Ken-ichi Komine

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg‐EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf‐dependent, antibody‐independent and complement‐mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.

Induction of Nitric Oxide Production Mediated by Tumor Necrosis Factor Alpha on Staphylococcal Enterotoxin C-Stimulated Bovine Mammary Gland Cells

ABSTRACT Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-α in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-α in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Predominant subpopulations of T lymphocytes in the mammary gland secretions during lactation and intraepithelial T lymphocytes in the intestine of dairy cows.

Lymphocytes obtained from mammary gland secretions (MGS) during lactation or the dry period of dairy cows were simultaneously analyzed and compared to ileal intraepithelial lymphocytes (IEL) and peripheral blood lymphocytes (PBL) using monoclonal antibodies (mAb) specific for bovine leukocyte differentiation antigens. The T-lymphocytes of MGS during lactation and those in IEL were predominantly CD8(+), while T-cells in MGS during the dry period were predominantly CD4(+). In addition, the proportion of gamma delta T-cells in MGS during lactation and IEL was fairly high. A large percentage of CD8(+) cells and T-cells coexpressed the activation molecule, ACT2, yielding a high proportion of ACT2(+) CD8 T-cells and ACT2(+) gamma delta T-cells, in MGS during lactation and IEL. However, both types of cells were found at an extremely low level in MGS during the dry period and in PBL. Thus, the predominant T-cell populations in MGS during lactation are phenotypically similar to those in IEL in the intestine.

Inflammatory responses of bovine polymorphonuclear neutrophils induced by staphylococcal enterotoxin C via stimulation of mononuclear cells.

ABSTRACT To elucidate the pathological roles of staphylococcal enterotoxin C (SEC) in bovine staphylococcal mastitis, a histopathological analysis of SEC-inoculated mammary glands was performed. SEC-inoculated mammary glands exhibited interstitial inflammation, and the leukocytes that migrated into the gland were predominantly polymorphonuclear neutrophils (PMN). In the gland cistern tissues dissected from SEC-inoculated mammary glands, epithelial cellular degeneration was observed. We also investigated the physiological effects of SEC on PMN in vitro. PMN migration was induced by culture supernatant of SEC-stimulated peripheral blood mononuclear cells (S-PBMC sup) but not by that of nonstimulated PBMC (N-PBMC sup). The concentration of interleukin-8 was significantly (P < 0.05) higher in S-PBMC sup than N-PBMC sup, and a significantly (P < 0.05) higher mRNA expression of growth-regulated oncogenes was detected in SEC-stimulated PBMC than in nonstimulated PBMC. Milk PMN collected from SEC-inoculated mammary glands produced more than 2 times the amount of superoxide at 1 day postinoculation (dpi) than at 0 dpi in the presence of phorbol 12-myristate 13-acetate (PMA). PMN cultured with S-PBMC sup for 24 h also produced significantly (P < 0.05) larger amounts of superoxide than those cultured with N-PBMC sup in the presence of PMA. Moreover, S-PBMC sup induced the long-time survival of PMN. These results indicate that SEC induces the activation of PMN via the stimulation of mononuclear cells.

Differential gene expression of cytokine and cell surface molecules in T cell subpopulation derived from mammary gland secretion of cows

Problem:  As T cell subpopulations in the mammary gland secretion (MGS) of cows dynamically vary through the lactation cycle, their functional analysis is important to understand the mammary immune responses.

Effects of Sodium Dichloroisocyanurate, Sodium Hypochlorite and Formaldehyde on Activated Sludge

The effects of disinfectants on activated sludge were studied using pretreated swine feces as a waste water. The disinfectants used were sodium dichloroisocyanurate (SDIC), sodium hypochlorite (SHC) and formaldehyde (FA). The COD and transparency of the effluent, accumulation of MLSS and faunal characteristics of mircroorganisms (protozoa and metazoa) were investigated. The critical concentrations of SDIC, SHC and FA at which the effluent COD and transparency were deteriorated were 5, 1 and 25mg/l respectively, and those at which the accumulation of MLSS was reduced were 5, 5 and 100mg/l respectively. The concentrations of these disinfectants at which the microorganisms were adversely affected were 2.5, 1 and 10mg/l respectively. Carchesium, Trachelophyllum, Aspidisca, Vorticella and Aeolosoma were highly susceptible to these disinfectants. The results obtained indicated that FA is suited for the safest achievement of waste water treatment by the activated sludge process, and SDIC and SHC come next to FA in the order named. The results also suggested the necessity of controlling the concentrations of SDIC, SHC and FA not to exceed 5, 1 and 25mg/l/day respectively for preventing deterioration of the purification efficiency. Jpn. J. Zootech. Sci., 60 (9): 857-864, 1989