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Background: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level and play a key role in the control of innate and adaptive immune responses. For a subclinical infection such as bovine streptococcal mastitis, early detection is a great challenge, and miRNA profiling could potentially assist in the diagnosis and contribute to the understanding of the pathogenicity and defense mechanisms. We have examined the miRNA repertoire and the transcript level of six key immune genes [tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and transforming growth factor beta 1 (TGFβ1)] during the early phase response of bovine immature macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of small RNA libraries from 20 cultures of blood monocyte-derived macrophages exposed to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h in vitro and unchallenged controls was performed. Results: Analyzes of over 356 million high quality sequence reads, revealed differential expression of 17 and 44 miRNAs (P < 0.05) in macrophages infected with ST103 and ST12, respectively, versus unchallenged control cultures. We also identified the expression of 31 potentially novel bovine miRNAs. Pathway analysis of the differentially regulated miRNAs and their predicted target genes in the macrophages infected with ST12 revealed significant enrichment for inflammatory response and apoptosis, while significant enrichment for integrin and GABA signaling were found in ST103 infected macrophages. Furthermore, both bacterial strains regulated miRNAs involved in the alternative activation of macrophages. The transcript levels of TNF-α, IL-1β, IL-6, IL-8 and IL-10 were significantly up-regulated by both bacterial strains, however the expression of TGFβ1 was significantly down-regulated only by ST12. Conclusions: Our study identified pathogen-induced differential regulation of miRNAs controlling inflammation and polarization in bovine macrophages. This implies that miRNAs have potential to serve as biomarkers for early detection of bacterial infection. [ABSTRACT FROM AUTHOR]