Wang Di, Wei Yiyuan, Shi Liangyu, Khan Muhammad Zahoor, Fan Lijun, Wang Yachun, Yu Ying
Key Laboratory of Agricultural Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
Asian-Australas J Anim Sci. 2020 Feb 1;33(2):203-211. doi: 10.5713/ajas.18.0858. Epub 2019 Apr 15.
Staphylococcus aureus (S. aureus) is one of the major microorganisms responsible for subclinical mastitis in dairy cattle. The present study was designed with the aim to explore the DNA methylation patterns using the Fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) techniques in a S. aureus-infected mouse model.
A total of 12 out-bred ICR female mice ranging from 12 -13 weeks-old were selected to construct a mastitis model. F-MSAP analysis was carried out to detect fluctuations of DNA methylation between control group and S. aureus mastitis group.
Visible changes were observed in white cell counts in milk, percentage of granulocytes (GRN %), percentage of lymphocytes (LYM %), CD4+/ CD8+ ratio (CD4+/ CD8+), and histopathology of mice pre and post-challenge with S. aureus. These findings showed the uniformity and suitability of the S. aureus-infected mouse model. A total of 369 fragments was amplified from udder tissue samples from the two groups (S. aureus-infected mastitis group and control group) using eight pairs of selective primers. Results indicated that the methylation level of mastitis mouse group was higher than that in the healthy group. In addition, NCK-associated protein 5 (Nckap5) and transposon MTD were identified to be differentially methylated through secondary PCR and sequencing in the mastitis group. These outcomes might play an important role in the development of S. aureus mastitis.
Collectively, our study suggested that the methylation modification in Nckap5 and transposon MTD might be considered as epigenetic markers in resistance to S. aureus-infected mastitis and provided a new insight into S. aureus mastitis research in dairy industry and public health.
金黄色葡萄球菌是导致奶牛亚临床乳腺炎的主要微生物之一。本研究旨在利用荧光标记的甲基化敏感扩增多态性（F-MSAP）技术，在金黄色葡萄球菌感染的小鼠模型中探索DNA甲基化模式。
选取12只12 - 13周龄的远交ICR雌性小鼠构建乳腺炎模型。进行F-MSAP分析以检测对照组和金黄色葡萄球菌乳腺炎组之间DNA甲基化的波动情况。
在金黄色葡萄球菌攻击前后，观察到小鼠乳汁中的白细胞计数、粒细胞百分比（GRN%）、淋巴细胞百分比（LYM%）、CD4+/CD8+比值（CD4+/CD8+）以及组织病理学有明显变化。这些结果表明金黄色葡萄球菌感染的小鼠模型具有一致性和适用性。使用八对选择性引物从两组（金黄色葡萄球菌感染的乳腺炎组和对照组）的乳腺组织样本中扩增出总共369个片段。结果表明乳腺炎小鼠组的甲基化水平高于健康组。此外，通过二次PCR和测序鉴定出NCK相关蛋白5（Nckap5）和转座子MTD在乳腺炎组中存在差异甲基化。这些结果可能在金黄色葡萄球菌乳腺炎的发展中起重要作用。
总体而言，我们研究表明Nckap5和转座子MTD中的甲基化修饰可能被视为抵抗金黄色葡萄球菌感染乳腺炎的表观遗传标记，并为乳制品行业和公共卫生领域的金黄色葡萄球菌乳腺炎研究提供了新的见解。
