Metzger Stephanie A, Hernandez Laura L, Skarlupka Joseph H, Walker Teresa M, Suen Garret, Ruegg Pamela L
Department of Dairy Science, University of Wisconsin-Madison, Madison, WI, United States.
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States.
Front Vet Sci. 2018 Oct 9;5:247. doi: 10.3389/fvets.2018.00247. eCollection 2018.
The objective of this longitudinal cohort study was to describe the milk microbiota of dairy cow mammary glands based on inflammation status before and after the dry period. Individual mammary quarters were assigned to cohorts based on culture results and somatic cell count () at dryoff and twice in the first 2 weeks post-calving. Mammary glands that were microbiologically negative and had low SCC (< 100,000 cells/mL) at all 3 sampling periods were classified as Healthy ( = 80). Microbiologically negative mammary glands that had SCC ≥150,000 cells/mL at dryoff and the first post-calving sample were classified as Chronic Culture-Negative Inflammation (CHRON; = 17). Quarters that did not have both culture-negative milk and SCC ≥ 150,000 cells/mL at dryoff but were culture-negative with SCC ≥ 150,000 at both post-calving sampling periods were classified as Culture-Negative New Inflammation (NEWINF; = 6). Mammary glands with bacterial growth and SCC ≥ 150,000 cells/mL at all 3 periods were classified as Positive (POS; = 3). Milk samples were collected from all enrolled quarters until 150 days in milk and subjected to microbiota analysis. Milk samples underwent total DNA extraction, a 40-cycle PCR to amplify the V4 region of the bacterial 16S rRNA gene, and next-generation sequencing. Healthy quarters had the lowest rate of PCR and sequencing success (53, 67, 83, and 67% for Healthy, CHRON, NEWINF, and POS, respectively). Chao richness was greatest in milk collected from Healthy quarters and Shannon diversity was greater in milk from Healthy and CHRON quarters than in milk collected from glands in the NEWINF or POS cohorts. Regardless of cohort, season was associated with both richness and diversity, but stage of lactation was not. The most prevalent OTUs included typical gut- and skin-associated bacteria such as those in the phylum Bacteroidetes and the genera and . The increased sequencing success in quarters with worse health outcomes, combined with the lack of bacterial growth in most samples and the high PCR cycle number required for amplification of bacterial DNA, suggests that the milk microbiota of culture-negative, healthy mammary glands is less abundant than that of culture-negative glands with a history of inflammation.
这项纵向队列研究的目的是根据干奶期前后的炎症状态描述奶牛乳腺的乳汁微生物群。在干奶期以及产犊后前两周内两次，根据培养结果和体细胞计数（SCC）将各个乳腺象限分配到不同队列。在所有3个采样期微生物学检测为阴性且SCC较低（<100,000个细胞/毫升）的乳腺被分类为健康组（n = 80）。在干奶期和产后第一次采样时SCC≥150,000个细胞/毫升的微生物学检测为阴性的乳腺被分类为慢性培养阴性炎症组（CHRON；n = 17）。在干奶期没有同时具备培养阴性乳汁和SCC≥150,000个细胞/毫升，但在产后两个采样期均为培养阴性且SCC≥150,000的象限被分类为培养阴性新炎症组（NEWINF；n = 6）。在所有3个时期细菌生长且SCC≥150,000个细胞/毫升的乳腺被分类为阳性组（POS；n = 3）。从所有纳入的象限中采集乳汁样本直至产奶150天，并进行微生物群分析。乳汁样本进行总DNA提取、40个循环的PCR以扩增细菌16S rRNA基因的V4区域，以及下一代测序。健康象限的PCR和测序成功率最低（健康组、CHRON组、NEWINF组和POS组分别为53%､67%､83%和67%）。Chao丰富度在健康象限采集的乳汁中最大，香农多样性在健康和CHRON象限的乳汁中比在NEWINF或POS队列的腺体采集的乳汁中更大。无论队列如何，季节与丰富度和多样性均相关，但泌乳阶段则不然。最常见的操作分类单元包括典型的与肠道和皮肤相关的细菌，如拟杆菌门以及某些属的细菌。健康状况较差的象限测序成功率增加，再加上大多数样本中缺乏细菌生长以及扩增细菌DNA所需的高PCR循环数，表明培养阴性、健康乳腺的乳汁微生物群比有炎症史的培养阴性腺体的乳汁微生物群数量更少。
