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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGIC STRATEGIES TO IMPROVE MILK PRODUCTION EFFICIENCY AND CONTROL MASTITIS
2007 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.


4.Accomplishments
Developed a peptide for the treatment of Gram-negative bacterial infections of cattle. Most of the clinical infections and mortality associated with food-animal neonates result from Gram-negative bacteria and the ensuing host inflammatory response to the bacteria. Current strategies for the treatment of mastitis and systemic infections caused by Gram-negative bacteria remain suboptimal. Thus, new therapeutics are needed to control these infections. We have identified a peptide derived from human bactericidal/ permeability-increasing protein (BPI) that has antimicrobial and toxin neutralizing activity against an array of mastitis pathogens. This combined activity enables the peptide to both kill the pathogenic bacteria and neutralize the harmful toxins released by these bacteria. Because it has broad-spectrum activity and retains activity in serum, this peptide has therapeutic applicability to the treatment of an array of systemic infections in cattle. 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.

Established a therapeutic application for a Toll-like receptor agonist in the treatment of Staphylococcus aureus-induced mastitis. Staphylococcus aureus (S. aureus) are one of the most prevalent bacteria to cause mastitis and intramammary infections caused by these bacteria are difficult to treat and can persist throughout the lifetime of the animal. We have demonstrated that bacterial lipopolysaccharide, which activates the host immune receptor Toll-like receptor-4, can reduce intramammary S. aureus bacterial concentrations by 10-fold. This finding provides evidence for the therapuetic application of Toll-like receptor-4 agonists to the treatment of mastitis and offers a new treatment option for the management of this disease. 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.

Developed and validated assays for measuring bovine neutrophil reactive oxygen species. Neutrophil-derived reactive oxygen species (ROS), which are released in response to infection, can cause injury to host tissues. Because there is a need for anti-inflammatory compounds that can minimize injury to the host without impairing the host’s ability to clear harmful bacterial pathogens, development of assays that can specifically measure bovine ROS are useful for screening and identifying novel compounds with such activity. We have now developed and validated assays that can discriminate between intracellular ROS that are critical to the control of infection and extracellular ROS that can damage host tissues. These assays provide a means for measuring key molecules critically involved in the innate immune response of the bovine mammary gland during mastitis. Further, these methods are currently being used to identify novel anti-inflammatory compounds. 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.

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.


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

Number of new CRADAs and MTAs3
Number of active CRADAs and MTAs5
Number of invention disclosures submitted2
Number of non-peer reviewed presentations and proceedings17

Review Publications
Burvenich, C., Bannerman, D.D., Lippolis, J.D., Peelman, L., Nonnecke, B.J., Kehrli, Jr., M.E., Paape, M.J. 2007. Cumulative physiological events influence the inflammatory response of the bovine udder to Escherichia coli infections during the transition period. Journal of Dairy Science. 90(E.Suppl.):E39-E54.

Kauf, A.C., Rosenbusch, R.F., Paape, M.J., Bannerman, D.D. 2007. Innate Immune Response to Intramammary Mycoplasma bovis Infection. Journal of Dairy Science. 90(7):3336-3348.

Kauf, A.C., Vinyard, B.T., Bannerman, D.D. 2007. Effect of intramammary infusion of bacterial lipopolysaccharide on experimentally induced staphylococcus aureus intramammary infection. Res. Vet. Sci. 82(1):39-46.

Kim, F., Pham, M., Luttrell, I., Bannerman, D.D., Tupper, J., Thaler, J., Hawn, T.R., Raines, E.W., Schwartz, M.W. 2007. Toll Like Receptor-4 Mediates Vascular Inflammation and Insulin Resistance in Diet-Induced Obesity. Circulation Research. 100(11):1589-1596.

Paape, M.J., Wiggans, G.R., Bannerman, D.D., Thomas, D.L., Sanders, A.H., Contreras, A., Moroni, P., Miller, R.H. 2007. Monitoring goat and sheep milk somatic cell counts. Small Ruminant Research. 68(1-2):114-125.

Rinaldi, M., Moroin, P., Leino, L., Paape, M.J., Bannerman, D.D. 2006. Effect of cis-urocanic acid on bovine neutrophil generation of reactive oxygen species. Journal of Dairy Science. 89(11):4188-4201.

Rinaldi, M., Moroni, P., Paape, M.J., Bannerman, D.D. 2007. Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species. Vet.Immunol. Immunopathol. 115(1-2):107-125.

Sohn, E.J., Paape, M.J., Bannerman, D.D., Connor, E.E., Fetterer, R.H., Peters, R.R. 2007. Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8. Vet. Res. 38(1):95-108.

Last Modified: 4/17/2014
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