Multilocus sequence typing characterizes diversity of Ureaplasma diversum strains, and intra-species variability induces different immune response profiles.

BACKGROUND: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states. RESULTS: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors. CONCLUSION: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.

Ureaplasma diversum and Its Membrane-Associated Lipoproteins Activate Inflammatory Genes Through the NF-κB Pathway via Toll-Like Receptor 4.

Objectives:Ureaplasma diversum is a pathogen of cows that may cause intense inflammatory responses in the reproductive tract and interfere with bovine reproduction. The aims of this study were to evaluate the immune response of bovine blastocysts and macrophages to U. diversum infection and to evaluate the invasion capacity of this microorganism in bovine blastocysts. Methods: Viable and heat-inactivated U. diversum strains ATCC 49782 and CI-GOTA and their extracted membrane lipoproteins were inoculated in macrophages in the presence or absence of signaling blockers of Toll-Like Receptor (TLR) 4, TLR2/4, and Nuclear Factor KB (NF-κB). In addition, the same viable U. diversum strains were used to infect bovine blastocysts. RNA was extracted from infected and lipoprotein-exposed macrophages and infected blastocysts and assayed by qPCR to evaluate the expression of Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor Alpha (TNF-α), TLR2 and TLR4 genes. U. diversum internalization in blastocysts was followed by confocal microscopy. Results: Both Ureaplasma strains and different concentrations of extracted lipoproteins induced a higher gene expression of IL-1ß, TNF-α, TLR2, and TLR4 in macrophages (p < 0.05) when compared to non-infected cells. The used blockers inhibited the expression of IL-1ß and TNF-α in all treatments. Moreover, U. diversum was able to internalize within blastocysts and induce a higher gene expression of IL-1b and TNF- α when compared to non-infected blastocysts (p < 0.05). Conclusion: The obtained results strongly suggest that U. diversum and its lipoproteins interact with TLR4 in a signaling pathway acting via NF-kB signaling to stimulate the inflammatory response. This is the first study to evaluate the in vitro immunological response of macrophages and bovine blastocysts against U. diversum. These results may contribute to a better understanding of the immunomodulatory activity and pathogenicity of this infectious agent.