Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation.

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Colonization and local host response following intramammary Staphylococcus chromogenes challenge in dry cows.

Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

Mechanisms by which mastitis affects reproduction in dairy cow: A review.

Reproductive performance is a key factor in determining the profitability of dairy farm, which is affected by many factors such as environment and diseases. Mastitis is a common and important disease, which has caused huge economic losses to the dairy industries worldwide. Mammary gland infection causes immune responses, resulting in the abnormal secretion of cytokines and hormones and abnormal function of the reproductive system such as the ovary, corpus luteum, uterus and embryo. Cows with mastitis have delayed oestrus, decreased pregnancy rate and increased risk of abortion. The adverse effects of mastitis on reproductive performance are affected by many factors, such as occurrence time, pathogen and cow factors. This paper primarily reviews the progress in the effects and mechanisms of mastitis on reproductive performance, with emphasis on maternal transcriptome, genomic analysis, epigenetic modification, microbiota, inflammatory regulation and immune evasion mechanism of mastitis, aiming to provide directions for the prevention and control of mastitis in the future.

Going further post-RNA-seq: In silico functional analyses revealing candidate genes and regulatory elements related to mastitis in dairy cattle.

This study aimed to obtain a better understanding of the regulatory genes and molecules involved in the development of mastitis. For this purpose, the transcription factors (TF) and MicroRNAs (miRNA) related to differentially expressed genes previously found in extracorporeal udders infected with Streptococcus agalactiae were investigated. The Gene-TF network highlighted LOC515333, SAA3, CD14, NFKBIA, APOC2 and LOC100335608 and genes that encode the most representative transcription factors STAT3, PPARG, EGR1 and NFKB1 for infected udders. In addition, it was possible to highlight, through the analysis of the gene-miRNA network, genes that could be post-transcriptionally regulated by miRNAs, such as the relationship between the CCL5 gene and the miRNA bta-miR-363. Overall, our data demonstrated genes and regulatory elements (TF and miRNA) that can play an important role in mastitis resistance. The results provide new insights into the first functional pathways and the network of genes that orchestrate the innate immune responses to infection by Streptococcus agalactiae. Our results will increase the general knowledge about the gene networks, transcription factors and miRNAs involved in fighting intramammary infection and maintaining tissue during infection and thus enable a better understanding of the pathophysiology of mastitis.

Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux.

Staphylococcus aureus is a pathogen that is the causative agent of several human and veterinary infections and plays a critical role in the clinical and subclinical mastitis of cattle. Autophagy is a conserved pathogen defence mechanism in eukaryotes. Studies have reported that S aureus can subvert autophagy and survive in cells. Staphylococcus aureus survival in cells is an important cause of chronic persistent mastitis infection. However, it is unclear whether S aureus can escape autophagy in innate immune cells. In this study, initiation of autophagy due to the presence of S aureus was detected in bovine macrophages. We observed autophagic vacuoles increased after S aureus infection of bovine macrophages by transmission electron microscopy (TEM). It was also found that S aureus-infected bovine macrophages increased the expression of LC3 at different times(0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours). Data also showed the accumulation of p62 induced by S aureus infection. Application of autophagy regulatory agents showed that the degradation of p62 was blocked in S aureus induced bovine macrophages. In addition, we also found that the accumulation of autophagosomes promotes S aureus to survive in macrophage cells. In conclusion, this study indicates that autophagy occurs in S aureus-infected bovine macrophages but is blocked at a later stage of autophagy. The accumulation of autophagosomes facilitates the survival of S aureus in bovine macrophages. These findings provide new insights into the interaction of S aureus with autophagy in bovine macrophages.

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.

Staphylococcus aureus metal acquisition in the mastitic mammary gland.

Mastitis, resulting from mammary gland infection, is a common and painful disease associated with lactation. In addition to the impact on human and animal health, mastitis causes substantial economic losses in the dairy industry. Staphylococcus aureus is a frequent cause of mastitis worldwide. Despite significant progress in understanding S. aureus pathogenesis in general, much remains to be learned regarding virulence factors relevant in the context of mastitis. This review outlines the molecular mechanisms by which S. aureus acquires essential metals such as iron, zinc, manganese, copper, cobalt and nickel within lactating mammary glands, while exposing areas where our current knowledge is deficient. Increased understanding of how these factors facilitate bacterial survival in the lactating mammary gland can provide therapeutic targets for more effective mastitis prevention and treatment.

The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence.

Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

Prognostic potential of pre-partum blood biochemical and immune variables for postpartum mastitis risk in dairy cows.

BACKGROUND: Mastitis is the most frequent diseases for transition cows. Identification of potential biomarkers for diagnosis of mastitis is important for its prevention. Thus, this study was conducted to investigate blood variables related to lipid metabolism, oxidative stress and inflammation, and serum variables that are related to health in postpartum cows. RESULTS: Seventy-six healthy Holstein dairy cows at week 4 before calving were selected to collect blood samples from weeks - 4 to 4 weekly relative to calving, respectively. Milk yield and composition were recorded weekly. According to the cut-off of somatic cell counts (SCC) for diagnosis of mastitis, 33 cows with SCC ≥ 500,000 cells ml- 1, 20 cows with 200,000 cells ≤ SCC < 500,000 cells ml- 1, and 23 cows with SCC < 200,000 cells ml- 1 were defined as high, middle, and low SCC, respectively. Serum concentrations of ß-hydroxybutyrate were higher (P < 0.01) during all weeks, and non-esterified fatty acids were higher in high SCC than in low SCC cows from weeks - 3 to 2 relative to calving. Higher serum concentrations of superoxide dismutase (P < 0.01) and lower malondialdehyde levels (P < 0.01) in low SCC than in high SCC cows indicate that the latter suffered from oxidative stress. The difference analysis of the three groups suggested that none of the above-mentioned variables can be used as potential prognostic candidates. On the other hand, high SCC cows exhibited higher blood neutrophil to lymphocyte ratio (NLR, P < 0.01) and platelet to lymphocyte ratio (PLR, P < 0.01) than low SCC cows, with a higher NLR (P < 0.01) in middle SCC than in low SCC cows. The high SCC cows had lower levels of anti-inflammatory factors including IL-10 (P = 0.05), but higher levels of proinflammatory factors such as IL-6 (P < 0.01), TNF-α (P < 0.05), and PSGL-1 (P < 0.01) than low SCC cows. CONCLUSIONS: The significantly different NLR and PLR pre-partum between the middle and low SCC cows suggest their prognostic potential for postpartum mastitis risk.

An in silico evaluation of non-synonymous single nucleotide polymorphisms of mastitis resistance genes in cattle.

Recognition of deleterious non-synonymous single nucleotide polymorphism (SNPs) aids in the assessment of genetic basis of diseases and prediction of clinical phenotypes. In this study, data obtained from whole exome sequencing of Vechur cow using Illumina HiSeq 2500 platform is compared with that of crossbred cattle of Kerala. Sequence analysis of selected 18 mastitis resistant genes, evaluated the consequence of non-synonymous SNPs in these genes from both Vechur and crossbred cattle of Kerala, using sequence and structure-based computational tools such as SIFT, PROVEAN and I-MUTANT 2.0. Compared to Vechur cattle, incidence of missense deleterious mutation to effect protein functioning were relatively higher in crossbred cattle. These results on the type of genetic variants and its impact on normal functioning of a protein will assist to predict and enhance the disease resistance in cattle breeds.

Comparison of antibody repertories against Staphylococcus aureus in healthy and infected dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine.

Staphylococcus aureus is one of the pathogens most frequently isolated from cases of mastitis worldwide. To decrease the effect of S. aureus mastitis in dairy farming, alternative strategies for controlling mastitis are needed that depend on a better knowledge of cow-to-cow variations in S. aureus antibody production. The present study sought to explore the diversity of S. aureus antibodies produced by dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine. We obtained protein extracts from S. aureus isolates derived from persistent subclinical mastitis. Proteins were fractionated using 2-dimensional gel electrophoresis and Western blotting. Then, Western blotting membranes were exposed to sera from 24 dairy cows that had been divided into the following groups: vaccinated dairy cows that were infected with S. aureus, further subdivided according to whether they (a) remained infected by S. aureus or (b) recovered from the intramammary infection; unvaccinated dairy cows infected with S. aureus; and vaccinated healthy dairy cows with no history of S. aureus mastitis. Proteins found to be reactive by Western blot were identified by mass spectrometry (MALDI/TOF-TOF). Our most important finding was that F0F1 ATP synthase subunit α, succinyl-diaminopimelate desuccinylase, and cysteinyl-tRNA synthetase were potential candidate proteins for the prevention of S. aureus mastitis. This study strengthens the notion that variations among animals should not be ignored and shows that the heterogeneity of antibody production against anti-staphylococcal antigens in animals may enable the identification of new immunotherapy targets.

Clinical presentation and immune characteristics in first-lactation Holstein-Friesian cows following intramammary infection with genotypically distinct Staphylococcus aureus strains.

Staphylococcus aureus is an important cause of bovine mastitis, and intramammary infections caused by this pathogen are often characterized as mild, chronic, or persistent. The strains of Staph. aureus associated with mastitis belong to several distinct bovine-adapted bacterial lineages. Studies of host-pathogen interactions have demonstrated that significant differences exist between Staph. aureus strains and lineages in their ability to internalize and to elicit expression of chemokines and pro-inflammatory mediators in bovine cells in vitro. To determine the effect of bacterial strain on the response to intramammary infection in vivo, 14 disease-free, first-lactation cows were randomly allocated to 2 groups and challenged with Staph. aureus strain MOK023 (belonging to CC97) or MOK124 (belonging to CC151). Clinical signs of infection, as well as somatic cell count (SCC), bacterial load, IL-8 and IL-1ß in milk, anti-Staph. aureus IgG in milk and serum, anti-Staph. aureus IgA in milk, and white blood cell populations in milk and blood were monitored for 30 d after the challenge. Cows infected with MOK023 generally developed subclinical mastitis, whereas cows infected with MOK124 generally developed clinical mastitis. Milk yield was reduced to a greater extent in response to infection with MOK124 compared with MOK023 in the first week of the study. Significantly higher SCC, IL-8, and IL-1ß in milk as well as higher anti-Staph. aureus IgG and IgA in milk and anti-Staph. aureus IgG in serum were also observed in response to MOK124 compared with the response to MOK023. Higher proportions of neutrophils were observed in milk of animals infected with MOK124 than in animals infected with MOK023. Higher neutrophil concentration in blood was also observed in the MOK124 group compared with the MOK023 group. Overall, the results indicate that the outcome of mastitis mediated by Staph. aureus is strain dependent.

Cows selected for divergent mastitis susceptibility display a differential liver transcriptome profile after experimental Staphylococcus aureus mammary gland inoculation.

Infection and inflammation of the mammary gland, and especially prevention of mastitis, are still major challenges for the dairy industry. Different approaches have been tried to reduce the incidence of mastitis. Genetic selection of cows with lower susceptibility to mastitis promises sustainable success in this regard. Bos taurus autosome (BTA) 18, particularly the region between 43 and 59 Mb, harbors quantitative trait loci (QTL) for somatic cell score, a surrogate trait for mastitis susceptibility. Scrutinizing the molecular bases hereof, we challenged udders from half-sib heifers having inherited either favorable paternal haplotypes for somatic cell score (Q) or unfavorable haplotypes (q) with the Staphylococcus aureus pathogen. RNA sequencing was used for an in-depth analysis of challenge-related alterations in the hepatic transcriptome. Liver exerts highly relevant immune functions aside from being the key metabolic organ. Hence, a holistic approach focusing on the liver enabled us to identify challenge-related and genotype-dependent differentially expressed genes and underlying regulatory networks. In response to the S. aureus challenge, we found that heifers with Q haplotypes displayed more activated immune genes and pathways after S. aureus challenge compared with their q half-sibs. Furthermore, we found a significant enrichment of differentially expressed loci in the genomic target region on BTA18, suggesting the existence of a regionally acting regulatory element with effects on a variety of genes in this region.

Immune response in nonspecific mastitis: What can it tell us?

We analyzed a large number of immune response parameters from quarter milk samples with distinct bacteriological and quarter somatic cell count (qSCC) statuses. Furthermore, we sought to explore and identify displayed immune response patterns in milk samples from mammary glands with nonspecific mastitis. Thus, 92 quarter milk samples from 28 cows were stratified into 4 groups, as follows: (1) 49 culture-negative control quarters with a low qSCC (<1 × 105 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 105 cells/mL; so-called quarters with nonspecific mastitis) from 10 dairy cows; (3) 8 culture-positive quarters with low qSCC (noninflammatory quarters with low qSCC) from 5 dairy cows; and (4) 20 culture-positive quarters with high qSCC (so-called truly infected quarters) from 8 dairy cows. Using flow cytometry, we evaluated the percentage of milk neutrophils and their viability, intracellular reactive oxygen species production, phagocytosis, and the expression of CD62L, CD11b, and CD44 for each of the 4 quarter strata. Furthermore, the percentage of monocyte/macrophages, B cells, and T lymphocyte subsets were evaluated by flow cytometry. Milk samples from bacteriologically negative quarters (both with a low and elevated qSCC) had a lower qSCC than those with bacteriologically positive outcomes (both with a low and elevated qSCC). As expected, the healthy quarters showed the lowest percentage of neutrophils and also showed a higher percentage of milk monocytes/macrophages and lower percentage of T lymphocytes than truly infected quarters. The most prominent result of the present study is that quarters with nonspecific mastitis showed the highest percentage of milk CD4+ T lymphocytes. The healthy quarters had a lower percentage of apoptotic neutrophils than noninflammatory and truly infected quarters, although it did not differ from those from the quarters with nonspecific mastitis. Our study supports the role of differential cell counting in the diagnosis of mastitis, as the milk leukocyte populations markedly fluctuate under healthy and inflammatory conditions. Furthermore, an increase in milk CD4+ T cells was associated with nonspecific mastitis, suggesting an increase in this leukocyte subpopulation is correlated with low bacterial shedding. Our study allows us to go further in our understanding of mammary gland immunity, providing further insights on potential protective mammary gland immunity, which we hypothesize can open new avenues for the development of novel targets that can promote bovine udder health.

Staphylococcal enterotoxin M induced inflammation and impairment of bovine mammary epithelial cells.

Staphylococcus aureus is one of the major etiological pathogens of bovine mastitis. Its invasion into mammary epithelial cells has been proven to be a key event in the pathogenesis of mastitis. However, the specific pathogenic factors have not been clearly identified. Staphylococcus aureus often triggers infections by releasing virulence factor. Recent several studies reported that staphylococcal enterotoxin M was one of the most frequently found enterotoxin genes associated with bovine mastitis. Thus, the effect of staphylococcal enterotoxin M on inflammation and damage of the bovine mammary epithelial bovine mammary gland epithelial cell line (MAC-T) cells with 48 h treatment was explored in the present study. First, staphylococcal enterotoxin M protein was purified by a Ni-NTA spin column (GE Life Science, Westborough, MA). The levels of tumor necrosis factor-α, IL-6, and monocyte chemoattractant protein 1 (MCP-1) secretion were measured with the corresponding ELISA kits (R&D Systems, Abingdon, UK). Second, cell viability was assessed with a Cell Counting Kit-8 (Bioswamp, Wuhan, China) and the apoptotic percentage of cells was determined by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI; Beyotime, Nanjing, China) staining. Third, ATP concentration, reactive oxygen species (ROS) generation and lactate dehydrogenase (LDH) release were assayed with commercial kits, then mitochondrial membrane potential (ΔΨm) was estimated using fluorescent probe JC-1 (Beyotime). Finally, the production intercellular cell adhesion molecule-1 (ICAM-1), microtubule-associated protein 1A/1B-light chain 3 I/II (LC3 I/II), p62 (Proteintech, Rosemont, IL), and phosphorylation of IκBα, caspase 3, and mammalian target of rapamycin were detected by Western blot. The results showed that staphylococcal enterotoxin M induced inflammation of epithelial cells (upregulating tumor necrosis factor-α, IL-6, MCP-1, and ICAM-1 production) and activated NF-κB (promoting phosphorylation of IκBα). Furthermore, staphylococcal enterotoxin M impaired MAC-T cells via cell necrosis (enhancing LDH release), apoptosis (annexin V-FITC/PI stain, exacerbating oxidative stress, decreasing ΔΨm and intracellular ATP concentration, and activating caspase 3), but independent of autophagy (nonsignificantly increasing LC3-II, decreasing p62 expression, and activating mammalian target of rapamycin). Thereby, staphylococcal enterotoxin M induced the inflammatory property of bovine mammary epithelial cells by boosting cytokine, chemokine, and adhesion molecule production. Furthermore, it caused epithelial cell dysfunction via depressing cell viability and initiating cell necrosis and apoptosis. Because epithelial cells played important roles in orchestrating the inflammatory response and protecting bovine mammary tissue from mastitis, our results indicated that staphylococcal enterotoxin M may be associated with mastitis.

Role of Liver X Receptor in Mastitis Therapy and Regulation of Milk Fat Synthesis.

Mastitis is important disease that causes huge economic losses in the dairy industry. In recent years, antibiotic therapy has become the primary treatment for mastitis, however, due to drug residue in milk and food safety factors, we lack safe and effective drugs for treating mastitis. Therefore, new targets and drugs are urgently needed to control mastitis. LXRα, one of the main members of the nuclear receptor superfamily, is reported to play important roles in metabolism, infection and immunity. Activation of LXRα could inhibit LPS-induced mastitis. Furthermore, LXRα is reported to enhance milk fat production, thus, LXRα may serve as a new target for mastitis therapy and regulation of milk fat synthesis. This review summarizes the effects of LXRα in regulating milk fat synthesis and treatment of mastitis and highlights the potential agonists involved in both issues.

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BACKGROUND: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder. RESULTS: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01). CONCLUSION: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.