TLR9 DNA methylation and gene expression in cattle naturally infected with B. abortus.

Brucella abortus is a facultative intracellular bacterium that causes bovine brucellosis, a chronic infectious disease associated with reproductive failure and economic loss in livestock. The pathogen evades host immune responses by interfering with innate immune pathways, particularly those mediated by Toll-like receptors (TLRs). Among these, TLR9 plays a crucial role in recognizing unmethylated CpG motifs in bacterial DNA and triggering immune activation. This study aimed to investigate the expression level of the TLR9 gene and evaluate potential epigenetic regulation via DNA methylation within its coding sequence (CDS) in cattle naturally infected with B. abortus. Blood samples were collected from 35 infected and 30 healthy control animals confirmed by serological testing. TLR9 expression was assessed using quantitative real-time PCR, while DNA methylation of a CpG-rich region in the TLR9 CDS was analyzed by direct bisulfite sequencing. The results revealed a significant downregulation of TLR9 expression in infected cattle compared to controls (p < 0.05), suggesting a potential mechanism by which Brucella evades host immune surveillance. However, no significant differences in CpG methylation were detected between the groups. These findings suggest that DNA methylation within the analyzed region is unlikely to explain the reduced expression of TLR9. Further research is needed to explore these mechanisms and to better understand the epigenetic landscape shaping host responses to chronic Brucella infection.