Epigenetic insights into mastitis in Mediterranean Italian River buffalo using nanopore sequencing

Mastitis represents one of the main challenges in dairy buffalo farming, with significant implications for animal health, milk quality, and farm profitability. Among its different forms, subclinical mastitis is difficult to detect due to the absence of standardised diagnostic tools. Epigenetic mechanisms, such as DNA methylation, have been associated with immune response and disease susceptibility in cattle, but remain unexplored in buffaloes. This study explores genome-wide DNA methylation profiles in Mediterranean Italian River Buffaloes, using nanopore sequencing technology, to identify potential epigenetic signatures associated with mastitis resistance. CpG sites showed a unimodal distribution, with most sites exhibiting high methylation levels. A total of 22 differentially methylated cytosines (DMCs) were identified, with 68% showing hypomethylation in the control group and 32% showing hypermethylation. Genomic annotation revealed that hypermethylated DMCs were predominantly located in intronic regions, while hypomethylated DMCs were largely enriched in distal intergenic regions. This study is the first to investigate DNA methylation changes associated with mastitis in Mediterranean Italian River Buffalo using nanopore sequencing. Distinct epigenetic patterns were identified between healthy and mastitic animals. This study provides a first epigenetic overview of mastitis in buffaloes and lays the groundwork for future investigations with larger cohorts to validate and extend these observations. Given the small sample size, these findings should be considered exploratory, but offer insights into the molecular basis of mastitis and may support the development of new diagnostic tools based on validated epigenetic signatures.
Genome-wide DNA methylation profiling in buffalo using nanopore sequencing
Identification of 22 DMCs associated with mastitis in buffalo
Detection of different epigenetic patterns between groups

乳房炎是奶水牛养殖面临的主要挑战之一，对动物健康、牛奶品质以及牧场盈利能力均具有重大影响。其中亚临床型乳房炎因缺乏标准化诊断工具，难以被检测。表观遗传机制（epigenetic mechanisms）如DNA甲基化（DNA methylation）已被证实与牛的免疫应答及疾病易感性相关，但在水牛中尚未得到探索。本研究利用纳米孔测序（nanopore sequencing）技术，对意大利地中海型河流水牛开展全基因组DNA甲基化谱分析，以期鉴定与乳房炎抗性相关的潜在表观遗传标记。

CpG位点（CpG sites）呈现单峰分布，绝大多数位点表现出高甲基化水平。本研究共鉴定出22个差异甲基化胞嘧啶（differentially methylated cytosines，DMCs），其中68%在对照组中呈低甲基化状态，32%呈高甲基化状态。基因组注释结果显示，高甲基化的DMCs主要位于内含子区域，而低甲基化的DMCs则显著富集于远端基因间区。

本研究是首个利用纳米孔测序技术探究意大利地中海型河流水牛乳房炎相关DNA甲基化变化的研究。研究明确了健康水牛与患乳房炎水牛之间存在截然不同的表观遗传模式。本研究首次对水牛乳房炎的表观遗传特征进行了系统性概述，为后续通过更大样本队列验证并拓展上述发现奠定了基础。鉴于本研究样本量较小，上述发现仅可视为探索性结果，但可为解析乳房炎的分子机制提供新视角，也有助于基于经过验证的表观遗传标记开发新型诊断工具。

1. 利用纳米孔测序技术开展水牛全基因组DNA甲基化谱分析
2. 鉴定水牛中与乳房炎相关的22个DMCs
3. 检测组间不同的表观遗传模式