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.
For Objective 1, the retirement of key personnel with the essential scientific expertise resulted in no progress on demonstrating that effluent milk from mastitic cows can be used with luciferin/luciferase and peptidoglycan hydrolases in a luminometer to detect gram positive bacteria at a sensitivity of 100 CFU/ml. Similarly, the retirement of key personnel resulted in no progress on developing a strep suis animal model in collaboration with the National Animal Disease Center in Ames, Iowa). Limited progress was made on the cell-transplantation-based improvement of disease resistance in mice (Subobjectives 1.B.1). Five NIH/3T3 cell lines transfected with the Swine Influenza Virus Nucleoprotein (SIV-NP) expression vector were established and cryopreserved, but their expression of the recombinant protein remains to be confirmed before their injection (transplantation) into non-syngenic mice begins. Alginate encapsulated ST-GFP cells were injected subcutaneously into Swiss Webster mice for assessments of the cells’ survival following transplantation and for detection of an immune response by the mice to GFP over 3 week post-transplantation (Subobjective 1.B.2). However, these experiments were interrupted by the COVID-19 maximum telework order. Progress was made on additional subobjectives in Objective 1 as ARS scientists continued an international industry collaboration with Perstorp. A Perstorp representative provided tributyrate and valeric acid reagents for feeding experiments. The development and completion of a state of the art piglet microbiome facility and a swine metabolism room was accomplished in January 2020 and is currently in use for ongoing feeding experiments which have unfortunately been impeded by infectious outbreaks in the swine herd. Although progress could not be made on the designated experiments described in Objective 2, ARS scientists performed a pilot analysis of the metagenome of piglets throughout the weaning transition which will provide essential information when those experiments can be implemented. This longitudinal dataset is being analyzed to determine the metabolic changes in the cecum and colon during the weaning transition. Multiple meetings were held with the Statistics group at the Beltsville Agricultural Research Center (BARC) and with the University of Maryland to ensure that our proposed analyses were sound. Further training was attended through workshops with the National Institute of Health and the University of Maryland to enhance statistical analyses of metagenomic datasets. One publication is expected to come from this data within the next year. To continue to optimize financial expenditures, a contract with the University of Michigan Microbiome Core to isolate DNA, create libraries, and sequence samples was continued. A contract was continued for the CLC Microbiome Workshop Software that will permit statistical analysis of porcine data including microbiome, metagenome, and genomic datasets. An Oak Ridge Institute for Science and Education postdoctoral research fellow with extensive microbiome analysis experience continued to work in conjunction with ARS scientists to develop further analytical tools appropriate for fungal samples and to begin to analyze metagenome and full genome datasets. Analyses of the development of the bacteriome and mycobiome over time were completed, including inferred interactions between the bacteria and fungi in the piglet gut. These analyses resulted in two publications, one in Frontiers in Microbiology and one in Microorganisms. Expanding on the research tools necessary for Objective 2, ARS scientists isolated fungal species found in the feces of piglets and adult pigs in Beltsville, Maryland, identified, and cultured them to create a well-defined fungal mock community. This fungal mock community was created to investigate biases found from high throughput sequencing and the database used for analyzing fungal sequencing results. Preliminary studies investigating Illumina MiSeq platform results demonstrate that certain fungal species have enhanced sequencing bias over other fungal species and can result in skewed sequencing results. Further, database biases were shown that demonstrate that the choice of database can alter species identification. Scientists continue to work on additional analyses for publication. Further, the Symposium on Gut Health in Food Production Animals and the American Chemical Society Annual meeting were attended by multiple scientists from this project to expand our knowledge in the field and one scientist presented as an invited speaker. For Objective 3, the primary pig ileum explant culture system (established in FY2018/2019) was found to suffer from variability in its response to lipopolysaccharide challenge. The reason for the variability in the primary explant cultures was studied, but its source(s) has not yet been identified. Two unique pig small intestinal cell lines were created. The Pig Ileum-1 (PI-1) cell line has a goblet cell phenotype, i.e., at the RNA level it expresses mucin-2, and electron microscopy revealed the cells contained numerous, prominent mucus vesicles. This goblet cell phenotype makes the PI-1 cell line novel. The Pig Ileum-4 (PI-4) cell line is distinct from the PI-1 cell line in ultrastructure features and specific gene expression, but it remains to be fully characterized. 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-MARC to feed piglets Kazachstania slooffiae during the weaning transition to look for enhanced growth and health. These proposed experiments will be the first to investigate the role of this fungus in piglet growth, intestinal health, and changes in susceptibility to infection.
1. Genome sequencing of the piglet-associated fungus, Kazachstania slooffiae. The intestinal fungal population in animals has recently been recognized as a critical component of host health through its ability to interact with the host immune system and the host gut bacterial population. Weaning is a period of stress and environmental changes that lead to a predisposition to infection in piglets, making it a time point of interest for dietary interventions. ARS scientists at Beltsville, Maryland, isolated Kazachstania slooffiae, the most dominant post-weaning fungus in healthy piglets, optimized protocols to isolate high-quality fungal DNA, and were the first scientists to sequence this fungal genome. K. slooffiae has positive inferred interactions with beneficial bacteria in the piglet gut, suggesting a strong beneficial role in piglets; therefore, this genome is a critical first step for investigating the effects of this commensal in piglet growth and health. Because K. slooffiae is presumed to interact directly with the piglet gut and the bacteria present in the gut, ARS scientists are actively working to determine genetic components that result in fungal-host-bacterial interactions that can alter piglet health and growth. There is great promise that K. slooffiae can be utilized as a naturally-derived probiotic to enhance piglet growth.
Swift, S.M., Reid, K.P., Donovan, D.M., Ramsay, T.G. 2019. Thermophile lytic enzyme fusion proteins that target Clostridium perfringens. Antibiotics. https://doi.org/10.3390/antibiotics8040214.
Arfken, A.M., Foster Frey, J.A., Summers, K.L. 2020. Temporal dynamics of the gut bacteriome and mycobiome in the weanling pig. Microorganisms. https://doi.org/10.3390/microorganisms8060868.
Ramsay, T.G., Kahl, S., Long, J.A., Summers, K.L. 2019. Peripheral histamine and neonatal growth performance in swine. Domestic Animal Endocrinology. https://doi.org/10.1016/j.domaniend.2019.06.002.
Arfkin, A.M., Foster Frey, J.A., Ramsay, T.G., Summers, K.L. 2019. Yeasts of burden: exploring the mycobiome-bacteriome of the piglet GI tract. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.02286.