Bovine Mammary Gland Research Articles

Alachlor breaks down intracellular calcium homeostasis and leads to cell cycle arrest through JNK/MAPK and PI3K/AKT signaling mechanisms in bovine mammary gland epithelial cells

Alachlor is a widely used herbicide for the cultivation of various grains employed as food for cattle. The mechanisms leading to the toxic effects of alachlor on epithelial cells of the bovine mammary gland are not well known. Thus, this study was conducted to clarify the toxicological effects of alachlor on the immortalized epithelial cell line of the bovine mammary gland (MAC-T) cells. After treatment, many factors related to cell viability, proliferation, and cellular homeostasis were evaluated. Alachlor arrested cell cycle progression by blocking the expression of cyclin and cyclin-dependent kinases, and induced the breakdown of Ca2+ homeostasis. The cytosolic and mitochondrial levels of Ca2+ were also abnormally increased after the treatment of cells with alachlor, ultimately leading to the depolarization of mitochondrial membrane potential in MAC-T cells. The signaling cascade was found to be dysregulated by the abnormal phosphorylation of signaling molecules involved in PI3K/AKT (AKT, p70S6K, and S6) and MAPK/JNK (JNK and c-Jun) pathways. In these mechanisms, exposure to alachlor led to a reduction in the viability and proliferation of MAC-T cells. Altogether, the toxic effects of alachlor can lead to abnormal conditions in epithelial cells of the bovine mammary gland, which might hinder these cells from performing their main role, such as producing milk.

MiR-140 downregulates fatty acid synthesis by targeting transforming growth factor alpha (TGFA) in bovine mammary epithelial cells

Fat is an indispensable nutrient and basic metabolite for sustaining life, and milk is particularly rich in fatty acids, including a variety of saturated and unsaturated fatty acids. MicroRNA (miRNA) and mRNA play an important role in the regulation of milk fat metabolism in mammary gland tissue. It has been shown that lipid metabolism has a complex transcriptional regulation, but the mechanism by which milk fat synthesis is regulated through miRNA–mRNA interactions is poorly understood. In this study, we performed transcriptome sequencing with bovine mammary gland tissue in the late lactation (270 and 315 days after parturition) to identify the key gene that regulating milk fat metabolism. A total of 1 207 differentially coexpressed genes were selected, 828 upregulated genes and 379 downregulated genes were identified. The transforming growth factor alpha (TGFA) gene was selected as the target gene, and luciferase reporter assay, Western blotting and qRT-PCR were used for further study. The results demonstrated that miR-140 was an upstream regulator of TGFA, and miR-140 could inhibit (P<0.01) unsaturated fatty acid and triglyceride (TAGs) production in bovine mammary epithelial cells (BMECs). In contrast, TGFA promoted (P<0.01) unsaturated fatty acid and TAG production. Rescue experiments further indicated the miR-140/TGFA regulatory mechanism. Taken together, these results suggest that the miR-140/TGFA pathway can inhibit (P<0.01) milk fat metabolism and improve milk quality by genetic means.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

Effect of Streptococcus uberis on Gamma Delta T Cell Phenotype in Bovine Mammary Gland.

Simple SummaryBovine mastitis (inflammation of the mammary gland) is still an important problem for dairy farmers. This disease causes great financial losses across the world. The common method of treating mastitis is through the use of antibiotics. Antibiotic treatment should be minimized because of increasing antibiotic resistance. Streptococcus uberis (S. uberis) is one of the most important pathogens that causes bovine mastitis. This bacterium is able to hide and survive inside of epithelial cells. In this situation, antibiotic treatment is not efficient. Therefore, it is necessary to study the pathogenesis of mastitis that is caused by S. uberis to better understand how to treat this disease. In this study, we investigated a special type of lymphocytes—γδ T cells. The results of our study show that those cells may play a role in terminating inflammation in the mammary glands of cattle.In this study, we focused analyzing γδ T cells during bovine mammary gland inflammation induced by Streptococcus uberis. A mammary gland cell suspension was obtained using lavage 24, 48, 72, and 168 h after intramammary-induced infection. The proportion of lymphocytes increased during the entire week in which inflammation was present. The γδ T cells were also elevated during inflammation, reaching their peak at 72 h following induced inflammation. The percentage of apoptotic lymphocytes continually increased, with the highest proportion occurring 168 h after S. uberis infection. The results show that γδ T cells may be involved in the resolution of inflammation in bovine mammary glands, with the apoptosis of those cells potentially playing an important role.

The bovine milk microbiome – an evolving science

Improved access to genome based, culture independent methods has generated great interest in defining the bovine milk microbiome. Several comprehensive reviews of this subject have recently been published and the purpose of this short review is to consolidate current understanding of the relevance and biological significance of this emerging topic. In contrast to mucosal organs that contain rich and well-characterized culturable and nonculturable microbial communities, milk obtained from the healthy bovine mammary gland usually contains few or no viable bacteria. The low bacterial biomass of milk has created methodological challenges that have resulted in considerable variability in results of studies that have used genomic methods to define the microbiota of milk obtained from healthy or diseased mammary glands. While genomes from several bacterial genera are routinely identified from samples of milk, teat skin and the teat canal, the viability, origin, and function of these organisms is uncertain as environmental factors have been shown to strongly influence the composition of these bacterial populations. Possible sources of microbial DNA include bacteria introduced from skin or the environment, bacteria trapped in teat canal keratin or bacteria engulfed by phagocytes. Researchers have not achieved consensus about key concepts such as the presence of a core commensal milk microbiome or dysbiosis as part of a causal pathway disrupting udder health. Understanding of the bovine milk microbiome has been greatly impeded by a lack of standardized methods used to collect, process, and assess bovine milk samples. Sample collection is a critical first step that will determine the validity of results. To minimize contamination with external sources of bacterial DNA, teat sanitation methods used for collection of milk samples that will be subjected to extraction and amplification of bacteria DNA should far exceed aseptic techniques used for collection of milk samples that will be submitted for microbiological culture. A number of laboratory issues have yet to be resolved. Contamination of low biomass samples with bacterial DNA from laboratory reagents is a well-known issue that has affected results of studies using bovine milk samples and results of sequencing of negative controls should always be reported. Replication of experiments has rarely been performed and consistency in results are lacking. While progress has been made, standardization of methods and replication using samples originating from differing farm conditions are critically needed to solidify knowledge of this emerging topic.

Aclonifen induces bovine mammary gland epithelial cell death by disrupting calcium homeostasis and inducing ROS production

Herbicides play key roles in agriculture. Aclonifen is a diphenyl ether herbicide that is widely used for sunflower, potato, corn, and wheat crops. Since it has a long half-life, it is considered persistent and can easily accumulate in the environment. Therefore, livestock and humans are at risk of exposure to aclonifen. Importantly, aclonifen is toxic to several mammals such as rats, mice, and dogs. However, the toxicity of aclonifen in cattle remains unclear. Therefore, we sought to investigate its toxicity in cattle using bovine mammary gland epithelial cells (MAC-T). We found that aclonifen induces sub-G1 phase arrest and represses MAC-T proliferation. In addition, aclonifen caused mitochondrial dysfunction, as evidenced by excessive ROS production and loss of mitochondrial membrane potential. Furthermore, cytosolic and mitochondrial calcium homeostases were disrupted after aclonifen treatment. Moreover, aclonifen treatment caused alterations in the PI3K/AKT and MAPK signaling pathways, which are involved in the regulation of cell survival and death. In conclusion, aclonifen causes MAC-T cell death through mitochondrial dysfunction and the collapse of calcium homeostasis.

Fluroxypyr-1-methylheptyl ester causes apoptosis of bovine mammary gland epithelial cells by regulating PI3K and MAPK signaling pathways and endoplasmic reticulum stress

Fluroxypyr-1-methylheptyl ester (FPMH) is an auxin herbicide that is widely applied to crops and pastures to block growth of post-emergence weeds. Several studies have reported the toxicity of FPMH in aquatic vertebrates. However, the adverse impacts of FPMH on mammals, including domestic animals, have not been reported. The purpose of our current study is to assess the impact of FPMH on the bovine mammary system and milk production. To evaluate the toxicity of FPMH on the mammary glands of lactating cows, the bovine mammary gland epithelial cell line, MAC-T, was exposed to various concentrations (0, 5, 7.5, 10, 15, and 20 μM) of FPMH for 24 h, and then various assessments were performed. The results showed that FPMH dose-dependently reduced MAC-T cell viability following exposure to FPMH and induced mitochondrial depolarization and apoptosis. FPMH also modulated signaling through the PI3K and MAPK pathways. In addition, the expression levels of proteins related to endoplasmic reticulum (ER) stress were upregulated, indicating induction of ER stress, and calcium homeostasis was disrupted following FPMH treatment. In conclusion, our investigation suggests that FPMH may be toxic to the bovine mammary system and may decrease dairy production.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Chemerin Regulates Epithelial Barrier Function of Mammary Glands in Dairy Cows.

Simple SummaryThis study evaluated the production and the function of chemerin, known as a chemoattractant and an antimicrobial protein, with regard to mammary epithelial defense in cows. The result demonstrated that mammary epithelial cells express chemerin protein. Chemerin treatment promoted the proliferation of bovine mammary epithelial cells and protected epithelial cells from oxidative stress. Meanwhile, mammary chemerin production was elevated by mastitis, which was possibly attributed to inflammatory cytokines. Therefore, the present study demonstrates the supportive ability of chemerin for mammary epithelial tissue and its regulation by inflammatory stimuli, suggesting its role in mammary epithelial defense against pathogen infection in cows.Epithelial barrier function in the mammary gland acts as a forefront of the defense mechanism against mastitis, which is widespread and a major disorder in dairy production. Chemerin is a chemoattractant protein with potent antimicrobial ability, but its role in the mammary gland remains unelucidated. The aim of this study was to determine the function of chemerin in mammary epithelial tissue of dairy cows in lactation or dry-off periods. Mammary epithelial cells produced chemerin protein, and secreted chemerin was detected in milk samples. Chemerin treatment promoted the proliferation of cultured bovine mammary epithelial cells and protected the integrity of the epithelial cell layer from hydrogen peroxide (H2O2)-induced damage. Meanwhile, chemerin levels were higher in mammary tissue with mastitis. Tumor necrosis factor alpha (TNF-α) strongly upregulated the expression of the chemerin-coding gene (RARRES2) in mammary epithelial cells. Therefore, chemerin was suggested to support mammary epithelial cell growth and epithelial barrier function and to be regulated by inflammatory stimuli. Our results may indicate chemerin as a novel therapeutic target for diseases in the bovine mammary gland.

Expression of cytokines in dairy cattle mammary gland parenchyma during chronic staphylococcal infection

The study aim was to determine the expression of genes potentially related to chronic mastitis at the mRNA and protein levels, viz. chemokine C–C motif receptor 1 (CCR1), C–C motif chemokine ligand 2 (CCL2), C–C motif chemokine ligand 5 (CXCL5), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 18 (IL-18), in bovine mammary gland parenchyma. The study examines the differences in expression of selected genes between cows with chronic mastitis caused by coagulase-positive (CoPS) or coagulase-negative staphylococci (CoNS) and those with healthy udders (H). Samples were collected from the udder quarters from 40 Polish Holstein–Friesian cows; 54 of these samples were chosen for analysis based on microbiological analysis of milk taken two days before slaughter. They were categorized into three groups: CoPS (N = 27), CoNS (N = 14) and H (N = 13). The RNA expression was analyzed by RT-qPCR and protein concentration by ELISA. No differences in the mRNA levels of seven genes (TNFα, IL-18, CCR1, IL-1β, CCL2, IL-8, IL-6) and four proteins (TNFα, IL-18, CCR1, IL-1β) were identified between the CoPS and H groups. Higher transcript levels of CXCL5 (p ≤ 0.05) gene were noted in CoPS than in H. Compared to H, higher concentrations of IL-8 and CXCL5 (p ≤ 0.05) were observed in CoPS (0.05 < p < 0.1) and CCL2 (0.05 < p < 0.1) in CoNS, while lower levels of Il-6 were found in CoPS. This may suggest that during chronic mastitis the organism stops producing pro-inflammatory cytokines, probably to protect the host tissues against their damage during prolonged infection.

Directed Expression of Tracheal Antimicrobial Peptide as a Treatment for Bovine-Associated Staphylococcus Aureus-Induced Mastitis in Mice.

Bovine mastitis is perplexing the dairy industry since the initiation of intensive dairy farming, which has caused a reduction in the productivity of cows and an escalation in costs. The use of antibiotics causes a series of problems, especially the formation of bacterial antimicrobial resistance. However, there are limited antibiotic-free therapeutic strategies that can effectively relieve bacterial infection of bovine mammary glands. Hence, in this study, we constructed a mammary gland tissue-specific expression vector carrying the antimicrobial peptide of bovine-derived tracheal antimicrobial peptide (TAP) and evaluated it in both primary bovine mammary epithelial cells (pBMECs) and mice. The results showed that the vector driven by the β-lactoglobulin gene (BLG) promoter could efficiently direct the expression of TAP in pBMECs and the mammary gland tissue of mice. In addition, significant antibacterial effects were observed in both in vitro and in vivo experiments when introducing this vector to bovine-associated Staphylococcus aureus-treated pBMECs and mice, respectively. This study demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antimicrobial peptide both in in vitro and in vivo and will provide a new therapeutic strategy in the treatment of bovine mastitis.

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

Background: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. Methods: In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. Results: AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. Conclusion: Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.

Late gestation hyperthermia: epigenetic programming of daughter's mammary development and function

Exposure to stressors during early developmental windows, such as prenatally (i.e., in utero), can have life-long implications for an animal's health and productivity. The mammary gland starts developing in utero and, like other developing tissues and organs, may undergo fetal programming. Previous research has implicated factors, such as prenatal exposure to endocrine disruptors or alterations in maternal diet (e.g., maternal over or undernutrition), that can influence the developmental trajectory of the offspring mammary gland in postnatal life. However, the direct links between prenatal insults and future productive outcomes are less documented in livestock species. Research on in utero hyperthermia effects on early-life mammary development is scarce. This review will provide an overview of key developmental milestones taking place in the bovine mammary gland during the pre- and postnatal stages. We will showcase how intrauterine hyperthermia, experienced by the developing fetus during the last trimester of gestation, derails postnatal mammary gland development and impairs its synthetic capacity later in life. We will provide insights into the underlying histological, cellular, and molecular mechanisms taking place at key postnatal developmental life stages, including birth, weaning and the first lactation, that might explain permanent detriments in productivity long after the initial exposure to hyperthermia. Collectively, our studies indicate that prenatal hyperthermia jeopardizes the normal developmental trajectory of the mammary gland from fetal development to lactation. Further, in utero hyperthermia epigenetically programs the udder, and possibly other organs critical to lactation, yielding a less resilient and less productive cow for multiple lactations.

Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation

Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

N-acetyl-β-D-glucosaminidase biomarker quantification in milk using Ag-porous Si SERS platform for mastitis severity evaluation

Bovine mammary gland is susceptible to numerous bacterial infections resulting in inflammatory disease condition of bovine mastitis (BM) with a staggering economic impact on the dairy industry worldwide. Early BM detection is crucial for infection control within the dairy herd, which can be alleviated by antimicrobial therapy. N-acetyl-β-D-glucosaminidase (NAGase) is a prominent BM inflammatory biomarker secreted onto the blood circulation upon pathogenesis and then released into milk, capable of separating healthy quarters from subclinical and clinical BM cases. Herein, we report a sensitive, cost-effective and handy-to-use BM severity assay based on NAGase inherent content found in whole milk samples. Silver-coated porous Si (Ag-PSi) Fabry–Pérot interferometers were employed for quantifying the lysosomal activity in different milk qualities using surface-enhanced Raman spectroscopy (SERS). The optical response was proportional to the occurring pathogenesis induced by predominant bacteria found in dairy farms at different somatic cell levels. The optimized Ag-PSi SERS substrates were utilized for quantitative analysis of NAGase levels found at clinically relevant BM concentrations while presenting the detection limit of 0.27 μM min−1. The optical performances were associated with the conventional standardized approach in terms of recovery values (85–98%), thus presenting sufficient adaptability to complex matrix analysis as whole milk.

The inhibitory effect of trans-10,cis-12 conjugated linoleic acid on sterol regulatory element binding protein-1 activation in bovine mammary epithelial cells involved reduced proteasomal degradation of insulin-induced gene-1

Trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) is well recognized as a key CLA isomer responsible for the reduction in milk fat synthesis that leads to milk fat depression in dairy cows. Sterol regulatory element binding protein-1 (SREBP1) is a key transcription factor in bovine mammary gland coordinating transcription of the genes for fatty acid synthesis. SREBP1 activation requires the removal of insulin-induced gene-1 (Insig1) that serves as a repressor of SREBP1 in the endoplasmic reticulum (ER). We hypothesized that t10,c12 CLA reduced SREBP1 activation by delaying Insig1 degradation. In the present study, we used undifferentiated bovine mammary epithelial cells (MAC-T cells) and treated them with t10,c12 CLA for 6 h. We found that SREBP1 protein expression declined over 56% when cells were treated with 60 µM or greater concentration of t10,c12 CLA. Such inhibitory effects were also observed in the mRNA expression of SREBP1-regulated genes including SREBP1, fatty acid synthetase, stearoyl-CoA desaturase, and Insig1. Compared with no CLA group, 60 µM or higher concentration of t10,c12 CLA increased Insig1 protein expression over 2-fold in cells transfected with FLAG-tagged Insig1. This stimulatory effect was not specific to t10,c12 CLA but also other polyunsaturated fatty acids including cis-9,trans-11 CLA and linoleic acid. Oleic acid had no effect on Insig1 protein expression, whereas palmitic acid decreased Insig1 protein expression. Further investigation revealed that increased abundance of FLAG-Insig1 with t10,c12 CLA was due to the inhibition of the proteasomal degradation of Insig1. The t10,c12 CLA delayed the Insig1 decay when protein synthesis was blocked. Immunoprecipitation also confirmed that the interaction between ubiquitin-like domain-containing protein 8 and Insig1, the key step of removing Insig1 from ER and freeing SREBP1 for proteolytic processing, was inhibited by t10,c12 CLA, but not palmitic acid. These findings suggested that t10,c12 CLA played a role in regulating SREBP1 activation by reducing proteasomal degradation of Insig1. We concluded that stabilized Insig1 retained SREBP1 in the ER from activation, thus reducing lipogenic gene transcription.

Milk Macrophage Function in Bovine Leukemia Virus-Infected Dairy Cows.

The implications of bovine leukemia virus (BLV) on innate and adaptive immune responses have been widely investigated; however, the effects of BLV on mammary gland immunity require further investigation. The present study investigated the viability, phagocytic capacity, and intracellular production of reactive oxygen and nitrogen species (RONS) by macrophages in milk samples from dairy cows naturally infected with BLV with or without persistent lymphocytosis (PL). No effect of BLV infection in the overall number of macrophages per milliliter and in the percentage of viable macrophages among overall milk viable cells was found. Furthermore, BLV-infected dairy cows had a higher frequency of viable milk macrophages, while healthy animals had a tendency toward a higher percentage of apoptotic milk macrophages. The percentage of milk macrophages that phagocytosed Staphylococcus aureus in seronegative animals was higher than that in BLV-infected dairy cows. No effect of BLV infection on the intracellular RONS production and the intensity of phagocytosis by milk macrophages was observed. Thus, this study provides new insights into the implications of BLV infections in the bovine mammary gland.

Antimicrobial susceptibilities in dairy herds that differ in dry cow therapy usage

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy; DCT) is a central part of mastitis control programs and is one of the major indications for antimicrobial use in dairy cows. However, with increasing focus on prudent use of antimicrobials and concerns about emergence of antimicrobial resistance, the practice of treating every cow at the end of lactation with DCT is in question. This cross-sectional, observational study determined the minimum inhibitory concentrations (MIC) of 10 antimicrobials for coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates from milk samples from dairy cows with somatic cell counts >200,000 cells/mL in herds that had been organic for >3 yr (n = 7), or had used either ampicillin-cloxacillin DCT (n = 11) or cephalonium DCT (n = 8) in the preceding 3 yr. The organic herds were certified under the United States Department of Agriculture National Organic Program, meaning that there was no blanket DCT, and minimal use of antimicrobials in general, with a loss of organic status of the animal if treated with antimicrobials. Breakpoints (where available) were used to categorize isolates as resistant, intermediate, or susceptible to antimicrobials. The MIC distributions of isolates from different herd types were compared using binomial or multinomial logistic regression. Of 240 CNS isolates, 12.9, 0.8, 7.1, 32.6, and 1.2%, were intermediate or resistant to ampicillin, cephalothin, erythromycin, penicillin, and tetracycline, respectively. Of 320 Staph. aureus isolates, 29.0, 2.5, 1.2, and 34.9% were intermediately resistant or resistant to ampicillin, penicillin, erythromycin, and oxacillin, respectively. Of 184 Strep. uberis isolates, 1.1, 25.0, 1.6, and 1.6% were intermediately resistant or resistant to erythromycin, penicillin, pirlimycin, and tetracycline, respectively. Generally, the MIC of CNS and streptococcal isolates from organic herds were lower than isolates from herds using DCT. However, the differences in MIC distributions occurred at MIC below clinical breakpoints, so that the bacteriological cure rates may not differ between isolates of differing MIC. Bimodal distributions of MIC for ampicillin and penicillin were found in Staph. aureus isolates from organic herds, suggesting that isolates with a higher MIC are a natural part of the bacterial population of the bovine mammary gland, or that isolates with higher MIC have persisted within these organic herds from a time when antimicrobials had been used. Given these observations, further work is required to determine if exposure to DCT is causally associated with the risk of elevated MIC, and whether reduction or removal of DCT from herds would reduce the risk of elevated MIC of mastitis pathogens.

Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro

Communications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.