2008 Annual Report 1a.Objectives (from AD-416) The objectives of the research project plan are as follows: . 1)to identify disease resistance genes and immunological responses that influence the course of intramammary infection;. 2)to discover and evaluate effective biotherapeutics for the prevention and treatment of bovine mastitis; and. 3)to develop strategies that promote cell replacement in the bovine mammary gland. 1b.Approach (from AD-416) To identify disease resistance genes that influence the course of intramammary infection (Objective. 1)we will: . 1)compare differential innate immune response patterns and host gene expression profiles that are elicited in response to intramammary pathogens that are readily cleared from the gland versus those that establish chronic infection;. 2)determine whether experimentally-induced inflammation enhances clearance of mastitis pathogens that cause chronic subclinical mastitis; and. 3)compare the inflammatory and gene expression responses of primiparous versus multiparous cows. To discover and evaluate effective biotherapeutics for the prevention and treatment of bovine mastitis (Objective. 2)we will: . 1)test the efficacy of intramammary infusion of recombinant bovine sCD14 as a means to recruit neutrophils and promote clearance of E. coli;. 2)test the effectiveness of the organic irritant dextran at dry-off to prevent new intramammary infections;. 3)evaluate the anti-inflammatory and microbicidal activity of bovine bactericidal-permeability increasing protein (BPI) in various biological fluids as an initial indicator of its utility in the treatment of intramammary and systemic infections; and. 4)evaluate the ability of cis-urocanic acid to inhibit neutrophil-induced respiratory burst activity and injury to the mammary epithelium. To identify strategies that promote cell replacement in the bovine mammary gland (Objective. 3)we will focus on the biology of bovine mammary stem cells, which are crucial for the proliferation replacement of mammary epithelial cells. In prepubertal heifers, we will: . 1)identify mammary stem cells by their ability to retain bromodeoxyuridine label for an extended time and develop genetic markers for these cells, by isolating them from tissue using laser microdissection, and performing microarray analysis to identify markers that distinguish stem cells from non-stem cells; and. 2)we will evaluate methods to promote expansion of the stem cell population in vivo by modulating key signaling pathways. 3.Progress Report We initiated a project to evaluate the response to E. coli infection of the mammary gland by region within the mammary gland and by cell type. Mammary glands of cows were infected with E. coli and tissues harvested at two times after bacterial infusion, from different regions within the mammary gland. Tissues were obtained from the region of bacterial entry (teat) to the region that would next be exposed to bacteria, gland cistern, and then to the upper secretory regions of the gland. Microscopic dissections will be used to isolate different cell types and gene expression evaluated by microarray analysis. As described in accomplishments, we completed experiments demonstrating that in vivo treatment with xanthosine expands the mammary stem cell population in dairy heifers, evaluated regulation of myoepithelial cell differentiation, and established therapeutic use for cis-urocanic acid in reducing mastitis-induced tissue injury. All research progress addressed “Component 2: Genetic and Biological Determinants of Disease Susceptibility; Problem Statement 2A: Mastitis” in the 2007-2012 National Program 103 Action Plan. 4.Accomplishments 1. Completed an experiment demonstrating that in vivo treatment with xanthosine expands the mammary stem cell population in dairy heifers. Findings were presented at the national meeting of the ADSA/ASAS and provide a strong base for future research in this area. Increasing expansion of mammary stem cells has the potential to enhance proliferation and replacement of mammary epithelial cells. This strategy may provide a tool for increasing the replacement of cells damaged by mastitis and for increasing lactation efficiency. This accomplishment addresses “Component 2: Genetic and Biological Determinants of Disease Susceptibility; Problem Statement 2A: Mastitis” in the 2007-2012 National Program 103 Action Plan. 2. Established a therapeutic use for cis-urocanic acid in reducing mastitis-induced tissue injury. Demonstrated that therapeutic concentrations of cis-urocanic acid can be readily delivered to the mammary gland via intramammary infusion. Also demonstrated that two indices of tissue injury, NAGase activity and LDH concentration, are both lower in infected glands treated with cis-UCA than in those infused with a control solution. This accomplishment addresses “Component 2: Genetic and Biological Determinants of Disease Susceptibility; Problem Statement 2A: Mastitis” in the 2007-2012 National Program 103 Action Plan. 3. Evaluated the regulation of myoepithelial cell differentiation. Demonstrated that estrogen enhanced differentiation of myoepithelial cells in mammary tissue of prepubertal dairy heifers and that this effect may be mediated by decreased expression of an inhibitor of muscle development, myostatin. Increased knowledge of mammary gland growth and differentiation will be important for developing tools for increasing the replacement of cells damaged by mastitis and for increasing lactation efficiency. This accomplishment addresses “Component 2: Genetic and Biological Determinants of Disease Susceptibility; Problem Statement 2A: Mastitis” in the 2007-2012 National Program 103 Action Plan. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Review Publications Rice, C., Baldwin, R.L., Abbott, L., Hapeman, C.J., Capuco, A.V., Le, A.N., Bialek Kalinski, K.M., Bannerman, D.D., Hare Jr, W.R., Paape, M.J. 2007. Predicting Perchlorate Exposure in Milk From Concentrations in Dairy Feed. Journal of Agricultural and Food Chemistry. 55:8806-8813. Bannerman, D.D., Kauf, A.C., Paape, M.J., Springer, H.R., Goff, J.P. 2008. Comparison of Holstein and Jersey Innate Immune Responses to Escherichia coli Intramammary Infection. Journal of Dairy Science. 91(6):2225-2235. Chockalingam, A., Mckinney, C.E., Rinaldi, M., Zarlenga, D.S., Bannerman, D.D. 2007. A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis. Veterinary Microbiology. 125(1-2):80-90. Chockalingam, A., Zarlenga, D.S., Bannerman, D.D. 2007. Antimicrobial activity of bovine bactericidal-permeability-increasing protein-derived peptides against gram-negative bacterial isolated from the mok of cows with clinical mastitis. American Journal of Veterinary Research. 68(11):1151-1159. De Schepper, S., De Ketelaere, A., Bannerman, D.D., Paape, M.J., Peelman, L., Burvenich, C. 2008. The Toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Veterinary Research. 39(1):(article #5)1-23. Gong, P., Angelini, D.J., Yang, S., Xia, G., Cross, A.S., Mann, D., Bannerman, D.D., Vogel, S.N., Goldblum, S.E. 2008. Toll-like receptor 4 signaling is coupled to src family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. Journal of Biological Chemistry. 283(19):13437-13449. Ibeagha-Awemu, E.M., Lee, J.W., Ibeagha, A.E., Bannerman, D.D., Paape, M.J., Zhao, X. 2008. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells. Veterinary Research. 39(2):(article #11)1-12. Connor, E.E., Matukumalli, L.K., Chockalingham, A., Bannerman, D.D., Sonstegard, T.S., Thomas, E.D., Van Tassell, C.P. 2008. Identification of polymorphisms within the bovine BPI and analysis of their associations with somatic cell score in Holstein cattle. Journal of Dairy Science. 39:456-457. Bannerman, D.D., Springer, H.R., Paape, M.J., Kauf, A.C., Goff, J.P. 2008. Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows to Staphylococcus aureus intramammary infection. Journal of Dairy Research. 75:291-301. Meyer, M.J., Rhoads, R.P., Capuco, A.V., Connor, E.E., Boisclair, Y.R., Van Amburgh, M.E. 2007. Ontogenic and nutritional regulation of steroid receptor and igf-i transcript abundance in the prepubertal bovine mammary gland. Journal of Endocrinology. 195:59-66. Capuco, A.V. 2007. Identification of Putative Bovine Mammary Epithelial Stem Cells by Their Retention of Labeled DNA Strands. Experimental Biology and Medicine. 232:1381-1390. Li, R.W., Capuco, A.V. 2008. Canonical Pathways and Networks Regulated by Estrogen in the Bovine Mammary Glands. Biomed Central (BMC) Genomics. 8:55-68.