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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.

ABSTRACT. 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 Nterminal 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-γ, IL-4, IL-6, and IL-1α. However low Con A affinity Lf induced the production of TNFα, 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 TNFα, 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. KEY WORDS: concanavalin A, lactoferrin, mammary gland secretion, mastitis.

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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 Staphylococcus aureus is the major causative bacteria of bovine mastitis. S. aureus infections result in changes of T-cell subpopulations and proinflammatory cytokine and chemokine production (27,35). Moreover, lymphocytes and leukocytes stimulated with staphylococcal enterotoxins produce proinflammatory cytokines (36) and nitric oxide (17), which show inflammatory effects. In ruminants, many strains of S. aureus isolated from mammary gland (MG) secretions (MGSs) produce staphylococcal enterotoxin C (SEC) (16,20,33). It was reported that SEC induced clinical signs of bovine mastitis such as an increase in the number of leukocytes in the MG (16). SEC binds to the specific V␤ chain of the T-cell receptor via the major histocompatibility complex class II molecules of antigen-presenting cells such as macrophages and activates bovine T lymphocytes (5,18,36). Then, the activated T lymphocytes and macrophages produce proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, gamma interferon, and tumor necrosis factor alpha (TNF-␣) (35,36). Therefore, it is suggested that the superantigenic activity of SEC induces bovine mastitis (5, 36). Thus, it is indicated that inflammatory cytokines such as IL-1, IL-6, and TNF-␣ are produced by activated leukocytes and lymphocytes in the early stages of staphylococcal mastitis.Down-regulation of intracellular cytochrome P450 (CYP450) expression is caused by IL-1, 25), which are produced in response to certain toxins such as staphylococcal enterotoxin B (31), aflatoxin B1 (15), and Escherichia coli lipopolysaccharide (LPS) (19). These cytokines also induce the expression of the inducible isoform of nitric oxide (NO) synthetase (iNOS), which results in the production of NO in certain cells including hepatocytes, macrophages, endothelial cells, and leukocytes during the cellular response. Several studies have suggested that NO production is mediated by the down-regulation of CYP450 expression in cytokine and LPS models of inflammation, based on the ability of exogenously administered NO to down-regulate CYP450 gene expression and attenuate the down-regulation by NO inhibitors (13,14). Then, the production of NO is regulated by the downregulation of CYP450 and cause...