. Although it is well established that farnesol and tyrosol are produced by several yeast species, it is unknown whether the yeast K. slooffiae produces these molecules. Scientists have confirmed that K. slooffiae contains the genes necessary for production of these QSMs and will next utilize the newly developed HPLC method to assess whether K. slooffiae also produces these two molecules.

Renovation of the new farrowing barn (Building 203D) is ongoing. The electrical work is completed. Ventilation adjustments will be performed next week, with the plumbing installation to follow immediately. Contracts have been awarded for evaporative cooling installation and necessary metalwork for the farrowing crate installation. Sow facilities (east wing of 203) have undergone minor modifications to meet the requirements of the Animal Care Committee. The laboratory will purchase pregnant sows to reduce the time prior to starting experiments and with the expectation of farrowing in October, 2023. Requirements have been submitted to the Beltsville, Maryland, engineering office for the renovation of the east wing of Building 203 to house 36 sows and for the west wing to house 200 nursery pigs. **Accomplishments** **Review Publications**
                            *Davies, C.L., Summers, K.L., Arfken, A., Darwish, N., Chaudharil, A., Foster Frey, J.A., Schreier, L.L., Proszkowiec-Wegla, M.K. 2022. Temporal dynamics of the chicken mycobiome. Frontiers in Physiology. 13:1057810. https://doi.org/10.3389/fphys.2022.1057810.*
                                *Arken, A.M., Foster Frey, J.A., Carrillo, N.I., Dike, N.I., Onyeachonamm, O., Rivera, D.R., Davies, C.L., Summers, K.L. 2023. Porcine fungal mock community analyses: Implications for mycobiome investigations. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2023.928353.*


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