Objective 1) Develop alternative strategies to replace or reduce the use of conventional antibiotics for improved growth, animal health and product safety. A. Develop alternative antimicrobials to treat or prevent diseases in swine and dairy. B. Develop transgene-expressing cell transplantation methods to enhance growth rate and to treat or prevent diseases in swine. C. Develop effective dietary/nutritional regimens that can be implemented to maintain the healthful character of the gut of weanling swine. Objective 2) In order to develop alternatives to antibiotic growth promoters, identify mechanisms underlying the growth promoting effects of antibiotics in swine. A. Establish which microbial population distribution patterns are predictive of GI health and efficient nutrient utilization. B. Identify biomarkers of gut health and efficient nutrient utilization that are associated with specific changes in the metabolomic profile of the weanling pig gut. Objective 3) Develop and/or utilize molecular tools to understand the role of genes relevant to health, growth or intestinal function in swine and dairy with the goal of identifying targets for alternatives to antibiotic growth promotants. A. Establish in vitro approaches (intestinal pig cell lines) to model the role of specific metabolites or cytokines in gut nutrient absorption and gut immunological responses. B. Develop and apply site-specific gene modifying technologies to modify intestinal epithelial cell function and metabolism. C. Target specific bovine genes for editing that are relevant to health, milk production and milk quality.
The unifying theme of the project is to determine ways to reduce the use of antibiotics in farm animals. Foremost is investigating the growth promotant mechanism(s) of antibiotics in the context of the pig’s gut microbiome, metabolome and proteome. To this end, we will identify alternative products and methods to replace the use of antibiotics as growth promotants in pigs, and to mitigate mastitis in dairy cattle. One potential approach to limit the use of antibiotics in farm animals is to change the expression of the animal’s genes via gene-editing. Novel antimicrobials based on bacteriophage endolysins will be tested with young pigs and as a means of early mastitis detections in dairy cows. Another approach will be transplantation of transgenically modified pig cells that secrete specific proteins conferring disease resistance. Other studies will examine the effects of promising probiotics in weanling pigs for growth support in the critical preweaning period. Coupled with this will be an examination of the weanling pig’s gut microbiome with prebiotic feeding in comparison to antibiotics. The final objective will be to establish novel pig ileal cell culture lines. Improved in vitro models would enable faster evaluations of microbe/pig gut interactions and of nutrient absorption and inflammatory responses in screenings of probiotic efficacy. Consistent in vitro models also provide a platform for testing the expression and effects of gene-editing on pig small intestine cells.
This is the final report for project 8042-31440-001-00D which terminates September 30th. This project was a complete redirection of research focus from previous cycles, focusing on the developing intestinal tract in the pig. Two scientists retired (cell biologist, molecular biologist) and two scientists were hired during the project (microbiologist, bioinformatician). There are currently two unfilled positions. At the project onset, preliminary research by ARS scientists at Beltsville demonstrated peptidoglycan hydrolases (PGHs) functioning as endolysins could kill C. perfringens bacteria. The genes for PGHs from thermophilic bacteria were then isolated, since PGH enzymes from these bacteria should be tolerant to the high temperatures during feed processing. Heat tolerance was confirmed and the PGH genes were manipulated to generate multiple fusion proteins, fusing the thermophilic protein with cell wall binding proteins. These lytic peptides effectively killed C. perfringens as determined by Minimum Inhibitory Concentration (MIC) and Turbidity Reduction (TR) assays. A successful collaboration was developed with a commercial partner (CRADA) that led to identification of commercially viable fusion peptides and resulted in funding for a post-doctoral fellow. With the hiring of a microbiologist at the beginning of this project, in vitro and in vivo work began to validate methodologies for piglet fecal microbiome and mycobiome studies. Feces were collected from birth through day 35 and samples were cultured for fungal populations. In vitro plating techniques were validated and showed that piglets have a low-level of fungi present in their feces at birth that steadily declines until about 1 week of age. Fungal levels were below detectable levels from roughly week 1 until the point of weaning. At weaning, piglet fecal fungal levels increased 10-fold with the observation that Kazachstania slooffiae is the predominant fungus in the mycobiome of the post-weaning pig. DNA isolation techniques were optimized for the piglet fungus, Kazachstania slooffiae, which resulted in the publication of the first draft genome of this significant gut commensal fungus. Analyses are ongoing to assemble and annotate the full genome. Concurrently, the culture conditions and growth parameters were determined for K. slooffiae and its ability to grow as a biofilm was confirmed. These data were then tested in vitro against bacterial pig gut isolates to determine if the behavior of the fungi was altered by gut bacteria, and it was determined that molecules formed by the bacteria and secreted into the culture supernatant were capable of altering fungal biofilm development. These data provide the basis for future studies to not only assess this novel in vivo effect of bacterial-fungal interactions in the piglet gut, but also identify the molecules behind the cross-kingdom network. 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 to feed piglets Kazachstania slooffiae during the weaning transition to look for enhanced growth and health. These recently completed experiments are the first to investigate the role of this fungus in piglet growth, intestinal health, and changes in susceptibility to infection. Most recently, ARS scientists discovered that many changes occur in the ileal microbiome during the first week post-weaning. Pigs were weaned at d21 and ileal samples were collected at d1, d21, d24, d28 and d35 of age. The microbial population at d24 of age was different than at d21 or d28, while the d35 ileal microbiome was similar to the d28 microbiome. Previous studies have not examined the microbiome during the first week post weaning and thus the acute changes in the microbial population at this age were unknown. These very rapid changes in the microbiome between weaning (d21) and d24 of age and then to d28 of age could be correlated with overall growth rate in the piglets. Thus, manipulation of the microbiome through the use of prebiotics, probiotics, and/or neutraceuticals which replace in-feed antibiotics could maximize growth rate and reducing post-weaning growth lag. We are using both classical statistics and machine learning approaches to identify these therapeutic targets. We are collaborating with the University of Maryland Eastern Shore and AH Pharma to pursue similar work in poultry and were recently awarded a NIFA 1890 grant “Exploring the practical application of microbiome analysis as a tool to improve poultry production”. Work on this project was hampered by Covid, like all projects in ARS. However, this project was further limited by the slow pace of farm renovation. The Beltsville Agricultural Research Center swine farm was forced to depopulate in 2019 so that space could be renovated to permit the farrowing of sows in a facility not contaminated with biofilm. The renovated space for farrowing is not yet completed. The impact of modern animal facilities cannot be overstated. ARS scientists have garnered the interest of Universities and Industry because of a nursery room that was renovated in 2019 into a space specifically for housing of weanling pigs for microbiome studies that eliminates cross contamination between pens (treatment groups) and can be effectively decontaminated between experimental trials. There is no better facility in this country for performing microbiome research in post-weaning pigs. An industry collaborator is specifically providing us with probiotics unavailable to most labs because of this unique space. Creation of new space or renovation of existing space is urgently needed for the rest of the swine farm. ARS scientists at Beltsville are fortunate to have the support of the Center administration and the National Program staff for modernizing the farm. As this project ends and looking forward to the next project, the ARS scientists are excited and enthused at the potential research opportunities that would be available with space designed for the current research needs. Current Year Summary For Objective 2.A, a fungal mock community was created from fecal fungal isolates from piglets at BARC during the weaning transition. These isolates were identified and utilized to create a reproducible mock community that could be utilized to assess potential points of analytical biases during microbiome investigations. Genome copy numbers were also determined to establish their role in potential biases. This data was submitted to Frontiers in Cellular and Infection Microbiology special research topic: “Beyond Bacteria: Exploration of the Complete Microbiome”. Determination of the optimal machine learning model for predicting pig weights from amplicon-based microbiome data was initiated. A workflow in R was identified to test and evaluate three machine learning (ML) models: random forest, support vector machine, and L2 regularize logistic regression. Funding for an ARS-headquarters postdoctoral associate was obtained to assist with this research. For Sub-objective 2.B, fecal samples from healthy piglets were prepared and sent for mass spectrometry metaproteomic analysis. These analyses will determine the proteins present in bacterial and host cells in the feces of healthy piglets at two timepoints, at weaning and after weaning. Understanding the fecal metaproteome is important for understanding host health in the mucosa and other regions, and ultimately, markers from the fecal metaproteome may be developed to indicate good and poor swine growth at an early age. Additional experiments: 1) An Antimicrobial Resistance and Alternatives to Antibiotics Award from the Office of National Programs was received in FY20 to collaborate with scientists at US-MARC to investigate the effects of feeding K. slooffiae during the weaning transition on growth and health of piglets. Due to COVID restrictions, this experiment was postponed until the second quarter of FY21. These data are the first to investigate the role of this fungus in piglet growth, intestinal health, and changes in susceptibility to infections. Analyses are ongoing and 2 publications are expected to result from these data in the next fiscal year. 2) Glucagon-like peptide 2 (GLP2), is a powerful hormone produced in the digestive system that can improve the health and healing ability of the digestive system. ARS scientists used the pig intestinal cell line IPEC-J2 to demonstrate that GLP2 directly communicates with intestinal epithelium to enhance the availability of amino acids. GLP2 reduced both the synthesis and breakdown of cellular protein in IPEC-J2 cells, thereby increasing the available pool of amino acids for transport to serve as nutrients for muscle and bone synthesis. Additional metabolic analyses confirmed that the IPEC-J2 cell line is metabolically similar to the intestinal epithelium, further validating the IPEC-J2 cell line as a model for intestinal epithelium in the pig. ARS scientists virtually attended and presented at the Joint Agency Microbiome Symposium to learn new technologies utilized across agencies in the field of microbiome science. Other virtual meetings presented at this year were the Agricultural Genome to Phenome Initiative (AG2PI) Field Days, American Society of Microbiology Microbe Meeting, and the Intelligent Systems for Molecular Biology Annual Meeting. ARS scientists were also interview by the scientific podcast, “Swine Nutrition Blackbelt”, regarding porcine research at Beltsville Agricultural Research Center.
Summers, K.L., Arfken, A.M. 2022. The gut mycobiome and animal health. Springer Nature Applied Sciences. https://doi.org/10.1007/978-3-030-90303-9_6.
Baldwin, R.L., Liu, M., Connor, E.E., Ramsay, T.G., Liu, G., Li, C. 2021. Transcriptional reprogramming in rumen epithelium during the developmental transition of pre-ruminant to the ruminant in cattle. Animals. 11(10):2870. https://doi.org/10.3390/ani11102870.
Summers, K.L., Foster Frey, J.A., Arfken, A.M., Davies, C.L. 2021. Draft Genome Sequences of Kazachstania slooffiae, isolated from post-weaning piglet feces. Microbiology Resource Announcements. https://doi.org/10.1128/MRA.00198-21.