Location: Animal Biosciences & Biotechnology Laboratory
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
For Objective 1, the retirement of key personnel with the essential scientific expertise resulted in no significant progress on the development of a method to screen for gram positive bacteria in milk using a luminometer. A Cooperative Research and Development Agreement (CRADA) partner could not produce an adequately sensitive luminometer, essential to permitting detection of bacterial ATP utilization by ATPase, and the CRADA partner would not provide the device to this laboratory for independent testing. In addition, the retirement of key personnel resulted in a lack of knowledge and expertise to pursue developing animal models for C. perfringens testing of endolysins (enzymes that can digest the bacterial cell wall and that can kill this bacteria). However, great success was obtained in identifying and developing novel endolysins that can kill C. perfringens. Two particular lytic peptides were developed by either cleaving existing natural endolysins (a deletion mutant) or by fusion of two lytic domains from existing endolysins to create novel and highly active enzymes that can kill C. perfringens as determined by Minimum Inhibitory Concentration (MIC) and Turbidity Reduction (TR) assays. These proteins have been expressed and purified for further analysis to determine if these two novel endolysins can function as substitutes for antibiotics in the treatment or prevention of C. perfringens infection.
Progress was made on the cell-transplantation-based improvement of disease resistance in pigs. A swine influenza virus (SIV) nucleoprotein (NP) gene expression vector was transfected into National Institute of Health (NIH)/3T3 clone 7 cells and into ST swine testis cells. The SIV-NP producing cells are being transplanted into mice and pigs to theoretically produce an immune response against swine influenza nucleoprotein. Protocols for the preparation of alginate encapsulated cells were further tested and clumped cells were found to survive the best (in vitro) in 120 µM diameter sized alginate beads. A swine testis (ST) cell line that expresses green fluorescent protein (GFP) was used in the alginate bead/cell preparations so that the cells could be assessed for survival by fluorescent microscopy before transplantation. The alginate covering should protect the cells from the host immune response directed against the foreign cells after they are transplanted into the mouse or pig. The ST-GFP cells were transplanted 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 weeks post-transplantation.
An experiment was planned for this year to evaluate the potential for preweaning versus postweaning feeding pigs butyrate to reduce weaning stress and enhance gut function. This experiment requires individual housing of the animals to prevent cross contamination of their gut ecology. A consultation with Ag engineers was held in March 2018 to define the space, ventilation and temperature requirements for housing nursery pigs in Building 203. The renovation was delayed due to staffing shortages in the Facilities Office, who are responsible for writing the construction contract. A contract was finally awarded in December 2018, but was then cancelled due to the government shutdown. The contract has just been signed and the renovation should begin in the next several weeks, over a year since proposed. The butyrate experiment will be performed immediately upon completion of the renovation (estimate of August, 2019). One benefit of the proposed research is that ARS scientists have fostered an international industry collaboration with Perstorp, a major purveyor of animal feed supplements. A Perstorp representative visited BARC in February 2019 and provided butyrate reagents for the experiments during the weaning transition. Perstorp has expressed interest in funding future studies upon completion of the piglet microbiome facilities to investigate dietary supplementation of valeric acid during the weaning transition.
Objective 2 was met in establishing microbiome mathematical and statistical tools for research analysis. An Oak Ridge Institute for Science and Education (ORISE) postdoctoral research fellow with expertise in microbiome analysis was hired and worked in conjunction with ARS scientists to develop and implement microbiome analysis of swine fecal and gastrointestinal samples, resulting in a publication in the Journal of Animal Science.
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. Multiple meetings were held with the Statistics group at the Beltsville Agricultural Research Center (BARC) to ensure that our proposed mathematical analyses were sound. Further, the Microbiome Congress and the Symposium on Gut Health in Food Production Animals were attended to expand our knowledge in the field. A contract was established for the CLC Microbiome Workshop Software that will permit statistical analysis of porcine microbiome data.
For Objective 3, the pig ileum explant culture system established last year (FY2018) was modified to culture under 95% O2/5% CO2 to improve the survival and in vivo-like quality of the explants. Validation of this improvement was assessed by light microscopy and tumor necrosis factor-alpha (TNF-a) production (measured by enzyme-linked immunosorbent assay [ELISA]) in response to LPS challenge. The TNF-a response to decreasing amounts of LPS exposure was measured to find the lowest amount of LPS that gave the maximum TNF-a production from the explants. Nearly a dozen phytochemicals known for their anti-inflammatory or anti-bacterial properties were tested on the pig ileum explant cultures at the optimized LPS dosage. Also, explant cultures were treated with several plant-derived extracts to measure their survival and anti-inflammatory responses (TNF-a ELISA) to the plant extracts, with or without LPS treatment.
Work on gene editing of swine has not progressed because of delays in hiring key personnel. A post-doc position was advertised in October, 2018 but due to the government shutdown, no candidate could be interviewed. A candidate has now been selected and the background check is currently being completed prior to the post-doc beginning work on the project. An ARS scientist position specializing in gene editing has been written and approved. This position has been placed in the ARS hiring queue.
Accomplishments
1. Characterization of the porcine mycobiome (fungus). The microbiota of the gastrointestinal tract in animals is recognized as a critical component of host health. Recently, the significance of the mycobiome (fungus) has been recognized, but culture-independent studies are limited, especially in swine. Weaning is a period of stress, dietary changes, and a predisposition to infections, making it a time point of interest to industry. ARS scientists at Beltsville, Maryland, performed the first in-depth analysis of the mycobiome during the piglet weaning transition and they demonstrated a dynamic shift in fungal populations at the time of weaning, including the dominance of the fungus, Kazachstania slooffiae, in weaned piglets. This identification could be helpful in producing future interventions and dietary modifications to enhance piglet performance and increasing the productivity of the swine herd.
Review Publications
Talbot, N.C., Shannon, A.E., Garrett, W.M. 2019. Pancreatic duct-like cell line derived from pig embryonic stem cells: expression of uroplakin genes in pig pancreatic tissue. In Vitro Cellular and Developmental Biology - Animals. 55(4):285-301.
Swift, S.M., Donovan, D., Oakley, B.B., Waters, J.J., Rowley, D.T., Ramsay, T.G. 2018. Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens. FEMS Microbiology Letters. https://doi.org/10.1093/femsle/fny179.
Huang, D., Wang, L., Talbot, N.C., Huang, C., Tian, X., Zhang, M., Tang, Y. 0218. Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters. Stem Cells International. 13(10):e0203923.
Ramsay, T.G., Fernández-Fígares, I., Lachica, M., Martínez-Pérez, M. 2019. Conjugated linoleic acid and betaine affect lipolysis in pig adipose tissue explants. Animal-The International Journal of Animal Biosciences. https://doi.org/10.1017/S1751731119001186.
Rodriguez-Rubio, L., Donovan, D., Martinez, B., Rodriguez, A., Garcia, P. 2019. Peptidoglycan hydrolytic activity of bacteriophage lytic proteins in zymogram analysis. Veterinary Research. 1898:107-115.
Summers, K.L., Foster Frey, J.A., Ramsay, T.G., Arfken, A.M. 2019. The piglet mycobiome during the weaning transition: a ailot study. Journal of Animal Science. https://doi.org/10.1093/jas/skz182.
Seale, B., Dryder, D., Oakley, B.B., Brussow, H., Bikard, D., Rich, J.O., Miller, S., Devillard, E., Kwan, J., Bertin, G., Reeves, S., Swift, S.M., Raicek, M., Gay, C.G. 2018. Microbial-derived products as potential new antimicrobials. Veterinary Research. 49:66-78.
