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 Sub-objective 1.A, the retirement of key personnel with the essential scientific expertise resulted in no progress on a diagnostic luminometer to detect gram-positive bacteria. Similarly, the retirement of key personnel resulted in no progress in the testing of specific peptidoglycan hydrolases (PGH) in animal models. NIH/3T3 mouse cells were transfected with the swine influenza A nucleoprotein expression vector and five clonal cell lines containing the vector were established and cryopreserved. No progress was made on the cell-transplantation-based improvement of disease resistance in mice (Subobjective 1. B). The planned experiments were not possible because of the COVID-19 maximum telework policy. For Sub-objectives 2.A and 2.B, progress was made despite the challenges posed by the maximum telework policy. Preliminary metagenome analyses of the gut microbiome of piglets during the weaning transition were conducted, which will guide future metagenomic analyses related to pig performance. This information and the creation of analytical pipelines are essential to future experimental implementation. Further, DNA isolation techniques were optimized for the piglet fungal commensal, Kazachstania slooffiae, which resulted in submitting the first draft genome of this significant gut commensal fungus for publication. The genome data suggest a highly complex, potentially polyploid genome, and 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 gut bacteria altered the behavior of the fungi, 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 were published and provide the basis for future study to assess this novel in vivo effect of bacterial-fungal interactions in the piglet gut and identify the molecules behind the cross-kingdom network. For Objective 2.A, a fungal mock community was created from fecal fungal isolates from piglets at Beltsville Agricultural Research Center (BARC) during the weaning transition. These isolates were identified, five were chosen to investigate in more detail, and their 18S and ITS copy numbers were determined. Based on these defined fungi, a detailed assessment of fungal primers, PCR conditions, and other amplicon-based data was performed to provide better standard procedures when performing mycobiome studies. A manuscript is being prepared, and it is expected that these data will be published in the next fiscal year. For Objective 2.A, it was 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 not known. These very rapid changes in the microbiome between weaning (d21) and d24 of age and then to d28 of age could be correlated with the overall growth rate in the piglets. The relationship between specific bacterial families or genera with an average daily gain during the very early post-weaning period may be important for maximizing growth rate and reducing post-weaning growth lag. These changes in microbial distribution associated with higher growth rates could be potential targets for prebiotics/nutraceuticals. Another possibility is the use of specific bacteria families or genera that were correlated with growth as potential probiotics that serve as effective substitutes for antibiotics in promoting early post-weaning growth. An example of a potential probiotic is Clostridium scindens, the only bacteria analyzed in the microbiome that was positively correlated with the growth rate in the preweaning pig (R-squared = 0.604, P < 0.001). For Objective 2.B, the ontogeny of several important gut peptides from birth through the weaning transition was characterized. These gut peptides included glucagon-like peptide 1 (GLP1), GLP2, gastric inhibitory peptide (GIP), and serotonin (5HT). Plasma samples were collected at d1, d21, and d35 of age (n = 44), from birth through the weaning transition and into the nursery stage. The relationship of plasma concentrations of each peptide to the overall pig growth rate (average daily gain) was analyzed. None of these gut peptides (GLP1, GLP2, GIP, and 5HT) were correlated with growth rate (P > 0.05). Plasma 5HT was unaffected by age (P = 0.288). However, GLP1 and GLP2 plasma levels declined between birth and weaning (P < 0.001), while GIP declined between weaning (d21) and d35 of age (P < 0.001). Plasma insulin-like growth factor 1 (IGF1) is a well-characterized growth-promoting plasma peptide, and IGF1 was positively correlated with growth rate in these pigs (R-squared = 0.633, P < 0.0001). The data indicate that these gut peptides do not have an overall effect on growth rate but appear to respond to the nutritional and metabolic status of the pig between birth and the early post-weaning nursery stage of development. For Objective 3, the primary pig ileum explant culture system (established in FY18/19) exhibited variability in its response to lipopolysaccharide challenge, and investigations to correct this problem were interrupted by the COVID-19 maximum telework order. Further characterization of the Pig Ileum-1 (PI-1) cell line showed that the cell line was composed of at least two distinct epithelial cells types. One type was goblet-cell-like, as demonstrated by MUC2 gene expression, and the other cell type is undefined. The culture of pig ileum stem cells in monolayer culture (2D culture) was achieved. Optimal growth conditions of the stem cells are being investigated, and their ability to differentiate into the various epithelial cell types of the pig ileum remains to be characterized. 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 the 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. ARS scientists virtually attended the Keystone Microbiome Conference to learn new technologies in the field of microbiome science with an emphasis on maternal to offspring effects. This year, other virtual meetings were the Swine Research Forum and the National Bio and Agro-Defense Symposium to continue education in swine physiology. ARS scientists were invited to participate in the Agriculture and Agri-Food Canada (AAFC) – USDA joint workshop on Antimicrobial Resistance in Swine and were also interviewed by the scientific blog, “Curious Kocab”, regarding porcine research at Beltsville Agricultural Research Center.
Summers, K.L., Foster Frey, J.A., Arfken, A.M. 2021. Characterization of Kazachstania slooffiae, a proposed commensal in the porcine gut. The Journal of Fungi. 7(2):146. https://doi.org/10.3390/jof7020146.