基于高通量测序技术分析奶牛乳房炎关联微生物群落结构及多样性

doi:10.6048/j.issn.1001-4330.2022.04.029

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (4): 1025-1033.DOI: 10.6048/j.issn.1001-4330.2022.04.029

• 基因工程·草业·农产品分析检测·畜牧兽医 • 上一篇下一篇

基于高通量测序技术分析奶牛乳房炎关联微生物群落结构及多样性

刘肖利¹(), 程彪¹, 刘璐瑶¹, 李勤凡², 佟盼盼¹, 张毅¹, 刘英玉¹, 苏战强¹(), 李斌¹()

1.新疆农业大学动物医学学院,乌鲁木齐 830052
2.西北农林科技大学动物医学院,陕西杨凌 712100

收稿日期:2021-05-13 出版日期:2022-04-20 发布日期:2022-04-24
通信作者: 苏战强(1974-),男,新疆乌鲁木齐人,副教授,博士生导师,研究方向为动物源食品安全,(E-mail) szq00009@163.com;
李斌(1992-),男,新疆乌鲁木齐人,讲师,研究方向为动物病原微生物致病机理与防治,(E-mail) libin19921221@126.com
作者简介:刘肖利(1995-),女,河南淮阳人,硕士,研究方向为临床兽医学,(Email) 1316875742@qq.com
基金资助:
奶牛养殖提质增效关键技术集成示范(2020A01001-2);新疆褐牛健康养殖技术集成与产业化示范(2017B01001-2);2019年新疆农业大学-中国农业大学联合基金项目“新疆奶牛乳房炎防控关键技术研究与示范”(2019-7)

Analysis of the Structure and Diversity of Microbial Communities of Cow Mastitis based on High-throughput Sequencing

LIU Xiaoli¹(), CHENG Biao¹, LIU Luyao¹, LI Qinfan², TONG Panpan¹, ZHANG Yi¹, LIU Yingyu¹, SU Zhanqiang¹(), LI Bin¹()

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
2. College of Veterinary Medicine, Northwest A&F University, Yangling Shanxi 712100, China

Received:2021-05-13 Published:2022-04-20 Online:2022-04-24
Supported by:
Integration and Demonstration of Key Technologies to Improve the Quality and Efficiency of Dairy Cow Breeding(2020A01001-2);Xinjiang Brown Cattle Healthy Breeding Technology Integration and Industrialization Demonstration(2017B01001-2);2019 Xinjiang Agricultural University-China Agricultural University Joint Fund Project "Research and Demonstration of Key Technologies for the Prevention and Control of Dairy Cow Mastitis in Xinjiang"(2019-7)

摘要/Abstract

摘要：

【目的】研究奶牛乳房炎主要病原微生物及乳汁中微生物群系分布。【方法】对CJ和HZS 2个牛场的奶牛进行乳房炎检查,视觉和触觉等临床检查奶牛乳房及乳汁后,将2个场中确诊为临床乳房炎的奶牛随机采集乳汁样本各3份,并将非临床型奶牛的乳汁样本随机采集各1份,通过对乳房炎奶牛和非临床型奶牛乳样中细菌的16S rRNA基因V3-V4区的PCR扩增和高通量测序,分析临床奶牛乳汁样本和非临床型奶牛乳汁样本中微生物群落的多样性,并比较二者之间的差异性。【结果】6个临床乳房炎样本共获得25个门,47个纲,82个目,174个科,349个属;2个非临床型奶牛乳汁样本共获得23个门,38个纲,61个目,125个科,212个属。在临床乳房炎样本中,优势菌群分别是变形菌门(Proteobacteria)、无壁菌门(Tenericutes)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria);在非临床型样本中,优势菌群是变形菌门(Proteobacteria),其次是厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)。在属水平上,非临床型样本与乳房炎样本相比,检测到的优势菌群较少,且仅在HZS场乳房炎样本中检测到链球菌,CJ场乳房炎样本中检测到支原体。【结论】奶牛患乳房炎后菌群丰度明显升高,多样性变化显著;奶牛患乳房炎后会引起奶牛乳汁菌群失调,乳汁菌群结构分布和丰度变化与乳房炎的发生具有密切的联系。

关键词: 奶牛; 乳房炎; 微生物多样性

Abstract:

【Objective】 To study the main pathogenic microorganisms of dairy cow mastitis and the distribution of microbiota in milk.【Method】 The cows in the two farms of CJ and HZS were examined for mastitis.After visual and tactile examination of the udder and milk of the cows, 3 milk samples were randomly collected from the dairy cows diagnosed with clinical mastitis in the two farms each, and then 1 milk sample was collected from non-clinical dairy cows.Through PCR amplification and high-throughput sequencing of the 16S rRNA gene V3-V4 regions of bacteria in milk samples of mastitis dairy cows and non-clinical dairy cows, the diversity of microbial communities in clinical dairy milk samples and non-clinical dairy milk samples were analyzed and the differences were compared between the two.【Result】 The results showed that from the 6 clinical mastitis samples, a total of 25 phyla, 47 classes, 82 orders, 174 families, and 349 genera were obtained; from 2 non-clinical dairy cow milk samples, a total of 23 phyla, 38 classes, 61 Orders, 125 families, 212 genera were obtained.In clinical mastitis samples, the dominant bacterial groups were Proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, Actinobacteria; In non-clinical dairy milk samples, the dominant flora were Proteobacteria, followed by Firmicutes and Bacteroidetes.At the genus level, compared with mastitis samples, non-clinical dairy milk samples had fewer dominant bacterial groups, and only streptococcus was detected in HZS mastitis samples and Mycoplasma was detected in CJ mastitis samples.【Conclusion】 After cows suffer from mastitis, the abundance of the microbial flora is significantly increased, and the diversity changes significantly, indicating that the milk cows suffering from mastitis will cause the milk cow's milk flora to be imbalanced, and the changes in the structure and abundance of the milk flora are closely related to the occurrence of mastitis.

Key words: cow; mastitis; microbial diversity

中图分类号:

S852.61

刘肖利, 程彪, 刘璐瑶, 李勤凡, 佟盼盼, 张毅, 刘英玉, 苏战强, 李斌. 基于高通量测序技术分析奶牛乳房炎关联微生物群落结构及多样性[J]. 新疆农业科学, 2022, 59(4): 1025-1033.

LIU Xiaoli, CHENG Biao, LIU Luyao, LI Qinfan, TONG Panpan, ZHANG Yi, LIU Yingyu, SU Zhanqiang, LI Bin. Analysis of the Structure and Diversity of Microbial Communities of Cow Mastitis based on High-throughput Sequencing[J]. Xinjiang Agricultural Sciences, 2022, 59(4): 1025-1033.

图/表 7

表1 样本菌群Alpha多样性指数

Table 1 Microflora Alpha Diversity Index of Sample

样品 Sample	Chao1指数 Chao1 index	辛普森指数 Simpson index	香浓指数 Shannon-index	均匀度 Pielou_e	可见物种 Observed- species	覆盖度 Goods coverage (%)
CJ.LC1	462.288	0.121 018	0.743 671	0.084 275 3	453.3	99
CJ.LC2	1 348.25	0.827 401	4.431 91	0.429 205	1 283.5	99
CJ.LC3	2 622.23	0.957 049	6.647 99	0.586 655	2 578	99
CJ.ZC	1 148.38	0.650 92	3.454 96	0.340 844	1 125.6	99
HZS.LC1	2 513.94	0.972 411	7.153 58	0.636 186	2 426.2	99
HZS.LC2	1 779.43	0.970 484	6.836 3	0.636 326	1 714.3	99
HZS.LC3	478.182	0.092 715 3	0.581 873	0.065 990 2	451.2	99
HZS.ZC	1 024.69	0.956 733	5.680 18	0.572 007	975.7	99

图1 相似度为97%条件下各样本的稀释曲线

Fig.1 Rarefaction curves of each sample at cutoff level of 3%

图2 细菌门分类水平的比较注:HZS.ZC:HZS奶牛场非临床型乳汁样本;HZS.LC1:HZS奶牛场临床型乳房炎乳汁样本;HZS.LC2:HZS奶牛场临床型乳房炎乳汁样本;HZS.LC3:HZS奶牛场临床型乳房炎乳汁样本;CJ.ZC:CJ奶牛场非临床型乳汁样本;CJ.LC1:CJ奶牛场临床型乳房炎乳汁样本;CJ.LC2:CJ奶牛场临床型乳房炎乳汁样本;CJ.LC3:CJ奶牛场临床型乳房炎乳汁样本,下同

Fig.2 Comparison of bacteria groups at phylum level Note:HZS.ZC:Non-clinical milk samples from HZS dairy farm; HZS.LC1:Milk samples of clinical mastitis from HZS dairy farm; HZS.LC2:Milk samples of clinical mastitis from HZS dairy farm; HZS.LC3:Milk samples of clinical mastitis from HZS dairy farm; CJ.ZC:Non-clinical milk samples from CJ dairy farm; CJ.LC1:Milk samples of clinical mastitis from CJ dairy farm; CJ.LC2:Milk samples of clinical mastitis from CJ dairy farm; CJ.LC3:Milk samples of clinical mastitis from CJ dairy farm,the same as below

图3 细菌科分类水平的比较

Fig.3 Comparison of bacteria groups at family level

图4 细菌属分类水平的比较

Fig.4 Comparison of bacteria groups at genus level

图5 样本层级聚类树

Fig.5 Hierarchical clustering tree

图6 属水平物种组成热图注:红色色块代表该属在该样本中的丰度较其他样本高,蓝色色块代表该属在该样本中的丰度较其他样本低

Fig.6 Heat map of genus horizontal species composition Note:The red color block indicates that the abundance of this genus in this sample is higher than other samples, the blue color block means that the abundance of this genus in this sample is lower than other samples

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基于高通量测序技术分析奶牛乳房炎关联微生物群落结构及多样性

Analysis of the Structure and Diversity of Microbial Communities of Cow Mastitis based on High-throughput Sequencing

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