1Department of Animal Science,São Paulo State University (FCAV/UNESP),Jaboticabal,SP 14884-900,Brazil.
2Department of Exact Sciences,São Paulo State University (FCAV/UNESP),Jaboticabal,SP 14884-900,Brazil.
Animal. 2019 Aug;13(8):1651-1657. doi: 10.1017/S1751731118003324. Epub 2019 Jan 9.
Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.
水牛奶生产在世界范围内变得非常重要，但是，针对水牛的生物技术工具的研究很少。为了验证亚临床乳腺炎对乳成分的影响，并研究亚临床乳腺炎奶牛乳腺中的先天免疫系统，我们评估了患有和不患有亚临床乳腺炎的水牛乳中乳铁蛋白（LTF）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-8（IL-8）和 toll 样受体 2（TLR-2）和 4（TLR-4）基因的表达水平。采集奶样以测定奶成分：体细胞评分（SCS）、脂肪、蛋白质、乳糖、总固体和非脂固体（SNF），并从奶细胞中提取 RNA、合成互补 DNA，以及通过定量实时 PCR 定量表达谱。对于基因表达，使用目标基因的表达与两组之间管家基因的表达进行调整后的差异 Ct（ΔΔCt）来估计。进行线性回归分析以确定所研究的基因与奶成分之间的关系。亚临床乳腺炎导致水牛乳中的脂肪、乳糖和 SNF 发生变化，并且 TLR-2、TLR-4、TNF-α、IL-1β和 IL-8 基因在患有亚临床乳腺炎的水牛奶细胞中的信使 RNA 丰度上调，而 LTF 基因未表现出差异表达。线性回归分析的结果表明，TLR-2 基因表达最能解释 SCS 的变化，TNF-α 基因的 ΔCt 单位变化将导致 SCS 更高的增加。研究这些在乳腺中活跃的免疫功能基因对于表征在奶牛亚临床乳腺炎中发生的先天免疫的作用机制非常重要，并且可能有助于制定保护乳房健康的策略。
