Location: Animal Biosciences & Biotechnology Laboratory
2018 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
This project is a complete redirection of research focus from previous years for the scientists currently working on this project. While two scientists are working in completely new areas of research, the third scientist is newly hired. Thus progress primarily centered on developing the essential tools for studies that will occur in future years of the project.
For Sub-objective 1.A.1., a variety of filters were tested for the ability to lower the ATP content of the milk. Ahlstrom filter paper #141 could remove >75% of the ATP in the milk from somatic cells. Another way to remove the ATP from somatic cells is to selectively break these cells open and then use enzymes to eliminate the released ATP. Once this ATP is removed, this enzyme must also be removed so that it does not eliminate the ATP to be released later from the bacteria. Apyrase is an enzyme that is very efficient at breaking down ATP, but is difficult to eliminate from the reaction. One solution would be to add an inhibitor of apyrase to the reaction. We are currently screening several such inhibitors. A CRADA partner has recently demonstrated a newly engineered luminometer with the potential to provide the needed sensitivity for this diagnostic assay. However, ARS scientists have not had sufficient access to the device to test its sensitivity and performance consistency.
For Sub-objective 1.A.2, peptidoglycan hydrolases (PGHs) can be used to kill S. suis and C. perfringens bacteria and represent alternatives to traditional antibiotic treatment. As C. perfringens is commonly associated with infections of the intestine, it would be useful if PGHs could be incorporated into animal feed; however, this requires these enzymes to be resistant to the processing temperatures that occur with feed manufacture. To that end, genes for PGHs were found in the DNA of thermophilic bacteria. The proteins from these genes should be more resistant to heat as these bacteria are associated with hot springs. These genes were manipulated to generate a set of 15 recombinant proteins where thermophilic proteins were fused to cell wall binding proteins. These proteins were tested for their ability to kill C. perfringens strains isolated from chickens, cows, and swine. Some of the new recombinant proteins killed these C. perfringens strains and had increased resistance to a heat challenge. Further research on the utility of these proteins in treatment of animals will continue in a new collaboration with a commercial partner (CRADA). Similarly, a bioinformatic search identified genes for several PGHs that are predicted to target S. suis. Candidate genes were synthesized, and their encoded proteins were produced. Testing of these proteins for the ability to kill S. suis is currently ongoing with a collaborator at the University of Maryland.
For Sub-objective 1.B.1, 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 made. The vector will be used to put the SIV-NP gene into mouse and pig cells in vitro so that the cells can then produce the SIV nucleoprotein. The SIV-NP producing cells will be transplanted into mice and pigs to theoretically produce an immune response against swine influenza. A matching anti-SIV-NP antibody has been acquired to verify protein expression.
For Sub-objective 1.B.2, protocols for the preparation of alginate encapsulated cells were investigated. The alginate covering protects the cells from the host immune response directed against the foreign cells after transplantation into animals. 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 and after transplantation. Two methods for keeping track of the injection site (internally and externally) of the transplanted cells were tested in pigs: tattooing ink and red fluorescent microbeads. Both methods worked well and both will be used for locating the cells after subcutaneous injections of GFP-expressing or SIV-NP expressing cells into mice or pigs.
For Sub-objective 1.C., progress was made in validating in vivo and in vitro methodologies for future analysis of the gut microbiome in piglets. In vitro techniques were validated using the porcine jejunal cell line, IPEC-J2, and methodologies were validated for ELISA, RT-PCR, and gene expression in this cell line. Several cytokines, including IL-6 and IL-8 are elevated following exposure of IPEC-J2 cells to bacterial antigens such as peptidoglycan, lipoteichoic acid and LPS. Pretreatment with sodium butyrate, a short chain fatty acid, helped to mitigate the inflammatory reaction to these antigens.
In vivo methodologies were validated through the extraction of gastrointestinal tissues from 20 pigs. DNA and RNA extraction protocols were established from tissue samples to be used for future high-throughput sequencing with the University of Michigan Microbiome Core. In addition, primers have been designed and real-time quantitative PCR assays have been validated for quantifying the expression of more than 15 separate small intestine genes associated with gut health and function. ELISA assays have been validated for the gut/serum proteins in swine relevant to this project, while antibodies have been acquired and are currently in review for Western analysis using the iBright imaging system which permits rapid quantification of protein abundance.
Further in vitro and in vivo work was done to validate methodologies for piglet fecal microbiome 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. Fecal samples will be further analyzed by high throughput sequencing for microbiome analysis. Plasma samples from piglets in these studies demonstrated that poor-performing piglets have elevated histamine levels compared to littermates with normal growth rates.
For Sub-objective 2.A., two workshops were attended that focused on microbiome data analysis. The first workshop was the “R Workshop” sponsored by the University of Michigan in February 2018. The second workshop was the NEA Microbiome Conference in March 2018 that provided training. In addition, a professional relationship with a computational biologist was fostered for assistance with future data analysis. A contract was established to purchase CLC Microbiome Workshop Software that will permit statistical analysis of porcine microbiome data.
For Sub-objective 3.A., the culture of isolated and purified pig small intestine epithelial cells was investigated for establishing pig small intestine cell lines growing in monolayer culture. The surface epithelial cells of the intestinal villi were found to be very sensitive to detachment from their underlying stromal tissue, i.e., 99.99% die quickly after detachment. Various “substrates” (i.e., surfaces), including the use of mouse and pig “feeder-cells”, were tested for their effectiveness in supporting the growth and in vivo-like character of the pig intestine’s epithelial cells. Initial results indicate that the intestinal epithelial cell will attach to various substrates, but then will not proliferate on those substrates. No factors or hormones so far tested have improved the survival or stimulated the replication of the intestinal epithelial cells, relative to culture in serum-containing medium in ambient atmosphere.
Progress on a variety of pig small intestine “organoid” culture (explant culture of small, intact tissue) methods was made. Pig small intestine organoid cultures were examined by electron microscopy (EM) to assess cell survival and health after various culture times. EM analysis showed the organoid explant cultures remained healthy and retained their in vivo-like morphology for up to week in culture. The most promising culture method/in vitro model to date is free-floating or adherent organoid cultures. The cultured small intestine organoids of the pig are currently being characterized in comparison to intact in vivo tissue using histochemical and immunocytochemical methods, while additional samples are being collected for protein and gene expression analysis.
For Sub-objective 3.B., a targeting vector consisting of GFP has been generated with sequence flanking the last exon of the Villin gene to facilitate recombination and integration into last exon of Villin gene. A CRISPR (Clustered regularly interspersed palindromic repeats) single guide RNA targeting downstream of the last exon to facilitate double strand break and introduction of GFP sequence was generated and tested. Going forward, the targeting vector and the CRISPR ribonucleoproteins will be introduced into porcine cells and the targeted cells will be used to generate edited pigs via somatic cell nuclear transfer.
Accomplishments
1. Identification of elevated histamine levels in the plasma of poor-performing piglets. The period of weaning is a time of high stress in piglets and can result in some piglets performing poorly, costing swine producers more than $600 million dollars per year in lost production. Scientists at the Agricultural Research Service, Beltsville, Maryland, have documented a large increase in plasma histamine levels with weaning. This data suggests an allergic-type response may be preventing underperforming piglets from growing optimally. 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., Krasnec, K.V., Garrett, W.M., Shannon, A.E., Long, J.A. 2018. Finite cell lines of turkey sperm storage tubule cells: ultrastructure and protein analysis. Poultry Science. https://doi.org/10.3382/ps/pey208.
Ramsay, T.G., Stoll, M.J., Schreier, L.L., Shannon, A.E. 2017. Use of cross-fostering to enhance growth of pigs that are predicted to grow poorly based on plasma a-1 acid glycoprotein concentration. Open Journal of Animal Sciences. https://doi.org/10.4236/ojas.2018.81004.
Ramsay, T.G., Stoll, M.J., Shannon, A.E., Blomberg, L. 2018. Metabolomic analysis of longissimus from underperforming piglets relative to piglets with normal preweaning growth. Journal of Animal and Biotechnology. https://doi.org/10.1186/s40104-018-0251-3.
Verbree, C.T., Datwyler, S.M., Eichenseher, F., Meile, S., Donovan, D.M., Loessner, M.J., Schmelcher, M. 2017. Identification of peptidoglycan hydrolase constructs with synergistic staphylolytic activity in cow's milk. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.03445-16.
Talbot, N.C., Shannon, A.E., Phillips, C., Garrett, W.M. 2018. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31. In Vitro Cellular and Developmental Biology - Animals. 54:321-330.
Telugu, B.P., Sheets, T.P., Park, K., Park, C., Swift, S.M., Powell, A., Donovan, D.M. 2018. Targeted mutation of NGN3 gene disrupts pancreatic endocrine cell development in pigs. Journal of Animal Science. 8(1):3582. https://doi.org/10.1038/s41598-018-22050-0.
Ramsay, T.G., Elsasser, T.H., Caperna, T., Blomberg, L. 2018. a-1 acid glycoprotein inhibits insulin responses by glucose oxidation, protein synthesis and protein breakdown in mouse C2C12 myotubes. Animal. https://doi.org/10.1017/S1751731118001787.
