Intestinal microbiota of mastitis cows are distinct from those of healthy cows

To probe the link between gut microbiota and bovine mastitis, fecal microbiota from twelve 3–6-year-old cows diagnosed of mastitis were compared to twelve physically similar, age-matched, healthy cows that served as the control (Additional file 1: Table S1; Materials and method). Disease status was classified based on milk somatic cell count (SCC) plus clinical signs that include abnormal milk production and udder redness and swelling (Materials and method): average SCC of the diseased cows was 715-fold that of the healthy ones (Fig. 1a).

Fig. 1 Distinction between healthy and mastitis intestinal microbiota in cow. a Comparison of somatic cell count (SCC) between healthy and mastitis cows (logarithmic base of 10). b Principal coordinate analysis (PCoA) via Meta-Storm distances: distinct organismal composition between healthy (green) and mastitis (red) cows was revealed. Size of dots represents somatic cell count (logarithmic base of 10). F value is the PERMANOVA result of β-diversity. c Heatmap showing the 31 discriminating operational taxonomic units (OTUs) between healthy and mastitis cows (abundance shown as logarithm base of 10). d Principal component analysis (PCA) of functional genes (annotated by KEGG) between healthy and mastitis cows. e KEGG metabolic pathways that differentiate the healthy and diseased state are shown as a line map (red dots: enriched in mastitis cows; green dots: enriched in healthy controls; size of the dots: value of identified KO number divided by all KOs number in the specific pathway). f Correlation network of differential OTUs and KOs, with elliptical nodes (OTUs) colored based on genus, edges defined by the type of correlation (dash: negative; solid: positive) and hexagonal nodes (KOs) colored (blue or white) based on pathways Full size image

Between the healthy and mastitis cows, full-length 16S rRNA sequencing of stool DNA by the PacBio platform revealed identical α-diversity (via Shannon Index [13]), yet significant difference in β-diversity (via Meta-storms distance [14, 15]; F = 3.87, p = 0.003, PERMANOVA; Fig. 1b). Thirty-one bacterial operational taxonomic units (OTUs) (10 positively and 21 negatively correlated; adjusted p < 0.01, Wilcoxon rand-sum test; Additional file 2: Table S2A, Fig. 1c) were significantly associated with bovine mastitis: all are from the phyla of Firmicutes (Anaerostipes, Dorea, Lachnospiraceae, and Roseburia enriched in healthy cows, while Oscillospira and Ruminococcaceae enriched in mastitis) and Bacteroidetes (all enriched in healthy cows).

To identify mastitis-associated functional genes, 24 stool samples from the 12 sick and 12 healthy cows were each shotgun sequenced and compared based on profiles of encoded bacterial genes (Materials and method). Significant difference in β-diversity was also observed between the diseased and healthy cows (F = 4.08, p = 0.014, PERMANOVA, Fig. 1d), suggesting that in bovine mastitis both organismal and functional structure were altered in the gut microbiota. Biomarker analysis revealed 269 positively (n = 86) or negatively (n = 183) mastitis-associated KEGG Orthology (KO) (p < 0.05; Wilcoxon rank-sum test, Additional file 3: Table S3A, C), which were enriched in 42 metabolic pathways (Z > 1.6; Materials and method; Additional file 3: Table S3F). In mastitis cows, two pathways of (i) valine, leucine, and isoleucine biosynthesis and (ii) d-glutamine and d-glutamate metabolism were enriched, while the majority, i.e., the remaining 40 pathways, were depleted (Fig. 1e). The latter included vitamin B-related metabolic pathways, i.e., lipoic acid metabolism (all the three identified KOs in this pathway were less abundant, abbreviated as 3/3), vitamin B 6 (5/7), one carbon pool by folate (15/16), and thiamine metabolism (8/10). Vitamin B as a cofactor for many biochemical reactions may suppress inflammation [16], yet intestinal microbes are a major source of vitamin B in human and other mammalian hosts who are unable to synthesize them [17]. Thus, it is possible that mastitis is associated with a disorder of vitamin B metabolism in intestinal microbiota, which may deserve further investigation.

Pathways with lower abundance in mastitis cows, which presumably limit inflammation and protect intestinal mucosa, also included, e.g., lysine biosynthesis (13/16), fatty-acid biosynthesis (9/11), purine (45/60), and pyrimidine (41/49) metabolism (increased levels of the purine metabolite inosine can inhibit multi-organ inflammation in mice [18]) and selenocompound metabolism (6/9). Carbon metabolism including pyruvate metabolism (23/34), galactose metabolism (14/21), citrate cycle (18/24), and glycolysis/gluconeogenesis (22/32) was also less abundant, suggesting reduced carbon metabolic activity of gut microbiota in mastitis cows.

To probe the link between the mastitis-associated organisms and functional genes, a taxon-function interaction network was constructed based on abundance pattern of the disease-associated OTUs and KOs in the 24 animals [19] (Fig. 1f), where the edges that connect OTU nodes to all those KOs with whom linear correlation was found (Spearman correlation coefficient > 0.8, adjusted p < 0.01) indicate potential organism-function links. Two prominent OTU-clusters, all from Firmicutes Phylum and together accounting for 42% of disease-associated OTU, were found: one around Ruminococcaceae and Eubacterium and the other around Pseudobutyrivibrio and Lachnospiraceae. These OTUs are all positively or negatively correlated with carbon metabolism (Fig. 1f; Additional file 4: Table S4A). Specifically, Ruminococcaceae and Eubacterium, both enriched in mastitis, exhibit positive correlation with 121 KOs in propanoate (7/16; the number of KOs assigned to this pathways in the cluster/all identified KOs in this pathway) and butanoate (5/15) metabolism; purine (9/60) and pyrimidine (7/49) metabolism; and valine, leucine, and isoleucine degradation (5/12), suggesting implication of the activities from these Firmicutes in mastitis. Interestingly, a large functional-gene cluster of 41 KOs was positively correlated with Eubacterium and Ruminococcaceae while also negatively correlated with Pseudobutyrivibrio and Lachnospiraceae (both of which depleted in mastitis). This suggests that the shift in fine balance between these two specific OTU-clusters might underlie the health-to-mastitis conversion, where enrichment of Eubacterium and Ruminococcaceae and depletion of Pseudobutyrivibrio and Lachnospiraceae may result in upregulation of propanoate and butanoate metabolism, purine metabolism, valine, leucine, and isoleucine degradation.

Intestinal microbiota from mastitis cows, but not healthy cows, induced mastitis in germ-free mice, whereas probiotic intake alleviated mastitis

To test whether the structural and functional alteration of gut microbiota are a cause or a consequence of bovine mastitis, fecal microbiota from the 12 mastitis and 12 healthy cows were respectively pooled and then inoculated into adult, pregnant gnotobiotic mice via fecal microbiome transplantation (FMT; Materials and method). Among the 35 recipient mice, 11 underwent FMT from healthy cows (group H), 12 from mastitis cows (group M), while a third group of 12 (group P) was established where the mice underwent both FMT from mastitis cows and a 25-day regimen of probiotics intake after FMT (via intragastric administration of 5 × 108 cfu/day Lactobacillus casei; Materials and method).

Comparison of murine post-FMT inflammatory responses among the three groups revealed that gut microbiota from mastitis cows induced a much greater inflammatory (of mammary gland, liver, jejunum, and colon) response than those from healthy cows. On mammary gland surface, severe inflammation that corresponded to mastitis was observed in group M, yet no pathological changes were visually apparent in groups H or P (Fig. 2a). This was supported by histopathologic section that evaluates mammary gland tissue damage (e.g., mammary alveolus thickening, hyperemia, and edema) and extent of inflammatory cell infiltration (i.e., stained leukocyte cells) [20, 21]. For example, under hematoxylin-eosin (HE) staining, group M featured broken lobules of the mammary gland, damaged acinuses, and destroyed epithelial cells, with inflammatory cells including macrophages, neutrophils, and blood cells detected in the mammary lobule (Fig. 2b); in contrast, in group H, no pathological changes were apparent, while in group P the lobules were largely complete and the acinuses were mostly intact (suggesting mitigated histopathology). These findings were further supported by the increased immunohistochemical staining of mammary gland for CD45 in group M (CD45 as the first and prototypic receptor-like protein tyrosine phosphatase is expressed on all nucleated hematopoietic cells and plays a central role in adaptive immunity [22]): inflammatory cells such as macrophages and neutrophils were found in group M but not in groups H or P (i.e., suggesting an inflammatory response in group M; Fig. 2c). The observed migration of leukocytes from blood into mammary gland indicated a bacteria-induced cellular inflammatory response that was stimulated by secreted chemotactic and inflammatory mediators [23].

Fig. 2 Histological analysis of mouse tissues after FMT and probiotics intervention. a Pathological changes in mammary gland surface, where two abdominal mammary glands were swelling in the mastitis group of mice on day 25 after FMT. Breast of mice was highlighted by red circles. b Representative photomicrographs of hematoxylin-eosin stained mammary gland tissue (× 200 magnification). c CD45 immunohistochemical staining sections at × 400 magnification. d–f Representative photomicrographs of hematoxylin-eosin stained liver (× 200), jejunum (× 100), and colon tissue (× 100). g The injury score of mammary gland, liver, jejunum, and colon Full size image

The murine inflammation induced by diseased bovine intestinal microbiota seemed pervasive. HE staining of murine liver sections revealed blur of hepatic lobe, hyperemia, and ballooning degeneration of hepatocyte in group M, in contrast to the normal liver structures in group H and the recovered liver structures in group P (Fig. 2d). Pathological section of murine intestinal and colon revealed in group M severe disorder in mucosa structure (necrosis of epithelial cells, extension of the subepithelial space, and structural damage of villi); in contrast, group H mice exhibited normal intestinal mucosa with integral villi, while probiotics intake in group P significantly improved intestinal and colon histology, featuring alleviated swelling of mucosa, less subepithelial space expansion, and well-arranged villi structure (Fig. 2e, f). In fact, for each of the tissues tested, pathological grade of injury was significantly higher in group M than in either group H (p < 0.01) or group P (p < 0.01; Fig. 2g). To test whether bacteria on the breast surface can induce mastitis, three mice were transplanted with healthy cow feces and were administered with mastitis cow feces on the surface of their breast (Materials and Method). HE staining showed that no inflammation was present in the mammary glands of the three mice throughput the duration of experiment (Additional file 5: Figure S1).

To assess the activation of immunological signaling pathways in the murine mammary gland, a panel of nine key cytokines were assayed by Western blot at day 25th after FMT (Fig. 3a; the housekeeping gene of β-actin as control), which includes NF-κb and Iκb-β in the NF-κb signaling pathway; ERK, p38, and JNK in the MAPKs signaling pathway; DNA binding protein STAT3 (which responds to epidermal growth factor production and IL-6 secretion [24, 25]); membrane-bound bile acid receptor TGR (involved in regulating energy homeostasis and glucose metabolism [26]); CLC4 (essential regulator of cell volume and repair of epithelial damages [27]); and Akt (which phosphorylates and inhibits proapoptotic components of the intrinsic cell death machinery [28]). Group M reported much higher levels for seven of the nine cytokines than group H (e.g., NF-κB is 9.12-fold higher in Group M), except CLC4 (equivalent) and JNK (29.4-fold lower in Group M). In group P, levels of the cytokines fell between group M and group H (except CLC4 which showed little variation); notably, the 57.7%-lower NF-κB level in group P than group M indicated an anti-inflammatory effect of probiotics that is linked to inhibition of NF-κB pathway activation (Fig. 3a).

Fig. 3 Effect of probiotics (Lactobacillus casei) administration on mice that were predisposed to risk of mastitis. a Western blots for quantification of NF-κb, Iκb-β, ERK, p38, JNK, STAT3, TGR, CLC4, and Akt protein levels in mammary glands (n = 2 per group), with β-actin as internal control. b Quantification of inflammatory cytokines in various tissues or organs using ELISA (n = 7 per group). The assays were all performed for the three groups of mice at Day 25 after FMT. Asterisk indicates significant difference between two groups (p < 0.05; Student’s t test) Full size image

Furthermore, a number of murine inflammatory cytokines produced predominantly by activated macrophages [21], including tumor necrosis factor (TNF), interferon (INF), myeloperoxidase (MPO), and interleukins (IL), were assayed via ELISA in various murine tissues: (i) TNF-α, MPO, and IL-6 in mammary gland; (ii) IFN-γ, IL-4, IL-10, IL-17, lysozyme, and endotoxin in serum; (iii) IL-1β in colon; (iv) IL-6 and TNF-α in jejuna; and (v) IL-17 in spleen (Fig. 3b; Additional file 6: Table S5). Compared to group H, group M mice exhibited increased level of all the cytokines tested (p < 0.01), consistent with a much higher post-FMT inflammatory response in this group. Interestingly, for the majority of cytokines tested, their level in group P was higher than group H yet lower than group M, with the notable exceptions being at the mammary tissues, where MPO, IL-6, and TNF-α in group P were higher than or equivalent to those in group M (Fig. 3b). Considering that group P exhibited mitigated histopathology (plus absence of macrophages and neutrophils) in the murine mammary gland and reduced pathological grade of injury in other organs, upregulation of cytokine secretion (a key feature of augmented immune protection) that resulted from probiotics administration may have underlie the alleviated mastitis symptoms in group P. Moreover, the highest level of serum endotoxin found in group M suggested the possibility of access of gut bacteria to the blood system through hepatoenteral circulation, which contributed to the mammary gland inflammation.

Organismal and functional distinction of intestinal microbiota between health and mastitis hosts was amplified by the cow-to-mouse FMT

To mechanistically probe the distinct disease outcome among the three post-FMT murine groups, both full-length 16S rRNA gene amplicons and shotgun metagenomes were analyzed for stools of each of the 35 mice at day 25th after FMT (Materials and Method). The 16S rRNA amplicon analysis revealed that, despite identical α-diversity, distinction in β-diversity between group H and group M mice was highly significant and in fact much greater (F = 42.19, p = 0.001; PERMANOVA, Fig. 4a) than that between diseased and healthy cows (F = 3.87, Fig. 1b). Underlying the high degree of discrimination are 66 OTUs (Additional file 2: Table S2B) from the phyla of Firmicutes (35 of them), Bacteroidetes (30), and Actinobacteria (1). Most of the Firmicutes OTUs (e.g., those from Lactobacillus, Eisenbergiella, Lachnospiraceae_Group, and Eubacterium genera) were enriched in group H, yet most of the Bacteroidetes OTUs enriched in group M (Fig. 4b).

Fig. 4 Distinction between healthy and mastitis intestinal microbiota in the mice after FMT. a PCoA clustering of the organismal structure of microbiota based on Meta-Storm distance. Percentage of variation explained by each principal coordinate is indicated on the axes. b Heat map of the 66 differential OTUs between group M and group H of mice. Relative abundance was shown as log 10 based. c PCA of functional gene structure between group M and group H of mice. d Significantly changed pathways of murine gut microbiota between group M and group H of mice. e Correlation network of differential OTUs and KOs, which revealed the taxon-function links Full size image

Comparison of shotgun metagenomes, i.e., β-diversity based on encoded microbial genes (via cosine distance of KOs), suggested strong functional discrimination of group M from group H mice (F = 104.61, p = 0.001, Fig. 4c). Moreover, the taxon- and function-based schemes are highly consistent (p = 0.0004, Monte Carlo test; Procrustes analysis based on PC1 and PC2). Among the 25 discriminating pathways (from 3525 differentiating KOs; Fig. 4d, Additional file 3: Table S3B, D, G), 9 were enriched while 16 depleted in group M (as compared to Group H). The most prominent is the lower abundance in mastitis of bacterial chemotaxis (23/24; depleted KOs vs all KOs in the pathway) and flagellar assembly (35/36), as well as intestinal mucosa repair and pathogen resistance, i.e., propanoate metabolism (32/44), butanoate metabolism (37/46), and selenocompound metabolism (13/21). Consistent with the findings in bovine, pyruvate metabolism (23/34), galactose metabolism (14/21), citrate cycle (18/24), and glycolysis/gluconeogenesis (22/32) were all depleted in mastitis mice. However, contrary to bovine, those enriched in mastitis mice included two vitamin B pathways of thiamine (9/14) and biotin (10/16) metabolism, as well as the degradation pathways of glycosaminoglycan (10/11) and other glycans (11/14). Considering the anti-inflammatory effect of glycosaminoglycan in rat arthritis [29], these results suggest that murine mastitis (but not bovine) may be potentially linked to the reduction of glycosaminoglycan, which is caused by the higher degradative activity of murine microbiota.

The murine OTU-KO correlation network, by correlating between the mastitis-associated taxonomical and functional profiles of the murine fecal microbiota, revealed two prominent clusters (Fig. 4e; Additional file 4: Table S4B): one around Eubacterium which is positively linked to propanoate metabolism (18/44; KOs from the clusters/all identified KOs) and butanoate metabolism (13/46), and the other around Lachnospiraceae (relative abundance of these OTUs all decreased in group M mice) which was positively associated with bacterial chemotaxis (15/24) and flagellar assembly (20/36). Notably, Eubacterium OTUs stood out in both of the murine and bovine OTU-KO networks, yet their taxonomical identity and associated KOs (i.e., functional roles) were both distinct (Fig. 1f): in cow, these OTUs were linked to the purine and pyrimidine metabolism, yet in mouse a different set of Eubacterium OTUs was linked to propanoate and butanoate metabolism. Thus the same bacterial components can exhibit distinct functions within cow and mouse.

Mechanism of mastitis alleviation as induced by probiotics intake in mice

Although probiotics intake in parallel with FMT from diseased cows resulted in a significant relief of mastitis, taxonomical structures of group P microbiota were indistinguishable from those of group M (F = 0.81, p = 0.48; PERMANOVA, Fig. 5a), yet are distinct from those of group H (F = 33.02, p = 0.001; Fig. 5b). Indeed, taxonomical structure of group P is much more similar to group M than to group H (Fig. 5c, d). In group P, 16 OTUs were significantly changed (all with lower abundance) as compared to group M: 11 from Bacteroides and S24_7 (Bacteroidetes) and 5 from the genera of Dorea, Eubacterium, Oscillospira, and Erysipelatoclostridium (Fig. 5e; Additional file 2: Table S2C). Fifteen of these 16 OTUs (except OTU1105016) were also found in group H and depleted as compared to group M, suggesting in group P microbiota a certain degree of “recovery” in structure from the mastitis state to the healthy state.

Fig. 5 Influence of probiotics administration on structure and function of murine intestinal microbiota. a~c PCoA of organismal structures of microbiota among the three groups of mice. d Similarity of the microbiota in organismal structure based on Meta-Storm distance. e~g PCA of functional gene structure (based on KEGG annotation) among the three groups of mice. h Similarity of the microbiota in functional gene structure based on cosine distance of KOs. i Heat map of the 16 differential OTUs between group P and group M of mice. j Metabolic pathways that were significantly altered between group P and group M, and between group M and group H. Pathways that drove the microbiota toward healthy state after probiotics administration were highlighted via red font. Pathways upregulated in group P (as compared to group M) yet downregulated in group H (as compared to group M) were colored with black, which represent microbial pathways induced by probiotics intake yet did not drive the microbiota towards the healthy state. Those pathways that were altered in one comparison yet not in the other were colored as gray. k Degree of microbiota divergence among group P, group M, and group H of mice, in terms of organismal structure of microbiota, functional gene structure of microbiota, as well as the mastitis symptom of the host Full size image

Interestingly, the KO profile derived from shotgun metagenomes separated group P from either group M (F = 5.40, p = 0.012; Fig. 5f) or group H (F = 106.34, p = 0.001; Fig. 5g); however, consistent with the taxonomy-based relationship, group P is also more similar to group M than to group H (Fig. 5h, i). Between groups P and M, 986 KO terms (from 219 pathways) and 32 pathways were significantly changed (z > 1.6, enrichment analysis; Additional file 3: Table S3E, H). These pathways were all group-P enriched, except for glycosaminoglycan degradation (depleted; 7/11). In the carbon metabolism, probiotics intake leads to higher abundance of galactose metabolism (28/42), pyruvate metabolism (43/64), and glycolysis/gluconeogenesis (42/59). Pathways for intestinal mucosa repair and inflammatory suppression, including butanoate metabolism (31/45), propanoate metabolism (31/44), and selenocompound metabolism (15/18), were also stimulated by probiotics. Furthermore, bacterial chemotaxis (22/24) and flagellar assembly (35/36) became more abundant. These features between groups P and M are mostly consistent or conserved with the health-enriched features identified from the comparison between groups M and H (Fig. 5j), although a few non-conserved pathways related to vitamin B such as biotin metabolism (12/14), one carbon pool by folate (16/18), and pantothenate and CoA biosynthesis (21/23) were specifically enriched in group P versus group M. Collectively, these results suggest that the probiotic intake led to a functional shift of murine intestinal microbiota toward the healthy state, plus a significant degree of host-symptom relief, despite the lack of conservation in taxonomical structure between groups H and P (Fig. 5k).

OTU-KO correlation analysis revealed that the Bacteroidetes OTUs that distinguish group P from group M (which were less abundant in group P and represented 31% of total differential OTUs) were positively correlated with glycosaminoglycan degradation (Fig. 4e). For example, in the Bacteroidetes cluster, the group P-depleted KOs of K01565, K01132, and K01135 were all implicated in glycosaminoglycan degradation (Fig. 4e). Considering that glycosaminoglycan degrading activities were much higher in group M than in group H, it is possible that the probiotic intake reduced the extent of glycosaminoglycan degradation by inhibiting selected Bacteroidetes OTUs that underlie such degradative activities.

Amplification of disease effect by microbiota transplantation across two orders of mammals

The ability to recapitulate mastitis in germ-free mice via FMT from mastitis cows supports gut microbiota as a cause, instead of consequence, of mastitis. Interestingly, the cross-mammal-order microbiota transplantation resulted in not just conservation in mastitis symptom but also amplification of intestinal microbiota dysbiosis, as evidenced in the 3-fold amplification of divergence (OTUs) between healthy and mastitis microbiota (averaged distance of 0.104 between groups H and M in cow versus 0.312 in mouse; Fig. 6a, b) and 23-fold amplification of averaged distance (KOs) between healthy and mastitis (averaged distance of 0.017 between groups H and M in cow versus 0.389 in mouse; Fig. 6c, d).

Fig. 6 Comparison of mastitis-associated microbiota in cow and those in mouse. PCoA clustering (a) and relative similarity (b) of bovine and murine microbiota based on organismal structure (via Meta-Storm distance) were shown. Moreover, PCA (c) and relative similarity (d) based on functional gene structure (via cosine distance of KOs) were presented. e Unique and shared OTUs and KOs before and after FMT in cows and mice. f OTUs that were shared between cows and mice during the FMT from healthy cows to healthy mice (upper panel) or that from diseased cows to diseased mice (lower panel). Red-font highlighted are the mastitis-associated OTUs in mice. g Mastitis-associated pathways that were shared between cows and mice. Those that were enriched in both cows and mice were highlighted in red fonts Full size image

We next probed how such “amplification effect” occurred and in particular, why the very large difference between donor microbiota and xenomicrobiota ended up with similar disease outcome. In the gut microbiota of the murine recipients, majority of family-level taxa (91.8% for healthy pairs and 94.2% for mastitis pairs) were from those of the cow donors (Fig. 6e). However, only one genus-level taxon, of Lachnospiraceae Group, exhibited identical trend of enrichment (i.e., enriched in healthy microbiota as compared to diseased ones) between cow and mouse (Fig. 6f). Although Lachnospiraceae Group represented only 3% of bacterial abundance in mastitis cows and mastitis mice, they were predominant in healthy murine gut (40%, as compared to 5% in healthy bovine gut). Thus loss of Lachnospiraceae may be associated with mastitis and Lachnospiraceae appeared to be critical to a healthy host state.

From the functional perspective, between the 269 mastitis-associated KOs (and 42 such pathways) in cow and the 3525 mastitis-associated KOs (and 25 such pathways) in mouse, 83 KOs (and 6 pathways) are shared that also showed an identical trend of alteration between diseased and healthy hosts. These six pathways are all of lower abundance in mastitis, including TCA cycle, galactose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, lipopolysaccharide biosynthesis, and selenocompound metabolism. Notably, the degree of enrichment (Z score) for these six pathways was each higher in mice than in cows (Fig. 6g). On the other hand, the vast majority (93.5% in cow and 98.4% in mouse) of disease-associated KOs are not shared between cow and mouse. Moreover, distinction in the disease-associated KOs was profound: those in cow featured disease-specific enrichment of certain vitamin B metabolism pathways (Fig. 1f), while those in mouse were characterized by disease-specific depletion of bacterial chemotaxis and flagellar assembly (Fig. 4e). Together, these results suggest that in the cow-to-mouse FMT, recapitulation of mastitis symptom was accompanied by amplification of the distinction between healthy and diseased microbiota, plus a dramatic change of mastitis-associated OTUs and microbial functions (Fig. 7).