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United States Department of Agriculture

Agricultural Research Service

Research Project: Identification of Host Immune Factors and Intervention Strategies for Mastitis
2012 Annual Report


1a.Objectives (from AD-416):
Objective 1: Determine the host innate immune mechanisms that enable effective responses to and the clearance of infections in cattle based on the mammary gland. Objective 2: Define the molecular and cellular pathways affected by vitamin D in immune cells to establish levels necessary for immune management and cattle health both experimentally and in field situations.


1b.Approach (from AD-416):
Mastitis is both the most prevalent infectious disease in dairy herds and the most costly disease for dairy producers. Older cost estimates for mastitis are in the neighborhood of $2 billion per year for producers. Antibiotics are the mainstay for mastitis treatment and control. Dairy cattle with mastitis receive more antibiotic therapy for its prevention and treatment than for all other dairy cattle diseases combined. Valid concerns by consumers regarding antibiotic usage need to be addressed by research on non-antibiotic alternatives. A more complete understanding of the bovine immune system will be a necessary component in the goal of greater understanding of non-antibiotic alternatives. A significant proportion of clinical mastitis cases occur in cows in the weeks shortly after calving when the cow’s innate immune system is compromised, highlighting the important role of a fully functional immune system in the fight against mastitis. Understanding of the bovine immune system is hampered by the lack of reagents that are available to other species, such as humans and mice. However, new technologies, such as RNA sequencing, do not depend on species-specific reagents and allow for the investigation of molecular interaction in a way not previously possible. Although many immune functions are similar between species, there are significant differences and these differences can have a profound impact on how a specific species reacts to a pathogen. It is the goal of this research project to determine the changes in gene and protein expression in bovine immune cells during an infection. In addition, this research will look at approaches to modulate the immune response in a way to enhance its ability to fight pathogens. The goals of the proposed research are to use a multi-discipline approach to:.
1)Use RNA sequencing technology and proteomics to building a database of gene and protein expression data. To then model the expression data to molecular and cellular pathways that will lead to a better understanding of bovine innate immune responses to a mammary infection..
2)Advance our knowledge about the role of 25-hydroxyvitamin D3 [25(OH)D3] in maintaining innate immune function and test 25(OH)D3 as a non-antibiotic immune modulator of mammary innate immunity to reduce the severity and duration of an infection. Results of proposed research will be used to improve the model of bovine innate immune response by identifying novel signaling, transcriptional and post-transcriptional regulators of innate immunity.


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 affects bovine immune function. This year, we have published that intramammary vitamin D administration at the time of infection reduces the severity of disease. We have continued this work to test the efficacy of vitamin D treatment on chronic mastitis infections caused by S. aureus. Those data are currently being analyzed.

In addition we have published a survey proteomic study on milk exosomes. Evidence has shown that exosomes play a number of tissue specific roles in intracellular communication and immune function. These exosome functions may have implications in mammary health. Our data contribute to expansion of Omics databases related to mammary function and milk components. These will be keys to a greater understanding of lactation physiology and mammary health. We are continuing this work to include proteomic studies comparing milk components from milk isolated from clean and mastitic cows.


4.Accomplishments
1. Reduction of mastitis severity with use of vitamin D. ARS researchers at Ames, IA, have previously shown that vitamin D influences gene expression in bovine immune cells in the mammary gland. The milk compartment of the mammary gland is deficient in vitamin D, even if the levels are normal in the blood. Therefore, vitamin D therapy may uniquely affect the molecular and cellular pathways in immune cells in the mammary gland and may be an important non-antibiotic option for mastitis treatment. ARS scientists in Ames, Iowa found that the severity of infection in animals with mastitis was reduced by vitamin D. Data shows that vitamin D treatment reduces the severity of the infection. Vitamin D is a simple and natural immune stimulator that in combination with current antibiotics could become an effective therapy for mastitis, reducing the time and amount of antibiotics needed for treatment, and potentially curing mastitis infections resistant to antibiotic treatment alone. Reducing antibiotic use would alleviate environmental and health concerns, increasing consumer confidence and international trading opportunities.

2. Exosomes are microscopic parts of an animal’s cell that are secreted by cells into fluids such as blood, milk, urine and lung mucus. There is emerging data that exosomes are unique to the body fluid they come from, important for cellular communication and immune function, and are a source of diagnostic markers for animal diseases. These exosomes carry proteins as well as gene messages that can change cellular functions. For example, a diseases cell can produce exosomes that can modulate the function of immune cells. We use a sophisticated instrument to identify the exosome proteins in normal milk, highlighting those proteins whose role may be involved in directing the immune response. This benefit of this research is to provide a better understanding of the immune response to mastitis and to find diagnostic markers that may identify infected milk for greater consumer safety and confidence.


Review Publications
Nelson, C.D., Reinhardt, T.A., Lippolis, J.D., Sacco, R.E., Nonnecke, B.J. 2012. Vitamin D signaling in the bovine immune system: A model for understanding human vitamin D requirements. Nutrients. 4(3):181-196.

Lippolis, J.D., Reinhardt, T.A., Sacco, R.E., Nonnecke, B.J., Nelson, C.D. 2011. Treatment of an intramammary bacterial infection with 25-hydroxyvitamin D3. PLoS One. 6(10):e25479. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184989/pdf/pone.0025479.pdf.

Reinhardt, T.A., Lippolis, J.D., Nonnecke, B.J., Sacco, R.E. 2012. Bovine milk exosome proteome. Journal of Proteomics. 75(5):1486-1492.

Last Modified: 10/1/2014
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