Methylome-wide analysis of milk somatic cells upon subclinical mastitis in dairy cattle

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.