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United States Department of Agriculture

Agricultural Research Service

2010 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
An experiment was conducted to characterize gene expression in different cell types within the bovine mammary gland to gain insight into interactions among these cells during mammary growth and development. Cells from four locations within and near the growing mammary ducts of heifer calves were captured by microscopic dissection (laser microdissection) of histological tissue sections. Cells within the basal layer of the epithelium express a preponderance of genes that regulate cell differentiation, and enrichment of transcripts for stem cell markers. A non-coding maternally imprinted gene, worthy of future investigation, was identified in the basal epithelium. Gene expression by cells in the connective tissue surrounding mammary ducts was very different from that of cells isolated from connective tissue that is further from the epithelium.

Studies outlined in a subordinate project (USDA-CSREES-NRI grant 1265-32000-083-06R) are evaluating the impact of treating mammary glands of prepubertal heifers with a natural nucleoside (xanthosine). Impact of treating the prepubertal gland on mammary stem cell number and subsequent milk production are being evaluated.

A project to evaluate the temporal, regional and cell-specific responses to intramammary E. coli infection was completed and a manuscript published. Using a global analysis of gene expression, we demonstrated that the teat as an important first responder to infection. Future studies will utilize laser microdissection of histological tissue sections to isolate specific cell types of importance and to study their response to bacterial infection.

In addition to addressing milestones outlined in this project plan, we initiated and completed experiments to develop immortalized bovine mammary epithelial cells by using a nonviral passage of the hTERT-telomerase gene (see accomplishment). These cells are the first of a 2-component system under development to establish functional 3-D cultures of bovine mammary epithelial and endothelial cells. We also initiated development of immortalized endothelial cells; however, the fragile nature of these cells has delayed the final establishment of immortalized bovine endothelial cells. This work involves collaboration with the Angiogenesis Core Facility of NIH-NCI (Gaithersburg, MD).

The first dairy cow trial (72 cows) was completed to assess the prevalence and production impact of polymorphisms in the tumor necrosis factor-a gene promoter. Previous data from beef cattle suggested that the linked change from a “g” to an “a” (position -526) and “c” to “t” (position -701) genotype is associated with increased TNF-a mRNA expression in white blood cells as well as increased circulating concentrations of TNF-a in blood in vivo following repeated endotoxin challenge. In healthy dairy cows the presence of the polymorphisms was statistically linked to increased milk production parameters as might occur through increased immunosurveillance or more efficient tissue remodeling.

1. Identified location and potential markers for bovine mammary stem cells. Putative stem cells and control cells from different regions of the mammary gland were isolated and the expression of genes in these cells was evaluated at a comprehensive level. The data provided evidence that bovine mammary stem cells are located in the basal region of the mammary epithelium and more differentiated progenitor cells are located in suprabasal layers of the epithelium. Several candidate markers for mammary stem cells have been identified. Our approach was novel and definitive in that we were the first to isolate putative stem cells by a microscopic dissection procedure (laser microdissection) that permits isolation of cells from known positions within the mammary gland, rather than by enzymatically digesting the tissue to obtain single cells and then using cell sorting techniques. Once validated, our candidate markers for mammary stem cells will provide a tool to investigate methods to alter their activity. Because these cells are necessary for mammary growth and tissue repair, they provide important targets for enhancing milk production efficiency and udder health, and for meeting increasing demands to feed an expanding world population.

2. Developed a bovine mammary epithelial cell line without viral transformation. Primary bovine mammary epithelial cells were successfully immortalized using nonviral insertion of the hTERT-telomerase gene. We used this strategy because we believed the exogenous telomerase activity would maintain chromosome integrity, an essential component of conferring cell immortality. Telomerase is an enzyme that is necessary for maintenance of chromosome integrity. The relatively efficient immortalization of these cells was accomplished in collaboration with the NIH-NCI Angiogenesis Core Facility (Gaithersburg, MD). The cells will be made available for international distribution, through the American Type Culture Collection, and will facilitate study of bovine mammary cell physiology, as well as establishment of more complex intra- and inter-species model systems.

Review Publications
Daniels, K.M., Capuco, A.V., Mcgilliard, M.L., James, R.E., Akers, R.M. 2009. Effects of Milk Replacer Formulation on Measures of Mammary Growth and Composition in Holstein Heifers. Journal of Dairy Science. 92(12):5937-5950.

Rinaldi, M., Li, R.W., Bannerman, D.D., Daniels, K.M., Clover, C.M., Silva, M.V., Paape, M.J., Van Ryssen, B., Burvenich, C., Capuco, A.V. 2010. A sentinel function for teat tissues in dairy cows: Dominant innate immune response elements define early response to E. coli mastitis. Functional and Integrative Genomics. 10(1):21-38.

Soberon, F., Lukas, J.L., Vanamburgh, M.E., Capuco, A.V., Galton, D.M., Overton, T.R. 2010. The effects of increased milking frequency on metabolism and mammary cell proliferation in holstein dairy cows. Journal of Dairy Science. 93:565-573.

Last Modified: 9/1/2015
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