MAC-T Cells Research Articles

Persistence of a Staphylococcus aureus small colony variants ( S. aureus SCV) within bovine mammary epithelial cells

Persistent bovine Staphylococcus aureus mastitis is attributable to the versatility of this pathogen within the mammary gland environment and to the formation of small colony variants (SCVs) that can survive within host cells. Previous studies had shown that S. aureus SCV Heba3231, isolated from a cow with chronic mastitis, had invaded and persisted in primary bovine aortic endothelial cells but caused minimal deleterious effects. The objective of this study was to investigate the interaction of SCV Heba3231 with bovine mammary epithelial cells (MAC-T cells) compared to its parent strain 3231 and to prototype strain Newbould 305. Monolayer cells were infected with each strain at various multiplicity of infections (MOIs) for 1 and 3.5 h, followed by 20 min incubation with lysostaphin. Recovery of the SCV was significantly higher ( P < 0.05) after 3.8 h with MOI of 100 compared to recovery of strains 3231 and Newbould 305. Upon further incubation, viable SCV were detected up to 96 h while 3231 were not isolated at 24 h or later. Transmission electron microscopy demonstrated SCV uptake by MAC-T cells following a series of events similar to those for strain 3231. At 24 h, multiple SCV were seen within enclosed vacuoles, while the 3231 parent strain was released extracellularly and the monolayer cells were damaged. The ability of SCV Heba3231 to survive inside vacuoles could be related to up-regulation of protective mechanisms. These findings highlight the potential role of bovine mammary epithelial cells and S. aureus SCV in persistent bovine mastitis.

Activation of AMP-activated protein kinase (AMPK) inhibits fatty acid synthesis in bovine mammary epithelial cells

Activation of AMP-activated protein kinase (AMPK), a heterotrimeric energy-sensing protein, decreases lipid synthesis in liver tissue of various species; however, little is known about the role of AMPK in the regulation of fatty acid synthesis in bovine mammary epithelial cells. Here we report the presence of AMPK mRNA in MAC-T bovine mammary epithelial cells and mammary gland. Treatment of MAC-T with an AMPK activator dramatically decreased de novo fatty acid synthesis by inactivating acetyl-CoA carboxylase-α. Activation of AMPK also modified the mRNA expression of several lipogenic genes including fatty acid synthase, glycerol-3-phosphate acyltransferase, and fatty acid binding protein-3. Additionally, decreases in energy availability or rises in intracellular Ca 2+ most likely activated AMPK in MAC-T. These data suggest the presence of LKB1 and Ca 2+/calmodulin-dependent kinase kinase, two known AMPK kinases, in MAC-T. Identifying AMPK as a molecular target capable of modifying energy substrate utilization may result in the development of new technologies that increase milk production or modify milk composition during periods of increased energy demand.

Ricin A-chain requires c-Jun N-terminal kinase to induce apoptosis in nontransformed epithelial cells

Ricin is a toxin isolated from castor beans that has potential as a weapon of bioterrorism. This glycoprotein consists of an A-chain (RTA) that damages the ribosome and inhibits protein synthesis and a B-chain that plays a role in cellular uptake. Ricin activates the c-Jun N-terminal kinase (JNK) and p38 signaling pathways; however, a role for these pathways in ricin-induced cell death has not been investigated. Our goals were to determine if RTA alone could activate apoptosis and if the JNK and p38 pathways were required for this response. Comparable caspase activation was observed with both ricin and RTA treatment in the immortalized, nontransformed epithelial cell line, MAC-T. Ribosome depurination and inhibition of protein synthesis were induced in 2–4 h with 1 μg/ml RTA and within 4–6 h with 0.1 μg/ml RTA. Apoptosis was not observed until 4 h of treatment with either RTA concentration. RTA activated JNK and p38 in a time- and concentration-dependent manner that preceded increases in apoptosis. Inhibition of the JNK pathway reduced RTA-induced caspase activation and poly(ADP-ribose) polymerase cleavage. In contrast, inhibition of the p38 pathway had little effect on RTA-induced caspase 3/7 activation. These studies are the first to demonstrate a role for the JNK signaling pathway in ricin-induced cell death. In addition, the MAC-T cell line is shown to be a sensitive in vitro model system for future studies using RTA mutants to determine relationships between RTA-induced depurination, ribotoxic stress, and apoptosis in normal epithelial cells.

Expression of bovine granulocyte chemotactic protein-2 (GCP-2) in neutrophils and a mammary epithelial cell line (MAC-T) in response to various bacterial cell wall components

CXC chemokines are potential attractants for polymorphonuclear leucocytes (PMNs) and play an important role in resistance to infectious diseases, such as bovine mastitis. In this study, a bovine mammary epithelial cell line (MAC-T) and blood PMNs were stimulated with bacterial cell wall components of gram negative and gram positive bacteria, including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The expression of two CXC chemokines, interleukin (IL)-8 and granulocyte chemotactic protein (GCP)-2 was analysed by real-time PCR. High concentrations (1 or 10 μg/mL) of LPS and LTA, but not PGN, significantly increased the expression of GCP-2 and IL-8 in both MAC-T and PMNs. Biopsies from mammary glands of cattle with clinical Escherichia coli mastitis also had increased expression of GCP-2. Using an in vitro transepithelial migration assay, recombinant human GCP-2 (rhGCP-2) showed weak chemoattractant effects on bovine blood PMNs. It was concluded that PMNs and MAC-T cells can express GCP-2 in response to certain bacterial cell components during the course of mastitis.

P.6.01 Incentive role of amygdaloid CRF receptors in the reinforcing effects of psychoactive drugs in rats: a new concept

This experiment examined the effects of cold-pressed, terpeneless citrus-derived oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of bovine mammary cells (MAC-T). To determine minimum inhibitory concentration, we used the broth dilution method, using CDO concentrations range from 0.0125 to 0.4% with 2-fold dilutions. Growth inhibition was examined by adding 0.00, 0.05, 0.025, 0.0125, and 0.00625% CDO to 105 cfu/mL S. aureus in nutrient broth and enumerating colonies after serial dilution. In a 96-well plate, S. aureus (107 cfu/mL) was allowed to form a biofilm, treated with 0, 0.025, 0.5, or 1% CDO, and then was measured using a spectrophotometer. Cytotoxic effect on immortalized MAC-T cells was also examined at various concentrations of CDO using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We observed that the minimum inhibitory concentration of CDO to inhibit the growth of S. aureus in vitro was 0.025% CDO. A time kill curve for CDO action on S. aureus over 4 h was generated. The CDO completely eliminated S. aureus after 3 h of incubation at a concentration of 0.25%, or after 2 h of incubation at concentrations of 0.05%. It was also observed that CDO had no effect on preformed biofilms except at a concentration of 0.05%, in which a significant reduction in the measured absorbance was noted. In addition, the association and invasion of S. aureus to MAC-T cells were significantly inhibited after 1 h of treatment with CDO. Citrus-derived oil was also able to increase cellular proliferation of MAC-T cells at concentrations up 0.05% and had no effect at a concentration of 0.1% after 1 h. Our data suggests that CDO should be considered for further research as a preventive and therapeutic against bovine mastitis.

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models

Staphylococcus aureus produces a variety of virulence factors that allow it to cause a wide range of infections in humans and animals. In the latter, S. aureus is a leading cause of intramammary infections. The contribution of catalase (KatA), an enzyme implicated in oxidative stress resistance, and beta-toxin (Hlb), a haemolysin, to the pathogenesis of S. aureus is poorly characterized. To investigate the role of these enzymes as potential virulence factors in S. aureus, we examined the intracellular survival of DeltakatA, Deltahlb and DeltakatA Deltahlb mutants in murine macrophages (J774A.1) and bovine mammary epithelial cells (MAC-T), and their virulence in different murine and ovine models. Catalase was not required for the survival of S. aureus within either J774A.1 or MAC-T cells. However, it was necessary for the intracellular proliferation of the bacterium after invasion of MAC-T cells. In addition, catalase was not needed for the full virulence of S. aureus in mice. Deletion of the hlb gene had no effect on the intracellular survival of S. aureus in J774A.1 cells but did cause a slight increase in survival in MAC-T cells. Furthermore, like catalase, beta-toxin was not required for complete virulence of S. aureus in murine models. Unexpectedly, the DeltakatA Deltahlb mutant showed a notably increased persistence in both cell lines, and was significantly less virulent for mice than were the wild-type strain and single mutants. Most interestingly, it was also markedly attenuated in intramammary and subcutaneous infections in ewes and lambs.

Antibodies to Capsular Polysaccharide and Clumping Factor A Prevent Mastitis and the Emergence of Unencapsulated and Small-Colony Variants ofStaphylococcus aureusin Mice

The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated "escape mutants" and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.

Effect of CLA and Other C18 Unsaturated Fatty Acids on DGAT in Bovine Milk Fat Biosynthetic Systems

Production of dairy products with increased amounts of nutraceutic FA such as conjugated linoleic acid (CLA) represents a recent approach for dairy producers and processors to increase the value of their products. The effect of CLA and other FA on the expression of diacylglycerol acyltransferase-1 (DGAT-1) and DGAT-2, and DGAT activity were investigated in bovine mammary gland epithelial (MAC-T) cells. DGAT gene expression analyses were also conducted using bovine mammary gland tissue from dairy cows. In the studies with MAC-T cells, there were no significant effects of CLA isomers or other FA on DGAT1 expression, whereas all FA tested showed enhanced DGAT2 expression (P < 0.05 to P < 0.001), with alpha-linolenic acid (alpha-18:3) having the greatest effect. Additionally, DGAT2 expression was co-ordinated with expression of lysophosphatidic acid acyltransferase (LPAAT), an observation that was also apparent in mammary gland from lactating dairy cows. In contrast, treatment of MAC-T cells with trans-10, cis-12 18:2 or alpha-18:3 resulted in a significant (P < 0.05) decrease in overall DGAT enzyme activity, although the mechanisms resulting in these effects are unclear. Competition assays using microsomes from bovine mammary gland tissue and 1-[(14)C]oleoyl-CoA suggested that DGAT activity was more selective for oleoyl (cis-9 18:1)-CoA than cis-9, trans-11 18:2-, trans-10, cis-12 18:2- or cis-9, cis-12 18:2-CoA. Collectively, the results suggest the relationship between trans-10, cis-12 18:2 and reduced TAG production in bovine milk is not linked to the production of DGAT1 or DGAT2 transcripts, but probably involves effects of this CLA isomer at events beyond transcription, such as post-translational and/or enzyme activity effects.

Biosynthesis of selenoproteins in cultured bovine mammary cells

The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with ( 75Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 75Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of 75Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more 75Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.

Growth hormone, prolactin, and sodium acetate have complex and non‐uniform effects on the lipid distribution of a differentiated bovine mammary epithelial cell line

Non‐polar lipids (NPL) accumulate within the mammary epithelial cell during lactation; however there are no reported systematic studies that utilize NPL composition as a sign of differentiation. Additionally, there are no reported systematic studies on the response of a bovine mammary alveolar cell line (MAC‐T), to a range of lactogenic hormone concentrations, or different lipogenic substrates. In a 3 x 3 factorial arrangement, the effect of growth hormone (GH; 0, 10, 100 ng/ml), and prolactin (PRL; 0, 5, 10 μg/ml) on the lipid composition of MAC‐T was examined using thin layer chromatography. Using the optimal hormone treatment (10 ng/ml GH, 5 μg/ml PRL) for NPL synthesis, inclusion of 10 mM sodium acetate (NaOAc) was assessed to determine the effect of lipogenic substrate on lipid composition. GH had a significant effect on NPL, cholesterol, phosphatidylcholine, phosphatidylinostitol, and sphingomyelin (SM), while PRL only affected SM. NaOAc had a significant effect on NPL, phosphatidylethanolamine and SM. The proportion of NPL peaked with 10 ng/ml GH, but dropped to near‐control levels with 100 ng/ml GH. This study underscores the importance of testing a range of hormone concentrations when assessing lipogenic profile of MAC‐T cells.

Regulation of mammary parenchymal growth by the fat pad in prepubertal dairy heifers: role of inflammation-related proteins

In prepubertal heifers, the mammary parenchyma consists of epithelial and myoepithelial cells growing within a mammary fat pad (MFP). The MFP produces IGF-I that stimulates epithelial cell proliferation. In other species, adipose tissue expansion induces inflammation-related proteins (IRP), such as tumor necrosis factor alpha (TNFalpha), interleukin (IL)-6, IL-1beta transforming growth factor beta, monocyte chemoattractant protein 1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1). The MFP production of IRP may influence mammary development because they impair not only insulin but also IGF-I actions. Moreover, the MFP expansion seen with development and increased nutrition coincides with reduced parenchymal growth. Our first objective was to identify IRP capable of altering proliferation of bovine mammary epithelial cells. TNFalpha, but neither IL-6, IL-1beta MCP-1 nor PAI-1, inhibited basal and IGF-I-stimulated proliferation in MAC-T cells and primary cells isolated from heifers. Our second objective was to determine whether MFP expression of IRP changed in a manner consistent with inhibition of parenchymal growth. MFP expression was measured from 100 to 350 kg body weight (experiment 1) or at 240 kg body weight (experiment 2) in dairy heifers offered restricted or high planes of nutrition. In experiment 1, neither nutrition nor development altered MFP expression of TNFalpha. Nutrition increased MCP-1 and PAI-1 but only before MFP expansion and after cessation of allometric parenchymal growth. In experiment 2, nutrition increased TNFalpha and PAI-1, but not MCP-1. Thus, MFP expansion increases IRP production in cattle, but this is unlikely to contribute to reduced parenchymal growth observed with development or increased nutrition.

Growth Hormone Can Induce Expression of Four Major Milk Protein Genes in Transfected MAC-T Cells

Growth hormone (GH) can increase milk production in cattle, and this effect was thought to be mediated by an indirect mechanism because traditional ligand binding assays failed to detect GH binding sites in the mammary gland. However, recent findings that GH receptor (GHR) mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly act on these cells to affect milk production. Therefore, the objective of this study was to determine whether GH could affect milk protein gene expression, nutrient uptake, and cell proliferation in bovine mammary epithelial cells using the bovine mammary epithelial cell-derived MAC-T cells as a model. Native MAC-T cells had low expression of GHR. Thus, we transfected them with expression plasmids for GHR and signal transducer and activator of transcription 5 (STAT5), 2 key components of GHR signaling, to maximize their GH response. Growth hormone increased the expression of αS1-casein, αS2-casein, β-casein, and α-lactalbumin mRNA 16- to 117-fold in the transfected MAC-T cells, whereas it had no effect on the expression of κ-casein, β-lactoglobulin, or insulin-like growth factor I mRNA. Cotransfection analyses showed that GH also strongly induced reporter gene expression from αS1-casein, αS2-casein, β-casein, and α-lactalbumin gene promoters. Growth hormone had no effect on the uptake of 2-deoxyglucose, an unmetabolizable glucose analog, amino acids, or oleic acid; neither did it affect cell proliferation or death. These observations together with the fact that GH receptor mRNA and protein are expressed in the epithelial cells of the bovine mammary gland raise the possibility that GH might act directly on the mammary epithelial cells in cows to stimulate transcription of major milk protein genes, as part of the mechanism by which GH stimulates milk production.

Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. In this study, MAC-T cells (a bovine mammary epithelial cell line) were incubated in the presence or absence of increasing concentrations of LPS for 24 h. Expression of TLR2 and TLR4 were analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and flow cytometry, respectively. The mRNA of both receptors were up-regulated by all concentrations of LPS used (P<0.01). Similarly, flow cytometry with specific antibodies against TLR2 and TLR4 detected increased surface expression of these proteins. Furthermore, expression of downstream TLR4 signaling molecules was examined by qPCR following varying exposure times to 1 mug/mL of LPS. Results demonstrate that the required adaptor molecules and transcription factors were up-regulated in a time-dependent manner. Both the MyD88 dependent and independent pathways in TLR4 signaling were activated in MAC-T cells. Expression of TOLLIP increased in response to LPS as did the pro-apoptotic protease, CASP8. These results suggest that the bovine mammary epithelium possesses the necessary immune repertoires required to achieve a robust defense against E. coli. The current findings, coupled with previous findings that S. aureus ligands induce up-regulation of TLR4, may indicate a positive adaptation by mammary epithelial cells to effectively respond to different types of mastitis pathogens.

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial CellsIn Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.

Prolactin-Induced Activation of Nuclear Factor κB in Bovine Mammary Epithelial Cells: Role in Chronic Mastitis

We sought to determine whether prolactin (PRL) could influence the neutrophilic inflammation that characterizes chronic mastitis. Most of the genes encoding inflammatory proteins depend on the nuclear factor κB (NF-κB) for their expression. We addressed the hypothesis that immunomodulatory activities of PRL might arise from an increase in NF-κB activity. MAC-T cells, a bovine mammary epithelial cell line, were stimulated with increasing concentrations of bovine PRL (1, 5, 25, 125, and 1,000 ng/mL). Level of NF-κB binding activity was measured and mRNA was evaluated for IL-1β, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GMCSF), IFN-γ, and tumor necrosis factor (TNF)-α, cytokines known to require NF-κB for their maximal transcription. Prolactin activated NF-κB; maximal NF-κB activation was weaker with PRL than with TNF-α at 30 or 180min poststimulation. In addition, PRL significantly amplified, in a dose-dependent manner, mRNA expression of IL-1β, IL-6, IL-8, GMCSF, and TNF-α. We measured PRL concentrations in blood and milk from healthy and chronic mastitis-infected cows, and studied the relationship between the PRL concentration and the degree of inflammation in the mammary gland as indirectly assessed by somatic cell counts (SCC). Plasma PRL did not differ significantly between healthy and chronic mastitis-affected cows (63.7 and 67.5 ng/mL, respectively). Milk PRL concentration was significantly increased in chronic mastitis-affected quarters with the highest SCC, and had a positive significant correlation between SCC, as well as between the number of neutrophils present in milk samples. The present findings show that PRL promotes an inflammatory response in bovine mammary epithelial cells via NF-κB activation, and suggest a role for PRL in the pathogenesis of chronic mastitis.

Differential Abilities of Capsulated and Noncapsulated Staphylococcus aureus Isolates from Diverse agr Groups To Invade Mammary Epithelial Cells

Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.

Helenalin reduces Staphylococcus aureus infection in vitro and in vivo

Staphylococcus ( S.) aureus is a major udder pathogen causing bovine mastitis. Some pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), enhance extracellular and intracellular growth of S. aureus, indicating that the inflammatory process favors S. aureus infection. Helenalin is a sesquiterpene lactone with potent anti-inflammatory properties. This study was designed to evaluate the effects of helenalin on S. aureus infection. First, in vitro experiments were conducted. These studies revealed that proliferation of S. aureus in bovine mammary epithelial MAC-T cells treated in the presence or absence of TNF-α was markedly reduced in the presence of helenalin. Secondly, in vivo effects of helenalin were investigated. Lactating mice treated in the presence or absence of helenalin were challenged by the intramammary route with S. aureus and the bacteria in the mammary glands were counted 12 h after infection. Significantly less numbers of bacteria were recovered from the infected glands of helenalin-treated mice compared with untreated mice. Moreover, histological examination of mammary tissue from helenalin-treated mice that were challenged with S. aureus indicated that helenalin is able to significantly reduce leukocyte infiltration in the mammary gland following S. aureus inoculation. Our results show that helenalin reduces S. aureus intracellular growth and experimental S. aureus infection. We conclude that helenalin may be of potential interest in the treatment of S. aureus-induced mastitis in the bovine species.

Leptin Does Not Act Directly on Mammary Epithelial Cells in Prepubertal Dairy Heifers

The mammary gland of prepubertal dairy heifers consists of parenchyma expanding into the stroma, a matrix of connective and adipose tissue. High planes of nutrition increase stromal mass, but inhibit growth of parenchyma. The parenchyma consists of epithelial cells proliferating in response to growth factors such as insulin like growth factor-I (IGF-I). These observations have led to the hypothesis that elevated planes of nutrition increase leptin production, which in turn inhibits IGF-I-mediated epithelial cell proliferation. To assess this possibility, heifers were offered planes of nutrition sustaining average daily gains of 715 g/d (normal; NP) or 1,202 g/d (high; HP) from 42 d of age until slaughter at 240kg. At slaughter, HP heifers had 2-fold greater plasma leptin concentration and 3-fold greater leptin mRNA abundance in mammary stroma and parenchyma. To assess the causal nature between leptin and parenchymal development, the induction of signaling events and functional responses in the MAC-T cell line and in primary mammary epithelial cells by leptin was examined. Leptin did not induce phosphorylation of signal transducers and activators of transcription (STAT)3, STAT5, extracellular signal-regulated kinase (ERK1/2), or AKT/Protein kinase B. Consistent with its inability to signal, leptin did not alter basal- or IGF-I–stimulated thymidine incorporation or increase suppressors of cytokine signaling 3 (SOCS3) expression in these cells. Transcripts corresponding to the short leptin receptor form were present in mammary tissue, but those corresponding to the long signaling form were not detected in either mammary tissue or cells. In conclusion, elevated planes of nutrition increase leptin synthesis in mammary stroma, but leptin does not act directly on bovine mammary epithelial cells.

Adherent and invasive Escherichia coli are associated with persistent bovine mastitis

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6–63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.