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United States Department of Agriculture

Agricultural Research Service

Research Project: Identification of Host Immune Factors and Intervention Strategies for Mastitis
2013 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:
Immune cells were isolated from milk at time points after the cow was infected with a bacteria in the mammary gland to determine gene expression changes during an infection. We have completed the RNA sequencing of the immune cells isolated from milk of cows with mastitis. Our collaborators in Ireland are now processing the data to identify gene expression changes. The computer analyzing work with the messenger RNA (mRNA) has been completed and the work with the micro RNA (miRNA) is nearly completed. As previously reported, sufficient total cellular protein amounts for proteomic analysis was not possible because the entire sample went to RNA and DNA isolation. We anticipate that final processing of this data will begin in a few months and publication of the results shortly thereafter.

Staphylococcus aureus are bacteria that cause persistent mastitis infections in dairy cattle. We have infected cows with S. aureus and allowed the infection to become persistent. We then treated the animals with 25(OH) Vitamin D3. Data are currently being evaluated and some tests are yet to be run for this experiment. We anticipate final data analysis will be complete in the next year.


4.Accomplishments
1. Characterizing the proteome of mastitis caused by staphylococcus aureus. Bovine mastitis is an infectious disease of the mammary gland that costs the dairy industry 2 billion dollars per year and disease control requires new knowledge about the proteins involved. ARS researchers in Ames, IA characterized the proteome of mastitis milk caused by staphylococcus aureus. Approximately 3000 protein were identified and analyzed, and about 100 proteins were shown to have altered expression. Many of these protein with altered expression were found to be part of the immune system of the cows. These immune proteins were from neutrophils, an important immune cell in the fight against mastitis. The neutrophil proteins were associated with a relatively new bacteria killing mechanism. In this bacterial killing mechanism the neutrophils transfer their DNA to the outside of the cell forming a spider web like structure of sticky DNA. The bacteria get stuck to this DNA spider web where they are then exposed to anti-bacterial proteins that kill the bacteria. This DNA-protein combination, called NETs, can both physically trap and kill pathogens. In this study milk fat globules were found to be coated with NETs during mastitis, which can now be tested to determine if NETs retain functional antimicrobial activity when associated with milk fat globules. These findings may result in a new and better understanding of how the immune system fights or fails to effectively fight the economical important dairy production disease mastitis.


Review Publications
Boggess, M.V., Lippolis, J.D., Hurkman II, W.J., Fagerquist, C.K., Briggs, S.P., Gomes, A.V., Righetti, P., Bala, K. 2013. The need for agriculture phenotyping: “Moving from genotype to phenotype”. Journal of Proteomics. doi: http://dx.doi.org/10.1016/j.jptot.2013.3.021.

Casas, E., Leach, R.J., Reinhardt, T.A., Thallman, R.M., Lippolis, J.D., Bennett, G.L., Kuehn, L.A. 2013. A genome-wide association study identified CYP2J2 as a major gene controlling serum vitamin D in beef cattle. Journal of Animal Science. 91:3549-3556.

Reinhardt, T.A., Sacco, R.E., Nonnecke, B.J., Lippolis, J.D. 2013. Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis. Journal of Proteomics. 82:141-154.

Last Modified: 7/25/2014
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