Olga Wellnitz

Staphylococcus aureus and Escherichia coli Cause Deviating Expression Profiles of Cytokines and Lactoferrin Messenger Ribonucleic Acid in Mammary Epithelial Cells

Pathogens invading the mammary gland cause a complex signaling network that activates the early immune defense and leads to an outcome of inflammation symptoms. To examine the importance of mammary epithelial cells in these regulations and interactions resulting in a pathogen-related course of mastitis, we characterized the mRNA expression profile of key molecules of the innate immune system by quantitative real-time PCR. Mammary gland epithelial cells isolated on d 42 of lactation from 28 first-lactation Holstein dairy cows were cultured separately under standardized conditions and treated for 1, 6, and 24 h with heat-inactivated gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Both pathogens increased mRNA expression patterns of proteins involved in pathogen recognition such as Toll-like receptors and nuclear factor-kappa B, whereas gram-negatives acted as a stronger stimulus. Furthermore, this could be confirmed by the expression profile of the proinflammatory cytokines tumor necrosis factor alpha, IL-1 beta, IL-6, and chemokines such as IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted). Remarkably, at a low level of mRNA expression after 1 h of treatment these cytokines and chemokines were expressed at a significantly higher level in Staphyloccocus aureus than in Escherichia coli affected cells. Lactoferrin showed a deviating expression pattern to pathogen stimulation (i.e., at the 1-h measuring point Escherichia coli induced a higher mRNA expression, whereas the highest level was reached after 24 h of stimulation with Staphylococcus aureus). Complement factor 3 was the only measured factor that responded equally to both microorganisms. Our data emphasize the role of mammary epithelial cells in the immune defense of the udder and confirm their contribution to pathogen-related different courses of mastitis.

The innate immune response of the bovine mammary gland to bacterial infection.

Intra-mammary (IM) bacterial infection in cattle can result in clinical outcomes that range from being acute and life-threatening to those that are chronic and sub-clinical. The typical bacteria involved in IM bacterial infections activate the mammary immune system in different ways which can influence the severity of the outcome. A clear understanding of the mechanisms that activate and regulate this response is central to the development of effective preventative and treatment regimes. This review focuses on the different immune responses of the bovine mammary gland to common mastitis-causing pathogens. There is special emphasis on comparing the responses to Escherichia coli and Staphylococcus aureus infections, as these are typically associated, respectively, with acute/severe and chronic/sub-clinical forms of the disease.

Inhibition of milk ejection in cows by oxytocin receptor blockade, α-adrenergic receptor stimulation and in unfamiliar surroundings

Inhibition of milk ejection in cows by oxytocin receptor blockade (Atosiban) and alpha-adrenergic receptor stimulation (phenylephrine) prior to prestimulation was compared with inhibition of milk ejection in unfamiliar surroundings. In addition, Atosiban and phenylephrine were administered after a 1 min prestimulation or 1 min after the start of milking. Oxytocin concentrations increased during milking in all treatments. The spontaneously removed milk fraction (before oxytocin was injected) was similar for Atosiban and phenylephrine treatments and in unfamiliar surroundings, but lower than in controls. Peak flow rates were similar in all treatments, but reduced as compared with controls when phenylephrine and Atosiban were administered before prestimulation. Peripheral (Atosiban, phenylephrine) and central (unfamiliar surroundings) inhibition of milk ejection reduced the amount of available milk similarity. Drug treatments resulted in similar peak flow rates; however, teats were contracted after phenylephrine administration but not after Atosiban. The inhibition induced by Atosiban could be abolished by oxytocin injection, but not induced by phenylephrine, which was antagonized by alpha-adrenergic receptor blockade. These results indicate that inhibition of milk ejection through activation of alpha-adrenergic receptors is based on blockade of milk flow into the cistern, but not through the teats.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle.

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 μg of LPS or 20 μg of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-α (TNFα), IL-1β, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFα. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFα in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFα, IL-1β, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of β- and α(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α(s1)- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous β-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 μg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows.

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cows immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.

Atosiban, an oxytocin receptor blocking agent: pharmacokinetics and inhibition of milk ejection in dairy cows

Plasma concentrations of the oxytocin receptor blocking agent Atosiban were measured at 2, 4, 10, 15 and 20 min after injection of 5, 10, 20 and 50 micrograms Atosiban/kg body weight in six dairy cows. The half life of Atosiban was 18 min and the total body clearance was 3301 ml/min. Intramammary pressure (IMP) within the teat cistern was measured in six cows before and after i.v. injection of 0 or 20 micrograms Atosiban/kg body weight and repeated injections of 0.2 or 0.5 i.u. oxytocin. IMP was also measured in eleven cows after injection of 0, 10 or 50 micrograms Atosiban/kg body weight: in seven during oxytocin infusions, in four after oxytocin injections in successively increasing dosages (0.05, 0.1, 0.2, 0.5, 1 and 10 i.u.). The occurrence of milk ejection was indicated by a rise in IMP. After injection of 20 micrograms Atosiban/kg body weight, 0.2 i.u. oxytocin did not induce an IMP rise before 48 min, whereas 0.5 i.u. oxytocin induced an IMP rise within 4 min. The time from the start of infusion until the beginning of the IMP rise and the duration of IMP rise during oxytocin infusions both increased, whereas the IMP rise itself was diminished by increasing Atosiban dosages. The amount of injected oxytocin necessary to induce an IMP response increased with increasing Atosiban dosages. Atosiban was shown to have a powerful effect in inhibiting milk ejection in dairy cows.

The Inflammatory Response of Primary Bovine Mammary Epithelial Cells to Staphylococcus aureus Strains Is Linked to the Bacterial Phenotype

Staphylococcus aureus is a major mastitis-causing pathogen in dairy cows. The latex agglutination-based Staphaurex test allows bovine S. aureus strains to be grouped into Staphaurex latex agglutination test (SLAT)-negative [SLAT(−)] and SLAT-positive [SLAT(+)] isolates. Virulence and resistance gene profiles within SLAT(−) isolates are highly similar, but differ largely from those of SLAT(+) isolates. Notably, specific genetic changes in important virulence factors were detected in SLAT(−) isolates. Based on the molecular data, it is assumed that SLAT(+) strains are more virulent than SLAT(−) strains. The objective of this study was to investigate if SLAT(−) and SLAT(+) strains can differentially induce an immune response with regard to their adhesive capacity to epithelial cells in the mammary gland and in turn, could play a role in the course of mastitis. Primary bovine mammary epithelial cells (bMEC) were challenged with suspensions of heat inactivated SLAT(+) (n = 3) and SLAT(−) (n = 3) strains isolated from clinical bovine mastitis cases. After 1, 6, and 24 h, cells were harvested and mRNA expression of inflammatory mediators (TNF-α, IL-1β, IL-8, RANTES, SAA, lactoferrin, GM-CSF, COX-2, and TLR-2) was evaluated by reverse transcription and quantitative PCR. Transcription (ΔΔCT) of most measured factors was induced in challenged bMEC for 6 and 24 h. Interestingly, relative mRNA levels were higher (P<0.05) in response to SLAT(+) compared to SLAT(−) strains. In addition, adhesion assays on bMEC also showed significant differences between SLAT(+) and SLAT(−) strains. The present study clearly shows that these two S. aureus strain types cause a differential immune response of bMEC and exhibit differences in their adhesion capacity in vitro. This could reflect differences in the severity of mastitis that the different strain types may induce.