高通量测序解析乳房-肠道-环境菌群与奶牛乳房炎关联性

Bovine mastitis is the most prevalent disease worldwide, caused by complex factors. The idea of modern medicine is that the occurrence and development of the disease is closely related to the change of microbial communities. In Hohhot, i.e., milk city of China, several large-scale dairy farms will be surveyed to determine the tested cow herds with sample sizes by field investigation. On the basis of America California Mastitis Test(CMT), the tested cow herds will be divided into negative group of bovine mastitis/health, weakly positive group of bovine mastitis / mild clinical mastitis, strongly positive group of bovine mastitis/severe clinical mastitis. According to bovine mastitis associated microbial communities from mammary gland, gut, housing and the surrounding environments, metagenomic DNA from milk, manure and soil samples will be extracted directly, respectively. Based on high-throughput sequencing of 16S rDNA amplicons and bioinformatics technique, the abundance and compositions of bacterial communities, diversity from individual samples will be analyzed, to explore the phylogenetic characteristics of lineages (from phylum to class, order, family, genus, species) in a certain bacterial community and the similarity of communities among all samples. The dynamic distribution of dominant and unique communities from milk, manure and soil samples will be explored, to uncover the response of bacterial communities from mammary glands and guts to different groups of bovine mastitis(i.e., negative, weakly positive, strongly positive groups of bovine mastitis) as well as the impact of bacterial communities from different environmental soils(e.g., soils from housing and the surrounding environments) on the occurrence and development of bovine mastitis, to elucidate the relationship among bacterial communities from milk, manure and soil samples, to decipher whether gut and soil microbiomes cause bovine mastitis, to mine the target bacterial communities causing mastitis in dairy cows. This will provide the scientific basis for early prevention and control as well as accurate diagnosis and treatment of mastitis in dairy cows.

奶牛乳房炎全球盛行诱因复杂。现代医学认为，疾病的发生发展与菌群变化密切相关。本研究以中国乳都呼和浩特市规模化奶牛场为研究区域，通过实地考察确定受试牛群及样本量，依据美国加州乳房炎试验将受试牛群分为乳房炎阴性/健康组、乳房炎弱阳性/轻度乳房炎组、乳房炎强阳性/重度乳房炎组。针对奶牛乳房炎发生可能相关的乳房、肠道、圈舍及周边环境菌群，直接提取乳汁、粪便及土壤宏基因组DNA。通过16S rDNA高通量测序及生物信息学分析乳样、粪样及土样细菌群落组成、丰度、多样性，搞清典型群落系统发育谱系特征和全部样品间群落相似性。探究乳汁、粪便、土壤特有菌群和优势菌群的分布动态，搞清乳房及肠道菌群对乳房炎阴性、弱阳性、强阳性的响应以及环境菌群变化对乳房炎发生发展的影响，阐明乳源、粪源及土源菌群间关系，揭秘肠道及环境菌群能否诱发奶牛乳房炎，挖掘诱发奶牛乳房炎的靶标菌群，为奶牛乳房炎的早期防控和精确诊治提供科学依据

奶牛乳房炎全球盛行诱因复杂。现代医学认为，疾病的发生发展与菌群变化密切相关。本项目通过牛乳CMT检测及临床症状观察筛选出健康奶牛组、乳房炎弱阳性组和强阳性组。通过16S rRNA 基因高通量测序和生物信息学分析研究乳样、粪样、饲料及土壤样品菌群多样性、组成、丰度及分布特征，探究乳房菌群、内源性肠道菌群和外源性饲料-土壤菌群三者之间的联系及其与奶牛乳房炎发生的关联性。主要结果：【1】健康乳样菌群多样性最低(Shannon 0.79-1.49，ACE 227-274)，乳房炎弱阳性乳样菌群多样性居中(Shannon 1.96-2.20，ACE 315-318)，乳房炎强阳性乳样菌群多样性最高(Shannon 2.39-2.87，ACE 294-403)；阳性乳样中硬壁菌(Firmicutes)、放线菌(Actinobacteria)组成相对丰富，两者在阳性乳样菌群中的相对丰度分别高达12.04%、5.77%， 而在健康乳样菌群中的相对丰度分别低至0.20%、0.05%，说明乳样菌群可有效区分健康和乳房炎奶牛；【2】健康与乳房炎奶牛粪便菌群多样性差异不明显(Shannon 5.39-5.78，ACE 1273-1454)，健康奶牛和乳房炎奶牛粪样菌群均以Firmicutes和拟杆菌 (Bacteroidetes)为主，说明粪样菌群不能区分健康和乳房炎奶牛；【3】外源性饲料-土壤菌群与乳样菌群的相似度分别高达63.3%和60.6%，而内源性肠道粪样菌群与乳样菌群的相似度低至6.7%，推测饲料及畜舍土壤微生物可能是引起奶牛乳房炎菌群多样性增加的重要因素；【4】深入研究发现奶牛品种差异、饲料土壤差异、母系差异、乳区差异分别解释牛乳菌群变异的67%、45%、43%、28%， X品种牛乳与B组饲料和2号土壤以α-变形菌(Alphaproteobacteria)谱系为主、Y品种牛乳与G组饲料和7号土壤以Firmicutes谱系为主，Z品种牛乳与D组饲料和4号土壤均以Actinobacteria谱系为主，推测先天的奶牛品种差异可能是引起乳房炎关联乳腺菌群多样性增加的关键因素，后天的饲养环境及畜舍卫生环境差异可能是引起乳房炎关联乳腺菌群多样性增加的重要因素。本项目研究成果能为奶牛乳房炎的早期防控和精确诊治提供科学依据，亦能为日后探明奶牛乳房炎发病机理及制定有效防治措施提供有价值的研究资料