A fiber-enriched diet alleviates Staphylococcus aureus-induced mastitis by activating the HDAC3-mediated antimicrobial program in macrophages via butyrate production in mice.

Mounting evidence suggests that the gut microbiota plays an important role in the pathogenesis of mastitis, an important disease affecting the health of lactating women and the development of the dairy industry. However, the effect of the regulation of the gut microbiota by dietary components on mastitis development remains unknown. In this study, we found that a fiber-enriched diet alleviated Staphylococcus aureus (S. au)-induced mastitis in mice, which was dependent on the gut microbiota as depletion of the gut microbiota by antibiotics abolished this protective effect. Likewise, fecal microbiota transplantation (FMT) from high-inulin (HI)-treated mice (HIF) to recipient mice improved S. au-induced mastitis in mice. Consumption of an HI diet and HIF increased fecal short-chain fatty acid (SCFA) levels compared with the control group. Moreover, treatment with SCFAs, especially butyrate, alleviated S. au-induced mastitis in mice. Mechanistically, consumption of an HI diet enhanced the host antimicrobial program in macrophages through inhibiting histone deacetylase 3 by the production of butyrate. Collectively, our results suggest that modulation of the gut microbiota and its metabolism by dietary components is a potential strategy for mastitis intervention and serve as a basis for other infectious diseases.

Hexadecanamide alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting inflammatory responses and restoring blood-milk barrier integrity.

Subacute ruminal acidosis (SARA) has been demonstrated to promote the development of mastitis, one of the most serious diseases in dairy farming worldwide, but the underlying mechanism is unclear. Using untargeted metabolomics, we found hexadecanamide (HEX) was significantly reduced in rumen fluid and milk from cows with SARA-associated mastitis. Herein, we aimed to assess the protective role of HEX in Staphylococcus aureus (S. aureus)- and SARA-induced mastitis and the underlying mechanism. We showed that HEX ameliorated S. aureus-induced mastitis in mice, which was related to the suppression of mammary inflammatory responses and repair of the blood-milk barrier. In vitro, HEX depressed S. aureus-induced activation of the NF-κB pathway and improved barrier integrity in mouse mammary epithelial cells (MMECs). In detail, HEX activated PPARα, which upregulated SIRT1 and subsequently inhibited NF-κB activation and inflammatory responses. In addition, ruminal microbiota transplantation from SARA cows (S-RMT) caused mastitis and aggravated S. aureus-induced mastitis, while these changes were reversed by HEX. Our findings indicate that HEX effectively attenuates S. aureus- and SARA-induced mastitis by limiting inflammation and repairing barrier integrity, ultimately highlighting the important role of host or microbiota metabolism in the pathogenesis of mastitis and providing a potential strategy for mastitis prevention.

Lactobacillus reuteri inhibits Staphylococcus aureus-induced mastitis by regulating oxytocin releasing and gut microbiota in mice.

Mastitis is the most frequent disease of cows and has well-recognized detrimental effects on animal wellbeing and dairy farm profitability. With the advent of the postantibiotic era, alternative antibiotic agents, especially probiotics, have received increasing attention in the treatment of mastitis. Based on research showing that Lactobacillus reuteri (L. reuteri) has anti-inflammatory effects, this study explored the protective effects and mechanisms of L. reuteri against mastitis induced by Staphylococcus aureus (S. aureus) in mice. First, mice with S. aureus-induced mastitis were orally administered L. reuteri, and the inflammatory response in the mammary gland was observed. The results showed that L. reuteri significantly inhibited S. aureus-induced mastitis. Moreover, the concentration of oxytocin (OT) and protein expression of oxytocin receptor (OTR) were measured, and inhibition of OTR or vagotomy reversed the protective effect of L. reuteri or its culture supernatant (LCS) on S. aureus-induced mastitis. In addition, in mouse mammary epithelial cells (MMECs), OT inhibited the inflammation induced by S. aureus by inhibiting the protein expression of OTR. It was suggested that L. reuteri protected against S. aureus-induced mastitis by releasing OT. Furthermore, microbiological analysis showed that the composition of the microbiota was altered, and the relative abundance of Lactobacillus was significantly increased in gut and mammary gland after treatment with L. reuteri or LCS. In conclusion, our study found the L. reuteri inhibited the mastitis-induced by S. aureus via promoting the release of OT, and treatment with L. reuteri increased the abundance of Lactobacillus in both gut and mammary gland.

The rumen microbiota contributed to the development of mastitis induced by subclinical ketosis.

BACKGROUND: Mastitis is a serious disease which affects animal husbandry, particularly in cow breeding. The etiology of mastitis is complex and its pathological mechanism is not yet fully understood. Our previous research in clinical investigation has revealed that subclinical ketosis can increase the number of somatic cell counts (SCC) in milk, although the underlying mechanism remains unclear. Recent studies have further confirmed the significant role of mastitis. RESULTS: In this study, we aimed to examine the SCC, rumen microbiota, and metabolites in the milkmen of cows with subclinical ketosis. Additionally, we conducted a rumen microbiota transplant into mice to investigate the potential association between rumen microbiota disturbance and mastitis induced by subclinical ketosis in dairy cows. The study has found that cows with subclinical ketosis have a higher SCC in their milk compared to healthy cows. Additionally, there were significant differences in the rumen microbiota and the level of volatile fatty acid (VFA) between cows with subclinical ketosis and healthy cows. Moreover, transplanting the rumen microbiota from subclinical ketosis and mastitis cows into mice can induce mammary inflammation and liver function damage than transplanting the rumen flora from healthy dairy cows. CONCLUSIONS: In addition to the infection of mammary gland by pathogenic microorganisms, there is also an endogenous therapeutic pathway mediated by rumen microbiota. Targeted rumen microbiota modulation may be an effective way to prevent and control mastitis in dairy cows.

Aryl hydrocarbon receptor activation by Lactobacillus reuteri tryptophan metabolism alleviates Escherichia coli-induced mastitis in mice.

The intestinal microbiota has been associated with the occurrence and development of mastitis, which is one of the most serious diseases of lactating women and female animals, but the underlying mechanism has not yet been elucidated. Aryl hydrocarbon receptor (AhR) activation by microbiota tryptophan metabolism-derived ligands is involved in maintaining host homeostasis and resisting diseases. We investigated whether AhR activation by microbiota-metabolic ligands could influence mastitis development in mice. In this study, we found that AhR activation using Ficz ameliorated mastitis symptoms, which were related to limiting NF-κB activation and enhancing barrier function. Impaired AhR activation by disturbing the intestinal microbiota initiated mastitis, and processed Escherichia coli (E. coli)-induced mastitis in mice. Supplementation with dietary tryptophan attenuated the mastitis, but attenuation was inhibited by the intestinal microbiota abrogation, while administering tryptophan metabolites including IAld and indole but not IPA, rescued the tryptophan effects in dysbiotic mice. Supplementation with a Lactobacillus reuteri (L. reuteri) strain with the capacity to produce AhR ligands also improved E. coli-induced mastitis in an AhR-dependent manner. These findings provide evidence for novel therapeutic strategies for treating mastitis, and support the role of metabolites derived from the intestinal microbiota in improving distal disease.

Retinoic acid ameliorates low-grade endotoxemia-induced mastitis by limiting inflammatory responses in mice.

Mastitis is a serious disease for humans and animals, which causes huge economic losses in the dairy industry and is hard to prevent due to the complex and unclear pathogenesis. Subacute ruminal acidosis (SARA) has contributed to the development of mastitis by inducing ruminal dysbiosis and subsequent low-grade endotoxemia (LGE), however, how ruminal metabolic changes regulate this progress is still unclear. Our previous study revealed that cows with SARA had increased ruminal retinoic acid (RA) levels, a metabolic intermediate of vitamin A that plays an essential role in mucosal immune responses. Hence, the aim of this study was to investigate the protective effect of RA on LGE-induced mastitis and the underlying mechanisms in mice. The results showed that RA alleviated LGE-induced mastitis, as evidenced by RA significantly reduced the increase in mammary proinflammatory cytokines and improved blood-milk barrier injury caused by LGE. In addition, RA increased the expression of tight junction proteins, including ZO-1, occludin and claudin-3. Furthermore, we found that RA limited the mammary inflammatory responses by inhibiting the activation of NF-κB and NLRP3 signaling pathways. These findings suggest that RA effectively alleviates LGE-induced mastitis and implies a potential strategy for the treatment and prevention of mastitis and other diseases.

Phytosphingosine alleviates Staphylococcus aureus-induced mastitis by inhibiting inflammatory responses and improving the blood-milk barrier in mice.

Mastitis is one of the common diseases in dairy cows which threatens the health of cows and impacts on economic benefits seriously. Recent studies have been showed that Subacute Ruminal Acidosis (SARA) increased the susceptibility of cow mastitis. SARA leads the disturbance of the rumen microbiota, and the rumen bacterial disordered community is an important endogenous factor of cow mastitis. That is to say, cows which suffer from SARA have a disordered rumen microbiota, a prolonged decline in ruminal PH and a high level of lipopolysaccharide (LPS) in the rumen, blood. Therefore, ruminal metabolism is closely related to the rumen microbiota. However, the specific mechanism of SARA and mastitis still not clear. We found an intestinal metabolite according to the metabonomics, which is correlated to inflammation. Phytophingosine (PS), a product from rumen fluid and milk of the cows which suffer from SARA and mastitis. It has the effect of killing bacteria and anti-inflammatory. Emerging evidences indicate that PS can alleviate inflammatory diseases. However, how PS affects mastitis is largely unknown. In this study, we explored the concrete role of PS on Staphylococcus aureus (S. aureus) -induced mastitis in mice. We found that PS obviously decreased the level of the proinflammatory cytokines. Meanwhile, PS also significantly relieved the mammary gland inflammation caused by S. aureus and restored the function of the blood-milk barrier. Here, we showed that PS increased the expression of the classic Tight-junctions (TJs) proteins including ZO-1, Occludin and Claudin-3. Moreover, PS improves S. aureus-induced mastitis by inhibiting the activation of the NF-κB and NLRP3 signaling pathways. These data indicated that PS relieved S. aureus-induced mastitis effectively. This also provides a reference for exploring the correlation between the intestinal metabolism and inflammation.

ZEA mediates autophagy through the ROS-AMPK-m-TOR pathway to enhance the susceptibility of mastitis induced by Staphylococcus aureus in mice.

Mastitis is an inflammatory response of the mammary tissue caused by pathogenic bacterial infections, especially Staphylococcus aureus (S. aureus). Zearalenone (ZEA) is one of the common mycotoxins in moldy feed, which usually affects the cow's resistance to pathogenic microorganisms. However, it is not well understood whether ZEA affects the development of mastitis. Therefore, this study aimed to investigate the role of ZEA in the development of S. aureus-induced mastitis in mice. The results showed that administered daily by gavage for one week of ZEA (40 mg/kg) aggravated the severity of mastitis induced by S. aureus. Furthermore, we found that ZEA promotes the adhesion and invasion of S. aureus into mouse mammary epithelial cells (MMEC) by activating autophagy, and the activation of autophagy mediated by ROS-AMPK-m-TOR pathway. Taken together, the results showed that ZEA enhances S. aureus-induced mastitis susceptibility through activating autophagy mediated by ROS-AMPK-mTOR signaling pathway.

Probiotic Enterococcus mundtii H81 inhibits the NF-κB signaling pathway to ameliorate Staphylococcus aureus-induced mastitis in mice.

Mastitis is part of the aggressive diseases that affecting the development of dairy farming. Lactic acid bacteria (LAB), an important microbiological agent of gastrointestinal flora, can effectively promote the development of the immune system. Herein, the objectives of this study is to explore the protective role of LAB on Staphylococcus aureus(S. aureus)-induced mastitis in mice. 88 strains of suspected LAB were isolated from the milk of healthy dairy cows. Antibacterial activity was screened, and the 16S rRNA sequence analysis showed that the bacteria were Enterococcus mundtii H81 (E. mundtii H81). Furthermore, the model of mastitis has been established by nipple duct injection of S. aureus in mice, while E. mundtii H81 was treated 2 h before S. aureus injection. Twenty-four hours later of S. aureus infection, the mammary gland tissues were collected. The pathological changes of the mammary gland were observed by H&E staining. The levels of TNF-α and IL-1ß were measured by ELISA and the myeloperoxidase (MPO) activity was measured by the MPO assay kit. We also observed the changes of nuclear transcription factor kappa B (NF-κB) by using western blotting. The results showed that E. mundtii H81 pretreatment reduced neutrophil infiltration, and significantly reduce the secretion of TNF-α and IL-1ß, down-regulate the phosphorylation of p65 NF-κB and IκB, and the expression of tight junction protein Claudin 3 and ZO-1 was up-regulated. Collectively, our findings showed that E. mundtii H81 protects mammary gland from S. aureus-induced mastitis, which may be a candidate of treatment for mastitis infected by S. aureus.

Kynurenic acid ameliorates lipopolysaccharide-induced endometritis by regulating the GRP35/NF-κB signaling pathway.

Endometritis is a serious reproductive disease in mammals that commonly results in reproductive loss and even permanent infertility. Kynurenic acid (KYNA) is the main bioactive metabolite of tryptophan degradation and exhibits neuroprotective and anticonvulsant properties. However, little is known about the role of KYNA in achieving endometritis remission. This study investigated the protective effects and mechanisms of KYNA using a mouse model of against lipopolysaccharide (LPS)-induced endometritis. The endometritis model was induced by an intrauterine injection of LPS, and KYNA was intraperitoneally injected before and two hours after LPS treatment. Twenty-four hours after LPS administration, pathological changes in uterine tissues were observed by hematoxylin- and eosin (H&E) staining. The levels of the inflammatory factors, TNF-α and IL-1ß, were measured by ELISA. The myeloperoxidase (MPO) activity in uterine tissues was detected using MPO kits and immunohistochemistry. Furthermore, the expression of signaling pathway proteins and tight junction proteins occludin and ZO-1 in uterine tissues was detected by western blot. KYNA prominently inhibited uterine pathological injury and neutrophil infiltration and restricted the secretion of TNF-α and IL-1ß in the uteri of subjects with endometritis. Furthermore, KYNA upregulated the levels of the tight junction proteins (TJPs)occludin and ZO-1 in the uterus. In vitro, KYNA inhibited LPS-induced TNF-α and IL-1ß production, and NF-κB activation in mouse endometrial epithelial cells (mEECs). In addition, KYNA increased the expression of G protein-coupled receptor 35 (GPR35) and inhibition of GPR35 reversed the anti-inflammatory effects of KYNA. In conclusion, KYNA protected against LPS-induced endometritis by maintaining epithelial barrier permeability and suppressing proinflammatory responses via the GRP35/NF-κB signaling pathway.

DNaseI protects lipopolysaccharide-induced endometritis in mice by inhibiting neutrophil extracellular traps formation.

Endometritis is an inflammatory of the inner lining of the uterus caused by bacterial infections that affect female reproductive health in humans and animals. Neutrophil extracellular traps (NETs) have the ability to resist infections that caused by pathogenic invasions. It has been proved that the formation of NETs is related to certain inflammatory diseases, such as mastitis and chronic obstructive pulmonary disease (COPD). However, there are sparse studies related to NETs and endometritis. In this study, we investigated the role of NETs in lipopolysaccharide (LPS)-induced acute endometritis in mice and evaluated the therapeutic efficiency of DNaseI. We established LPS-induced endometritis model in mice and found that the formation of NETs can be detected in the mice uterine tissues in vivo. In addition, DNaseI treatment can inhibit NETs construction in LPS-induced endometritis in mice. Moreover, myeloperoxidase (MPO) activity assay indicated that DNaseI treatment remarkably alleviated the inflammatory cell infiltrations. ELISA test indicated that the treatment of DNaseI significantly inhibited the expression of the proinflammatory cytokines TNF-α, and IL-1ß. Also, DNaseI was found to increase proteins expression of the uterine tissue tight junctions and suppress LPS-induced NF-κB activation. All the results indicated that DNaseI effectively inhibits the formation of NETs by blocking the NF-κB signaling pathway and enhances the expression of tight junction proteins, consequently, alleviates inflammatory reactions in LPS-induced endometritis in mice.

Sodium butyrate alleviates lipopolysaccharide-induced endometritis in mice through inhibiting inflammatory response.

Endometritis is commonly occurred in dairy cows after calving and results in a great deal of property damage. Although numerous studies have been performed to find the therapeutic agents for endometritis, the incidence of this disease remains high. Short-chain fatty acids (SCFAs), the major metabolic products of anaerobic bacteria fermentation in the gut, have been reported to exhibit anti-inflammatory properties. Therefore, the purpose of this study was to investigate the protective effects and mechanisms of sodium butyrate (SB) on lipopolysaccharide (LPS)-induced endometritis in mice. The mice were administered by intraperitoneal injection of SB at 1 h before LPS injection. 24 h later, the uterus tissues were collected. Hematoxylin and eosin (H & E) stained sections of uterus were used to determine the degree of the damage. Uterine myeloperoxidase (MPO) activity was used to analyze neutrophil granulocytes concentration. The levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were measured by ELISA. The activation of the NF-κB signaling pathway proteins were detected by Western blot analysis. The results showed that SB significantly attenuated the pathological injury of the uterus tissues. SB also suppressed LPS-induced MPO activity and the production of inflammatory cytokines TNF-α and IL-1ß. Furthermore, Western blot analysis showed that SB inhibited the activation of NF-κB signaling pathway. In addition, SB could inhibit histone deacetylases. In summary, SB protects against LPS-induced endometritis through HDAC inhibition.

Dimethyl itaconate protects against lippolysacchride-induced mastitis in mice by activating MAPKs and Nrf2 and inhibiting NF-κB signaling pathways.

Mastitis, as the main disease to affect the dry dairy cow with the characterized by increasing number of somatic cells in milk and reducing milk production, has been known as one of the most serious expensive disease for the dairy industry. Escherichia coli (E.coli), a gram negative bacterial, have normally been considered to be an opportunistic pathogen that can invade the mammary gland sometimes to cause inflammatory diseases. Lippolysacchride (LPS), as the co-cell wall component of the Escherichia coli (E.coli), is the main virulence factors to induce acute inflammation. Itaconate is an endogenous metabolite which has recently been reported to regulate the macrophage function and has the ability to reduce the secretion of pro-inflammatory cytokines, such as IL-6 and IL-12. Here, the aim of this study is to investigate the protective role of dimethyl itaconate (DI)-the membranepermeable derivative of itaconate, on LPS-induced mastitis in mice. To establish the model of mastitis, mice 5-7 day after delivery were utilized by nipple duct injection of LPS, while DI was treated 24h intraperitoneally before LPS injection. Further, the hematoxylin-eosin (H&E) staining was used to evaluate the pathological changes of the mammary gland, the inflammatory cytokines of TNF-α and IL-1ß and the myeloperoxidase (MPO) activity were also measured respectively by enzyme-linked immunosorbent assay (ELISA) and MPO assay kit. To clarify the underling mechanisms of the protective role of DI on mastitis, the MAPKs, NF-κB and Nrf2 signaling pathways were detected via western blotting. The results demonstrated that DI markedly decreased the pathological injury of mammary, and considerably reduced the production of TNF-α and IL-1ß, as well as up-regulated the Nrf2, HO-1, phosphorylation of p38 and ERK, but down-regulated TLR4 and phosphorylation of p65 NF-κB. Our research recommended that DI ameliorated LPS-induced mastitis which highlights itaconate may as a potential candidate to protect against mastitis.

Gut/rumen-mammary gland axis in mastitis: Gut/rumen microbiota-mediated "gastroenterogenic mastitis".

BACKGROUND: Mastitis is an inflammatory response in the mammary gland that results in huge economic losses in the breeding industry. The aetiology of mastitis is complex, and the pathogenesis has not been fully elucidated. It is commonly believed that mastitis is induced by pathogen infection of the mammary gland and induces a local inflammatory response. However, in the clinic, mastitis is often comorbid or secondary to gastric disease, and local control effects targeting the mammary gland are limited. In addition, recent studies have found that the gut/rumen microbiota contributes to the development of mastitis and proposed the gut/rumen-mammary gland axis. Combined with studies indicating that gut/rumen microbiota disturbance can damage the gut mucosa barrier, gut/rumen bacteria and their metabolites can migrate to distal extraintestinal organs. It is believed that the occurrence of mastitis is related not only to the infection of the mammary gland by external pathogenic microorganisms but also to a gastroenterogennic pathogenic pathway. AIM OF REVIEW: We propose the pathological concept of "gastroenterogennic mastitis" and believe that the gut/rumen-mammary gland axis-mediated pathway is the pathological mechanism of "gastroenterogennic mastitis". KEY SCIENTIFIC CONCEPTS OF REVIEW: To clarify the concept of "gastroenterogennic mastitis" by summarizing reports on the effect of the gut/rumen microbiota on mastitis and the gut/rumen-mammary gland axis-mediated pathway to provide a research basis and direction for further understanding and solving the pathogenesis and difficulties encountered in the prevention of mastitis.

Mental workload assessments of aerial photography missions performed by novice unmanned aerial vehicle operators.

BACKGROUND: Mental workload is one of the important variables in understanding human performance in drone operation. OBJECTIVE: To test the effects of gender, age group, flight route, and altitude on the flight performance and mental workload of the novice drone operators. METHODS: Ten male and ten female participants without prior drone operating experience joined. They were split into two age groups. After attending a training, the participants operated a drone to perform photo taking missions under flight route and altitude conditions. The weighted NASA Task Load Index (TLX), Modified Cooper-Harper (MCH) scale, heart rate, and interbeat interval were measured to assess the mental workload of the participants. Flight time to complete the mission was adopted to indicate flight performance. RESULTS: The effect of age group was significant (p < 0.05) on flight time, weighted TLX score, and MCH score. Flight route and altitude were not significant on the two subjective ratings and two cardiac measures. CONCLUSION: The flight performance of younger participants was significantly better than that of their older counterpart. The effects of both the flight route and altitude on the perceived mental workload of the drone operators were insignificant. Both the weighted NASA TLX and MCH scales were appropriate in measuring the mental workload of the novice drone operators.

Research on the air supply adjustment technology of breath-following powered air-purifying respirators.

In the hope of reducing the air supply flow of the powered air-purifying respirator (PAPR) and extending the service life of the filter, a breath-following powered air-purifying respirator (BF-PAPR) that can dynamically adjust the air supply flow according to the breathing flow is proposed. The BF-PAPR changes the air supply flow by adjusting the speed of the variable-frequency centrifugal fan according to the air velocity at the half mask outlet (vhm) monitored by the modular wind speed transmitter. In the study, the air supply flow adjustment model of the BF-PAPR is developed. It is found that the filtration resistance barely influences vhm. In addition, under the same mean inhalation flow, the minimum outlet air velocity increases first and then decreases with the increase of the duty cycle variation coefficient (λ), while the maximum outlet air velocity decreases first and then increases. Moreover, the minimum air supply flow of the BF-PAPR is achieved when the standard value of the air velocity is 13.4 m/s and the value of λ is 1. The BF-PAPR can reduce the air supply flow by 6.5%-8.6% and the energy consumption by approximately 20% compared with the PAPR, which is beneficial for reducing the usage cost and extending the continuous working time.

The Prevention Effect of Bacillus subtilis on Escherichia coli-Induced Mastitis in Mice by Suppressing the NF-κB and MAPK Signaling Pathways.

Mastitis, common inflammation of the mammary gland, caused by various factors, is a challenge for the dairy industry. Escherichia coli (E. coli), a Gram-negative opportunistic pathogen, is one of the major pathogens causing clinical mastitis which is characterized by reduced milk production and recognizable clinical symptoms. Bacillus subtilis (B. subtilis) has been reported to have the ability to limit the colonization of pathogens and has immune-stimulatory effects on epithelial cells. The purpose of this study was to explore the preventive role of B. subtilis H28 on E. coli-induced mastitis in mice. The mastitis model was established by nipple duct injection of E. coli into mice, while B. subtilis H28 was utilized 2 h before E. coli injection. Furthermore, pathological changes in the mammary gland were evaluated by hematoxylin-eosin (H&E) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6). We also observed changes in Toll-like receptor 4 (TLR4), nuclear transcription factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) expression by using western blotting. The results revealed that B. subtilis H28 pretreatment reduced neutrophil infiltration in the mammary gland tissues, significantly decreased the secretion of TNF-α, IL-1ß, and IL-6, and downregulated the activation of TLR4 and the phosphorylation of p65 NF-κB, IκB, p38, and ERK. In conclusion, our results indicated that B. subtilis H28 can ameliorate E. coli-induced mastitis and suggest a new method for the prevention of mastitis.

Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice.

Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism.

Sialic acid exacerbates gut dysbiosis-associated mastitis through the microbiota-gut-mammary axis by fueling gut microbiota disruption.

BACKGROUND: Mastitis is one of the most severe diseases in humans and animals, especially on dairy farms. Mounting evidence indicates that gastrointestinal dysbiosis caused by induction of subacute ruminal acidosis (SARA) by high-grain diet consumption and low in dietary fiber is associated with mastitis initiation and development, however, the underlying mechanism remains unknown. RESULTS: In the present study, we found that cows with SARA-associated mastitis have altered metabolic profiles in the rumen, with increased sialic acids level in particular. Consumption of sialic acid (SA) in antibiotic-treated mice, but not healthy mice, induced marked mastitis. SA treatment of antibiotic-treated mice also induced mucosal and systemic inflammatory responses, as evidenced by increased colon and liver injuries and several inflammatory markers. In addition, gut dysbiosis caused by antibiotic impaired gut barrier integrity, which was aggravated by SA treatment. SA potentiated serum LPS level caused by antibiotic treatment, leading to increased activation of the TLR4-NF-κB/NLRP3 pathways in the mammary gland and colon. Moreover, SA facilitated gut dysbiosis caused by antibiotic, and especially enhanced Enterobacteriaceae and Akkermansiaceae, which correlated with mastitis parameters. Fecal microbiota transplantation from SA-antibiotic-treated mice mimicked mastitis in recipient mice. In vitro experiments showed that SA prompted Escherichia coli growth and virulence gene expression, leading to higher proinflammatory cytokine production in macrophages. Targeting the inhibition of Enterobacteriaceae by sodium tungstate or treating with the commensal Lactobacillus reuteri alleviated SA-facilitated mastitis. In addition, SARA cows had distinct ruminal microbial structure by the enrichment of SA-utilizing opportunistic pathogenic Moraxellaceae and the depletion of SA-utilizing commensal Prevotellaceae. Treating mice with the specific sialidase inhibitor zanamivir reduced SA production and Moraxellaceae abundance, and improved mastitis in mice caused by ruminal microbiota transplantation from cows with SARA-associated mastitis. CONCLUSIONS: This study, for the first time, indicates that SA aggravates gut dysbiosis-induced mastitis by promoting gut microbiota disturbance and is regulated by commensal bacteria, indicating the important role of the microbiota-gut-mammary axis in mastitis pathogenesis and suggesting a potential strategy for mastitis intervention based on gut metabolism regulation. Video Abstract.

Enterogenic Stenotrophomonas maltophilia migrates to the mammary gland to induce mastitis by activating the calcium-ROS-AMPK-mTOR-autophagy pathway.

BACKGROUND: Mastitis is an inflammatory disease of the mammary gland that has serious economic impacts on the dairy industry and endangers food safety. Our previous study found that the body has a gut/rumen-mammary gland axis and that disturbance of the gut/rumen microbiota could result in 'gastroenterogenic mastitis'. However, the mechanism has not been fully clarified. Recently, we found that long-term feeding of a high-concentrate diet induced mastitis in dairy cows, and the abundance of Stenotrophomonas maltophilia (S. maltophilia) was significantly increased in both the rumen and milk microbiota. Accordingly, we hypothesized that 'gastroenterogenic mastitis' can be induced by the migration of endogenous gut bacteria to the mammary gland. Therefore, this study investigated the mechanism by which enterogenic S. maltophilia induces mastitis. RESULTS: First, S. maltophilia was labelled with superfolder GFP and administered to mice via gavage. The results showed that treatment with S. maltophilia promoted the occurrence of mastitis and increased the permeability of the blood-milk barrier, leading to intestinal inflammation and intestinal leakage. Furthermore, tracking of ingested S. maltophilia revealed that S. maltophilia could migrate from the gut to the mammary gland and induce mastitis. Subsequently, mammary gland transcriptome analysis showed that the calcium and AMPK signalling pathways were significantly upregulated in mice treated with S. maltophilia. Then, using mouse mammary epithelial cells (MMECs), we verified that S. maltophilia induces mastitis through activation of the calcium-ROS-AMPK-mTOR-autophagy pathway. CONCLUSIONS: In conclusion, the results showed that enterogenic S. maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calcium-ROS-AMPK-mTOR-autophagy pathway to induce mastitis. Targeting the gut-mammary gland axis may also be an effective method to treat mastitis.