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United States Department of Agriculture

Agricultural Research Service

2009 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
Mastitis is the most common disease that affects the dairy industry. Our work is designed to better understand the basic mechanisms of the immune response to this disease and how we can help the dairy farmer better protect their herds. We have determined that vitamin D can affect bovine immune cell gene expression. However, unlike human immune cells, the affect of vitamin D on bovine immune cells may affect signaling mechanisms more than pathogen killing mechanisms. The work to date in this area has been shared at several scientific meetings in the form of posters and talks.

We have published the protein expression changes of 1,000 E. coli proteins that were grown in unprocessed milk or laboratory media. Many of the proteins that showed significant expression changes are potential targets for novel therapies. This work was published in a leading proteomics journal and been shared with stakeholders at scientific meetings.

Work on neutrophil extracellular traps has concluded. We are exploring ways to continue this work.

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. These antibodies were used in confirmed proteomics data showing expression differences in milk and colostrum.

1. Effect of Vitamin D on Immune Function. 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 and 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 determine that a number of genes that are affected by vitamin D and a number of genes that are not. In contrast to humans, none of the bovine genes that generate small antibacterial proteins are affected by vitamin D concentration, which may be an important difference between human and bovine innate immune function. 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.

2. Changes in Bacterial Protein Expression When Grown in Milk. 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. 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 published 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. Many of these proteins have known or implied functions that affect their ability to cause an infection. Determination of the proteins necessary for bacteria growth in milk could lead to rational therapeutics specific for environmental causes of mastitis in dairy cows.

6.Technology Transfer


Review Publications
Lippolis, J.D., Bayles, D.O., Reinhardt, T.A. 2009. Proteomic changes in Escherichia coli when grown in fresh milk versus laboratory media. Journal of Proteome Research. 8(1):149-158.

Lippolis, J.D., Reinhardt, T.A. 2008. Centennial Paper: Proteomics in animal science. Journal of Animal Science. 86(9):2430-2441.

Last Modified: 8/28/2016
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