Expression profiling of microRNA from peripheral blood of dairy cows in response to Staphylococcus aureus-infected mastitis. Expression profiling of microRNA from peripheral blood of dairy cows in response to Staphylococcus aureus-infected mastitis

Purpose: To identify the expression profiling of miRNA in peripheral blood of dairy cows in response to S. aureus-infected mastitis and explore the biomarkers for early diagnosis of S. aureus-infected mastitis. Methods: RNAseq technology was used to determine the expression profiles of microRNA (miRNA) from peripheral blood of Chinese Holstein cows infected with S. aureus at 0, 1, 3, 5, and 7 days. Results: Ttal of 288 differentially expressed miRNAs (DIE-miRNA) including 108 known and 180 novel predicted miRNAs, involved in 10 immune system-related signaling pathways. Compare with the 0 dpi, the number of DIE-miRNAs in 1, 3, 5, and 7 dpi groups were 12, 21, 75, and 48, respectively. It was also found that the expression variation of up-regulated expression of miR-320a, miR-19a, and miR-19b as well as down-regulated expression of miR-143, miR‑205, and miR‑24 reached a significant level on the 5 dpi and 7 dpi. However, at different times after S. aureus infection, miR-1301 was significantly up-regulated in peripheral blood. miR-2284r was significantly down-regulated. Conclusion: miR-1301 and miR-2284r might be the new blood biomarkers for S. aureus-infected dairy cow mastitis. The above results laid a new foundation for the research and development of molecular diagnosis and biological therapy technology for S. aureus-infected mastitis in dairy cow. Overall design: The experimental animals in this study were lactating 2 to 3-year-old half-sib Chinese Holstein cows, which were obtained from a farm in Yangling, Shaanxi, China. They had the same feeding and management environment. The body conditions of the dairy cows were measured three weeks before the experiment. All individuals had no symptoms of mastitis, and the somatic cell count (SCC) of milk was less than 200,000/mL. The udders and mammilla of the experimental dairy cows were cleaned and sterilized with 75% ethanol, and 5 mL of 10^5 CFU/mL S. aureus (ATCC 25923) suspension was injected into the udders of the experimental dairy cows through the mammary duct with a milk-passing needle. On the 7th day of induction, the mammary gland tissues were collected using painless surgery for hematoxylin-eosin (HE) staining and pathological analysis for mastitis model identification. On the day 0 (control), 1, 3, 5, and 7 after S. aureus infection, peripheral blood was collected from the jugular vein, and blood samples of different individuals with the same induction time were mixed and used to perform RNAseq.

研究目的：鉴定奶牛外周血微小核糖核酸（microRNA, miRNA）在金黄色葡萄球菌（Staphylococcus aureus, S. aureus）感染性乳腺炎中的表达谱，并探索用于该类乳腺炎早期诊断的生物标志物。
研究方法：采用RNA测序（RNAseq）技术，对感染金黄色葡萄球菌的中国荷斯坦奶牛外周血中的微小RNA表达谱进行检测，采样时间点设为感染后0、1、3、5及7天。
研究结果：共计鉴定得到288个差异表达miRNA（DE-miRNA），其中包含108个已知miRNA及180个新预测miRNA，这些差异表达miRNA涉及10条免疫系统相关信号通路。与感染后0天（0 dpi）组相比，感染后1、3、5、7天组的差异表达miRNA数量分别为12、21、75及48个。此外，研究发现miR-320a、miR-19a与miR-19b的上调表达，以及miR-143、miR-205和miR-24的下调表达，在感染后5天和7天均达到显著水平。在金黄色葡萄球菌感染后的不同时间点，外周血中miR-1301呈显著上调表达，而miR-2284r则呈显著下调表达。
研究结论：miR-1301与miR-2284r或可作为奶牛金黄色葡萄球菌感染性乳腺炎的新型血液生物标志物。上述研究结果为奶牛金黄色葡萄球菌感染性乳腺炎的分子诊断及生物治疗技术研发提供了全新基础。
实验设计：本研究的实验动物为2~3岁泌乳期半同胞中国荷斯坦奶牛，取材自中国陕西杨凌某养殖场，所有个体饲养管理环境一致。实验前3周对奶牛体况进行评估，所有受试个体均无乳腺炎症状，牛乳体细胞计数（Somatic Cell Count, SCC）低于200,000个/mL。首先用75%乙醇对实验奶牛的乳房及乳头进行清洁消毒，随后通过泌乳针经乳腺导管向奶牛乳腺内注入5 mL浓度为10^5 CFU/mL的金黄色葡萄球菌（ATCC 25923）菌悬液。感染诱导第7天，采用无痛手术采集乳腺组织，通过苏木精-伊红（HE）染色及病理分析鉴定乳腺炎模型。分别在金黄色葡萄球菌感染后0天（对照组）、1、3、5及7天采集颈静脉外周血，将相同诱导时间点的不同个体血液样本混合后进行RNA测序。