SARA induces the system inflammatory response in dairy cows
In order to exclude the effect of experimental period on milk composition, we detected the milk composition of another eight Hosltein cows with the same standards of the cows that used to establish SARA model (lactation days and weight similar and non-pregnant cows). The results showed that there were no significant changes in milk yield, milk fat, milk protein, lactose, urea nitrogen and SCC of healthy cows during the experiment period (Fig. 1). To evaluate the HCD-induced SARA model in cows, we measured the dry matter intake (DMI) and milk yield weekly. After 8 weeks of feeding an HCD, the DMI and milk production were significantly reduced when compared to the baseline from before HCD feeding (see supplementary fig. 1A-B). In addition, the pH of the rumen fluid was obviously reduced, and a pH value < 5.8 was sustained for more than 3 h at different periods of time in the cows fed an HCD for 8 weeks (see supplementary Table S1), indicating that SARA was efficiently provoked[21]. In addition, levels of pH in feces were also significantly reduced, but there were no changes in the blood in SARA cows compared to the control cows (see supplementary Table S1). Abundance of evidence proved that a large number of LPS-derived rumen of SARA cows can be translocated into the bloodstream, and lead to systemic inflammatory response[22]. In the present study, the levels of LPS in rumen fluid, feces, and tail vein significantly all increased in cows happened SARA (Fig. 2A-C). To evaluate the host system inflammatory response, we tested the clinical health parameters, including rectal temperature, pulse rate, and respiratory rates, has not changed in SARA cows (see supplementary Table S2). However, the numbers of neutrophils in blood has markedly increased (see supplementary Table S3), and the levels of Ca and PHOS in plasma were significantly reduced in cows suffered from SARA (see supplementary Table S4). In addition, inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-17, as well as the levels of acute phase proteins serum amyloid-A (SAA) in plasma all significantly increased in SARA cows (Fig. 2D-H). Plasma levels of ALB, GLB, A/G, TP, and GLU were assessed as a indicator of the metabolism and inner organ status of animal[23]. The higher levels of ALB and the lower levels of GLU (see supplementary fig. S2C and S1G), but another has no changes in plasma were observed in SARA cows (see supplementary fig. S1D-F). The liver is an important metabolic and immune organ of ruminants, it plays an important role in removing the LPS in bloodstream[24]. On the contrary, over-production of LPS also can promotes the occurrence of inflammatory reactions and the aggregation of immune cell, and thus interferes with the metabolism of substances in the liver[25]. SARA cows showed a severe pathological damage, including inflammatory cells infiltration and liver cells injury ballooning (Fig. 2I-J), and up-regulated the level of AST (Fig. 2K). LPS entering the blood from the rumen needs to cross the rumen wall, thus, the rumen epithelial permeability was assessed in cows from control and SARA. Hispathological changes suggested that rumen epithelium of SARA was incomplete, and greater numbers of immune cells infiltrated into the rumen epithelium of SARA cows (Fig. 2M-N). Furthermore, the tight junction proteins of rumen barrier testing indicated that the proteins expression of Claudin-1, Claudin-3, Occludin, and ZO-1 all significantly reduced in SARA cows when compared to the control cows (Fig. 2O). Moreover, histopathologic changes of intestine tissues indicated that gut epithelium desquamation and severe cellular damage in SARA cows when compared to the control cows, and SARA cows showed significantly higher epithelial damage scores in intestines (Fig. 2P-Q). In addition, the expression of Claudin-1, Claudin-3, Occludin, and ZO-1 were all significantly reduced in SARA cows when compared to the control cows (Fig. 2R). These results suggested that cows SARA increase the permeability of the rumen epithelial, and induces rumen-derived LPS is released into the bloodstream, damaging the liver function and inducing systemic inflammation.
SARA induces the inflammatory response in the mammary gland by increasing blood-milk barrier permeability in dairy cows
Milk composition analysis showed that the contents of milk fat, milk protein, fat/protein ratio and dry matter were markedly reduced (Fig. 3A-C and Fig. 3E). The content of lactose in milk from SARA cows was unchanged (Fig. 3D) and urea nitrogen significantly increased when compared to the mil from control cows (Fig. 3F). Importantly, SCC and SAA, two important indicators of mastitis, also significantly increased after cows suffered from SARA (Fig. 3G-H). In addition, the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were obviously increased in the milk (see supplementary fig. S2A-C) and the mammary glands (Fig. 3I-K) from cows suffering from SARA. Histopathological analysis from SARA cows showed a large amount of inflammatory cell infiltration, thickening of the alveolar wall, and mammary gland destruction in the mammary gland and a higher inflammation score (Fig. 3L-M). Furthermore, we assessed whether cow mastitis induced by SARA was associated with LPS migration from the rumen to the mammary gland via the bloodstream. The levels of LPS in the lacteal veins, milk and mammary glands were all significantly increased in cows with SARA (Fig. 3N-P). LPS is a strong activator of innate immune responses and is recognized by TLR4, which then activates activated NF-κB to induce the transcription of TNF-α, IL-1β, and IL-6. The expression of TLR4, phosphorylation NF-κB p65, and phosphorylation IκBα were upregulated in the mammary glands of SARA compared with control cows (Fig. 3Q). The blood-milk barrier is a specific structure that plays an important role in preventing foreign matter from the blood or external environment entering the mammary gland[26]. To evaluate the permeability of the blood-milk barrier in control and SARA cows, we detected the tight junction proteins that make up the blood-milk barrier. The expression of Claudin-1, Claudin-3, Occludin, and ZO-1 was significantly reduced in SARA cows (Fig. 3R). In addition, blood-derived proteins in milk, including IgG and lactate dehydrogenase (LDH), serve as indicators of the integrity of the blood-milk barrier[27]. The production of IgG and LDH obviously increased both in the blood and milk of SARA cows when compared to the control cows (see supplementary fig. S2D-G). These results suggest that SARA of cows leads to rumen-derived LPS entering the mammary gland through blood circulation, damaging the blood-milk barrier, and inducting inflammation of the mammary gland in cows.
Dissimilarity of milk, rumen fluid, feces, and blood microbiota in cows from control and SARA
To explore the development of mastitis induced by SARA was associated with the changes with microbiota, we collected milk, rumen fluid, feces, and blood samples from the same 8 Holstein dairy cows on day 0 and 8 weeks after feeding an HDC to detect the community of microbiota using the V4 region of the bacterial 16S ribosomal RNA (rRNA) gene amplified PCR from all 64 samples. 16S rRNA sequencing resulted in 6,835,632 reads, with an average of 77677.64 ± 2614.504 reads (standard error of the mean) from each sample. The estimators of community richness (chao 1, and ace) and richness (shannon index and simpson) of milk, rumen fluid, and feces in SARA cows were significantly reduced compared to the control cows, but there were no significant effects on the richness of blood (see supplementary fig. S3). The Top 10 phyla and the top 30 genera in relative abundance were used to analyze the bacterial community structure of rumen fluid, milk, feces, and blood of cows. At the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, and Tenericutes were the four dominant phyla in all samples by relative abundance (Fig. 4A and supplementary Table S5). T-test analysis showed that in the rumen fluid microbiota, Proteobacteria was significantly increased in SARA cows, while Firmicutes, Tenericutes and other low abundance phyla, including Spirochaetes, Gracilibacteria, Euryarchaeota, Fibrobacteres, and Kiritimatiellaeota were significantly reduced in SARA when compared to the control cows (Fig. 4B). The changes of milk microbiota was similar to the changes of rumen microbiota, as shown in Proteobacteria was significantly increased, while other phyla, including Firmicutes, Bacteroidetes, Tenericutes, and Actinobacteria were significantly reduced in SARA cows when compared to the control cows (Fig. 4C). In feces microbiota, the relative abundance of Proteobacteria significantly increased, while the relative abundance of Melainabacteria was reduced in SARA cows when compared to the control cows (Fig. 4D). However, there are no detected changed significantly phyla in blood between control and SARA cows (see supplementary Table S6).
To test the microbiota elements at the genus level, the sequence showed that Stenotrophomonas, Bacteroides, Exiguobacterium, and Sphingononas were the most prominent genera in all simples (Fig. 5A and Table S7). Significant differences in the genus of milk, rumen fluid, feces, and blood between control and SARA cows were further assessed by t-test analysis. Stenotrophomonas and Succinivibrio were increased, while the other ten genes were reduced in rumen from SARA cows compared to those from control cows (Fig. 5B). The changes of milk microbiota was also similar to the changes of the rumen microbiota, as shown in the relative abundance of Stenotrophomonas, Sphingomonas and Brevnudimonas were obviously elevated, while other detected 14 genera all were reduced in milk from SARA cows compared with those from control cows (Fig. 5C). Microbiota in feces from cows indicated that except Stenotrophomonas, other genera, including Succinivibrio , unidentified-Prevatellaceae, unidentified-Erysipelotrichaceae, Unidentified-Clostridiales, Roseburia, Oscillibacter, Anaeroplasma, and Marvinbryantia were significantly increased, and other detected five genera were reduced in the SARA group (Fig. 5D). In addition, there are no significantly changes in microbiota in blood from SARA and control cows (see supplementary Table S8). These results showed that the changes of the microbiota of milk and rumen fluid samples after SARA have similarities. It is indicates that there may be a certain correlation between milk microbiota and rumen microbiota of dairy cows.
Similarity of bacterial community composition in the milk, rumen fluid, feces, and blood of control and SARA cows
To explore the correlation of microbiota structure in milk, rumen fluid, feces, and blood, we carried out a similarity analysis on the microflora of four different samples from control cows. Although the rumen fluid and feces are the most similar at the level of diversity of microbiota (Fig. 6A and supplementary fig. S4A), the milk and rumen fluid present the most similar microbiota abundance (Fig. 6B and supplementary fig. S4B). In addition, a principal coordinate analysis (PCoA) plot, based on the bray curtis distance matrices, indicated that the microbiota composition of milk was more similar to the microbiota composition of rumen fluid (Fig. 6C). Interestingly, nonmetric multidimensional scaling (NMDS) ordination performed on the Bray-Curtis dissimilarity showed that the bacterial community profiles of rumen fluid was closer to those of milk in SARA cows than in control cows (Fig. 6D). To confirm the results in the NMDS, the microbiota community dissimilarity was also evaluated by MRPP, which was based on the Bray-Curtis distance matrices. Application of MRPP to milk and rumen fluid and milk and feces indicated that rumen fluid (HM-HR vs SM-SR, expected-delt = 0.621 vs 0.446), but not feces microbiota (HM-HF vs SM-SM, expected-delt = 0.649 vs 0.683), were more similar to milk microbiota in SARA cows when compared to the control cows (Fig. 6E-F). These results suggest that the major changes in the bacterial community profile of milk, rumen fluid, feces, and blood during SARA lead to more similar community profiles among milk and rumen fluid.
Stenotrophomonas from the rumen maybe a factor to induce inflammation in the mammary gland in cows suffering from SARA
To evaluate whether a unique bacterial was associated with the occurrence of mastitis by analyzing core genera shared in milk, rumen fluid, feces, and blood in both control and SARA cows using Venn diagrams. A total of 772 core genera in control cows and 824 core genera in SARA cows overlapped among the milk, fluid rumen, feces, and blood samples (Fig. 7A-B). This is indicated that changes in bacterial abundance is more important for the development of mastitis than unique differences in bacterial communities. Furthermore, core genera accounted for 13.87 % of all milk bacteria in control cows and 21.25 % in SARA cows, 18.87 % of all rumen fluid bacteria on control cows and 22.26 % on SARA cows, 25.76 % of all feces bacteria on control cows and 25.46 % on SARA cows, 22.37 % of all blood bacteria on control cows and 25.46 % on SARA cows (Fig. 7C). This suggested that some bacteria may migrate among the milk, rumen fluid, and blood, resulting in a consistent change in the proportion of the shared core genera in these samples after cow suffered SARA.
To identify the specific bacteria that may be translocated from rumen to mammary gland via blood and be associated with mammary gland inflammation, we performed a biomarker analysis using linear discriminant analysis (LDA=4.0) effect size (LEfSe) and a cladogram generated from LEfSe analysis on the microbiota community of milk, rumen fluid, feces, and blood. At geneus levels, only Only Stenotrophomonas and Sphingomonadaceae were enriched in milk from SARA cows (Fig. 8A-B). Additionally, the relative abundance of the Stenotrophomonas in rumen fluid and feces all significantly increased in SARA cows when compared to the control cows (Fig. 8C-F). However, there was no detection of Stenotrophomonas enrichment in the blood of SARA cows, and we did not isolated the Stenotrophomonas in the blood (data has no shown). This results suggested that the consistency of the changes in the milk microbiota and rumen microbiota caused by SARA may not be due to the migration of Stenotrophomonas through the blood. However a large amount of Stenotrophomonas derived from the rumen may be associated with the development of mastitis. To rule out the possibility that mastitis in SARA cows is caused by common pathogenic microorganisms in the environment, we tested the relative abundance of common mastitis pathogens, including Staphylococcus, Streptococcus, and Escherichia coli, in the milk of control and SARA cows. The results showed that the abundance of Staphylococcus, Streptococcus, and Escherichia coli was reduced in SARA cows we observed when compared to the control cows (see supplementary fig. S5A-D). These results indicated that the development of mastitis in SARA cows may be caused by endogenous pathogens, such as Stenotrophomonas, rather than by common pathogen infections.
Treatment of Stenotrophomonas. maltophilia (S. maltophilia) induced mastitis in mice
To detect the relationship of elevated Stenotrophomonas in the rumen of SARA cows and the occurrence of mastitis, we detected the effected of S. maltophilia (the only species of the Stenotrophomonas) on the mastitis in mice. The results showed that gavaged of S. maltophilia results in the damages of mammary gland tissues (Fig. 9A-B), and increased the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the mammary glands (Fig. 9C-E). In addition, to detect whether S. maltophilia on the mammary gland surface can induce mastitis, the feces from mice that gavaged S. maltophilia were collected and smeared on the surface of healthy mice. H&E staining and ELISA analysis showed that no inflammation was present in the mammary glands of the mice smeared with feces from the mice that gavaged S. maltophilia duration of experiment (Fig. 9A-E). These results suggested that gut-derived S. maltophilia is an important factors to induce the development of mastitis.
Increased severity to S. aureus mastitis in cows suffering from SARA
To evaluate the effect of SARA on the severity of bovine mastitis , we tested the milk composition in cows of control and SARA after infection with S. aureus. Milk composition analysis demonstrated that milk fat was reduced after S. aureus infusion, and the contents were recovered when the 24 h after S. aureus treatment. However, a significant difference in SARA-positive and SARA-negative was only found 4 h after the S. aureus infusion (Fig. 10A). Moreover, infusion of S. aureus results in increased milk protein content in both SARA-positive and SARA-negative cows. A difference was found 24 h after S. aureus treatment in SARA-positive and SARA-negative cows (Fig. 10B). Furthermore, the lactose and dry matter content was significantly reduced after S. aureus infection in both SARA-positive and SARA-negative cows. However, no difference was found in SARA-positive vs SARA-negative until 24 h after S. aureus infusion (Fig. 10C-D). Infusion of S. aureus elevated the levels of SCC until the end of the observation time. More SCC was also detected in SARA-positive than in SARA-negative animals 12 h after S. aureus infusion, although there was no significant difference (Fig. 10E). Further research was conducted to detect the load of S. aureus in milk of SARA-positive and SARA-negative after receiving the S. aureus challenge. As shown in Fig. 10F, the bacterial loads in milk 4 h after S. aureus infusion reached the maximum, and then the loads of S. aureus were gradually reduced due to the clearance effect of the mammary gland on S. aureus. However, there were significantly higher bacterial loads in the milk of SARA-positive cows than that of SARA-negative cows from 12 h until 24 h after S. aureus challenge. These results suggested that cow rumen microbiota disturbance induced by SARA may increase the severity to mastitis by reducing the clearance ability of the mammary gland to S. aureus infection.