interleukin-8 and Mastitis--Bovine

The staphylococcal bi-component leukotoxins constitute a family included in the super-family of the beta-sheet-structured pore-forming toxins. They may be produced by Staphylococcus aureus and by Staphylococcus intermedius and their target cells vary according to the molecules. The mode of action proceeds by the sequential binding of the class S proteins, then by that of the class F proteins at the surface of the membranes. Then, the activation of cellular calcium-channels precedes the pore formation which seems to be sensitive to several monovalent cations. The cell response is inflammatory and includes the neosynthesis as well as the secretion of leukotriene B4, interleukin -8, histamine. The injection of leukotoxins to rabbits generates cell chemotaxis , vasodilatation, and tissue necrosis. The association of the production of leukotoxins with clinical syndromes concerns several aspects of the pathology of S. aureus, and confers to these leukotoxins an important role of virulence factors.

Topics: Animals; Bacterial Proteins; Bacterial Toxins; Calcium Channels; Cations, Divalent; Cattle; Cell Membrane Permeability; Chemotaxis, Leukocyte; Cross Infection; Erythrocytes; Exotoxins; Female; Hemolysin Proteins; Histamine Release; Humans; Interleukin-8; Ion Transport; Leukocidins; Leukotriene B4; Male; Mastitis, Bovine; Models, Biological; Necrosis; Neutrophils; Rabbits; Staphylococcal Infections; Staphylococcus aureus; T-Lymphocytes; Vasodilation; Virulence; Vitreous Body

The ELR(+) CXC chemokines are critical for protective neutrophil responses to most bacterial infections, but nevertheless can contribute importantly to the pathogenic effects of many inflammatory responses. We recently engineered a series of high affinity CXCL8/IL-8 antagonists, one of which, CXCL8((3-73))K11R/G31P, binds very strongly to neutrophils via the CXCR1 and CXCR2. Herein we show in competitive 125I-ligand binding assays that bovine CXCL8((3-73))K11R/G31P has an affinity for neutrophils that is 2-3 orders of magnitude higher than that of CXCL8/IL-8. Furthermore, when used at approximately 0.5 nM, CXCL8((3-73))K11R/G31P inhibited by 50% the chemotactic responses of neutrophils to 129 nM CXCL8/IL-8, but it also blocked chemotactic responses to the alternate ELR-CXC chemokines CXCL1/GRO alpha and CXCL5/ENA-78. Furthermore, CXCL8((3-73))K11R/G31P could inhibit by 93-97% the spectrum of neutrophil chemotactic activities present within wash fluids from clinical bacterial pneumonia or experimental endotoxin-induced mastitis lesions. Finally, intramuscular or subcutaneous application of CXCL8((3-73))K11R/G31P (75 micro g/kg) reduced by up to 97% neutrophil infiltration into intradermal endotoxin challenge sites in cattle, and prevented their circulating neutrophils from responding to CXCL8/IL-8 or ENA-78 in vitro. This data thus encourages further investigation of the potential impact of this novel antagonist on ELR-CXC chemokine-driven inflammatory disorders.

Topics: Animals; Cattle; Chemokines, CXC; Chemotactic Factors; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Endotoxins; Interleukin-8; Mastitis, Bovine; Neutrophils; Pasteurellosis, Pneumonic; Peptide Fragments; Skin Tests; Time Factors

This study aimed to determine the effect of lipopolysaccharide (LPS)-induced mastitis with or without nonsteroidal anti-inflammatory drug (NSAID) on dairy cows' clinical, physiological, and behavioral responses in the milking parlor and freestalls as well as the specificity (Sp) and sensitivity (Se) of behavioral responses in detecting cows with LPS-induced mastitis. Twenty-seven cows received an intramammary infusion of 25 µg of Escherichia coli LPS in 1 healthy quarter. Following LPS infusion, 14 cows received a placebo (LPS cows), and 13 cows received 3 mg/kg of body weight of ketoprofen i.m. (LPS+NSAID cows). Cow response to the challenge was monitored at regular intervals from 24 h before to 48 h postinfusion (hpi) through direct clinical observations, markers of inflammation in milk, and via point-in-time direct behavioral observations in the barn and at milking. In LPS cows, infusion induced a significant increase of plasma cortisol levels at 3 and 8 hpi, milk cortisol levels at 8 hpi, somatic cell counts from 8 to 48 hpi, IL-6 and IL-8 at 8 hpi, milk amyloid A (mAA) and haptoglobin at 8 and 24 hpi, rectal temperature at 8 hpi, and respiratory rate at 8 hpi. Their rumen motility rate decreased at 8 and 32 hpi. Compared with before the challenge, significantly more LPS cows stopped feeding/ruminating and pressed their tail between their legs at 3 and 5 hpi, increased feeding/ruminating at 24 hpi, and had the tendency to be less responsive, dropping their head, and dropping their ears at 5 hpi. At milking, compared with before challenge, significantly more LPS cows lifted their hooves at forestripping at 8 hpi. The 2 groups showed similar patterns of response for milk cortisol, somatic cell count, respiratory rate, mAA, haptoglobin, and IL-6, IL-1β, and IL-8. Compared with LPS cows, LPS+NSAID cows had significantly lower plasma cortisol levels at 3 hpi, their rectal temperature decreased at 8 hpi, their rumen motility rate increased at 8 and 32 hpi, and their heart rate increased at 32 hpi. Compared with LPS cows, a significantly larger proportion of LPS+NSAID cows were feeding/ruminating, a lower proportion had ears down at 5 hpi, and a larger proportion lied down at 24 hpi. At milking, whatever the phase of milking, for "hoof to belly," 9 out of 14 cows did not show this behavior before infusion (Sp = 64%) and 14/14 did not kick during pre-infusion milking (Sp = 100%). Regarding sensitivity, at maximum, 5 cows out of 14 (Se = 36%) displayed "hoof t

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Behavior Observation Techniques; Cattle; Cattle Diseases; Escherichia coli; Female; Haptoglobins; Hydrocortisone; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mastitis, Bovine; Milk; Pain; Serum Amyloid A Protein

This study investigated the mRNA of immune factors expressed by milk somatic cells from 72 healthy lactating Holstein cows on 1 farm. Milk samples were collected aseptically from the right front mammary gland before milking. The milk samples that had a negative reaction to the California mastitis test were used to analyze the mRNA of immune factors. Cows were divided into 2 groups based on the detection of bacteria in milk samples: positive group (. Cette étude a examiné l’ARNm des facteurs immunitaires exprimés par les cellules somatiques du lait de 72 vaches Holstein en lactation en bonne santé dans une ferme. Des échantillons de lait ont été prélevés aseptiquement du quartier avant droit de la glande mammaire avant la traite. Les échantillons de lait ayant eu une réaction négative au test de mammite de Californie ont été utilisés pour analyser l’ARNm des facteurs immunitaires. Les vaches ont été divisées en deux groupes sur la base de la détection de bactéries dans les échantillons de lait : groupe positif (

Topics: Animals; Arginase; Cattle; Cattle Diseases; Female; Immunologic Factors; Interleukin-8; Lactation; Ligands; Mammary Glands, Animal; Mastitis, Bovine; Milk; RNA, Messenger

Klebsiella pneumoniae (K. pneumoniae) is one of the main environmental pathogens causing bovine mastitis. The incidence of bovine mastitis caused by K. pneumoniae is increasing worldwide. Selenium is an essential trace element that has multiple physiological functions, such as antioxidant and anti-inflammatory activities. Therefore, this study aimed to verify whether selenomethionine (SeMet) could contribute to alleviating the inflammatory injury and oxidative damage induced by K. pneumoniae. Bovine mammary epithelial cells were cultured in vitro and pretreated with 4 μM SeMet before being infected with K. pneumoniae. Western blot analysis was used to detect the expression of the related proteins in the NF-κB and Nrf2 signaling pathways. The gene expression levels of IL-1β, IL-6, IL-8, TNF-α, Nrf2, Keap1, NQO-1 and HO-1 were detected using RT-qPCR. The levels of MDA, GSH-PX, SOD, CAT and T-AOC were detected by commercial assay kits. Flow cytometry was used to determine the level of intracellular ROS, and immunofluorescence was used to detect the nuclear localization of Nrf2 protein. Briefly, SeMet downregulated the phosphorylation levels of IκBα and p65 proteins and the gene expression levels of IL-1β, IL-6, IL-8 and TNF-α were also decreased. Moreover, the protein and gene expression levels of Nrf2, NQO-1 and HO-1 were upregulated, and the nuclear expression of Nrf2 protein was also promoted, which enhanced the activity of antioxidant enzymes. In conclusion, SeMet protected BMECs from inflammatory injury and oxidative stress induced by K. pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.

Topics: Animals; Antioxidants; Cattle; Female; Interleukin-6; Interleukin-8; Kelch-Like ECH-Associated Protein 1; Klebsiella pneumoniae; Mastitis, Bovine; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Selenomethionine; Signal Transduction; Tumor Necrosis Factor-alpha

Staphylococcus aureus (S. aureus) is a major mastitis-causing pathogen in dairy cows. Dairy cows with mastitis suffer from a decrease in milk yield and protein content. Chlorogenic acid (CGA) is a natural product with anti-inflammatory effects. In this study, we examined the function and mechanism of CGA with regard to its anti-inflammatory effects and evaluated its protective function in milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were cultured with and without infection by S. aureus and CGA, and extracellular inflammatory cytokines and amino acids in the medium and milk proteins were determined by ELISA. The function of IL-10RA in anti-inflammatory processes and of SF-1 in milk protein synthesis was assessed by gene silencing. The activity of mTORC1, NF-κB, and STAT5 was examined by western blot. S. aureus caused intracellular infection and upregulated TNF-α, IL-1β, IL-6, and IL-8, whereas uptake of amino acids and milk protein synthesis were suppressed. CGA mitigated the S. aureus-induced inflammatory response and milk protein synthesis in vitro and in vivo. CGA alleviated S. aureus-induced inhibition of mTORC1 and STAT5 and upregulated IL-10 and IL-10RA. In addition, SF-1 was predicted to be a transcription factor of the milk protein-encoding genes α-LA, β-LG, and CSN2. S. aureus downregulated SF-1 and CGA reversed the decline in milk protein synthesis due to SF-1 knockdown. Thus, CGA mitigates the inflammatory response that is induced by S. aureus and protects the uptake of amino acids and milk protein synthesis in BMECs.

Topics: Animals; Anti-Inflammatory Agents; Cattle; Chlorogenic Acid; Cytokines; Epithelial Cells; Female; Interleukin-10; Interleukin-6; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Mechanistic Target of Rapamycin Complex 1; Milk Proteins; Staphylococcal Infections; Staphylococcus aureus; STAT5 Transcription Factor; Tumor Necrosis Factor-alpha

Streptococcus agalactiae is one of the main pathogens associated with bovine mastitis. The invasion of S. agalactiae in bovine mammary epithelial cells (BMECs) has been implicated as a key event in the pathogenesis of mastitis. Matrine is known for its various pharmacological activities, such as immune response regulation and anti-inflammation. The primary aim of the research was to investigate the preventive effect of matrine on S. agalactiae-induced inflammation in BMECs along with underlying molecular mechanisms. Our data showed matrine at the concentrations of 50-100 μg/mL promoted BMECs proliferation without infection, and decreased cytotoxicity induced by S. agalactiae. Subsequently, BMECs were pre-treated with matrine (50, 75, or 100 μg/mL) for 24 h, followed by the infection with S. agalactiae for an additional 6 h. Pretreatment with matrine followed by S. agalactiae treatment decreased cell apoptosis of BMECs. Also, pretreatment of matrine to BMECs prevented the invasion of S. agalactiae. The mRNA abundances of IL-1β, IL-6, IL-8, and TNF-α were down-regulated in S. agalactiae-infected cells pretreated with matrine. In addition, the greater ratios of protein NF-κB p-p65/p65, p-IκBα/IκBα, p-38/38, and p-ERK/ERK induced by S. agalactiae were attenuated due to matrine treatment. Furthermore, pretreatment of BMECs with matrine impeded the degradation of TAK1 induced by S. agalactiae infection. These results suggest matrine could be a potential modulator in immune response of the mammary gland. In conclusion, matrine prevents cellular damage due to S. agalactiae infection by the modulation of NF-κB and MAPK signaling pathways and pro-inflammatory cytokine production.

Topics: Animals; Cattle; Epithelial Cells; Female; Interleukin-6; Interleukin-8; Mammary Glands, Animal; MAP Kinase Signaling System; Mastitis, Bovine; Matrines; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Streptococcus agalactiae; Tumor Necrosis Factor-alpha

An extended milking interval of 24 h (24-h milking interval (24h-MI)) constitutes the acute phase of cow adaptation to once-daily milking (ODM). A recent trial including 724 24h-MI challenges demonstrated that milk yield responses to this acute phase of ODM are highly variable (from+22% to -52% of milk yield when switching to the 24h-MI, mean=-25.3%) and that factors such as stage of lactation parity and milk yield level influenced cows' responses but did not account for all individual variability. Additional traits related to physiological, immune and behavioural adaptation were measured on a subset (96 observations) of this data set. This study aimed to determine (1) the relationship of these traits with cows' milk yield responses, (2) their ability - combined with previously identified traits - to help predict milk yield responses to 24h-MI (adaptive profiles). The 24h-MI challenge consisted of three successive periods: one control week of twice-daily milking (cTDM), one single day of 24h-MI and then 13 days of TDM (pTDM). Milk yield responses to the 24h-MI (corrected for effects of stage of lactation, parity, milk yield level and milk yield) were related to physiological traits measured during cTDM (milk flow rate, presence or absence of interleukin-8) and to their changes during the 24h-MI (absolute increase in milk flow rate and relative udder distension). Analysis of associations between milk yield responses, stage of lactation, parity, milk yield level, proteolysis, udder expansion and immune traits found three adaptive cow profile clusters. Cows in cluster 1 had a less compliant udder than cows in cluster 2, and they lost more milk during the 24h-MI than cluster-2 and cluster-3 cows. After resuming twice daily-milking (TDM), cluster-2 cows fully recovered the milk they had lost during the 24h-MI. On the opposite, cluster-3 cows did not recover the milk they lost, likely due to udder inflammation during cTDM, as suggested by elevated concentrations of interleukin-8 in their milk. These results combining new traits with stage of lactation, parity and milk yield level constitute a first step towards predicting individual cow responses to a 24h-MI.

Topics: Animals; Cattle; Dairying; Female; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Milk; Proteolysis; Time Factors

Mastitis caused by environmental pathogens such as Escherichia coli is highly problematic to the dairy industry because it incurs substantial cost and tends to be difficult to manage. An effective innate immune response by the host is key to controlling infection, but it should also limit collateral damage to the mammary gland. Between-animal differences in mastitis severity have been attributed to variability in the innate response. In the current study, we used primary dermal fibroblast as a model to rank animals based on composite expression of the toll-like receptor 4 gene (TLR4) and lipopolysaccharide (LPS)-induced IL-8 and IL-6 protein production. Animals ranked as high and low responders (HR and LR, respectively) were then infected with the P4 strain of E. coli to determine how difference in rank would affect response to mastitis. All animals developed an acute response to the infection with varying degrees in severity; however, HR animals had an elevated somatic cell count and fever response at 12 h post-infection and greater production of milk IL-8 at 24 h post-infection. The HR animals were also significantly more capable of limiting bacterial growth. No differences in post-infection milk production or concentrations of milk BSA were measured. The current study indicates that HR animals have an early upregulation in their innate response that is beneficial for bacterial clearance; however, they are equally susceptible to tissue damage caused by an exuberant response to the infection. The dermal fibroblast may be used in conjunction with other cell types to determine how the innate response is regulated to mitigate unnecessary injury to the mammary gland while still effectively clearing the pathogen.

Topics: Animals; Cattle; Cell Count; Dairying; Escherichia coli; Escherichia coli Infections; Female; Fibroblasts; Gene Expression Regulation; Immunity, Innate; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mastitis, Bovine; Milk; Toll-Like Receptor 4

Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are constituted by a single cell population. Our aim was to test whether an explant of heifer gland, where the tissue structure is maintained, might be a valid model to investigate the innate immune response to infection. The study was carried out on 2mm

Topics: Animals; Cattle; Cells, Cultured; Epithelial Cells; Female; Gene Expression Regulation; Immunity, Innate; Interleukin-1beta; Interleukin-6; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Staphylococcus aureus; Tissue Culture Techniques; Tumor Necrosis Factor-alpha

Interleukin 8 is a proinflammatory chemokine involved in neutrophil recruitment and activation in response to infection and also in the resolution of inflammation. Our previous studies identified a number of genetic polymorphisms in the bovine IL8 promoter region which segregate into two haplotypes, with balanced frequencies in the Holstein-Friesian (HF). We subsequently showed that these haplotypes confer divergent IL8 activity both in vitro in mammary epithelial cells and in vivo in response to LPS. In this study, we hypothesised that the balanced frequency of IL8 haplotype in HF could be explained by divergent selection pressures acting on this locus. To address this hypothesis, an association study was carried out aiming to identify a putative link between the IL8 haplotype and somatic cell score (SCS) in 5746 Holstein-Friesian dairy cows. In addition, the basal and inducible promoter activity of the two IL8 haplotypes was characterised in bovine endometrial epithelial (BEND) cells and in monocyte-derived macrophages. Results showed a significant association between IL8 haplotype 2 (IL8-h2) with increased SCS (P<0.05). Functional analysis showed that the same haplotype was a more potent inducer of IL8 expression in BEND cells in response to LPS and TNFα stimulation. In contrast, co-transfection of the BEND cells with a DNA construct encoding a bovine herpesvirus 4 antigen, induced significantly higher IL8 expression from IL8-h1. The present study sheds light on the molecular mechanisms underlying selection for SCS and provides evidence that the balanced frequencies of the two IL8 haplotypes in HF cattle may occur as a result of opposing directional selection pressures of both bacterial and viral infection.

Topics: Animals; Cattle; Endometrium; Female; Haplotypes; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Polymorphism, Genetic; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.

Topics: Animals; Asymptomatic Infections; Cattle; Cell Count; Female; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-8; Leukocyte Count; Mammary Glands, Animal; Mastitis, Bovine; Recombinant Proteins; Staphylococcal Infections

Rearing of indigenous Tharparkar (TP) cows (native of arid Thar deserts) under high humid conditions (>75 % humidity) has increased the incidence of mammary infections in them. A study was undertaken to see the number, activity, and expression of milk neutrophils isolated from healthy and mastitic cows. There was a significant (P < 0.05) influx in milk somatic cell counts (SCC) and neutrophils in sub-clinical and clinical mastitis cows. No change was observed in the phagocytic activity (PA) of milk neutrophils between healthy and sub-clinical mastitis (SCM) cows, but these activities decreased significantly (P < 0.05) in clinical cases. Chemotactic activity showed a significant difference between all the groups. Lactose varied significantly (P < 0.05) between healthy, sub-clinical, and clinical mastitis (CM) cows. Expression of chemokine receptor (CXCR1) was more in mastitis cows and also higher as compared to CXCR2. No change was observed in cluster of differentiation molecule (CD62L) among all the three groups of TP cows. Expression of interleukin (IL-8) and CD11b was low in healthy cows, increased significantly (P < 0.05) in both sub-clinical and mastitis cows. This study indicates that low producing TP cows are also prone to mammary infections when reared under semi-arid conditions.

Topics: Animal Husbandry; Animals; Cattle; Cell Count; Droughts; Female; Incidence; India; Interleukin-8; Lactose; Mastitis, Bovine; Milk; Neutrophils; Receptors, Interleukin-8A; Tropical Climate

The single nucleotide polymorphisms (SNPs) in the 5' upstream of bovine IL8 gene were investigated in 810 Chinese Holstein cows from 35 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The Real-time PCR and Western blot were used to detect the mRNA and protein levels of genotype Chinese Holstein dairy cows. The results showed that one SNP -105G>A was detected, designating three genotypes (GG, GA and AA) with respective frequencies of 0.38, 0.46, and 0.16. The significant association of the SNP -105G>A with somatic cell score (SCS) was identified. Genotype GG had a significantly lower SCS than genotype GA or AA (P < 0.01), and the relative mRNA expression and protein level of GG was found to be the highest. These results suggest that the genotype GG may be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein dairy cows.

Topics: Animals; Cattle; China; Female; Genetic Association Studies; Genetic Markers; Genotype; Interleukin-8; Mastitis, Bovine; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; RNA, Messenger

Staphylococcus aureus is a common cause of chronic mammary gland infections in dairy cattle. However, the inflammatory response and duration of infection following pathogen exposure is variable between individual animals. To investigate interanimal differences in immune response, dermal fibroblast cultures were established from skin biopsies collected from 50 early lactation Holstein cows. The fibroblasts ability to produce IL-8 in response to a 24-h treatment with a synthetic toll-like receptor 2/6 agonist (Pam2CSK4) was used to assign a response phenotype to the animals. Five high-responding and 5 low-responding animals were then selected for an intramammary challenge with S. aureus to evaluate differences in the inflammatory response, chronicity of infection, and development of antibodies to the pathogen. All animals exhibited clinical symptoms of mastitis at 24h postchallenge. Animals previously classified as high responders experienced a greater inflammatory response characterized by elevated levels of milk somatic cell count, IL-8, and BSA following the challenge compared with low responders. In addition, antibodies toward the challenge strain of S. aureus reached higher levels in whey from the challenged gland of high responders compared with low responders. Despite the antibody response, all 5 high responders were chronically infected for the 6-wk duration of the study, whereas 2 of the low responders cleared the infection, although 1 of these did become reinfected. The observed differences between animals classified as low and high responders based on their fibroblast responsiveness suggests that this cell type can be used to further examine the causes of interanimal variation in response to mammary infection.

Topics: Animals; Cattle; Cattle Diseases; Cell Count; Female; Fibroblasts; Interleukin-8; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 6

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

Topics: Animals; Antibiosis; Bacterial Adhesion; Cattle; Cell Line; Endocytosis; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Escherichia coli; Interleukin-8; Lactococcus lactis; Mastitis, Bovine; Probiotics; Staphylococcus aureus

Metabolic adaptations during negative energy and nutrient balance in dairy cows are thought to cause impaired immune function and hence increased risk of infectious diseases, including mastitis. Characteristic adaptations mostly occurring in early lactation are an elevation of plasma ketone bodies and free fatty acids (nonesterified fatty acids, NEFA) and diminished glucose concentration. The aim of this study was to investigate effects of elevated plasma β-hydroxybutyrate (BHBA) at simultaneously even or positive energy balance and thus normal plasma NEFA and glucose on factors related to the immune system in liver and mammary gland of dairy cows. In addition, we investigated the effect of elevated plasma BHBA and intramammary lipopolysaccharide (LPS) challenge on the mammary immune response. Thirteen dairy cows were infused either with BHBA (HyperB, n=5) to induce hyperketonemia (1.7 mmol/L) or with a 0.9% saline solution (NaCl, n=8) for 56 h. Two udder quarters were injected with 200 μg of LPS after 48 h of infusion. Rectal temperature (RT) and somatic cell counts (SCC) were measured before, at 48 h after the start of infusions, and hourly during the LPS challenge. The mRNA abundance of factors related to the immune system was measured in hepatic and mammary tissue biopsies 1 wk before and 48 h after the start of the infusion, and additionally in mammary tissue at 56 h of infusion (8h after LPS administration). At 48 h of infusion in HyperB, the mRNA abundance of serum amyloid A (SAA) in the mammary gland was increased and that of haptoglobin (Hp) tended to be increased. Rectal temperature, SCC, and mRNA abundance of candidate genes in the liver were not affected by the BHBA infusion until 48 h. During the following LPS challenge, RT and SCC increased in both groups. However, SCC increased less in HyperB than in NaCl. Quarters infused with LPS showed a more pronounced increase of mRNA abundance of IL-8 and IL-10 in HyperB than in NaCl. The results demonstrate that an increase of plasma BHBA upregulates acute phase proteins in the mammary gland. In response to intramammary LPS challenge, elevated BHBA diminishes the influx of leukocytes from blood into milk, perhaps by via modified cytokine synthesis. Results indicate that increased ketone body plasma concentrations may play a crucial role in the higher mastitis susceptibility in early lactation.

Topics: 3-Hydroxybutyric Acid; Acute-Phase Proteins; Animals; Cattle; Cell Count; Energy Metabolism; Fatty Acids, Nonesterified; Female; Interleukin-10; Interleukin-8; Ketone Bodies; Ketosis; Lactation; Lipopolysaccharides; Liver; Mammary Glands, Animal; Mastitis, Bovine; Milk; Serum Amyloid A Protein; Up-Regulation

To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows.. 5 lactating Red Holsteins.. Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 μg of lipopolysaccharide [LPS], 100 μg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1β in milk somatic cells were analyzed.. Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1β, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1β in milk somatic cells increased after LPS infusion and was unaffected by prednisolone.. Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1β during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Topics: Analysis of Variance; Animals; Cattle; Cell Count; DNA Primers; Female; Gene Expression Regulation; Interleukin-1beta; Interleukin-8; Lactation; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Milk; Prednisolone; Real-Time Polymerase Chain Reaction; Time Factors; Tumor Necrosis Factor-alpha

Streptococcus uberis is an important cause of intramammary infection in dairy cattle. Strains of Strep. uberis appear to differ in their ability to cause disease based on previous epidemiological studies. We explored the pathogenicity of 2 strains of Strep. uberis, where one strain represented a putatively host-adapted type based on its ability to cause persistent infection and to spread from cow to cow in a lactating herd. This type was part of a clonal complex that is commonly associated with bovine mastitis. The other strain, which was isolated from a transient infection in a single animal in the same herd and did not belong to any known clonal complex, was selected as putatively nonadapted type. Cows (6 per strain) were experimentally challenged in a single hind quarter and the adjacent hind quarter was used as mock challenged control quarter. Both strains showed an equal ability to grow in the milk of challenge animals in vitro. All cows that were challenged with the putatively host-adapted strain developed clinical signs of mastitis, including fever and milk yield depression as well as elevated somatic cell count due to influx of polymorphonuclear leucocytes and lymphocytes. The cytokine response followed a specific order, with an increase in IL-1β, IL-6, and IL-8 levels at the time of first SCC elevation, followed by an increase in IL-10, IL-12p40, and tumor necrosis factor-α levels approximately 6h later. In 4 of 6 animals, IL-17A was detected in milk between 57 and 168 h postchallenge. The increase in IL-17A levels coincided with inversion of the prechallenge CD4(+)-to-CD8(+) T lymphocyte ratio, which was observed from 96 h postchallenge. This was followed by normalization of the CD4(+)-to-CD8(+) ratio due to continued increase of the CD8(+) concentration up to 312 h postchallenge. Spontaneous resolution of infection was observed in 5 animals and coincided with a measurable IL-17A response in 4 animals, suggesting that IL-17 may be involved in the resolution of intramammary infection. With the exception of minor elevation of IL-8 levels, no clinical, cytological, or immunological response was detected in quarters challenged with the nonadapted strain. The observed strain-specific pathogenicity was consistent across animals, implying that it is determined by pathogen factors rather than host factors.

Topics: Animals; Cattle; Cell Count; Electrophoresis, Gel, Pulsed-Field; Female; Interleukin-17; Interleukin-1beta; Interleukin-6; Interleukin-8; Lymphocyte Count; Mastitis, Bovine; Milk; Streptococcal Infections; Streptococcus

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-inflammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cytokines; Epithelium; Female; Interleukin-1beta; Interleukin-6; Interleukin-8; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Recombinant Fusion Proteins; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Mastitis is a frequent disease and considerable problem for the global dairy industry. Identification of solutions leading to the development of new control strategies is therefore of high importance. In this study, we have integrated genomic data from genome-wide association mapping in cattle with transcriptomic data from microarray studies of several mastitis pathogens and host species in vitro and in vivo. To identify significant candidate pathways directly and indirectly involved in the immune response to mastitis, ingenuity pathway analysis (ipa) and database for annotation, visualization and integrated discovery bioinformatic (david) were applied. Several candidate pathways were found. Of great interest are IL-17 and IL-8 signalling pathways, responsible for the recruitment and migration of inflammatory cells into tissue during inflammation and infection. These results may emphasize further functional studies for identification of factors contributing to resistance to mastitis pathogens in cattle.

Topics: Animals; Cattle; Chromosome Mapping; Female; Genome-Wide Association Study; Interleukin-17; Interleukin-8; Macrophages; Mammary Glands, Animal; Mastitis, Bovine; Neutrophils; Oligonucleotide Array Sequence Analysis; Quantitative Trait Loci; Signal Transduction

Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.

Topics: Animals; Cattle; Cells, Cultured; Chemokine CCL5; Cytokines; Epithelial Cells; Female; Gene Expression Regulation, Bacterial; Interleukin-1beta; Interleukin-8; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; RNA, Messenger; Severity of Illness Index; Streptococcal Infections; Streptococcus; Tumor Necrosis Factor-alpha

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90 μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48 h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo.

Topics: Animals; Biomarkers; Cattle; Cells, Cultured; Epithelial Cells; Female; Gene Expression Regulation; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; RNA, Messenger; Trans Fatty Acids

Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection.. Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the S. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against S. aureus intramammary infection.. Intramammary infection of dairy cattle with S. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.

Topics: Animals; Area Under Curve; Cattle; Female; Haptoglobins; Interferon-gamma; Interleukin-8; Mastitis, Bovine; Milk; Proteomics; Staphylococcal Infections; Staphylococcus aureus; Transforming Growth Factor beta; Up-Regulation

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1β and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cow's response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.

Topics: Animals; Cattle; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Female; Fibroblasts; Inflammation; Interleukin-6; Interleukin-8; Lipopeptides; Lipopolysaccharides; Mastitis, Bovine; Phenotype; Skin

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.

Topics: Animals; Cattle; Cell Line; Chemokine CXCL6; Chemotactic Factors; Epithelial Cells; Escherichia coli; Female; Humans; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Neutrophils; Peptidoglycan; Recombinant Proteins; Teichoic Acids

Enhancement of the diseased mammary gland immunity and therapeutic potential of hydro-methanolic extract of Tinospora cordifolia (T. cordifolia; stem) in bovine subclinical mastitis was investigated. Somatic cell count (SCC), total bacterial count (TBC), phagocytic activity, and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase activity and Interleukin-8 (IL-8) level were evaluated after intramammary infusion of hydro-methanolic extract (stem) of T.cordifolia in diseased cows. The qualitative analysis of the extract revealed the presence of polysaccharide, phenol, alkaloid, and protein. Intramammary infusion of hydro-methanolic extract of T. cordifolia treatment initially enhanced the SCC; thereafter, significant reduction in cell count (P < 0.05) was observed on day 15 of the treatment period, however, reduction in TBC was observed from day 3 onwards. The phagocytic activity of milk polymorphonuclear cells enhanced in the diseased cows treated with the T. cordifolia extract. Similarly, the lysosomal enzyme content of the milk polymorphonuclear cells enhanced significantly (P < 0.05) in diseased cows treated with T. cordifolia. The IL-8 level in milk serum also increased significantly (P < 0.05) in diseased cows treated with the herb extract. The results suggest that the hydro-methanolic extract of T.cordifolia (stem) possesses antibacterial and immunomodulatory properties. In the present study, the biological activity of the Tinospora cordifolia extract at standardized dose against bovine subclinical mastitis is reported for the first time. Development of alternative therapy with medicinal plants is an option for livestock farmers who are not allowed to use the conventional allopathic drugs under certain farming system or cannot afford to use allopathic drugs.

Topics: Acid Phosphatase; Animals; Cattle; Cell Count; Female; Interleukin-8; Mastitis, Bovine; Milk; Muramidase; Neutrophils; Peroxidase; Phagocytosis; Phytotherapy; Plant Extracts; Plant Stems; Tinospora

We examined if and how mammary epithelial cells (MECs) calibrate and confine the intensity of an inflammatory response elicited by different concentrations of mastitis pathogens. Therefore we quantified in primary bovine MEC the effect of different E. coli pathogen concentrations upon the abundance of mRNA molecules encoding factors of immune defence. Induced synthesis of the mRNAs encoding tumor necrosis factor alpha and interleukin 8 both clearly correlated with the E. coli dose 1h after stimulation. Also the decay rate of those mRNAs reflected the pathogen load. The higher the concentration of E. coli, the faster and stronger was the up regulation and also the subsequent degradation of those particular mRNA species. Modulation of the mRNA concentration of tristetraprolin, a factor crucially involved in the mRNA degradation, followed the same pattern. In contrast, extent and kinetics of increasing the mRNA concentrations of serum amyloid A3 and lingual antimicrobial peptide were almost independent of the pathogen dose. We show that MEC perceive the information about the different pathogen concentrations and convert this signal into a calibrated synthesis of pro-inflammatory cytokines. We suggest that selective degradation of the mRNA molecules encoding those inflammatory cytokines contributes significantly to prevent an overshooting immune response in the udder.

Topics: Animals; beta-Defensins; Cattle; Epithelium; Escherichia coli; Female; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; RNA, Messenger; Tumor Necrosis Factor-alpha

The objective of the current observational study was to determine the potential associations between cow factors, clinical mastitis (CM) etiology, and concentrations of select acute phase proteins and cytokines in milk from affected quarters of cows with CM. Cows with CM (n=197) were grouped based on systemic disease severity, milk culture result, parity, days in milk (DIM), previous CM occurrence, and season of the year when CM occurred. Concentrations of lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), BSA, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-8, IL-10, IL-12, transforming growth factor (TGF)-alpha, and TGF-beta and activity of lactate dehydrogenase (LDH) were evaluated. Differences in the least squares means log(10) transformed concentrations of these proteins were compared using multiple linear regression mixed models. The milk concentrations of LBP, Hp, IL-1beta, IL-10, and IL-12, and activity of LDH in milk were higher in cows with moderate to severe versus mild systemic disease. The concentrations of Hp, BSA, IL-1beta, and IL-10 in milk were higher in cows with a gram-negative versus gram-positive milk culture result. Season of the year when CM occurred was associated with the concentration of all proteins evaluated except for IL-1beta and IL-12. Concentrations were higher in the winter versus summer except for Hp and TGF-beta, for which the opposite was true. Concentrations of LBP, IL-10, and IL-12, and LDH activity in milk were associated with DIM group. Except for LBP, these proteins were lower in cows with CM during the first 60 DIM versus those in mid or later lactation. Interferon-gamma, TNF-alpha, and IL-8 were undetectable in 67, 31, and 20% of samples, respectively. Detection of IFN-gamma and IL-8 was associated with season, and detection of TNF-alpha and IL-8 was associated with systemic disease severity. The current study provides the most comprehensive report of milk concentrations of innate immune response proteins in cows with naturally occurring CM and identifies factors that potentially influence those concentrations. Further investigation into the seasonal variation of cytokine production and its potential effect on the outcome of CM is warranted. Furthermore, the results of this study provide useful data for planning future studies examining the role of the innate immune response in CM.

Topics: Acute-Phase Proteins; Animals; Cattle; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-1beta; Interleukin-8; L-Lactate Dehydrogenase; Mastitis, Bovine; Milk; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Mastitis is one of the most costly diseases to the dairy farming industry. Conventional antibiotic therapy is often unsatisfactory for successful treatment of mastitis and alternative treatments are continually under investigation. We have previously demonstrated, in two separate field trials, that a probiotic culture, Lactococcus lactis DPC 3147, was comparable to antibiotic therapy to treat bovine mastitis. To understand the mode of action of this therapeutic, we looked at the detailed immune response of the host to delivery of this live strain directly into the mammary gland of six healthy dairy cows. All animals elicited signs of udder inflammation 7 h post infusion. At this time, clots were visible in the milk of all animals in the investigation. The most pronounced increase in immune gene expression was observed in Interleukin (IL)-1beta and IL-8, with highest expression corresponding to peaks in somatic cell count. Infusion with a live culture of a Lc. lactis leads to a rapid and considerable innate immune response.

Topics: Animals; Cattle; Cell Count; Female; Gene Expression; Interleukin-1beta; Interleukin-8; Lactococcus lactis; Mammary Glands, Animal; Mastitis, Bovine; Milk; Vaccination

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.

Topics: Animals; Cattle; CD36 Antigens; Cell Line; Cloning, Molecular; Female; Flow Cytometry; Humans; Interleukin-8; Lipopolysaccharides; Mastitis, Bovine; Nitric Oxide; Peptidoglycan; Receptors, Pattern Recognition; Reverse Transcriptase Polymerase Chain Reaction; RNA; Streptococcal Infections; Streptococcus agalactiae; Teichoic Acids; Toll-Like Receptor 2; Transfection

Mastitis is one of the most prevalent diseases in cattle and remains among the most costly diseases to the dairy industry. Various surveys have indicated a greater prevalence of and risk for mastitis in Holstein cows than in Jersey cows. The innate immune system comprises the immediate host defense mechanisms that respond to infection, and differences in the magnitude and rapidity of this response are known to influence susceptibility to and clearance of infectious pathogens. The reported differences in the prevalence of mastitis between Holstein and Jersey cows may suggest the occurrence of breed-dependent differences in the innate immune response to intramammary infection. The objective of the current study was to compare the acute phase and cytokine responses of Holstein and Jersey cows following intramammary infection by the bacterial pathogen Escherichia coli, a leading cause of clinical mastitis. All cows in the study were in similar stages of lactation, of the same parity, subjected to the same housing and management conditions, and experimentally infected on the same day with the same inoculum preparation. Before and after infection, the following innate immune parameters were monitored: bacterial clearance; febrile response; induction of the acute phase proteins serum amyloid A and lipopolysaccharide-binding protein; alterations in total and differential white blood cell counts; changes in milk somatic cell counts and mammary vascular permeability; and induction of the cytokines IFN-gamma, IL-1beta, IL-8, IL-12, and tumor necrosis factor-alpha. Overall innate immune responses were similar between the 2 breeds; however, temporal differences in the onset, cessation, and duration of several responses were detected. Despite these differences, intramammary clearance of E. coli was comparable between the breeds. Together, these data demonstrate a highly conserved innate immune response of Holstein and Jersey cows to E. coli intramammary infection.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Animals; Breeding; Carrier Proteins; Cattle; Cytokines; Disease Susceptibility; Escherichia coli; Escherichia coli Infections; Female; Immunity, Innate; Interferon-gamma; Interleukin-1; Interleukin-12; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Membrane Glycoproteins; Prevalence; Risk Factors; Time Factors

The effects of interleukin-8 (IL-8) on bovine mammary functions such as milk protein secretion and the blood-milk barrier during mammary involution were evaluated. Following the final milking, recombinant bovine (rb) IL-8 (5 or 25 microg) and a saline placebo were individually infused into the left- and right-front teat cisterns of 6 cows, respectively. Three cows without treatment at the final milking were also used as controls. Mammary secretions and blood were collected at -24, 0, 10, 24, 72, 168, 336, and 720 h after infusion. In the mammary glands infused with 25 microg of rbIL-8, the increases in somatic cell counts and in the concentrations of serum albumin, IgG1 and IgG2, and the decreases in the concentrations of alpha- and beta-casein and beta-lactoglobulin were greater than in the control glands. In the mammary glands infused with 5 microg of rbIL-8, compared to the glands infused with 25 microg of rbIL-8, these changes were moderate. These results indicate that rbIL-8 impairs the integrity of the blood-milk barrier and suppresses milk-specific protein secretions. In the cows infused with 25 microg of rbIL-8, the rectal temperature and serum haptoglobin level were transiently elevated after the infusion, showing that intramammary infusion of rbIL-8 could elicit systemic inflammation.

Topics: Acute-Phase Proteins; Animals; Cattle; Cell Count; Female; Haptoglobins; Infusions, Parenteral; Interleukin-8; Lactation; Mammary Glands, Animal; Mastitis, Bovine; Milk; Milk Proteins; Random Allocation; Recombinant Proteins; Time Factors

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G-->C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca++]i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca++]i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.

Topics: Animals; Calcium; Calcium Signaling; Cattle; Chemotaxis, Leukocyte; Dairying; Female; Genotype; Interleukin-8; Mastitis, Bovine; Neutrophils; Phenylurea Compounds; Receptors, Interleukin-8; Receptors, Interleukin-8A; Receptors, Interleukin-8B

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

Topics: Animals; Anti-Inflammatory Agents; Antibodies, Monoclonal; Base Sequence; Cattle; Dexamethasone; Electrophoretic Mobility Shift Assay; Epithelial Cells; Female; In Vitro Techniques; Infliximab; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Molecular Sequence Data; Mucous Membrane; NF-kappa B; Tumor Necrosis Factor-alpha

Mastitis, a mammary gland inflammation in response to bacterial infection, is a major problem in the dairy industry. We found that cows susceptible to mastitis have a three-base insertion in a glycine-coding stretch of the gene for forebrain embryonic zinc finger-like (FEZL), a transcription factor with a role in neuronal development. Mastitis induces FEZL expression in mammary glands, and induced FEZL promotes expression of the axon-attracting molecule semaphorin 5A (SEMA5A) through a GCAG sequence. FEZL also induces SEMA5A expression in susceptible cattle but at a lower level than in resistant cattle. Enhanced SEMA5A induces expression of at least nine genes related to the host's immune response, including TNF-alpha and IL-8. We propose that susceptibility to mastitis results from an impaired immune response due to the lower transcription activity of susceptible FEZL. Our results provide an avenue to select for genetic improvement of resistance to mastitis and suggest that the FEZL-SEMA5A pathway might control both neuronal development and innate immunity.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Base Sequence; Binding Sites; Cattle; Cell Line; DNA; Female; Gene Expression; Genetic Linkage; Haplotypes; Interleukin-8; Mastitis, Bovine; Molecular Sequence Data; Nerve Tissue Proteins; Prosencephalon; Receptors, Immunologic; Recombinant Proteins; Signal Transduction; Transcription Factors; Transfection; Tumor Necrosis Factor-alpha; Zinc Fingers

Summary The effect of intramammary injection of recombinant bovine interleukin-8 (rbIL-8, 1 mg/10 ml of saline) on quarter milk levels of somatic cell count (SCC), chemiluminescence (CL) activity and counts of total bacteria and Staphylococcus aureus (S. aureus) was investigated, using 10 Holstein cows with an early stage or a late stage of subclinical mastitis naturally infected with S. aureus. In the late-stage group, milk SCC and CL activity had significant rises with maximum levels at 6 h, following maintained high levels thereafter post-cytokine injection. The counts in milk total bacteria and S. aureus were insignificantly decreased, being increased back on day 7 post-cytokine injection. Thus, the cytokine was inefficient for the late-stage subclinical mastitis. However, in the early-stage group milk SCC and CL activity declined to under pre-injection levels on day 7 after marked and significant rises at 6 h and day 1 post-cytokine injection. The milk total bacterial count decreased significantly on days 0.25 and 2. Furthermore, the milk S. aureus count was decreased significantly on days 1, 2, 3 and 7 by the cytokine injection. These results suggest that the rbIL-8 has a potential as a therapeutic agent of the subclinical mastitis of dairy cows, if the cytokine is applied at an initial stage of infection.

Topics: Animals; Cattle; Cell Count; Colony Count, Microbial; Female; Injections; Interleukin-8; Luminescent Measurements; Mammary Glands, Animal; Mastitis, Bovine; Milk; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

Infection of the bovine mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Greater understanding of the initial host response to infection may lead to more accurate selection of resistant animals or to novel prophylactic or therapeutic intervention strategies. The epithelial cell plays a role in the host response by alerting the immune system to the infection and providing a signal as to where the infection is located. To understand this process better, a cDNA microarray approach was used to search for potential signals produced by mammary epithelial cells in response to exposure to Escherichia coli lipopolysaccharide (LPS). Total RNA from separate cultures of epithelial cells from 4 Holstein cows was harvested 6 h after LPS challenge or control conditions. For each cow, RNA from control or LPS-exposed cells was transcribed to cDNA and labeled with Cy3 or Cy5, then pooled and applied to a bovine total leukocyte (BOTL) microarray slide containing 1278 unique transcripts. Dye reversal was used so that RNA from two of the control cultures was labeled with Cy3 while RNA from the other two control cultures was labeled with Cy5. From the resulting microarray data we selected 4 of the 9 genes significantly (P < 0.02) induced (>1.25-fold) in response to LPS exposure for more detailed analysis. The array signal intensity for 3 of these genes, RANTES/CCL5, IL-6 and T-PA, was relatively low, but quantitative real-time RT-PCR (Q-RT-PCR) analysis revealed that they were induced 208-fold, 10-fold and 3-fold, respectively. The gene that showed the greatest fold induction by microarray analysis (2.5-fold) was CXCL5. This gene had a relatively strong signal intensity on the array and was easily detected by northern blot analysis, which indicated a 10-fold induction. This cell culture model system provides evidence for an important role of the mammary epithelial cell in initiating the innate response to infection.

Topics: Animals; Cattle; Cell Culture Techniques; Chemokine CXCL5; Chemokines, CXC; Epithelial Cells; Escherichia coli Infections; Female; Gene Expression Profiling; Gene Expression Regulation; Interleukin-8; Leukocytes; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections

Acute Escherichia coli mastitis is one of the major sources of economic loss in the dairy industry due to reduced milk production, treatment costs, discarded milk, and occasional fatal disease. Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as adjunctive therapy to antibiotics. The objective of the current study was to evaluate the effect of carprofen treatment following infusion of Escherichia coli into the mammary glands of primiparous cows during the periparturient period. Severity of mastitis was scored based on the average milk production in the uninfected quarters on d +2 postinoculation and a clinical severity score. Carprofen was administered intravenously at 9 h postchallenge, when clinical signs of mastitis appeared. In previous work, efficacy of NSAIDs was mainly evaluated using clinical symptoms. In the present study, the effect of carprofen on innate immune response was also assessed by quantification of inflammatory mediators. All primiparous cows reacted as moderate responders throughout the experimental period. Primiparous cows were intramammarily inoculated with 1 x 10(4) cfu of E. coli P4:O32 in 2 left quarters. Analysis of blood and milk parameters, including IL-8, complement component C5a, lipopolysaccharide-binding protein (LBP), soluble CD14, prostaglandin E2, and thromboxane B2 was performed from d 0 to d +6 relative to intramammary inoculation. Rectal temperature in carprofen-treated animals was lower than in control animals at 3 and 6 h posttreatment. Treatment also restored the decreased reticulorumen motility that occurs during E. coli mastitis to preinfection levels faster than in control animals. Carprofen treatment resulted in an earlier normalization of the clinical severity score. Eicosanoid (prostaglandin E2 and thromboxane B2) production in milk tended to be inhibited by carprofen. No significant differences in the kinetic patterns of somatic cell count, IL-8, complement component C5a, LBP, and soluble CD14 were observed. In conclusion, carprofen treatment improved general clinical condition by effective antipyrexia and restoration of reticulorumen motility but did not significantly inhibit eicosanoid production. Carprofen treatment did not result in a significant decrease of chemotactic inflammatory mediators, IL-8 and C5a, and early innate immune molecules, sCD14 and LBP. Therefore, major modulatory effects from NSAID administration were not observed in this mastitis model, although a larger study mig

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carbazoles; Cattle; Cell Count; Colony Count, Microbial; Complement C5a; Dinoprostone; Escherichia coli Infections; Female; Hematocrit; Interleukin-8; Lactation; Leukocyte Count; Lipopolysaccharide Receptors; Mastitis, Bovine; Milk; Parity; Pregnancy; Thromboxane B2

Bovine mastitis continues to be the most detrimental factor for profitable dairying. Recent research conducted within our laboratory has identified a genetic marker in the CXCR2 gene associated with mastitis susceptibility. The objective of the present study was to evaluate the migratory ability of neutrophils from cows with different CXCR2 +777 genotypes. Neutrophils isolated from peripheral blood of 30 Holstein cows were tested for in vitro migration and adhesion molecule expression. Cows with the CC or GC genotype at CXCR2 +777 showed significantly lower neutrophil migration to recombinant human interleukin-8 (rhIL-8) than cows with the GG genotype (P < 0.05). Cows with the CC genotype at CXCR2 +777 also showed decreased neutrophil migration to zymosan-activated serum compared to these same cows (P < 0.05). Decreased upregulation of CD18 expression was observed after stimulation with rhIL-8 in cows expressing the CXCR2 +777 CC genotype compared to cows expressing the GG genotype (P < 0.05). A similar trend was observed for CD11b (P < 0.10). However, no difference in CD62 downregulation was observed with respect to genotype. These results provide initial evidence for a phenotypic association between a single nucleotide polymorphism and neutrophil function in dairy cows, as well as potential insight into specific mechanisms affected in cows more susceptible to mastitis.

Topics: Animals; Cattle; CD11b Antigen; CD18 Antigens; Cell Adhesion Molecules; Cell Movement; Female; Genetic Predisposition to Disease; Genotype; Interleukin-8; Mastitis, Bovine; Neutrophils; Receptors, Interleukin-8B; Up-Regulation

To define the cytokine response of a cultured mammary gland epithelial cell line (ie, Mac-T) when incubated with Escherichia coli or its products.. Mac-T cells and E coli from cows with mastitis.. Mac-T cells were incubated with E coli or its products. The cytokine response of Mac-T cells to these treatments was quantified by measuring mRNA content of interleukin (IL)-1alpha, IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha by use of a quantitative reverse transcriptase-polymerase chain reaction assay. The amount of TNF-alpha secreted was also measured.. Treatment with E coli or its products resulted in significant increases in IL-1alpha, IL-8, and TNF-alpha mRNA content in Mac-T cells. This increase was reversible when culture filtrate was incubated with polymyxin B. The amount of IL-1beta mRNA in Mac-T cells increased only slightly over baseline after treatment with E coli or its products, but this increase was not diminished by incubation of E coli filtrate with polymyxin B.. Incubation of Mac-T cells with E coli or its products resulted in increased amounts of IL1alpha, IL-8, and TNF-alpha mRNA. Inhibition of this response by incubation of culture filtrate with polymyxin B suggested that lipopolysaccharide was the main bacterial product that stimulated the cytokine response. The small increase in IL-1beta content in Mac-T cells incubated with E coli or its products suggested that this cytokine had a smaller role in the Mac-T cell response to E coli.

Topics: Animals; Cattle; Cell Line; Culture Media; Cytokines; Dose-Response Relationship, Drug; Epithelial Cells; Escherichia coli; Female; Interleukin-1; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1beta (IL-1beta), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

Topics: Acute-Phase Proteins; Animals; Blood Cell Count; Body Temperature; Capillary Permeability; Carrier Proteins; Cattle; Cell Count; Cell Division; Complement C5a; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Female; Immunity, Innate; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-12; Interleukin-8; Lactation; Lipopolysaccharide Receptors; Mammary Glands, Animal; Mastitis, Bovine; Membrane Glycoproteins; Milk; Neutrophils; Serum Albumin, Bovine; Staphylococcus aureus; Tumor Necrosis Factor-alpha

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.

Topics: Acute-Phase Proteins; Animals; Carrier Proteins; Cattle; Female; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-8; Kinetics; Klebsiella Infections; Klebsiella pneumoniae; Lipopolysaccharide Receptors; Mastitis, Bovine; Membrane Glycoproteins; Milk; Solubility; Tumor Necrosis Factor-alpha

Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.

Topics: Animals; Apolipoproteins; Blotting, Northern; Cattle; Cell Culture Techniques; Cryopreservation; Epithelial Cells; Female; Interleukin-8; Lactoferrin; Lipopolysaccharides; Mastitis, Bovine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus aureus

The objective of the current study was to evaluate the dynamics of infection and the immunological response to varying numbers of Escherichia coli injected into the mammary glands of primiparous cows during the periparturient period. Primiparous cows have been shown to be more resistant to intramammary E. coli challenge, and an increase of the inoculum dose by 2 log10 units induced a more rapid clinical response and clearance of the organisms. Recognition of lipopolysaccharide (LPS) is a key event in the innate immunity response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). Primiparous cows were inoculated with 1 x 10(4) (Group A; n=8) or 1 x 10(6) (Group B; n=8) cfu E. coli P4:O32 in their 2 left quarters during the periparturient period. Clinical examination and analysis of blood and milk parameters, including IL-8, complement fragment 5a (C5a), LBP, and soluble CD14 (sCD14), were performed from d -4 to d +3 relative to infection. Primiparous cows in Group B initiated a more rapid clinical response following intramammary infection (IMI), resulting in typical clinical signs and changes in blood and milk parameters approximately 3 h earlier compared with primiparous cows in Group A. Based on average milk production in the noninfected quarters on d +2 postinoculation, all heifers reacted as moderate responders. Distinct differences in the kinetic patterns of rectal temperature, somatic cell count (SCC), IL-8, C5a, LBP, and sCD14 were observed between both groups during the early phase of inflammation. Both C5a and IL-8 increased before cellular influx into the infected glands, followed by increases in sCD14 and LBP. In conclusion, primiparous cows are able to clear an intramammary E. coli infection efficiently. Moreover, increasing the inoculum dose induces a more rapid inflammatory reaction, mainly because of early activation of the innate host immune response.

Topics: Animals; Cattle; Cell Count; Colony Count, Microbial; Complement C5a; Escherichia coli; Escherichia coli Infections; Female; Immunity, Innate; Interleukin-8; Lipopolysaccharide Receptors; Mastitis, Bovine; Milk; Parity; Random Allocation

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4h after infusion (p.i.). Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i. The level of IL-1beta increased at 4h p.i. in milk, but was not detected in lymph. Interferon-gamma was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.

Topics: Animals; Cattle; Cytokines; Endotoxins; Female; Interleukin-1; Interleukin-8; Leukocyte Count; Lymph; Mastitis, Bovine; Milk; Time Factors; Tumor Necrosis Factor-alpha

Bacterial mastitis is accompanied by a drastic increase in milk somatic cell count (SCC), with neutrophils being the predominant cell type found in the infected quarters. Accumulation and activation of neutrophils at the site of infection require local expression of many inflammatory genes encoding adhesion molecules, chemokines and cytokines. Most of the inflammatory genes contain binding sites for the nuclear factor kappaB (NF-kappaB) within their promoter and therefore partly depend on NF-kappaB for their expression. We thus hypothesized that an increase in NF-kappaB activity in the mammary gland could contribute to development of the neutrophilic inflammation that characterizes mastitis. In an attempt to verify this hypothesis, we first assessed milk cells from healthy and acute and chronic mastitis-affected cows for NF-kappaB activity using electrophoretic mobility shift assays. We next studied the relationships between the intensity of NF-kappaB activity in these cells and the degree of udder inflammation. Active NF-kappaB complexes were undetectable in milk cells from healthy cows, whereas high levels of NF-kappaB activity were always found in cells from cows with acute mastitis. In milk cells obtained from chronic mastitis-affected cows, NF-kappaB activity varied from low to high. Finally, the level of NF-kappaB activity measured in milk cells from chronic mastitis-affected cows was not correlated to SCC or to the proportion of neutrophils present in milk samples, but was highly correlated with the expression level of interleukin-8 and granulocyte/macrophage colony-stimulating factor, two NF-kappaB-dependent cytokines crucially involved in initiation and perpetuation of neutrophilic inflammation. These results suggest that NF-kappaB might play a role in mastitis pathogenesis.

Topics: Animals; Cattle; Cell Count; Electrophoretic Mobility Shift Assay; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immunoblotting; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Milk; Neutrophils; NF-kappa B

The purpose of this study was to determine whether soluble CD14 (sCD14) in milk was affected by stage of lactation, milk somatic cell count (SCC), presence of bacteria, or lipopolysaccharide (LPS)-induced inflammation. Milk samples from 100 lactating cows (396 functional quarters) were assayed for sCD14 in milk to determine effects of stage of lactation, SCC, and intramammary infection. The concentration of sCD14 was highest in transitional milk (0 to 4 d postpartum) and in milk with high SCC (> 750,000 cells/ml). Most of the infected quarters (> 80%) were infected by coagulase-negative staphylococci and yeast. No difference was found between noninfected and infected quarters. One quarter of six healthy lactating cows was challenged with 100 microg LPS in order to study the kinetics of sCD14 during an LPS-induced inflammation. Milk samples were collected at various intervals until 72 h after injection. Rectal temperature, milk tumor necrosis factor-alpha, and interleukin-8 increased immediately after challenge. The increase in sCD14 paralleled the increase in SCC, peaked at 12 h, and started to decline after 24 h. Serum leakage, as characterized by the level of bovine serum albumin in milk, peaked at 4 h and then gradually decreased. All parameters remained at basal levels in control quarters throughout the study. In vitro experiments indicated that neutrophils released sCD14 in response to LPS in a dose-dependent manner. The results indicate that the concentration of sCD14 was significantly increased in milk after LPS challenge. The increase was not likely due to serum leakage. Instead, infiltrated neutrophils might be the main source of increased sCD14 in milk during inflammation.

Topics: Animals; Cattle; Cell Count; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Female; Interleukin-8; Kinetics; Lactation; Lipopolysaccharide Receptors; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Milk; Neutrophils; Solubility; Tumor Necrosis Factor-alpha

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.

Topics: Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Female; Interleukin-1; Interleukin-8; Leukocyte Count; Mammary Glands, Animal; Mastitis, Bovine; Milk; Streptococcal Infections; Streptococcus; Tumor Necrosis Factor-alpha

Several species of gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and various species of Enterobacter, are common mastitis pathogens. All of these bacteria are characterized by the presence of endotoxin or lipopolysaccharide (LPS) in their outer membrane. The bovine mammary gland is highly sensitive to LPS, and LPS has been implicated, in part, in the pathogenesis of gram-negative mastitis. Recognition of LPS is a key event in the innate immune response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). The objective of the current study was to determine whether LBP levels increased in the blood and mammary gland following LPS challenge. The left and right quarters of five midlactating Holstein cows were challenged with either saline or LPS (100 microg), respectively, and milk and blood samples collected. Basal levels of plasma and milk LBP were 38 and 6 microg/ml, respectively. Plasma LBP levels increased as early as 8 h post-LPS challenge and reached maximal levels of 138 microg/ ml by 24 h. Analysis of whey samples derived from LPS-treated quarters revealed an increase in milk LBP by 12 h. Similar to plasma, maximal levels of milk LBP (34 microg/ml) were detected 24 h following the initial LPS challenge. Increments in milk LBP levels paralleled a rise in soluble CD14 (sCD14) levels and initial rises in the levels of these proteins were temporally coincident with maximal neutrophil recruitment to the inflamed gland. Because LBP and sCD14 are known to enhance LPS-induced host cell activation and to facilitate detoxification of LPS, these data are consistent with a role for these molecules in mediating mammary gland responses to LPS.

Topics: Acute-Phase Proteins; Animals; Carrier Proteins; Cattle; Escherichia coli; Female; Interleukin-8; Kinetics; Lactation; Leukocyte Count; Lipopolysaccharide Receptors; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Membrane Glycoproteins; Milk; Neutrophils

Standard therapies including administration of potent antibiotics, aggressive fluid resuscitation and metabolic support have not been successful in relieving symptoms and reducing mortality associated with acute coliform mastitis. It is important to understand the pathophysiological response of the mammary gland to coliform infections when designing preventive or therapeutic regimens for controlling coliform mastitis. Our laboratory has previously shown that macrophages and polymorphonuclear neutrophils in milk express CD14 on their cell surface. In this study, we found that soluble CD14 (sCD14) is present in milk whey as a 46kDa protein reacted with anti-ovine CD14 antibody. Additional functional studies found that: (1) under serum-free condition, complexes of LPS-recombinant bovine soluble CD14 (rbosCD14) induced activation of mammary ductal epithelial cells (as measured by changes in interleukin-8 (IL-8) mRNA level by competitive RT-PCR) at low concentrations of LPS after 6 or 24h incubation (1-1000ng/ml), whereas LPS alone did not induce activation of mammary ductal epithelial cells at the same concentrations, and (2) intramammary injection of low concentrations of LPS did not increase concentration of leukocytes in milk. In contrast, LPS-rbosCD14 complex containing the same concentration of LPS increased the concentration of leukocytes in the injected mammary gland at 12 and 24h post-injection. These results indicate that rbosCD14 sensitizes mammary epithelial cells to low concentrations of LPS in vitro and in vivo. Endogenous sCD14 in milk may be important in initiating host responses to Gram-negative bacterial infections.

Topics: Animals; Antibodies, Monoclonal; Cattle; Epithelial Cells; Escherichia coli Infections; Female; Immunization; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Milk; Recombinant Proteins; Sheep; Solubility; Transcription, Genetic

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.

Topics: Animals; Antibodies, Blocking; Cattle; Chemical Fractionation; Chemotaxis, Leukocyte; Female; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Milk Proteins; Neutrophils; Whey Proteins

To define causes of increased susceptibility to coliform mastitis after parturition.. 12 healthy Holstein cows assigned to 2 groups. Group-1 cows (n = 6) had calved between 6 and 10 days earlier. Group-2 cows (n = 6) were in midlactation.. Cows from each group were paired and challenge exposed with Escherichia coli in 1 mammary gland. Mastitis severity was determined by bacterial concentration in milk, pyrexia, and milk production. Measures of host defense were neutrophil chemotaxis, adhesion molecule expression, leukocyte recruitment, and cytokine production.. After challenge exposure, group-1 cows had more rapid E coli growth, higher peak bacterial concentration, and higher fever. Leukocyte recruitment was poor in 1 group-1 cow that had peracute mastitis. In contrast, leukocyte recruitment in 5 other group-1 cows began sooner than that in group-2 cows. In these group-1 cows, prechallenge-exposure milk somatic cell counts (SCC) were significantly lower than those in group-2 cows. Prechallenge-exposure SCC were correlated to stimulated CD18 expression (R2 = 0.79), and both measures correlated inversely with bacterial growth rate (R2 = -0.75). Values for tumor necrosis factor alpha, interleukin 1, and interleukin 8 in group-1 cows after challenge exposure were greater than or equal to those in group-2 cows.. Weak leukocyte recruitment to the mammary gland is associated with increased severity of coliform mastitis. Impaired production of cytokines measured is not a cause of increased susceptibility to coliform mastitis in early lactation.. Low milk SCC after calving may increase susceptibility to severe coliform mastitis.

Topics: Animals; Cattle; Cytokines; Escherichia coli Infections; Female; Interleukin-1; Interleukin-8; Labor, Obstetric; Lactation; Mastitis, Bovine; Milk; Neutrophils; Pregnancy; Tumor Necrosis Factor-alpha