1a. Objectives (from AD-416):
The objectives of the research project plan are as follows: 1) to evaluate the genotypic and biologic basis of susceptibility to experimentally induced mastitis by Gram-positive and Gram-negative bacteria; 2) to determine the relationship between mastitis and nitro-oxidative stress and develop innovative alternatives to reduce tissue damage; 3) to develop strategies for modulating somatic stem cells to promote the replacement of mammary epithelial cells damaged by mastitis.
1b. Approach (from AD-416):
To evaluate the genotypic and biologic basis of susceptibility to experimentally induced mastitis by Gram-positive and Gram-negative bacteria (Objective 1) we will: 1) evaluate the response of dairy cows with divergent estimated breeding value for somatic cell score, and presumptive differences in mastitis susceptibility and/or responsiveness, to experimental challenge with Gram-negative bacteria; 2) evaluate the mastitis susceptibility of cows that differ with regard to single nucleotide polymorphisms in proinflammatory cytokine promoters, which influence the innate immune response. To determine the relationship between mastitis and nitro-oxidative stress and to further develop innovative alternatives to reduce tissue damage (Objective 2) we will focus on the relationship between oxygen-nitrogen adducts that are generated to combat invading pathogens and damage to mammary tissue, as reflected by increased protein nitration, and develop targeted biochemical interventions to reduce generation of nitrated proteins. We will: 1) evaluate the extent of protein nitration in mammary glands of control and experimentally infected mastitic cows; 2) evaluate the capacity of ' - and d -tocopherols to limit protein nitration during experimentally-induced mastitis by virtue of their ability to neutralize oxygen-nitrogen adducts. To develop strategies for modulating somatic stem cells to promote the replacement of mammary epithelial cells damaged by mastitis (Objective 3) we will: 1) evaluate the ability of xanthosine to promote the expansion of mammary stem cells and study the genomic response to xanthosine treatment in order to uncover critical regulatory pathways and stem cell modulators; 2) evaluate potential markers for bovine mammary stem cells that we identified in previous transcriptome analyses (This will facilitate future mammary stem cell research); 3) Evaluate the ability of regulators of the p53 pathway, or other stem cell stimuli, to promote repair of mammary tissue that is damaged by mastitis.
3. Progress Report:
Laser excision of putative mammary stem cells and control cells from histological sections led to identification of genes that are more highly expressed in mammary stem cells. Initial evaluation of these stem cell biomarkers is promising, as cells that express the markers are present in the appropriate location and abundance in mammary tissue sections and the stem cell population appears to be dynamic during the lactation cycle. Increased knowledge of bovine mammary stem cells may permit altering their activity to promote the repair of mammary tissue that is damaged by mastitis and to enhance milk production efficiency. Manuscripts reporting this work were submitted for publication. Reactive oxygen and nitrogen products are synthesized by immune and non-immune cells in response to infection. Although production of these reactants is needed to destroy mastitis-causing pathogens, they can damage the milk-secreting cells of the mammary gland. We characterized the distribution of these reactive compounds in response to experimentally induced intramammary infection. The production of reactive molecules was unevenly distributed within the tissue. Distribution patterns of the highly reactive peroxynitrate and resulting nitrated proteins coincided with areas of mitochondrial tissue damage. Strategies targeting the reactivity of peroxynitrite are a focus of the current project plan. Objectives for renewal of the current project plan include an evaluation of physiological and genetic factors that influence incidence and severity of mastitis. (1) We identified animals with differing physiological responses to injection of endotoxin that should equate with increased and decreased susceptibility to mastitis. The initial trial to identify these animals used yearling Holstein heifers and their lifetime mastitis incidence will be monitored. (2) Genetic profiling is being used to identify animals that possess the above physiological responses. The populations will be used to evaluate the relationship between this genotype and mastitis susceptibility. Endophyte-infected fescue contains ergot alkaloids that inhibit prolactin secretion. This in turn is thought to reduce mammary differentiation and milk yield. Effect of feeding cows a diet containing endophyte-infected fescue, during the two months prior to calving and the first week after calving, are being evaluated. Although milk production was initially reduced in cows consuming endophyte-infected feed and in the positive controls (receiving injections of a purified ergot alkaloid), ultimately production of treated cows exceeded that of control cows. Initial histological evaluation indicates that mammary tissue of treated cows was analogous to that of control cows. Additional laboratory analyses, including an analysis of the mammary stem cell population, will provide insight into underlying physiological mechanisms and possible management options.
1. Development of protocol to increase the population of mammary stem cells. ARS scientists have developed a protocol to expand the population of mammary stem cells. The protocol is based upon infusion of the natural nucleotides xanthosine or inosine through the teat canal into the mammary gland. In vivo studies have shown that treatment increases the number of putative mammary stem cells and in vitro studies confirm that treatment with xanthosine leads to the exponential growth of mammary stem cells. This protocol was used by Canadian scientists to increase milk production in transgenic goats by 62%. Results have been published and are expected to engender significant research and industry interest.
Elsasser, T.H., Romo, G.A., Eastridge, J.S., Kahl, S., Martinez, A., Li, C., Sartin, J.L., Cuttitta, F., Fayer, R., Solomon, M.B. 2011. Protein Tyrosine Nitration in Chronic Intramuscular Parasitism: Immunohistochemical evaluation of Relationships Between Nitration, Fiber Types, and Ubiquitin. Journal of Physiology. 1:10-18.