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United States Department of Agriculture

Agricultural Research Service

2009 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
Putative stem cells from different regions of the mammary gland were isolated, the RNA amplified and transcriptome profiling initiated using microarray analysis. Gene expression by these cells will be compared with control epithelial cells. This will provide data to test the hypothesis that the cells under investigation are stem cells and should provide potential biological markers for their further study. Analysis of these cells is based upon our development of methodology (described in accomplishments) that permits the isolation of quality RNA from cells in tissue sections that were labeled with the synthetic nucleotide, bromodeoxyuridine, immunologically detected and isolated by laser microdissection.

As described in accomplishments, the initial phase of a project to evaluate the temporal, regional and cell specific responses to intramammary E. coli infection was completed. Using microarray analysis, the study identified the teat as an important first responder to infection. A manuscript was submitted for publication. Future studies will utilize laser microdissection of tissue sections to study the response of specific cell types within the bovine mammary gland to bacterial infection.

Additional experiments under Objective 1 (To identify disease resistance genes and immunological responses that influence the course of intramammary infection), were initiated to evaluate the relationship between sensitivity to lipopolysacharide (endotoxin) challenge and mastitis susceptibility. Previous research in beef cattle showed a correlation between a specific variation in the TNF-alpha gene and a high likelihood for a severe response to an immune challenge. A coliform mastitis challenge was performed on 12 dairy heifers with the relevant TNF-alpha gene variant. Two of three relevant genotypes were represented in the population of heifers challenged with E. coli. The relationship between variation in the TNF-alpha gene and patterns of oxidative stress markers and somatic cell counts are being evaluated. Two research centers have also sought to collaborate on this project. Data on 72 cows in one collaborative herd have been genotyped with regard to the TNF-alpha gene polymorphism. These data are being correlated with production data and somatic cell counts. A subpopulation of these animals is scheduled for coliform mastitis challenge in the start of the next fiscal year.

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.

1. Identified potentially new means to fight mastitis in dairy cows. Novel means to fight mastitis (infections of the mammary gland) in dairy cattle are needed by dairy producers to improve animal health and profitability of milk production. Scientists from the Bovine Functional Genomics Laboratory in Beltsville, MD studied changes in gene expression over time and among different regions of bacterial-infected mammary glands. Their data provided strong support for an important immune surveillance role played by the teat and the first documentation of an immune response by the teat to bacterial infection. These results provide a basis for future research to enhance the cow's response to mastitis pathogens and suggest that the teat may provide a site for immunization against bacterial infections.

2. Established laboratory techniques to recover RNA from mammary stem cells. Stem cells are unique in that they can develop into any cell type in the body. Scientists in the Bovine Functional Genomics Laboratory in Beltsville, MD have identified potential stem cells from mammary gland of dairy cattle based on their staining characteristics. They have also developed methods to recover RNA from these stained cells in order to study gene expression in this unique cell population. This work will permit ongoing efforts to characterize mammary stem cells and to develop additional molecular markers that will aid in their identification. The ability to identify and study gene expression in mammary stem cells will promote efforts to improve lactation efficiency and tissue regeneration following tissue damage resulting from infections of the mammary gland. Furthermore, the methodology is applicable to study of somatic stem cells in other tissues.

6.Technology Transfer

Number of Other Technology Transfer1

Review Publications
Aitken, S.L., Karcher, E.L., Rezamand, P., Gandy, J.C., Vandehaar, M.J., Capuco, A.V., Sordillo, L.M. 2009. Evaluation of Antioxidant and Proinflammatory Gene Expression in Bovine Mammary Tissue during the Transition Period. Journal of Dairy Science. 92:589-598.

Capuco, A.V., Connor, E.E., Wood, D.L. 2008. Regulation of Mammary Gland Sensitivity to Thyroid Hormones during the Transition from Pregnancy to Lactation. Experimental Biology and Medicine. 233(10):1309-1314.

Follett, R.F. 2007. Economic and Societal Benefits of Soil Carbon Management: Cropland and Grazing Land Systems. In: Kimble, J.M., Rice, C.W., Reed, D.R., Mooney, S., Follett, R.F. and Lal, R. editors. Soil Carbon Management: Economic, Environmental, and Societal Benefits. London, England: CRC Press, Taylor and Francis Group. p. 99-128.

Hill, S.R., Knowlton, K.F., Daniels, K.M., James, R.E., Pearson, R.E., Capuco, A.V., Akers, R.M. 2008. Effects of Milk Replacer Composition on Growth, Body Composition, and Nutrient Excretion in Pre-weaned Holstein Heifers. Journal of Dairy Science. 91(8):3145-3155.

Zhou, Y., Capuco, A.V., Jiang, H. 2008. Involvement of Connective Tissue Growth Factor (CTGF) in Insulin-like Growth Factor-I (IGF1) Stimulation of Proliferation of a Bovine Mammary Epithelial Cell Line. Domestic Animal Endocrinology. 35(2):180-189.

Daniels, K.M., Mcgilliard, M.L., Meyer, M.J., Van Amburgh, M.E., Capuco, A.V., Akers, R.M. 2009. Effects of Body Weight and Nutrition on Histological Mammary Development in Holstein Heifers. Journal of Dairy Science. 92:499-505.

Capuco, A.V., Akers, R.M. 2009. The origin and evolution of lactation. Journal of Biology. 8:37.

Boehmer, J.L., Bannerman, D.D., Shefcheck, K.J., Ward, J.L. 2008. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis. Journal of Dairy Science. 91(11):4206-4218.

Cates, E.A., Connor, E.E., Mosser, D.M., Bannerman, D.D. 2008. Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-kappaB activation and apoptosis. Comparative Immunology Microbiology and Infectious Diseases. 32(6):477-490.

Bannerman, D.D. 2008. Pathogen-dependent induction of cytokines and other soluble inflammatory mediators during intramammary infection of dairy cows. Journal of Animal Science. 87:10-25.

Rinaldi, M., Ceciliani, F., Lecchi, C., Moroni, P., Bannerman, D.D. 2008. Differential effects of alpha 1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production. Veterinary Immunology and Immunopathology. 126:199-210.

Rinaldi, M., Moroni, P., Paape, M.J., Bannerman, D.D. 2008. Differential alterations in the ability of bovine neutrophils to generate extracellular and intracellular reactive oxygen species during the periparturient period. The Veterinary Journal. 178:208-213.

Last Modified: 4/16/2014
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