MtuA,a lipoprotein receptor antigen from Streptococcus uberis,is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.     

Proteomics and pathway analyses of the milk fat globule in sheep naturally infected by Mycoplasma agalactiae provide indications of the in vivo response of the mammary epithelium to bacterial infection

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.     

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence

During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as “nutritional immunity.” This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell “hummingbird” phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle.     

Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes

A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25–0.5 mM) reduces ∼50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), β-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.     

The mammary gland as a target organ for infection with caprine arthritis-encephalitis virus

A herd of 15 dairy goats with a previous history of neurological disease in kids was screened for caprine arthritis-encephalitis (CAE) virus infection. Virus was isolated from the PBL of all animals as well as milk from lactating does. All animals had antibody detected by ELISA and gel immuno-diffusion. Two animals from this herd were examined for responses to infection. One was an adult female with poor milk production and the other was a 3-month-old female kid which developed acute neurological disease. Both animals had a chronic mastitis compatible with a viral aetiology, and CAE virus was isolated from the mammary tissue of both. The mammary gland must thus be considered an important target organ for virus infection and dissemination.     

Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Farrerol regulates antimicrobial peptide expression and reduces Staphylococcus aureus internalization into bovine mammary epithelial cells

Mastitis, defined as inflammation of the mammary gland, is an infectious disease with a major economic influence on dairy industry. Staphylococcus aureus is a common gram-positive pathogen that frequently causes subclinical, chronic infection of the mammary gland in dairy cows. Farrerol, a traditional Chinese medicine isolated from rhododendron, has been shown to have anti-bacterial activity. However, the effect of farrerol on S. aureus infection in mammary epithelium has not been studied in detail. The aim of this study was to investigate the effect of farrerol on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus. The expression of antimicrobial peptide genes by bMEC were assessed in the presence or absence of S. aureus infection. Our results demonstrated that farrerol (4–16 μg/ml) reduced > 55% the internalization of S. aureus into bMEC. We also found that farrerol was able to down-regulate the mRNA expression of tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin 5 (BNBD5) in bMEC infected with S. aureus. The Nitric oxide (NO) production of bMEC after S. aureus stimulation was decreased by farrerol treatment. Furthermore, farrerol treatment suppressed S. aureus-induced NF-κB activation in bMEC. These results demonstrated that farrerol modulated TAP and BNBD5 gene expression in mammary gland, enhances bMEC defense against S. aureus infection and could be useful in protection against bovine mastitis.     

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.     

Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection

An enterotoxin D (SED)-producing strain of Staphylococcus aureus was used to infect one mammary gland of each of 17 lactating dairy cows. All glands became infected and shed bacteria over a sampling period of 3 weeks. Serum and milk antibodies specific for SED were monitored by an enzyme-linked immunosorbent assay for 12 weeks. Elevated anti-SED antibodies were detected in all cows after infection, and immunoglobulin of the G2 subclass comprised most of the specific serum response. SED was detected in mastitic milk samples from two cows at levels of 5 to 10 ng/ml. An in vitro lymphocyte proliferation assay showed that SED at levels below 10 pg/ml induced proliferation of bovine lymphocytes and that sheep antiserum specific for SED neutralized this proliferative response. Sera obtained from the cows pre- and postinfection inhibited lymphocyte proliferation at SED concentrations of 10 and 50 ng/ml, respectively. The addition of SED to whole blood or to isolated neutrophils had no significant effect on neutrophil function in vitro. The results show that SED is secreted during mammary gland infection, is mitogenic for bovine lymphocytes, and stimulates the production of specific antibodies.     

Destruction and repair of mammary gland parenchyma of cows infected with foot-and-mouth disease

Bovine mammary gland secretory epithelial cells show signs of infection as early as three days after exposure of lactating host dairy cows to pigs infected with foot-and-mouth disease. Isolated foci of necrotic alveoli, as observed by scanning electron microscopy, increased in area and number with time. Infection of individual milk synthesizing cells progressed to involve the entire secretory epithelium. Alveolar luminal contents, in contrast to those in control preparations, consisted of heavy concentrations of cellular debris and connective tissue fragments; few leukocytes were observed. Involutionary changes occurred 10 days after cow exposure. Significant evidence of repair in necrotic areas was present at 15 days, but limited repair occurred earlier. Characteristics of replacement secretory epithelium made it possible to differentiate it from older mammary gland parenchyma.     

Muramyl Dipeptide Synergizes with Staphylococcus aureus Lipoteichoic Acid To Recruit Neutrophils in the Mammary Gland and To Stimulate Mammary Epithelial Cells

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.Staphylococcus aureus is a pathogen frequently isolated from mastitis milk or mammary abscesses in the lapine, ovine, bovine, and human species (4, 5, 23, 41, 53). The recognition of S. aureus by the mammary gland is not as well known as the recognition of Escherichia coli, another major pathogen for the mammary gland. The E. coli outer membrane lipopolysaccharide (LPS) has been shown to be the major pathogen-associated molecular pattern (MAMP) recognized by the mammary gland through interactions with Toll-like receptor 4 (TLR4), which is expressed by bovine mammary epithelial cells (MEpC) (bMEpC) (18, 24). LPS has been extensively used to mimic mastitis due to E. coli, and it was shown that the responses that it provokes are in many respects representative of those induced by live bacteria (7). The counterpart of LPS, as a proinflammatory bacterial agonist of the mammary gland innate immune system, has not yet been established for S. aureus. This pathogen releases various MAMPs during infection, and two of these have been the subject of many studies. Lipoteichoic acid (LTA) was shown to be an important pattern for immune recognition of S aureus (66). LTA signals through TLR2 (56), a receptor which is expressed in the mammary gland, in particular by bMEpC (18, 24). We have recently established the ability of LTA to elicit an intense inflammatory response in the bovine mammary gland (49). Another major staphylococcal MAMP is the minimal bioactive constituent of the bacterial peptidoglycan MurNac-l-Ala-d-iso-Gln (MDP). A major sensor of MDP is the nucleotide-binding oligomerization domain 2 (NOD2) protein, which is encoded by the caspase recruitment domain 15 gene (CARD15) (17). NOD2 is expressed mainly by two cell types that are exposed to microorganisms that produce peptidoglycan, i.e., antigen-presenting cells and epithelial cells (25, 63).During infection, host-pathogen interactions are very complex and difficult to unravel. The use of isolated MAMPs makes it possible to decipher the components of the innate immune response at play in the mammary gland. Information gained from in vitro studies of the responses of relevant cells to the same bacterial agonists would increase our understanding of udder-pathogen interactions. The in vitro data can be compared with the immune response to experimentally induced infections. Similarities and differences are expected to point to important bacterial agonists of the host response and to important facets of this response. In turn, the new knowledge acquired can be used to devise new approaches with a view to modulating the inflammatory and the immune responses in the mammary gland. As information on the immune response of the mammary gland and mammary epithelial cells to staphylococcal MAMPs is relatively scarce, we decided to use two major staphylococcal MAMPs to induce mastitis and to stimulate MEpC. Epithelial cells, which line the lumen of the mammary gland, are the first and most abundant cells to be in contact with invading bacteria and their secreted products. Bovine MEpC have been shown to react to MAMPs such as LPS and LTA (62, 72).We investigated the ability of MDP to trigger an inflammatory response in the bovine mammary gland. As it was reported previously that MDP and LTA exert synergistic effects on a variety of cells (30, 63), we sought to determine whether these NOD2 and TLR2 agonists could induce additive or synergistic effects in vivo in the mammary gland and in vitro on bMEpC. Our results indicate that MDP induces neutrophilic inflammation and synergizes with LTA in the mammary gland to recruit neutrophils. Moreover, MDP and LTA synergized to induce the secretion of neutrophil-oriented chemokines by bMEpC but did not induce the secretion of the major proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). These new findings support the hypothesis that MDP and LTA are likely to play an important part in the initiation of inflammation during S. aureus mastitis. They contribute to an improved understanding of the detection of S. aureus by the mammary gland.     

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

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 1β (IL-1β), 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.     

CCL28 involvement in mucosal tissues protection as a chemokine and as an antibacterial peptide

CCL28 chemokine is expressed by epithelial cells of various mucosal tissues. This chemokine binds to CCR3 and CCR10 receptors and plays an essential role in the IgA antibody secreting cells (IgA-ASC) homing to mucosal surfaces and to lactating mammary gland as well. In addition, CCL28 has been shown to exert a potent antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi. Using the pig model, we investigated the expression of both CCR10 and CCR3 receptors in a large panel of mucosal tissues. RT-PCR analysis revealed the expression of CCR3 and CCR10 mRNA in salivary glands, nasal mucosae, Peyer’s patches, small and large intestine, suggesting the presence of leucocytes expressing these receptors within these tissues. CCR10 mRNA was observed in sow mammary gland at late gestation with an increasing level during lactation. Recombinant porcine CCL28 protein was produced and mass spectrometry analysis revealed antimicrobial chemokines features such as a high pI value (10.2) and a C-terminal highly positively-charged region. Using a viable count assay, we showed that CCL28 displayed antimicrobial activity against enteric pathogens and was effective in killing Salmonella serotypes Dublin and Choleraesuis, enteroinvasive Escherichia coli K88 and non-pathogenic E. Coli K12. The potent antimicrobial function of CCL28 combined with its wide distribution in mucosal tissues and secretions suggest that this protein plays an important role in innate immune protection of the epithelial surfaces.     

Subversion of antimicrobial calprotectin (S100A8/S100A9 complex) in the cytoplasm of TR146 epithelial cells after invasion by Listeria monocytogenes

Expressed by squamous mucosal keratinocytes, calprotectin is a complex of two EF-hand calcium-binding proteins of the S100 subfamily (S100A8 and S100A9) with significant antimicrobial activity. Calprotectin-expressing cells resist invasion by Porphyromonas gingivalis, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium (S. typhimurium). To understand the interactions between calprotectin and invasive bacteria, we studied the distribution of calprotectin in the cytoplasm of TR146 epithelial cells. In response to L. monocytogenes, calprotectin mobilized from a diffuse cytoplasmic distribution to a filamentous pattern and colocalized with the microtubule network. Listeria more frequently invaded cells with mobilized calprotectin. Calprotectin mobilization was listeriolysin O-dependent and required calcium (extracellular and intracellular) and an intact microtubule network. In the presence of preformed microtubules in vitro, the anti-Listeria activity of calprotectin was abrogated. To facilitate intraepithelial survival, therefore, Listeria mobilizes calprotectin to colocalize with cytoplasmic microtubules, subverting anti-Listeria activity and autonomous cellular immunity.     

Sinus‐like dilatations of the mammary milk ducts,Ki67 expression,and CD3‐positive T lymphocyte infiltration,in the mammary gland of wild European rabbits during pregnancy and lactation

Sinus‐like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E‐cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14‐positive cells, supported by a thin layer of fibrous tissue. Multi‐segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3‐positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus‐like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus‐like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.     

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Metabolism of immunoglobulin A in lactating mice: origins of immunoglobulin A in milk

The metabolism of albumin and IgA was studied in normal and lactating mice. Lactation resulted in significant changes in the metabolism of these proteins. The serum albumin concentration was lowered from 47 mg/ml in normals to 24 mg/ml in lactating mice. However, only a slight decrease in the serum concentration of IgA was observed during lactation. The proportion of polymeric and monomeric IgA in serum and milk was evaluated by gel exclusion chromatography. The onset of lactation led to a rise in the proportion of polymeric IgA (P-IgA) in serum from 37% to 51%. The proportion of P-IgA in milk was 65% and remained constant throughout lactation. P-IgA and albumin were shown to be efficiently transferred from the serum of lactating mice into their milk. Serum decay studies were performed to evaluate the turnover of the serum pools during lactation. The rates of disappearance from the serum of isotopically labeled albumin and P-IgA were observed to increase dramatically during lactation, suggesting that both of these two milk proteins might be derived at least in part from the serum. The sites of synthesis of milk IgA (local vs. extra-mammary gland) were evaluated by determining the extent of dilution of isotopically labeled serum IgA during transport through the mammary gland into the milk. Early in lactation, the majority of the IgA in mouse milk appeared to be derived from distant sites and transferred via the blood to the mammary gland. However, by day 8 of lactation, the isotopically labeled P-IgA in milk was significantly.diluted by the IgA synthesized in the mammary gland. Albumin and IgG were not diluted by local synthesis indicating that these proteins were exclusively serum-derived.     

Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 microg of LPS, 0.3 microg of LPS plus 100 micro g of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 microg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.     

奶牛乳腺肥大细胞的粘膜免疫学特征

应用改良甲苯胺蓝染色法 (MTB )和阿尔辛蓝 番红鉴别染色法 (AB S )对静止期和活动期奶牛乳腺肥大细胞的粘膜免疫学性质进行了研究。MTB染色结果显示 ,静止期乳腺中的肥大细胞主要分布于各级输乳管上皮基膜区 ,小血管周围也有肥大细胞分布。在活动期 ,肥大细胞主要分布于腺泡上皮基膜附近 ,上皮内也有极少数肥大细胞分布 ,小叶间结缔组织中的肥大细胞则分布于小叶间导管或输乳管上皮基膜区。AB S染色证实 ,乳腺肥大细胞只被阿尔辛蓝染成蓝色 ,而不被结缔组织型肥大细胞的标志性染料番红着染。这表明 ,乳腺肥大细胞是一种典型的粘膜型肥大细胞 (MMC ) ,并参与构成乳腺粘膜免疫的第一道防线。