探討荷蘭乳牛消化道微生物組與乳房炎之關聯性

Abstract

乳房炎被認為是造成酪農業產業巨大經濟損失的疾病，儘管其後發展出各種防治乳房炎的方法，但抗生素治療和牧場衛生管理仍是牧場中防治乳房炎的主要策略。然而，病原菌抗藥性及控制乳房炎的效率低落都一再促使人們研究替代方案，希望能維持動物健康。目前已有諸多研究指出消化道微生物組對動物健康狀態之變化也非常敏感，但是泌乳牛的消化道微生物組成與乳房炎之間關聯性的相關研究仍相當稀少。因此，本研究的目的即是評估荷蘭乳牛消化道微生物組成與乳房炎之間的相關性。本研究選用16頭泌乳牛，並將其分為健康泌乳牛組別和乳房炎泌乳牛組別，接著利用次世代基因定序法以及高效能液相層析串聯式質譜儀各別分析消化道微生物組與消化道代謝體。根據次世代基因定序結果可得知，健康與乳房炎泌乳牛兩組組內的消化道菌相之物種豐富度與多樣性相似，而瘤胃優勢細菌菌屬為Prevotella_1 (8.58%) ，其次依序為Succinivibrionaceae_UCG-001 (7.87%) 、Ruminococcaceae_NK4A214_group (6.60%) 、Christensenellaceae_R-7_group (5.75%) 、Ruminococcus_2 (2.87%) 與Ruminococcus_1 (2.86%) ；瘤胃優勢原蟲屬則是Entodinium (56.39%) 、Isotricha (9.57%) 、Diplodinium (2.50%) 與Dasytricha (2.50%) ；糞便優勢細菌菌屬為SMB53 (4.66%) ，其次依序是Butyrivibrio (3.02%) 、Oscillospira (3.01%) 、5-7N15 (2.59%) 與Clostridium (2.16%) 。 更進一步，本研究以偏最小平方判別分析法 (partial least squares discriminant analysis, PLS-DA) 分析兩組間瘤胃與糞便消化道微生物相是否具有組間差異，所得結果顯示出兩組之間的瘤胃細菌菌相有完全的分群效果，即為兩組別間瘤胃細菌菌相組成有所差異。因此，本研究即利用線性判別分析效力量鑑別分析 (linear discriminant analysis effect size analysis, LEfSe) 從菌門階層開始比較分析並逐步限縮至菌種層級，找出乳房炎與健康泌乳牛各自具生物標記功能之細菌菌群，首先在乳房炎泌乳牛中找到三個分類群分別是Bacillus (菌屬) 、Sharpea (菌屬) 與Bacillus anthracis (菌種) ；在健康泌乳牛中則找到七個分類群分別是Selenomonas_1 (菌屬) 、Fibrobacter (菌屬) 、Treponema_2 (菌屬) 、Ruminococcus_1 (菌屬) 、Ruminococcaceae_UCG-014 (菌屬) 、Ruminococcus flavefaciens (菌種) 與Treponema saccharophilum (菌種) 。為了深入探討生物標記菌與乳房炎指標 (血清介白素-6與乳體細胞數) 之關聯性，本研究利用Spearman秩相關係數 (Spearman’s correlation coefficient) 分析可得知，血清介白素-6與乳中體細胞數跟乳房炎組生物標記 (Bacillus、Sharpea與Bacillus anthracis) 呈現正相關性；與健康組生物標記 (Ruminococcaceae_UCG-014、Ruminococcus flavefaciens與Treponema saccharophilum) 呈現負相關性。另外，透過代謝體分析可得知，共21種化合物在健康泌乳牛的含量高於乳房炎泌乳牛 (P < 0.05) ，最後本研究亦利用Spearman秩相關係數分析瘤胃細菌與瘤胃代謝物之相關性。 目前本研究找出具有潛力的生物標記菌群以及泌乳牛中消化道微生物組與乳房炎之相關性，本研究或許能在防治乳房炎的策略上提供一個全新的方向。

Mastitis is considered to be an economically ruinous disease in dairy production. Despite the exploiting therapies, antibiotics and hygiene management are the major strategies against bovine mastitis in herd. However, the concerns for antibiotic resistance of pathogens and low efficacy to control mastitis caused by specific pathogens motivate the developments of alternative treatments to maintain animal health. It is well known that the gut microbiome is also sensitive to health status of ruminant. However, rare studies have investigated the association between gut microbiome and mastitis in dairy cows. Therefore, the aim of this study was to evaluate the correlation between gut microbiome and mastitis in Holstein dairy cows. Sixteen dairy cows were selected and equally separated into healthy and mastitis groups. Gut microbiome and metabolome were analyzed using next generation sequencing (NGS) technology and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), respectively. NGS results computed alpha diversity to describe the complexity in gut microbiota, and community richness and diversity indices were not different between groups. The most dominant bacterial families in rumen were Succinivibrionaceae_UCG-001 (7.87%), Ruminococcaceae_NK4A214_group (6.60%), Christensenellaceae_R-7_group (5.75%), Ruminococcus_2 (2.87%), and Ruminococcus_1 (2.86%). The most predominant protozoal genera in rumen were Entodinium (56.39%), Isotricha (9.57%), Diplodinium, and Dasytricha (2.50%). The most dominant bacterial families in feces were SMB53 (4.66%), Butyrivibrio (3.02%), Oscillospira (3.01%), 5-7N15 (2.59%), and Clostridium (2.16%). Furthermore, partial least squares discriminant analysis (PLS-DA) plot showed that ruminal microbiota at bacteria level was clearly separated between healthy and mastitis groups. Next, we investigated the bacterial biomarkers using linear discriminant analysis effect size (LEfSe) analysis, which was applied to screen the given OTUs as biomarkes in each group from phylum to species level. In mastitis group, biomarkers were three taxa including Bacillus (genus), Sharpea (genus) and Bacillus anthracis (species). In healthy group, biomarkers were seven taxa including Selenomonas_1 (genus), Fibrobacter (genus), Treponema_2 (genus), Ruminococcus_1 (genus), Ruminococcaceae_UCG-014 (genus), Ruminococcus flavefaciens (species), and Treponema saccharophilum (species). To better understand the correlations between biomarkers and mastitis factors (serum Interleukin-6 level and milk somatic cells), the Spearman’s correlation coefficient analysis was used in the present study. The results revealed that serum Interleukin-6 level and milk somatic cells were positively associated with Bacillus, Sharpea and Bacillus anthracis but negatively correlated with Ruminococcaceae_UCG-014, Ruminococcus flavefaciens and Treponema saccharophilum. On the other hand, the metabolomics analysis indicated that the signal intensity of 21 compounds compared using Mann-Whitney U test were higher in healthy group (P < 0.05). Finally, the relationship between ruminal microbiota and metabolites was also evaluated by Spearman’s correlation coefficient. The present study demonstrated the potential biomarkers and the relevancy between gut microbiome and mastitis in dairy cows. Our findings could further provide a novel perspective for therapeutic strategies against mastitis.