Selenium Counteracts Tight Junction Disruption and Attenuates the NF-κB-Mediated Inflammatory Response in Staphylococcus aureus-Infected Mouse Mammary Glands.

Tight junctions (TJs) are the key determinant of barrier function in the mammary gland, with their disruption being associated with the pathogenesis and progression of mastitis, especially in the case of Staphylococcus aureus (S. aureus) infection. This study investigated whether selenium (Se) could attenuate S. aureus-induced mastitis by inhibiting inflammation and protecting mammary gland TJs in mice. The expression profiles of S. aureus-infected gland tissues derived from the gene expression omnibus dataset were analyzed. We found cytokine production, cell junctions, the nuclear transcription factor-κB (NF-κB) signalling pathway, and inflammatory responses associated with the differentially expressed genes, as revealed by Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses. Se reduced the mRNA expression and production of inflammatory cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and decreased phosphorylation levels of the NF-κB complex. Moreover, Se alleviated structural damage and microvillus injury in mammary glands. Immunohistochemical staining revealed that Se increased the expression of Claudin-3; Western blot analysis revealed increased protein levels of Occludin and Tricellulin in the group supplemented with dietary Se. In summary, Se counteracted TJ disruption and attenuated NF-κB-mediated inflammatory responses in S. aureus-infected mouse mammary glands.

Molecular characterization of multi-drug-resistant Staphylococcus aureus in mastitis bovine milk from a dairy farm in Anhui, China.

Mastitis is an economically important disease in the dairy industry, which is caused by various infectious pathogens. There is limited information known about the situation of drug resistance and virulence factors of Staphylococcus aureus (S. aureus) in mastitis bovine milk in Anhui. Therefore, a total of 125 fresh milk samples from clinically mastitis-positive bovine animals were collected. The bacteria pathogens were identified via bacterial culture, Gram staining, biochemical analysis, DNA extraction, 16s rRNA amplification, and phylogenetic analysis. Drug resistance analyses were performed through drug-resistant genes and virulence genes amplification. Results showed that a total of 24.8% (31/125) bacterial isolates were isolated and identified as S. aureus by Gram straining, biochemical reactions, and 16 s rRNA genes blasting. Multiple sequence alignment analysis found that the current isolates were highly similar (96.9-100.0%) to previous isolates. Phylogenetic analysis demonstrated that S. aureus was similar with MK809241.1 isolated from food in China and wCP030426.1 isolated from a person in the United States. The bacterial isolates were detected resistant to 11 antibiotics, such as Penicillin G, SXT, Ciprofloxacin, Norfloxacin, Polymyxin B, Levofloxacin, Chloramphenicol, Clindamycin, Clarithromycin, Erythromycin, and Spectinomycin. Drug-resistant genes of blaZ, ermC, rpoB, and ant (4')-la were successfully amplified. Virulence genes of hla, nuc, clfa, and eta were found in S. aureus bacteria. The current study isolated S. aureus from milk samples and revealed its drug-resistant situation, drug-resistant genes, and virulence genes. Hence, regular monitoring of S. aureus in milk samples from dairy cows may contribute to the prevention and treatment of public health concerns causing bacteria in this region.

Comparative analysis of gut microbiota in healthy and diarrheic yaks.

BACKGROUND: Yak (Bos grunniens) mainly inhabiting Tibet Plateau, displayed a high incidence of diarrhea due to harsh living environment and nutritional deficit. Gut microbial community has been reported to be closely related to many diseases including diabetes, obesity and inflammatory bowel disease, but information regarding diarrheic influence on gut microbiota in yaks remains scarce. Here, this study was performed to investigate the gut bacterial and fungal alternations of diarrheic yaks. RESULTS: Results revealed that the gut bacterial and fungal communities of diarrheic yaks showed a distinct decline in alpha diversity, accompanied by significant shifts in taxonomic compositions. Specifically, diarrhea caused a distinct increase in the relative abundance of 1 phylum and 8 genera as well as a distinct decrease in 3 phyla and 30 genera. Fungal taxonomic analysis indicated that the relative richness of 1 phylum and 2 genera dramatically increased, whereas the relative richness of 2 phylum and 43 genera significantly decreased during diarrhea. Surprisingly, 2 bacterial genera and 5 fungal genera even cannot be detected in the gut microbiota of diarrheic yaks. CONCLUSIONS: In summary, this study indicated that the gut bacterial and fungal compositions and diversities of yaks altered significantly during diarrhea. Moreover, these findings also contribute to understanding the gut microbial composition and diversity of yaks and developing strategies to alleviate and prevent diarrhea from gut microbial perspective.

Selenium Attenuates S. aureus-Induced Inflammation by Regulation TLR2 Signaling Pathway and NLRP3 Inflammasome in RAW 264.7 Macrophages.

This study aimed to investigate the effects of selenium (Se) on the expression of Toll-like receptor (TLR) 2 and pyrin domain-containing protein (NLRP)3 inflammasome in macrophages infected by Staphylococcus aureus (S. aureus). RAW 264.7 macrophages were treated with 2 µmol/L Na2SeO3 for 12 h before infection with S. aureus for 2 h. Through Western blot, qRT-PCR, and ELISA analysis, the core molecules of TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages were detected. Results showed that Se significantly reduced the elevated mRNA expression of TLR2, myeloid differentiation factor-88 (Myd88), NLRP3, Caspase-recruitment domain (ASC), and Caspase-1 induced by S. aureus. Furthermore, compared with I group, the protein expression of TLR2, Myd88, NLRP3, ASC, and Caspase-1 were suppressed in T group. In addition, the mRNA and protein expression of interleukin-1 beta (IL-1ß) induced by S. aureus were also decreased after Se treatment. In conclusion, Se inhibits S. aureus-induced inflammation by suppressing the activation of the TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages.

Black Lycium barbarum polysaccharide attenuates LPS-induced intestine damage via regulation gut microbiota.

Lycium barbarums are traditionally used as a homology of medicinal plants in China with a potent role in metabolism and immunomodulation. The current study was performed to explore the attenuation effect and microbiota regulation of Lycium barbarum polysaccharide (BLBP) on lipopolysaccharide (LPS)-induced intestine damage in mice. A total of 70 mice were randomly divided into five groups; negative control (GA), LPS (GB), both treated with an equal volume of normal saline, and BLBP treatment groups GC (100 mg/kg), GD (200 mg/kg), and GE (400 mg/kg) via gavage for 19 days. On Day 19, mice in groups GB, GC, GD, and GE were treated with 10 mg/kg LPS for 24 h and euthanized to collect intestine samples for pathological examination and microbiota sequencing. The results showed a non-significant difference in body weight gain among the five mouse groups; however, mice in the GC and GE groups showed decreased weight gain. An H&E examination revealed that the integrity of intestinal villi was destroyed by LPS, while BLBP supplement alleviated intestinal damage with an increase in villus height and a decrease in crypt depth. A total of over 59,000, 40,000, 50,000, 45,000, and 55,000 raw sequences were found in groups GA, GB, GC, GD, and GE, respectively. LPS challenge decreased alpha diversity indexes significantly (p < 0.05), while a non-significant difference was found between different BLBP treatment groups and the GA group. A total of 8 phyla and 13 genera were found among five mouse groups, and BLBP partly restored the bacterial abundance in mice. LPS changed 282 metabolic pathways in KEGG L2, 77 metabolic pathways in KEGG L3, and 205 metabolic pathways in MetaCyc, respectively. The BLBP-supplemented groups, especially GE, showed reverse effects on those metabolic pathways. The current study revealed that BLBP can effectively decrease intestinal damage through the regulation of intestinal microbiota, which may provide new insights for the prevention of intestinal disease using food and medicine homologous of Lycium ruthenicum.

Contents and yields of copper, iron, manganese and zinc would be affected by lucerne age and cut.

BACKGROUND: Lucerne is a perennial legume forage, which can produce multiple cuts in 1 year. Microelements play fundamental roles in the function, maintenance and adaptation to the environment for lucerne growth. However, the role of the accumulation of copper (Cu), iron (Fe), manganese (Mn) and Zinc (Zn), which vary with lucerne ages or cuts, has not been previously determined. Therefore, a hypothesis on the Cu, Fe, Mn and Zn in lucerne varying with age and cut was tested. METHODS: A total of 11, 8, 5, 4 and 1 year old lucerne (Medicago sativa Longdong) were selected as the material (until 2012 year), and samples were taken as three cuts at the cutting periods (early flowering stage) in 2012. Then, the contents and yields of Cu, Fe, Mn and Zn in lucerne were measured and calculated. RESULTS: The highest contents of Cu, Fe, Mn and Zn in lucerne were found in the 1 year old among the five ages, at the 3rd cut compared to the other two cuts, and in the leaf among the three organs. The highest yields of Cu, Fe, Mn and Zn were found in the older ages (11 and 8 years old), at the 3rd cut, and in the root among the three organs. The most positive correlations were found between contents, yields and biomass. CONCLUSIONS: The hypothesis was supported by the results. And the contents and yields of lucerne Cu, Fe, Mn and Zn were affected by the age, cut and organ. Furthermore, the yields of lucerne Cu, Fe, Mn and Zn were determined by their contents and lucerne biomass.

Short Communication: Effects of Dietary Selenium Supplementation on Selenium Deposition and Antioxidant Status in Postpartum Mice.

This study aimed to investigate the effects of dietary selenium during pregnancy on the selenium deposition and antioxidant enzymes in postpartum mouse serum, liver, and mammary gland. Eighty BALB/c pregnant mice were randomly divided into four groups: CG (Se-deficient basal diet, n = 20), LG (0.05 mg/kg Se-supplemented diet, n = 20), MG (0.1 mg/kg Se-supplemented diet, n = 20), and HG (0.2 mg/kg Se-supplemented diet, n = 20). Four days after parturition, all mice were euthanized. The selenium deposition and antioxidants enzymes in serum, liver, and mammary gland were detected. Results show that with increasing selenium supplementation, the selenium deposition and activation of T-AOC, T-SOD, and GSH-Px increased, meanwhile the concentration of MDA decreased in serum, liver, and mammary gland. Therefore, this study suggested selenium was mainly deposited in the liver, and dietary selenium during pregnancy might improve the antioxidant status in postpartum animals.

Selenium Plays an Anti-Inflammatory Role by Regulation NLRP3 Inflammasome in Staphylococcus aureus-Infected Mouse Mammary Gland.

Selenium is an essential micronutrient that plays an important role in immunity. However, the mechanism that Selenium modulates mastitis is not fully clear. In this experiment, we investigated whether selenium can inhibit the activation of the NLRP3 inflammasome in a mouse model of Staphylococcus aureus-induced mastitis. Eighty BALB/c female mice were fed with experimental Selenium deficiency basal diet for 2 weeks to achieve the purpose of selenium consumption until pregnancy. Pregnant mice were randomly divided into four groups (control group; selenium supplement group; Staphylococcus aureus infection group and Staphylococcus aureus infection after selenium supplement group). Twenty-four hours after challenging, all mice were euthanized and mammary tissue samples were aseptically collected. Through pathological staining, western blot analysis, real-time fluorescence quantitative polymerase chain reaction analysis, and enzyme-linked immunosorbent assay, the regulation effect of Selenium on NLRP3 inflammasome was detected. The result showed that compared with the control group, selenium significantly inhibited the expression of NLRP3, ASC, Caspase-1, Caspase-1 p20, and Pro-IL-1ß (p < 0.01). Meanwhile the mRNA expression and release of IL-1ß was suppressed in the treatment group compared with Staphylococcus aureus infection group (p < 0.01). Therefore, these results suggest that dietary selenium can attenuate Staphylococcus aureus mastitis by inhibition of the NLRP3 inflammasome.

Effect of active dry yeast on lactation performance, methane production, and ruminal fermentation patterns in early-lactating Holstein cows.

This study was conducted to examine the effect of active dry yeast (ADY) supplementation on lactation performance, ruminal fermentation patterns, and CH4 emissions and to determine an optimal ADY dose. Sixty Holstein dairy cows in early lactation (52 ± 1.2 DIM) were used in a randomized complete design. Cows were blocked by parity (2.1 ± 0.2), milk production (35 ± 4.6 kg/d), and body weight (642 ± 53 kg) and assigned to 1 of 4 treatments. Cows were fed ADY at doses of 0, 10, 20, or 30 g/d per head for 91 d, with 84 d for adaptation and 7 d for sampling. Although dry matter intake was not affected by ADY supplementation, the yield of actual milk, 4% fat-corrected milk, milk fat yield, and feed efficiency increased quadratically with increasing ADY supplementation. Yields of milk protein and lactose increased linearly with increasing ADY doses, whereas milk urea nitrogen concentration and somatic cell count decreased quadratically. Ruminal pH and ammonia concentration were not affected by ADY supplementation, whereas ruminal concentration of total volatile fatty acid increased quadratically. Digestibility of dry matter, organic matter, neutral detergent fiber, acid detergent fiber, nonfiber carbohydrate, and crude protein increased quadratically with increasing ADY supplementation. Supplementation of ADY did not affect blood concentration of total protein, triglyceride, aspartate aminotransferase, and alanine aminotransferase, whereas blood urea nitrogen, cholesterol, and nonesterified fatty acid concentrations decreased quadratically with increasing ADY supplementation. Methane production was not affected by ADY supplementation when expressed as grams per day or per kilogram of actual milk yield, dry matter intake, digested organic matter, and digested nonfiber carbohydrate, whereas a trend of linear and quadratic decrease of CH4 production was observed when expressed as grams per kilogram of fat-corrected milk and digested neutral detergent fiber. In conclusion, feeding ADY to early-lactating cows improved lactation performance by increasing nutrient digestibility. The optimal ADY dose should be 20 g/d per head.

Characterization of Staphylococcus aureus Isolates From Cases of Clinical Bovine Mastitis on Large-Scale Chinese Dairy Farms.

Bovine mastitis is a prevalent disease that causes serious economic problems globally in the dairy industry. Staphylococcus aureus is an important pathogen of bovine mastitis. This study was conducted to characterize S. aureus isolates from clinical bovine mastitis cases in large-scale dairy herds in China. S. aureus was isolated from 624 clinical mastitis cases and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 62 S. aureus isolates were obtained. Cluster analysis, genetic diversity, quantification of biofilm formation, antimicrobial resistance, and detection of virulence genes were performed on these isolates of S. aureus. Eight isolates harbored the mecA gene and were sensitive to oxacillin. MALDI-TOF MS cluster analysis revealed that the 62 isolates were divided into three major clusters (I, II, III) and eight main groups (A-H) at the distance level of 700. The agr II was the most prevalent (56.5%). The 62 S. aureus isolates were assigned to seven spa types. The most common spa type was t529(58.1%), followed by t2196 (14.5%), t518 (14.5%), t571(6.5%), t034 (3.2%), t2734 (1.6%), and t730 (1.6%). Five STs were identified from seven representative isolates as follows: ST630/CC8, ST97/CC97, ST50, ST398, and ST705. All isolates had the ability to form biofilm. Antimicrobial resistance was most frequently observed to ciprofloxacin (29%), followed by penicillin (24.2%), and streptomycin (9.6%). All isolates harbored the fnbA, clfB (100%), icaA, and icaD genes. This study provides the basis for the development of bovine mastitis prevention program on large-scale dairy farms.

Selenium ameliorates Staphylococcus aureus-induced inflammation in bovine mammary epithelial cells by inhibiting activation of TLR2, NF-κB and MAPK signaling pathways.

BACKGROUND: Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMECs) is considered an important pathogenic mechanism for the establishment of mastitis. Given the interesting link between selenium (Se) status and mastitis, our objective was to prove that Se was essential to suppress pro-inflammatory mediators, in part, by modulation of Toll-like receptor2 (TLR2), nuclear factor kappaB (NF-κB) and mitogen activated protein kinase (MAPK) signal transduction pathway in bMECs. RESULTS: Results showed that Se (0~ 16 µM) did not affect the growth of bMECs. The mRNA expression of TLR2, Myeloid differentiation factor 88 (Myd88), Interleukin-1 receptor-associated kinase4 (Irak4), Interleukin-1 receptor-associated kinase1 (Irak1) and TNF receptor-associated factor6 (Traf6) in TLR2 signal pathway were increased or significantly increased by S. aureus. Se played an important role in regulating the genes expression of TLR2, Myd88, Traf6 but not in controlling the expression of Irak4 and Irak1. In addition, Se exerted strong inhibitory effects on the genes expression of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) induced by S. aureus. To further investigate the possible signaling mechanisms involved in the processes, we analyzed the role of MAPK and NF-κB signaling pathway in inflammation response in S. aureus-stimulated bMECs in vitro. Results showed that the phosphorylation of inhibitory kappaB alpha (IκBα), p65, p38 and extracellular regulated protein kinase (Erk) were significantly increased in S. aureus-stimulated bMECs. It indicated that S. aureus activated NF-κB and MAPK signaling pathway. We also examined the effects of Se on the phosphorylation of IκBα, p65, p38 and Erk in NF-κB and MAPK signaling pathway, which have well been proved to control the synthesis and release of pro-inflammatory mediators during inflammation. The findings are exciting, that pretreatment with Se (4, 8 µM) significantly suppressed the phosphorylation of IκBα, p65, p38 and Erk. CONCLUSIONS: These results suggest that Se down-regulates inflammatory mediators TNF-α, IL-1ß and IL-6 gene expressions via TLR2, NF-κB and MAPK signaling pathway in S. aureus-stimulated bMECs, which may be responsible for the anti-inflammatory effect of Se.

Selenium inhibits Staphylococcus aureus-induced inflammation by suppressing the activation of the NF-κB and MAPK signalling pathways in RAW264.7 macrophages.

Inflammation is the hallmark of Staphylococcus aureus (S. aureus)-induced mastitis. Given the interesting relationship between selenium levels and inflammation, this study aimed to demonstrate that selenium modulated the inflammation reaction by suppressing the nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signalling pathways. RAW264.7 macrophages were treated with three different concentrations (1µmol/l, 1.5µmol/l, and 2µmol/l) of Na2SeO3 for 12h before infection with S. aureus for 6h, 8h, and 10h. The results showed that selenium significantly reduced the mRNA expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). Furthermore, the release of TNF-α, IL-1ß, and IL-6 was decreased significantly with selenium supplementation. In addition, selenium influenced the NF-κB signalling pathway by suppressing the activation of NF-κB p65 and degradation of inhibitory kappa-B (IκB). Selenium also suppressed extracellular regulated protein kinase (Erk), c-Jun N-terminal kinase (Jnk), and p38 phosphorylation through the MAPK signalling pathway. In conclusion, selenium played an anti-inflammation role in RAW264.7 macrophages infected with S. aureus by suppressing the activation of the NF-κB and MAPK signalling pathways.