2008 Annual Report 1a.Objectives (from AD-416) Develop proteomic profiles of immune cells during the acute and chronic stages of mastitis and under different physiologic conditions known to be associated with immune suppression, such as parturition or nutritional imbalances. Identify proteins that are up- or down-regulated during these immunosuppressive states. Initiate studies into the function of these differentially regulated proteins. Develop proteome profiles of mastitis-causing bacteria isolated from in vivo conditions and in bacteria grown in vitro or in lab counterparts that are not considered highly virulent. Identify proteins that are up- or down-regulated across bacteria studied in each ecological state and initiate studies into the role these differentially regulated proteins play in the establishment of chronic infection of the mammary gland. Studies of immunomodulators to test their effectiveness at preventing disease by imposing them on a controlled reproducible mastitis challenge model. In the course of these studies we will discover and develop immunological reagents for the bovine and relevant wildlife animal species that will facilitate the discovery of innovative immunointervention strategies. 1b.Approach (from AD-416) The approach to this research project is through experimentation discover new insights into the mechanisms of how the bovine dairy cow immune system fights diseases. Failure of the dairy cow immune system results in numerous diseases, of which mastitis is the predominate disease resulting in an economic burden to the industry. Our research will investigate the host immune system – pathogen interaction with the goal of enhancing immune function. To accomplish this goal we will study how immune system functions are affected by nutritional status. We will also study a newly described immune system function in the context of the dairy cow. In addition, we will investigate how pathogenic bacteria adapt to the in vivo environment and escape immune clearance. Finally, we will add to the large animal immunological reagent toolbox, to aid in study of immunological questions in dairy animals for the entire research community. 3.Progress Report All work in progress is involved in research that is designed to give us a better understanding of the disease process of mastitis in dairy cattle. We have determined that bovine immune cells do convert Vitamin D into a form that can alter gene expression. We are currently working to determine which genes are regulated by Vitamin D. We will be using cutting-edge technologies, such as microarray and proteomics to answer this question. The work to date in this area has been shared at several scientific meetings in the form of posters and talks. We have obtained and have begun to analyze the large dataset of proteins whose expression changes when an E. coli strain that causes mastitis is grown in milk. We will work to define those proteins that may be good targets for disrupting bacterial viability in the mammary gland. This work has also been shared with stakeholders at two scientific meetings. Work on neutrophil extracellular traps has continued. However, this work has been impeded by the variability from animal to animal. This has required us to move to our contingency plans in our project plan. Antibodies specific for two very important immune molecules have been generated and purified. These two molecules, known as toll-like receptor (TLR) 2 and 4 are important receptors for bacterial cell components. When TLR 2 or 4 bind to the appropriate bacterial component, intracellular signals occur that begin an immune response. Ongoing are attempts to use these antibodies in experiments to quantify the amounts of TLR on various cell types. This research is addressed in the Action Plan for National Program 103 Animal Health under Component 2: Animal Genomics and Immunology; Problem Statement 2A: Mastitis. Work done by this research team is directly related to several of the Outputs described in the Action Plan: Effective vaccines to prevent mastitis; New biotherapeutic platforms based on protective host proteins to induce the cow’s innate immune response; and New management and nutritional schemes to prevent metabolic stresses contributing to immunosuppressive states in the dairy cow. 4.Accomplishments 1. Effect of Vitamin D on Immune Function. Recent discoveries have shown the importance of Vitamin D on the function of immune cells. Human immune cells can convert Vitamin D into a form that is able to alter gene expression and some of these genes are involved in antibacterial processes. Human studies have shown that insufficient levels of Vitamin D in the blood can affect the ability of immune cells to kill bacteria. We wish to determine if bovine immune cells can convert Vitamin D into a form that will alter gene expression in bovine immune cells. The goal of this work is to determine the role of Vitamin D in the health and well-being of a dairy cow and to determine that level of Vitamin D necessary for full immune cell function. We have determined that bovine immune cells do convert Vitamin D into a form that changes gene expression in the cells. This has been accomplished by using molecular biology techniques that allow us to show that a gene that is dependent on the active form of Vitamin D, was expressed. The health and well-being of dairy cows is of great concern to the dairy industry and consumers of U.S. dairy goods. Better understanding of the basic functions of the immune system of the cow will help us to develop therapeutics to solve disease problems common to the dairy industry. This research may also give farmers information about how much supplementation of their animal feed is necessary for a fully functional immune system in cows. This research is addressed in the Action Plan for National Program 103 Animal Health under Component 2: Animal Genomics and Immunology; Problem Statement 2A: Mastitis. 2. Changes in Bacterial Protein Expression when Grown in Milk. Mastitis is a problem that costs the dairy industry $2 billion a year. The most common cause of mastitis in dairy cattle is coliform bacteria, such as E. coli. Our research goal is to identify as many proteins as possible that are up or down regulated when the bacteria is grown in milk. Milk has many antibacterial factors that make bacterial growth difficult. Our hypothesis is that the bacteria will change the expression of a number of proteins that will then allow the bacteria to grow in milk. Knowledge of which proteins that may be necessary for bacterial growth in milk could lead to targeted therapeutics and a new generation of treatments for mastitis. We have gathered and analyzed a proteomic dataset of expression changes of 1,000 proteins in bacteria when grown in milk compared to laboratory media. Hundreds of proteins were shown to have significant expression changes in the two media. Determination of the proteins necessary for bacteria growth in milk could lead to rational therapeutics specific for environmental causes of mastitis in dairy cows. This research is addressed in the Action Plan for National Program 103 Animal Health under Component 2: Animal Genomics and Immunology; Problem Statement 2A: Mastitis. Work done by this research team is directly related to several of the Outputs described in the Action Plan: Effective vaccines to prevent mastitis; New biotherapeutic platforms based on protective host proteins to induce the cow’s innate immune response; New management and nutritional schemes to prevent metabolic stresses contributing to immunosuppressive states in the dairy cow. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Other Technology Transfer 3 Review Publications Kimura, K., Goff, J.P., Schmerr, M.J., Stabel, J.R., Inumaru, S., Yokomizo, Y. 2008. Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum. Journal of Dairy Science. 91(5):1852-1864. Lippolis, J.D. 2008. Immunological signaling networks: integrating the body's immune response. Journal of Animal Science. 86(14 Suppl):E53-E63.