concanavalin-a and Mastitis--Bovine

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cephalosporins; Concanavalin A; Cytokines; Drug Therapy, Combination; Electrophoresis, Gel, Two-Dimensional; Female; Lactoferrin; Mastitis, Bovine; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus

We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the "commensal" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4

Topics: Animals; Antibodies, Monoclonal; Cattle; Cattle Diseases; Cell Proliferation; Concanavalin A; Female; Interleukin-17; Leukocytes, Mononuclear; Mastitis, Bovine; Milk; Phytohemagglutinins; Staphylococcal Infections; Staphylococcus aureus

We examined the effective diagnostic indicator using the concanavalin A (Con A) low-affinity lactoferrin (Lf) to mastitic drying cows. The concentrations of both Lf and Con A low-affinity Lf in mammary gland secretions (MGSs) were lower than normal MGSs at the early and middle dry periods and colostrums. On the other hand, the levels of Con A low-affinity Lf in MGSs increased following the appearance of mastitis symptoms, and decreased when the mastitic symptoms were cured. Moreover, IgG1 concentrations of colostrums decrease on the quarters where a high level of Con A low-affinity Lf was determined after the onset of dry period. These results suggest that this method could be used as a useful indicator to mastitic drying cows.

Topics: Animals; Cattle; Colostrum; Concanavalin A; Female; Immunoglobulin G; Lactoferrin; Mastitis, Bovine

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4(+):CD8(+)T cell ratio and generation of an atypical CD8(+)T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4(+):CD8(+)T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8(+)T cells compared to CD4(+)T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2- biased microenvironment, and together with the inversion of the bovine CD4(+):CD8(+)T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.

Topics: Animals; Apoptosis; Cattle; CD4-CD8 Ratio; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Concanavalin A; Cytokines; Enterotoxins; Female; Lymphocyte Activation; Mastitis, Bovine; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections; Staphylococcus aureus

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

Topics: Amino Acid Sequence; Animals; Cattle; Concanavalin A; Epithelial Cells; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Molecular Sequence Data; Pancreatic Elastase; Staphylococcal Infections

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cattle; Cattle Diseases; Chromatography, Affinity; Concanavalin A; Cytokines; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Female; Immunoelectrophoresis, Two-Dimensional; Iron; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Molecular Sequence Data; Oxygen; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, Protein; Staphylococcus aureus; T-Lymphocytes; Tumor Necrosis Factor-alpha

To examine whether specific immunity in the mammary gland and blood of dairy cattle was enhanced after primary inoculation with a test antigen keyhole limpet hemocyanin (KLH) and recombinant bovine interferon-gamma (rBoIFN-gamma) as an adjuvant.. Holstein dairy cows received IM injections of KLH in conjunction with saline solution (n = 4), Freund's incomplete adjuvant (FIA; n = 4), or rBoIFN-gamma (n = 3). Milk and blood samples were collected during a 1-month period and analyzed for KLH antibody content. Isolated blood mononuclear cells were examined for their ability to proliferate and produce interleukin 2 (IL-2) after mitogen and/or KLH stimulation in vitro. The phenotype of isolated blood mononuclear cells also was characterized through flow cytometric analysis.. The adjuvant rBoIFN-gamma induced serum KLH antibody titers similar to those induced by administration of FIA. However, FIA induced significantly more KLH antibodies in milk. Administration of rBoIFN-gamma enhanced T-lymphocyte activity, as indicated by the greater expression of high-affinity IL-2 receptors and the increased response to the mitogens concanavalin A, phytohemagglutinin, and IL-2, compared with FIA or saline treatment. Lymphocyte and monocyte movement from the blood also was altered after rBoIFN-gamma treatment, which can have a profound influence on secondary immune responses, such as antibody production.. rBoIFN-gamma may safely enhance specific immunity in the bovine mammary gland and may be an effective adjuvant in mastitis immunization protocols.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Cattle; Cell Division; Concanavalin A; Female; Flow Cytometry; Freund's Adjuvant; Hemocyanins; Interferon-gamma; Interleukin-2; Lymphocyte Activation; Lymphocytes; Mammary Glands, Animal; Mastitis, Bovine; Mitogens; Phenotype; Random Allocation; Recombinant Proteins

Mitogenic responses of peripheral blood lymphocytes in naturally occurring clinical puerperal metritis and mastitis were investigated. Glucose consumption index (GCI) values for phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) in the puerperal metritic cows and mastitic cows were significantly lower than those in the healthy cows. Suppression of lymphocyte blastogenesis was correlated to an increased concentration of serum ammonia in the puerperal metritic cows, and of alpha 1-acid glycoprotein (alpha 1AG) in the mastitic cows. Lymphocyte blastogenesis in the mastitic cows was also correlated to the serum concentration of vitamin E. These findings indicate that the puerperal metritic and mastitic cows are associated with impaired lymphocyte blastogenesis.

Topics: Ammonia; Animals; Aspartate Aminotransferases; Cattle; Cattle Diseases; Cholesterol; Concanavalin A; Female; gamma-Glutamyltransferase; Glycolysis; Hydrocortisone; Ketone Bodies; Lymphocyte Activation; Lymphocytes; Mastitis, Bovine; Orosomucoid; Phytohemagglutinins; Pokeweed Mitogens; Puerperal Disorders; Reference Values; Uterine Diseases; Vitamin E

Mononuclear cells were isolated from bovine blood by density gradient centrifugation to determine variation in mitogen-induced mononuclear cell activity throughout the nonlactating period. In a preliminary study, optimum concentrations of Concanavalin A, phytohemagglutinin, Salmonella typhimurium lipopolysaccharide, and three Escherichia coli lipopolysaccharides were determined using six cows as blood donors. Concanavalin A, phytohemagglutinin, and E. coli 0111:B4 lipopolysaccharide were selected for further studies. Mitogenic responses of blood mononuclear cells from five cows were evaluated at drying off, 14 to 16 and 28 to 30 d of involution, 12 to 14 d prepartum, and at parturition. Concanavalin A-treated cells exhibited greater blastogenic activity than phytohemagglutinin-treated cells. Response of cells to Concanavalin A increased slightly through 28 to 30 d of involution and decreased markedly at parturition. Blastogenic activity of cells treated with phytohemagglutinin decreased throughout the nonlactating period and was lowest at parturition. Activity of lipopolysaccharide-treated mononuclear cells increased through 28 to 30 d of involution. However, response of mononuclear cells to lipopolysaccharide was minimal compared with response to Concanavalin A and phytohemagglutinin. Variation in peripheral blood mononuclear cell activity throughout involution may parallel mammary gland mononuclear cell activity, affecting susceptibility of the mammary gland to new intramammary infections.

Topics: Animals; Cattle; Concanavalin A; Escherichia coli; Female; In Vitro Techniques; Leukocytes, Mononuclear; Lipopolysaccharides; Mastitis, Bovine; Phytohemagglutinins; Salmonella typhimurium