lactoferrin and Staphylococcal-Infections

Severe infections represent the main cause of neonatal mortality and morbidity. Strategies of proven effectiveness in reducing the incidence of infection in neonatal intensive care units (NICUs) include hand hygiene practices and prevention of central venous catheter-related bloodstream infections. In recent years, new strategies have been developed to prevent infections in NICU including prevention of neonatal sepsis with lactoferrin, the use of heparin for the prevention of CRBSIs, the judicious use of antibiotics and chemoprophylaxis, prevention of invasive fungal infections with fluconazole, the use of specific anti-staphylococcal immunoglobulins, and the early identification of infants at higher risk of infection with the use of specific markers (mannose-binding lectin). This review will focus on these new strategies and on their role in clinical practice in order to further reduce the incidence of infection in NICU.

Topics: Antibiotic Prophylaxis; Antifungal Agents; Cross Infection; Heparin; Humans; Immunotherapy; Infant, Newborn; Infant, Newborn, Diseases; Infection Control; Intensive Care Units, Neonatal; Lactoferrin; Staphylococcal Infections; Staphylococcal Vaccines

Lactoferrin, an iron-binding glycoprotein, is a cell-secreted mediator that bridges innate and adaptive immune function in mammals. It is a pleiotropic molecule that directly assists in the influence of presenting cells for the development of T-helper cell polarization. The aim of this review is to provide an overview of research regarding the role of lactoferrin in maintaining immune homeostasis, in particular as a mediator of immune responses to infectious assault, trauma and injury. These findings are critically relevant in the development of both prophylactic and therapeutic interventions in humans. Understanding these particular effects of lactoferrin will provide a logical framework for determining its role in health and disease.

Topics: Animals; Humans; Immunity, Cellular; Immunity, Innate; Immunologic Factors; Lactoferrin; Oxidative Stress; Receptors, Cell Surface; Staphylococcal Infections; Systemic Inflammatory Response Syndrome

The widespread use of antibiotics has lead to the increased presence of pathogens that are less susceptible to their antibacterial effect. Lactoferrin (Lf) is naturally produced by the mammary gland. Lactoferrin is the main whey protein in human milk and is also present in cow's milk but at a much lower concentration than in human milk. This protein appears to have many biological functions, including antibacterial and antiinflammatory activities. The best-known effect of Lf is to bind iron that is essential for bacterial growth. However, the cationic nature of this protein also appears to be important for the antimicrobial activity of this protein. Lactoferrin has a weak antibacterial effect when used alone, but interestingly, Lf appears much more effective when used at low concentration in combination with several antibiotics. The most striking observation is that Lf increases the inhibitory activity of penicillin up to 4-fold in most penicillin-susceptible Staphylococcus aureus strains, whereas this increase was 4- to 16-fold in penicillin-resistant strains. Indeed, Lf reduces beta-lactamase activity in S. aureus strains producing this enzyme. Transcription of beta-lactamase gene is dramatically repressed in the presence of Lf. We evaluated the efficacy of intramammary treatments containing penicillin G or bovine Lf (bLf), or both, to cure chronic mastitis caused by a clinical isolate of S. aureus highly resistant to beta-lactam antibiotics. In a first trial, mastitis was induced in lactating cows by injecting a low dose of S. aureus through the teat canal of all quarters. Bacterial cure rate was null for control quarters, 11.1% for bLf, 9.1% for penicillin, and 45.5% for the combination of bLf and penicillin. A second trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. Quarters were treated with 100,000 IU of penicillin G with or without 250 mg of bLf for 7 d. Bacterial cure rate was greater for the bLf + penicillin combination (33.3%) compared with penicillin alone (12.5%). In conclusion, bLf added to penicillin is an effective combination for the treatment of stable S. aureus infections resistant to beta-lactam antibiotics.

Topics: Animals; Anti-Bacterial Agents; beta-Lactam Resistance; Cattle; Drug Synergism; Enterobacteriaceae; Female; Immune System; Lactoferrin; Mastitis, Bovine; Penicillin G; Staphylococcal Infections; Staphylococcus

This paper discusses the nature of host resistance factors in human milk and epidemiologic studies regarding infections and mortality rates in breastfed and nonbreastfed babies. The defense factors and their proposed modes of action are: 1) a growth enhancer of lactobacilli, which interferes with intestinal colonization of enteric pathogens; 2) antistaphylococcal factors, which inhibit staphylococci; 3) secretory IgA and other immunoglobulins, which protect the gut and respiratory tract; 4) C4 and C3 (complement components; C3 fragments have opsonic, chemotactic, and anaphylatoxic activities); 5) lysozome, lysis of bacterial cell wall; 6) lactoperoxidase, killing of streptococci; 7) lactoferrin, kills microorganism by chelating iron, and 8) macrophages and lymphocytes, phagocytosis and cell-mediated immunity. Although it can be postulated that the breastfed infant's resistance to infection would be superior on account of the greater presence of these factors in human milk compared to cow's milk, little is known about the effects of these defense factors on the infant. Epidemiologic studies have reported on the lower morbidity and mortality rates of breastfed infants as compared to bottlefed infants. Other studies have focused on the protective effects of human milk upon the infant, but these have been inconclusive. In countries with poor sanitation and high infection rates, the incidence of bacterial infections is lowest in breastfed infants. The advantages of human milk however are difficult to demonstrate in societies with high standards of sanitation and low infection rates. Infection and mortality rates in infants have in fact declined in developed countries as the practice of breastfeeding declined. Until it is established that immunity to common pathogens is transmitted to the infant by human milk, it will not be known whether human milk does have protective effects.

Topics: Animals; Antibodies; Breast Feeding; Colostrum; Complement System Proteins; Escherichia coli; Fatty Acids; Growth Substances; Humans; Immunoglobulins; Infant; Infant, Newborn; Infections; Lactobacillus; Lactoferrin; Leukocytes; Mice; Milk, Human; Muramidase; Peroxidases; Staphylococcal Infections

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

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cattle Diseases; Complement C3; Female; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Staphylococcal Infections; Staphylococcus; Time Factors

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of β-lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin's method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ β-lactam antibiotic dose combinations and observed that at low doses (< 0.39 μM), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 μM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the β-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ β-lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from β-lactam-induced production of β-lactamase, which when sufficiently large reduces the population's ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using β-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of β-lactams.

Topics: Anti-Bacterial Agents; beta-Lactamases; beta-Lactams; Humans; Iron; Lactoferrin; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus aureus

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG

Topics: Animals; Cattle; Cell Count; Female; Genotype; Immunity; Immunoglobulin G; Interleukin-6; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Nitric Oxide; Saline Solution; Staphylococcal Infections; Staphylococcus aureus

The aim of this study was to characterize the effects of chronic S. aureus intramammary infection (IMI) on local innate and adaptive immune response during active involution. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. The levels of interleukin (IL)-1β, IL-6 and IL-4 were significantly higher in mammary secretions of S. aureus-infected quarters compared with uninfected at d 7, 14 and 21 of involution. Lactoferrin (Lf), total IgG and S. aureus specific IgG

Topics: Adaptive Immunity; Animals; Bodily Secretions; Cattle; Cytokines; Female; Immunoglobulin G; Immunologic Factors; Interleukin-1alpha; Interleukin-4; Interleukin-6; Lactation; Lactoferrin; Macrophages; Mammary Glands, Animal; Reactive Oxygen Species; Staphylococcal Infections; Staphylococcus aureus; Time Factors

Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against

Topics: Anti-Infective Agents; Escherichia coli; Escherichia coli Infections; Humans; Lactoferrin; Microbial Sensitivity Tests; Peptides; Staphylococcal Infections; Staphylococcus aureus

The objective of this study was to compare the dynamics of innate immune components after intramammary infusion of Staphylococcus aureus (SA) under conditions of high oestrogen and high progesterone in goats. In one group ("E-group"), controlled internal drug release (CIDR) devices were inserted intravaginally from days -11 to -4. Prostaglandin F2α was administered immediately after removal of the CIDR device at day -3, and then oestradiol benzoate (E) was injected intramuscularly once a day from days -2 to 3. Heat-inactivated SA was then administered via intramammary infusion to the left udder at day 0, whilst only saline was infused to the right udder as a control. In a second group ("P-group"), CIDR devices were inserted intravaginally from days -3 to 7 and SA was infused at day 0 in the same way as in the E-group. The milk yield and the concentration of innate immune components (somatic cell count (SCC), lactoferrin (LF), S100A7 and goat ß-defensin 1 (GBD-1)) in the milk were measured. Milk yield decreased drastically in both SA and control udders in the E-group, whereas the P-group exhibited increased milk yield in both SA and control udders. SCC increased after SA infusion in both E- and P-groups, although it was higher in the E-group than in the P-group. There was no significant change in LF concentration in the E-group, but a decrease was observed in the P-group. Concentrations of S100A and GBD-1 were significantly increased after SA infusion in the E-group but not in the P-group. These results suggest that E enhances the innate immune response induced by SA in the goat mammary gland. This effect may be due to the reduction in milk yield and upregulation of innate immune components.

Topics: Animals; beta-Defensins; Cell Count; Dinoprost; Estradiol; Female; Goat Diseases; Goats; Immunity, Innate; Lactation; Lactoferrin; Mammary Glands, Animal; Mastitis; Milk; Staphylococcal Infections; Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infection remains a serious hazard to global health. The use of immune modulatory therapy to combat infection is gaining an interest as a novel treatment alternative. Lactoferrin (LF), an iron binding protein with immune modulating properties, has the potential to modify the course of systemic MRSA infection. Specifically, LF is capable of limiting deleterious inflammatory responses while promoting the development of antigen specific T-cell activity. The efficacy of a novel recombinant mouse LF (rmLF) to protect against MRSA infection was examined in a mouse peritonitis model. BALB/c mice were infected with a lethal dose of MRSA and treated at 2h post-infection with rmLF. Effects of rmLF on MRSA-infected primary monocytes and granulocytes were analyzed for inflammatory mediators. The rmLF treated mice demonstrated a modest increase in survival of more than 24h, albeit with reduced bacteremia. Serum cytokines, IL-17 and IL-6, were significantly reduced post-challenge post-rmLF treatment. The rmLF led to a minor decrease in IL-1b, and a slight increase in TNF-a production. Preliminary investigation towards human clinical relevance was accomplished using human blood derived monocytes and granulocytes infected with MRSA and treated with homologous recombinant human LF (rhLF). Treatment with (rhLF) led to increased production of IFN-g and IL-2. The human cell studies also showed a concurrent decrease in TNF-a, IL-6, IL-1b, IL-12p40, and IL-10. These results indicate that the rmLF and rhLF have a high degree of overlap to modify inflammatory responses, although differences in activities were observed between the two heterologous recombinant molecules.

Topics: Animals; Bacterial Load; CHO Cells; Cricetulus; Cytokines; Granulocytes; Humans; Immunologic Factors; Lactoferrin; Methicillin-Resistant Staphylococcus aureus; Mice, Inbred BALB C; Monocytes; Peritonitis; Recombinant Proteins; Spleen; Staphylococcal Infections

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goat β-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn(125) but not Asn(232) is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn(232) to Gln(232) substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn(125) to Gln(125) substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn(125) of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.

Topics: Animals; Caseins; Cell Line; Cloning, Molecular; Colony Count, Microbial; Female; Glycosylation; Goat Diseases; Goats; Immunohistochemistry; Lactoferrin; Lysostaphin; Mastitis; Mutagenesis, Site-Directed; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus

The occurrence of multidrug-resistant or meticillin-resistant Staphylococcus aureus (MRSA) has become an important issue in clinics. This study evaluated a combinatorial treatment approach by using the well-documented antibacterial protein apo-bovine lactoferrin (apo-bLf) or its hydrolysate and specific probiotic supernatants for controlling MRSA infection. Clinical MRSA strains were isolated from different patient specimens. Apo-bLf-hydrolysate possessed stronger anti-MRSA activity than complete bLf in that it inhibited the growth of most MRSA strains tested in vitro. Otherwise, the supernatants produced by Lactobacillus fermentum (ATCC 11739), Bifidobacterium longum subsp. longum (ATCC 15707) and Bifidobacterium animalis subsp. lactis (BCRC 17394) inhibited the growth of various MRSA strains. Further, L. fermentum or B. animalis subsp. lactis supernatant plus apo-bLf or bLf-hydrolysate led to partially synergistic to synergistic growth-inhibitory activity against MRSA strains. However, L. fermentum and not B. animalis subsp. lactis or B. longum subsp. longum was observed to resist the antibacterial activity of both apo-Lf and bLf-hydrolysate. Therefore, it is suggested that L. fermentum could be the best candidate to be used with apo-bLf or bLf-hydrolysate as a live supplement against MRSA infections.

Topics: Animals; Anti-Bacterial Agents; Bifidobacterium; Cattle; Humans; Lactoferrin; Limosilactobacillus fermentum; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Probiotics; Protein Hydrolysates; Staphylococcal Infections

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

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Topics: Alternative Splicing; Animals; Cattle; Exons; Female; Gene Expression; Lactoferrin; Liver; Mammary Glands, Animal; Mastitis, Bovine; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Staphylococcal Infections; Staphylococcus aureus

The objectives of this study were to determine whether a synergistic effect could be obtained in vitro between bovine lactoferricin (B-LFcin) and antibiotics against Pseudomonas aeruginosa and Staphylococcus aureus isolates from ocular infections, and to evaluate the use of B-LFcin as an adjunct to the antibiotic treatment of corneal infection in vivo.. Chequerboard and time-kill assays were performed to investigate the combined effects of B-LFcin and conventional antibiotics, including ciprofloxacin, ceftazidime and gentamicin, against 17 strains of P. aeruginosa (8) and S. aureus (9) isolated from ocular infection and inflammation, and 1 reference strain of S. aureus. Corneas of C57BL/6 mice were topically challenged with a multidrug-resistant strain of P. aeruginosa. Nine hours post-challenge, mice were treated topically and hourly with either vehicle, B-LFcin, ciprofloxacin or ciprofloxacin containing B-LFcin for 8 h. Corneas were then clinically examined, and bacterial numbers and levels of myeloperoxidase (MPO) evaluated.. Synergy between B-LFcin and ciprofloxacin or ceftazidime was identified in most P. aeruginosa isolates, including multidrug-resistant strains, whereas no synergistic effect was seen between B-LFcin and gentamicin. Synergy was only observed with B-LFcin and ciprofloxacin against 2/10 S. aureus strains, and there was no synergy between B-LFcin and any of the other antibiotics tested. Combined B-LFcin and ciprofloxacin treatment significantly improved the clinical outcome, and reduced bacterial numbers and MPO in infected mouse corneas. B-LFcin alone was also able to reduce levels of MPO in infected corneas.. These findings indicate that B-LFcin may have advantages as an adjunct therapy with both antimicrobial and anti-inflammatory properties in the treatment of corneal infection.

Topics: Administration, Topical; Animals; Anti-Bacterial Agents; Cattle; Corneal Diseases; Drug Synergism; Lactoferrin; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Microbial Viability; Pseudomonas aeruginosa; Pseudomonas Infections; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

Six transgenic cows producing recombinant human lactoferrin (rhLf) in their milk and five normal cows at the same lactation stage were experimentally infected with Staphylococcus chromogenes to study the effect of a high concentration of lactoferrin in milk. Coagulase-negative staphylococci such as S. chromogenes have become very common as agents causing mild or subclinical mastitis. All transgenic cows became infected but showed no clinical signs, unlike the control cows, which developed mild clinical mastitis. Transgenic cows eliminated bacteria faster from the quarters than did the controls. Local clinical signs were milder, and the inflammatory reaction assessed by NAGase activity in the milk and by the concentration of milk amyloid A was lower in the transgenic cows. The mild response probably reflected the rapid elimination of bacteria. The milk concentration of rhLf remained constant throughout the study period, but the total concentration of bovine lactoferrin in the milk peaked in both groups at 46h post-challenge. Three cows, all in the control group, exhibited systemic acute phase response as increased concentrations of serum amyloid A in the blood circulation. Transgenic cows with a high concentration of human lactoferrin in their milk seemed to be protected from clinical disease and from prolonged inflammatory reaction, but not from experimental intramammary infection induced by S. chromogenes.

Topics: Acetylglucosaminidase; Animals; Animals, Genetically Modified; Cattle; Cell Count; Female; Humans; Lactation; Lactoferrin; Linear Models; Mastitis, Bovine; Milk; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus

Bacteria embedded in biofilms resist both antibiotics and host defense mechanisms. However, biofilms are not inherently protected against the attack of phagocytic cells. The weapons that polymorphonuclear neutrophils employ against implant infection are phagocytosis, degranulation, with release of antimicrobial molecules, and formation of Neutrophil Extracellular Traps (NETs). NETs contain DNA, histones, and neutrophil elastase, which enable neutrophils to fulfill their role of limiting both microbial spread and the collateral damage from granular contents. It is not yet clear whether the DNA released by neutrophils would support biofilm formation by adding to bacterial extracellular DNA (eDNA), an integral part of the biofilm extracellular matrix. In spite of the evidence of somewhat effective phagocytosis around an implant infection, biofilm-embedded staphylococci persist, tissue destruction occurs and, in the case of orthopedic implant infection, osteolysis prevails. The mechanism for tissue destruction is based on the infiltration and persistence at the site of infection of neutrophils which are unable to effectively perform phagocytosis, but able to inflict tissue damage and cause osteolysis by the release of proteolytic and collagenolytic enzymes. Phagocytosis thus has an ambiguous and ambivalent role: it carries out an antibacterial strategy and at the same time is responsible for osteolysis.

Topics: Biofilms; Cell Degranulation; DNA, Bacterial; Humans; Immunity, Innate; Lactoferrin; Leukocyte Elastase; Neutrophils; Phagocytosis; Prosthesis-Related Infections; Staphylococcal Infections; Staphylococcus aureus

To determine the antimicrobial activity of nisin F against Staphylococcus aureus in the respiratory tract.. The respiratory tract of nonimmunosuppressed and immunosuppressed Wistar rats were colonized with 4 x 10(5) viable cells of S. aureus K and then treated by administering 8192 arbitrary units (AU) nisin F intranasal. Symptoms of pneumonia were detected in the trachea and lungs of immunosuppressed rats that had not been treated with nisin F. The trachea and lungs of immunosuppressed rats treated with nisin F were healthy. No significant differences were recorded in blood cell indices. The antimicrobial activity of low concentrations nisin F (80-320 AU ml(-1)) was slightly stimulated by lysozyme and lactoferrin.. Nisin F inhibited the growth of S. aureus K in the respiratory tract of immunocompromised rats. Treatment with nisin F at 8192 AU proofed safe, as the trachea, lungs, bronchi and haematology of the rats appeared normal.. Nisin F is nontoxic and may be used to control respiratory tract infections caused by S. aureus. This is, however, a preliminary study with an animal model and need to be confirmed with studies on humans.

Topics: Administration, Intranasal; Animals; Bronchi; Drug Interactions; Humans; Immunocompromised Host; Lactoferrin; Lung; Male; Muramidase; Nisin; Pneumonia, Bacterial; Rats; Rats, Wistar; Respiratory Tract Infections; Staphylococcal Infections; Staphylococcus aureus; Trachea

Nosocomial infection with antibiotic-resistant strains is a major threat to critical care medicine. Selective decontamination of the digestive tract (SDD) is one of the strategies used to reduce ventilator-associated pneumonia and sepsis in critically ill patients. In the present study, we performed pathogenic challenges of the digestive tract in a transgenic milk-fed animal model to test whether porcine lactoferrin (pLF) is an effective SDD regimen.. Transgenic mice expressing recombinant pLF in their milk at a mean+/-SD concentration of 120+/-13.6 mg/L during the lactation stage fed normal CD-1 mice pups for 4 weeks. The pups were subsequently challenged with pathogenic Escherichia coli, Staphylococcus aureus, and Candida albicans.. Compared with the control groups fed wild-type (normal) milk, the groups fed pLF-enriched milk demonstrated statistically significant improvements in weight gain; lower bacterial numbers in intestinal fluid, blood, and liver; healthier microvilli in the small intestine; and alveoli in the lungs.. Our results showed that oral administration of pLF-enriched milk to mice led to broad-spectrum antimicrobial activity in the digestive tract and protected the mucosa of the small intestine from injury, implying that pLF can be used as an effective SDD regimen.

Topics: Animals; Animals, Newborn; Bacteremia; Bacterial Infections; Body Weight; Candidiasis; Cytokines; Escherichia coli Infections; Gastrointestinal Tract; Immunohistochemistry; Intestines; Lactation; Lactoferrin; Lung; Mice; Mice, Transgenic; Milk; Polymerase Chain Reaction; Recombinant Proteins; Staphylococcal Infections; Swine

The study aimed to describe the patterns and density of early tracheal colonization among intubated patients and to correlate colonization status with levels of antimicrobial peptides and inflammatory cytokines.. The was a prospective cohort study.. The study was conducted in medical and cardiovascular intensive care units of a tertiary referral hospital.. Seventy-four adult patients admitted between March 2003 and May 2006 were recruited for the study.. Tracheal aspirates were collected daily for the first 4 days of intubation using standardized, sterile technique and sent for quantitative culture and cytokines, lactoferrin and lysozyme measurements.. The mean acute physiology and chronic health evaluation (APACHE II) score in this cohort was 24 +/- 7. Proportion of subjects colonized by any microorganism increased over the first 4 days of intubation (47%, 60%, 70%, 70%, P = .08), but density of colonization for bacteria or yeast did not change significantly. No known risk factors predicted tracheal colonization on day 1 of intubation. Several patterns of colonization were observed (persistent, transient, new colonization, and clearance of initial colonization).The most common organisms cultured were Candida albicans and coagulase-negative Staphylococcus. Levels of cytokines, lactoferrin, or lysozyme did not change over time and were not correlated with tracheal colonization status. Four subjects (6%) had ventilator-associated pneumonia.. The density of tracheal colonization did not change significantly over the first 4 days of intubation in medical intensive care unit patients. There was no correlation between tracheal colonization and the levels of antimicrobial peptides or cytokines. Several different patterns of colonization may have to be considered while planning interventions to reduce airway colonization.

Topics: Adult; APACHE; Candidiasis; Case-Control Studies; Colony Count, Microbial; Cross Infection; Cytokines; Female; Humans; Inflammation; Intensive Care Units; Intubation, Intratracheal; Lactoferrin; Logistic Models; Male; Middle Aged; Multivariate Analysis; Muramidase; Pneumonia, Ventilator-Associated; Prospective Studies; Respiration, Artificial; Respiratory Mucosa; Risk Factors; Staphylococcal Infections; Statistics, Nonparametric; Suction; Time Factors; Trachea

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

Topics: Animals; Anti-Bacterial Agents; Apoproteins; Cattle; Cell Proliferation; Enterotoxins; Female; Flow Cytometry; HLA-DR4 Antigen; Humans; Interleukin-2; Jurkat Cells; Lactoferrin; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Serum Albumin; Staphylococcal Infections; Superantigens; T-Lymphocytes; Transferrin

Neonatal sepsis causes significant mortality and morbidity. Coagulase-negative staphylococci (CoNS) and Candida frequently cause neonatal sepsis at >72 h of age. Lactoferrin, which is present in human milk, is a component of innate immunity and has broad-spectrum antimicrobial activity. The synergistic effects of lactoferrin with antibiotics against neonatal isolates have not been systematically evaluated. Here, eight clinical strains (seven neonatal) of CoNS and three strains (two neonatal) of Candida albicans were studied. MIC50 and MIC90 values of human recombinant lactoferrin (talactoferrin; TLF), vancomycin (VAN) and nafcillin (NAF) against CoNS, and of TLF, amphotericin B (AMB) and fluconazole (FLC) against C. albicans, were evaluated according to established guidelines. Antimicrobial combinations of TLF with NAF or VAN against CoNS, and TLF with AMB or FLC against C. albicans, were evaluated by a checkerboard method with serial twofold dilutions. Synergy was evaluated by the median effects principle, and combination indices and dose reduction indices were reported at 50, 75 and 90% inhibitory effect at several drug-dose ratios. It was found that TLF acted synergistically with NAF and VAN against CoNS, and with AMB and FLC against C. albicans, at multiple dose effects and drug-dose ratios with few exceptions. In synergistic combinations, drug reduction indices indicated a significant reduction in doses of antibiotics, which may be clinically relevant. Thus TLF acts synergistically with anti-staphylococcal and anti-Candida agents commonly used in neonatal practice and is a promising agent that needs to be evaluated in clinical studies.

Topics: Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Candidiasis; Coagulase; Drug Synergism; Humans; Infant, Newborn; Lactoferrin; Microbial Sensitivity Tests; Recombinant Proteins; Sepsis; Staphylococcal Infections; Staphylococcus

Both lactoferrin (LF) and bacteriophages are potent antibacterial agents. LF is contained in the secretory fluids of mammals and bacteriophages are specific bacterial viruses.. The aim of this investigation was to determine whether combined treatment of infected mice may allow lowering the therapeutic dose of specific bacteriophages for Escherichia coli and Staphylococcus aureus.. CBA mice were infected intravenously (i.v.) with sublethal doses of E. coli or S. aureus and the specific T4 or A5 bacteriophages, respectively, were administered intraperitoneally (i.p.) or per os one hour following infection. The numbers of colony-forming units (CFUs) were determined in the livers after 24 hours.. Comparative administration of bacteriophages i.p. or per os showed that both routes of administration were equally efficacious in the protective action of bacteriophages. The bacteriophages were still very potent in reducing CFU numbers in the liver at a dose of 10(5)/mouse. Application of bovine lactoferrin (LF), 10 mg i.v., 24 h before infection, was also very effective in reducing CFU numbers. Using suboptimal (10(3)-10(4)) doses of bacteriophages and administration of LF, a more potent protective effect in reducing the CFU numbers in the infected mice was demonstrated. The combined effect of LF and bacteriophages in reducing CFU numbers was significantly higher than the effects of either agent alone. The study demonstrated that the combined application of LF and bacteriophages can significantly lower (1000 times) the effective dose of bacteriophages in reducing CFU numbers in infected mice.

Topics: Administration, Oral; Animals; Bacteriophage T4; Cattle; Colony Count, Microbial; Dose-Response Relationship, Drug; Escherichia coli; Escherichia coli Infections; Female; Injections, Intraperitoneal; Injections, Intravenous; Lactoferrin; Liver; Male; Mice; Mice, Inbred CBA; Staphylococcal Infections; Staphylococcus; Staphylococcus Phages

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.

Topics: Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Species Specificity; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus

The synthetic antimicrobial peptide representative of the first 11 N-terminal amino acids of human lactoferrin (hLF 1-11) kills multidrug-resistant Staphylococcus aureus (MRSA). This study displays antimicrobial activity of hLF 1-11, via various routes of administration, against MRSA infections in mice. Radiolabeling hLF 1-11 with technetium-99m ((99m)Tc-hLF 1-11) enables scintigraphic monitoring directly after administration. (99m)Tc-hLF 1-11 was taken up by the gall bladder, intestines, and kidneys. Most of the radioactivity was captured in the urinary bladder and about 1% of the injected dose accumulated into infected thigh muscles. At 2 or 24h after either intravenously, subcutaneously, intraperitoneally, or orally injected a single dose of 0.04 mg/kg hLF 1-11 in mice significantly reduced (20-60 times) the number of viable MRSA. In a dose-response setting in immunocompetent mice maximum bactericidal effects (10,000 times reduction) of intravenously injected (99m)Tc-hLF 1-11 was seen with 40 mg/kg whereas the same dose of orally administered (99m)Tc-hLF 1-11 induced about approximately 100 times reduction. In conclusion, intravenously and orally administrated (99m)Tc-hLF 1-11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues.

Topics: Alanine; Amino Acid Sequence; Amino Acid Substitution; Animals; Antimicrobial Cationic Peptides; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Resistance, Multiple, Bacterial; Humans; Lactoferrin; Mice; Molecular Sequence Data; Peptide Fragments; Quality Control; Radionuclide Imaging; Radiopharmaceuticals; Staphylococcal Infections; Staphylococcus aureus; Technetium; Tissue Distribution

Pathogens invading the mammary gland cause a complex signaling network that activates the early immune defense and leads to an outcome of inflammation symptoms. To examine the importance of mammary epithelial cells in these regulations and interactions resulting in a pathogen-related course of mastitis, we characterized the mRNA expression profile of key molecules of the innate immune system by quantitative real-time PCR. Mammary gland epithelial cells isolated on d 42 of lactation from 28 first-lactation Holstein dairy cows were cultured separately under standardized conditions and treated for 1, 6, and 24 h with heat-inactivated gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Both pathogens increased mRNA expression patterns of proteins involved in pathogen recognition such as Toll-like receptors and nuclear factor-kappa B, whereas gram-negatives acted as a stronger stimulus. Furthermore, this could be confirmed by the expression profile of the proinflammatory cytokines tumor necrosis factor alpha, IL-1 beta, IL-6, and chemokines such as IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted). Remarkably, at a low level of mRNA expression after 1 h of treatment these cytokines and chemokines were expressed at a significantly higher level in Staphyloccocus aureus than in Escherichia coli affected cells. Lactoferrin showed a deviating expression pattern to pathogen stimulation (i.e., at the 1-h measuring point Escherichia coli induced a higher mRNA expression, whereas the highest level was reached after 24 h of stimulation with Staphylococcus aureus). Complement factor 3 was the only measured factor that responded equally to both microorganisms. Our data emphasize the role of mammary epithelial cells in the immune defense of the udder and confirm their contribution to pathogen-related different courses of mastitis.

Topics: Animals; Cattle; Cytokines; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to beta-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to beta-lactam antibiotics.

Topics: Animals; Anti-Bacterial Agents; beta-Lactam Resistance; Cattle; Cell Count; Drug Synergism; Drug Therapy, Combination; Female; Lactoferrin; Mastitis, Bovine; Milk; Penicillins; Random Allocation; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

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

The therapeutic efficacy of an antimicrobial peptide, human lactoferrin 1-11 (hLF1-11), was investigated in a model of chronic methicillin-resistant Staphylococcus aureus (MRSA) (gentamicin susceptible) osteomyelitis in rabbits. We incorporated 50 mg hLF1-11/g or 50 mg gentamicin/g cement powder into a calcium phosphate bone cement (Ca-P) and injected it into the debrided tibial cavity, creating a local drug delivery system. The efficacy of hLF1-11 and gentamicin was compared to that of a sham-treated control (plain bone cement) (n=6) and no treatment (infected only) (n=5). The results were evaluated by microbiology, radiology, and histology. MRSA was recovered from all tibias in both control groups (n=11). On the other hand, hLF1-11 and gentamicin significantly reduced the bacterial load. Furthermore, no growth of bacteria was detected in five out of eight and six out of eight specimens of the hLF1-11- and gentamicin-treated groups, respectively. These results were confirmed by a significant reduction of the histological disease severity score by hLF1-11 and gentamicin compared to both control groups. The hLF1-11-treated group also had a significantly lower radiological score compared to the gentamicin-treated group. This study demonstrates the efficacy of hLF1-11 incorporated into Ca-P bone cement as a possible therapeutic strategy for the treatment of osteomyelitis, showing efficacy comparable to that of gentamicin. Therefore, the results of this study warrant further preclinical investigations into the possibilities of using hLF1-11 for the treatment of osteomyelitis.

Topics: Animals; Anti-Bacterial Agents; Bone Cements; Calcium Phosphates; Chronic Disease; Disease Models, Animal; Drug Carriers; Female; Gentamicins; Humans; Lactoferrin; Methicillin Resistance; Osteomyelitis; Peptide Fragments; Rabbits; Radiography; Staphylococcal Infections; Staphylococcus aureus; Tibia; Treatment Outcome

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human beta-defensin-1 and human beta-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (> or =80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human beta-defensins production and reduce susceptibility of the airway to bacterial infection.

Topics: Anti-Bacterial Agents; beta-Defensins; Cell Survival; Cells, Cultured; Erythromycin; Gene Expression; Humans; Lactoferrin; Methicillin Resistance; Muramidase; Pseudomonas Infections; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium; Staphylococcal Infections; Trachea

Based on our earlier observation that (99m)Tc-UBI 29-41, a radiolabeled peptide derived from ubiquicidin (UBI), discriminates between infections and sterile inflammatory processes, we considered the possibility that this tracer could be used for monitoring the efficacy of antibacterial agents in animals infected with Staphylococcus aureus.. We injected (99m)Tc-UBI 29-41 into S. aureus-infected mice after treatment with various doses of cloxacillin or erythromycin. At intervals thereafter, accumulation of the radiolabeled peptide at the site of infection was assessed by scintigraphy. When S. aureus was antibiotic resistant, we evaluated the efficacy of hLF 1-11, an antimicrobial peptide derived from human lactoferrin (hLF), in rats using (99m)Tc-UBI 29-41 and scintigraphy.. Decreasing amounts of radiolabeled peptide at the site of the S. aureus infection in animals correlated (r(2) > 0.81; P < 0.001) with increasing doses of cloxacillin in animals. An effective dose of erythromycin resulted in reduced (P = 0.023) accumulation of the radiolabeled peptide at the site of S. aureus infection in mice. In addition, we noted decreasing amounts of (99m)Tc-UBI 29-41 at the site of infection after administration of increasing doses of hLF 1-11 peptide in rats infected with antibiotic-resistant S. aureus. Furthermore, the number of viable bacteria decreased with increasing doses of cloxacillin or hLF 1-11 peptide, and a good correlation (r(2) > 0.80; P < 0.001) between the accumulation of (99m)Tc-UBI 29-41 and the number of viable (antibiotic-resistant) S. aureus at the site of infection was seen. In an attempt to explain these results, we found that these antibacterial agents do not affect the in vitro binding of (99m)Tc-UBI 29-41 to bacteria. Furthermore, this radiolabeled peptide bound to free bacteria and to cell-adherent but not phagocytized S. aureus, suggesting that at sites of infection mainly extracellular bacteria are targeted by (99m)Tc-UBI 29-41.. (99m)Tc-UBI 29-41 allows the monitoring of the efficacy of antibacterial agents in mice and rats with S. aureus infections.

Topics: Animals; Anti-Bacterial Agents; Cloxacillin; Erythromycin; Female; Lactoferrin; Male; Mice; Peptide Fragments; Radionuclide Imaging; Rats; Rats, Wistar; Ribosomal Proteins; Staphylococcal Infections; Staphylococcus aureus; Technetium

Previous studies on cyclophosphamide (CP)-immunocompromised mice showed accelerated reconstitution of immune system function following oral treatment with lactoferrin (LF). The aim of this investigation was to evaluate the ability of mice, treated with a sublethal dose of CP and given LF, to combat bacterial infections. Mice were injected with a single, intraperitoneal dose of CP (350 mg/kg body weight). One group of CP-treated mice was also given LF in drinking water (0.5% solution) for 14 days. Untreated and LF-treated mice served as controls. On day 15 following CP administration, mice were infected intravenously with 10(8) Escherichia coli or 5 x 10(7) Staphylococcus aureus. Twenty-four hours later, the number of colony-forming units (CFU) in spleens and livers were determined. Phenotypic analysis of blood leukocytes was determined, as well as the ability of splenic and peritoneal cells to produce IL-6 spontaneously and in the presence of lipopolysaccharide (LPS). Treatment with CP, or with CP and LF, led to profound reduction of E. coli CFU in the liver and the spleen; treatment with LF alone had significant inhibitory effects on organ enumerated CFU. S. aureus CFUs were also significantly reduced in spleens of mice treated with CP or CP/LF and, to a lesser degree, after LF alone. These effects were also significantly reduced in the livers. Analysis of blood cellular phenotype revealed total number of peripheral leukocytes was lower in the CP-treated group (52.6%) but not significantly different from control values in CP/LF and LF-treated groups (90.7% and 104.6%, respectively). Conversely, percentage of blood neutrophils was markedly elevated in CP and CP/LF groups--62% and 42.5% vs. 18.4% in controls. These findings were accompanied by production of IL-6 by splenic and peritoneal cells which was significantly increased in CP- and CP/LF-treated groups. It was concluded that the increased clearance of bacteria in the organs of mice treated with CP and CP/LF may result from a rise in the number of neutrophils infiltrating the organs and contributing to accelerated clearance of bacteria. The study also suggests that the ability of cells from CP- and CP/LF-treated mice to produce significantly more IL-6 may also contribute to increased resistance to infections. Lastly, together with our previous data, this study indicates that LF used to reconstitute the antigen-specific immune response in CP-treated mice does not impair their resistance to infection.

Topics: Animals; Cyclophosphamide; Escherichia coli Infections; Female; Immunosuppressive Agents; Interleukin-6; Lactoferrin; Leukocyte Count; Leukocytes; Lipopolysaccharides; Liver; Macrophages, Peritoneal; Male; Mice; Mice, Inbred CBA; Spleen; Staphylococcal Infections

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 concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.

Topics: Aging; Animals; Cattle; Cell Count; Corynebacterium; Corynebacterium Infections; Female; Lactation; Lactoferrin; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

Topics: Animals; Anti-Infective Agents; Cattle; Colony Count, Microbial; Drug Therapy, Combination; Female; Lactoferrin; Mastitis, Bovine; Mice; Neutrophils; Penicillin G; Phagocytosis; Random Allocation; Staphylococcal Infections; Staphylococcus aureus

To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS).. 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis.. Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL.. In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner.. Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cattle; Female; Glycyrrhizic Acid; Histamine; Infusions, Parenteral; Lactalbumin; Lactoferrin; Leukocytes; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Time Factors

The therapeutic effect of administering lactoferrin hydrolysate (LFH) into the mammary glands of cows with subclinical mastitis was evaluated. Seven millilitres of a preparation of LFH (7% protein) was infused into 35 quarters of 25 cows with subclinical mastitis. The numbers of bacteria in the milk from infected quarters decreased, and bacteria disappeared by the 14th day after the administration of LFH. The mean somatic cell counts (SCC) peaked one day after administration of LFH and the counts were significantly p < 0.01) decreased on days 7, 14 and 21 compared to those before the administration of LFH. The mean lactoferrin concentration in the milk peaked on days 2 or 3 and then gradually decreased to day 14, returning to the level before the administration of LFH. It appears that administration of LFH may have a therapeutic effect when infused into the quarters of cows with subclinical mastitis.

Topics: Animals; Cattle; Cell Count; Escherichia coli Infections; Female; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Milk; Staphylococcal Infections; Streptococcal Infections

Lactoferrin (Lf) is an iron-binding protein of external secretions and neutrophil secondary granules with antimicrobial and immunomodulatory activities. To further define these properties of Lf, we have investigated the response to Staphylococcus aureus infection in transgenic mice carrying a functional human Lf gene. The transgenic mice cleared bacteria significantly better than congenic littermates, associated with a trend to reduced incidence of arthritis, septicemia, and mortality. We identified two pathways by which S. aureus clearance was enhanced. First, human Lf directly inhibited the growth of S. aureus LS-1 in vitro. Second, S. aureus-infected transgenic mice exhibited enhanced Th1 immune polarization. Thus, spleen cells from infected transgenic mice produced higher levels of TNF-alpha and IFN-gamma and less IL-5 and IL-10 upon stimulation ex vivo with the exotoxin toxic shock syndrome toxin-1 compared with congenic controls. To confirm that these effects of Lf transgene expression could occur in the absence of live bacterial infection, we also showed that Lf-transgenic DBA/1 mice exhibited enhanced severity of collagen-induced arthritis, an established model of Th1-induced articular inflammation. Higher levels of stainable iron in the spleens of transgenic mice correlated with human Lf distribution, but all other parameters of iron metabolism did not differ between transgenic mice and wild-type littermates. These results demonstrate that human Lf can mediate both antimicrobial and immunomodulatory activities with downstream effects on the outcome of immune pathology in infectious and inflammatory disease.

Topics: Adjuvants, Immunologic; Animals; Arthritis, Experimental; Arthritis, Infectious; Cytokines; Humans; Iron; Lactoferrin; Liver; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; Mice, Transgenic; Spleen; Staphylococcal Infections; Staphylococcus aureus; Th1 Cells

This study compared the possibilities and limitations of 99mTc-labeled synthetic peptides derived from two human antimicrobial peptides, namely, ubiquicidin (UBI) and lactoferrin (hLF), for the scintigraphic detection of bacterial and fungal infections in mice and rabbits. The rationale of our approach was that selected peptides accumulate in infected areas but not in sterile inflammatory lesions, because they bind preferentially to microorganisms. 99mTc-labeled human neutrophil peptides (defensins), ciprofloxacin, and human polyclonal IgG were included as control agents.. 99mTc-labeled peptides and control agents were injected intravenously into animals that had been injected intramuscularly 18 h earlier with multidrug-resistant Staphylococcus aureus, Klebsiella pneumoniae, or fluconazole-resistant Candida albicans. Sterile inflammatory sites were induced by the injection of heat-killed microorganisms or lipopolysaccharide (LPS) into the thigh muscle. Up to 4 h after injection, the accumulation of 99mTc-labeled compounds in the infected/inflamed thigh muscles was determined using scintigraphic techniques and radioactivity counts in dissected tissues.. Scintigraphy revealed that 99mTc-labeled peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins, which showed preferential in vitro binding to microorganisms in a former study, accumulated at a significantly higher rate (P < 0.01) in bacterial and C. albicans infections in mice and rabbits than in inflamed tissues induced by heat-killed microorganisms or by LPS. No significant difference in the accumulation of 99mTc-labeled ciprofloxacin was observed between infected and sterile inflamed thigh muscles in mice.. 99mTc-labeled antimicrobial peptides UBI 29-41, UBI 18-35, UBI 31-38, hLF 1-11, and defensins accumulate significantly in tissues infected with gram-positive and gram-negative bacteria and C. albicans. Significantly lower (P < 0.01) accumulation of these peptides occurs in sterile inflamed tissues. These data indicate that the peptides preferentially tag microorganisms at the site of infection, which is in agreement with their preferential binding to the microorganisms in vitro and in vivo. 99mTc-labeled ciprofloxacin does not distinguish between infections and sterile inflammatory lesions, which implies that its specificity for the detection of bacterial infections is not warranted.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Infections; Candidiasis; Ciprofloxacin; Defensins; Drug Resistance, Multiple; Immunoglobulin G; Inflammation; Klebsiella Infections; Lactoferrin; Male; Mice; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Ribosomal Proteins; Staphylococcal Infections; Staphylococcus aureus; Technetium

The in vitro antistaphylococcal activity of lactoferrin and the antibiotic resistance of clinical Staphylococcus aureus isolates obtained from three different sites of infection were examined. Antibiotic, but not lactoferrin resistance correlated with selective antibiotic pressure, and nosocomial and most community isolates were antibiotic resistant, whereas only a third of each group was resistant to lactoferrin. The antimicrobial activity of lactoferrin, both in defined medium and in normal human plasma serum, was dependent upon its ferrochelating properties. Therapeutic approaches based on the use of ferrochelating agents such as lactoferrin combined with antimicrobial drugs may help to counteract the reduced efficacy of current antibiotics.

Topics: Anti-Bacterial Agents; Community-Acquired Infections; Drug Resistance, Multiple, Bacterial; Humans; Iron; Iron Chelating Agents; Lactoferrin; Staphylococcal Infections; Staphylococcus aureus

To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis.. Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days.. DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival.. Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

Topics: Administration, Topical; Animals; Arthritis; Arthritis, Infectious; Collagen; Disease Models, Animal; Injections, Intra-Articular; Joints; Lactoferrin; Male; Mice; Mice, Inbred DBA; Staphylococcal Infections; Staphylococcus aureus; Tissue Distribution

Human and bovine lactoferrins (Lfs) and bovine lactoferrin hydrolysate (LH) were assessed in vitro and in vivo for their antibacterial effects on Staphylococcus aureus. Lactoferrins showed weak in vitro antibacterial activity while Fe-saturated Lfs and LH showed no activity. Lactoferrin-treated mice (1 mg, i.v.) when injected i.v. with 10(6) staphylococci, showed 30-50% reduction in kidney infections, and viable bacterial counts in the kidneys decreased 5-12-fold. The inhibitory effect was dose-dependent up to 1 mg Lf. Lactoferrins were effective when given 1 day prior to the bacterial challenge, after which there was no significant effect even at doses up to 5 mg. Apo- and Fe-saturated forms of human and bovine Lfs were all equally effective, while LH was not protective. Human and bovine Lfs with different degrees of iron saturation (9-97%) were found to be equipotent. Feeding mice with 2% bLf in drinking water also reduced the kidney infections by 40-60%, and viable bacterial counts, 5-12-fold. The results suggest a potential for the use of Lfs as natural antibacterial proteins for preventing bacterial infections.

Topics: Animals; Cattle; Colony Count, Microbial; Humans; Iron; Kidney; Kidney Diseases; Lactoferrin; Mice; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus aureus

The lactoferrin (LF) concentration in the milk from dairy cows with clinical mastitis was determined to evaluate the relationship between the LF concentration (LFC) in milk and the non-specific defensive capability of the udder. The mean LFC in 368 milk samples from 319 cows with clinical mastitis was significantly higher (p < 0.01) than that of normal cows. The mean LFC in milk from quarters infected with Mycoplasma bovis or Staphylococcus aureus was significantly higher (p < 0.05) than that of quarters infected with coagulase-negative staphylococci (CNS). In Escherichia coli mastitis, the level of LFC in milk from cows with peracute mastitis was significantly lower (p < 0.01) than that from cows with acute mastitis. In cases of mastitis due to E. coli, the mean LFC in milk from cows that needed more than 10 days to recover from the mastitis or were not cured was significantly lower (p < 0.05) than that for cows which took less than 10 days to be cured. The mean LFC in milk from cows with peracute E. coli mastitis was significantly lower (p < 0.05) than that for cows with mastitis associated with environmental streptococci or CNS, although these low LF levels were somewhat increased after 46 h from the occurrence of mastitis. These results suggest that the decreased levels of LF in peracute E. coli mastitis may be associated with the progress of inflammation in the early phase of mastitis.

Topics: Animals; Cattle; Escherichia coli; Escherichia coli Infections; Female; Immunodiffusion; Lactoferrin; Mastitis, Bovine; Milk; Mycoplasma; Mycoplasma Infections; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus

In a prospective study of 65 patients with S. aureus septicemia, the clinical value of measuring serum IL-6 and lactoferrin levels was assessed and compared with CRP levels and WBC count. 20/65 (31%) patients had a CRP value < or = 100 mg/l on admission and 10 (50%) and 11 (55%) of these had serum levels of IL-6 > 100 pg/ml or lactoferrin > 2.0 mg/l, respectively. 41/64 (64%) patients had a WBC count < or = 15.0 x 10(9)/l and the corresponding figures for increased IL-6 and lactoferrin values were 29 (71%) and 21 (51%) patients, respectively. The high concentrations of IL-6 and lactoferrin on admission decreased rapidly during the hospital stay, better reflecting the clinical course than CRP and WBC count. Patients with endocarditis showed higher IL-6 levels and body temperatures both on admission and during the first days of hospitalization compared with patients without endocarditis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bacteremia; Biomarkers; Blood Sedimentation; C-Reactive Protein; Child; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-6; Lactoferrin; Leukocyte Count; Male; Middle Aged; Prospective Studies; Sensitivity and Specificity; Staphylococcal Infections

A total of 103 Staphylococcus aureus strains isolated from bovine mastitis were tested for bovine lactoferrin binding in a 125I-labeled protein binding assay. More than 85% of the strains demonstrated high to moderate and a few showed little or no binding. Bovine lactoferrin binding to S. aureus cells was high when grown on blood, nutrient, or proteose-peptone agar, but the binding capacity was low with cells grown on salt rich media, in skim milk, or in broth. The kinetics of 125I-labeled bovine lactoferrin binding required approximately 90 min for complete saturation with optimal interaction in the pH range 4.0 to 7.0. The lactoferrin-staphylococci interaction was specific with a high affinity (association constant, Ka 14 x 10(6) L/mol). Scatchard plot analysis estimated the number of binding sites per cell at 7200 on strain SA-340. Unlabeled bovine lactoferrin effectively displaced the binding of the labeled ligand to strain SA-340 in a dose-dependent manner. Bovine lactoferrin binding was inhibited or displaced by human lactoferrin. Various plasma, connective tissue, or mucosal secretory proteins tested did not inhibit lactoferrin-staphylococci interaction. Bovine lactoferrin binding components on SA-340 were resistant to glycolytic enzymes and moderately susceptible to proteolytic digestion. Two proteins with an estimated molecular weight of approximately 92 and 67 kDa were identified as bovine lactoferrin binding components of S. aureus strain SA-340.

Topics: Animals; Binding, Competitive; Cattle; Culture Media; Lactoferrin; Mastitis, Bovine; Receptors, Cell Surface; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus strains (n = 100) isolated from bovine mastitis were classified according to the presence of capsular polysaccharide serotype 5 (n = 46), type 8 (n = 26), and non-5/8 (n = 28). Strains from each type were tested for protein interaction in a 125I-labeled ligand binding assay. A majority of type 5 and type 8 strains showed a higher degree of binding to lactoferrin, fibronectin, and IgG than the non-5/8 strains. Fibrinogen binding was low in all serotypes. Most of the type 5 and non-5/8 strains bound less than 10% laminin, whereas type 8 strains bound laminin in the 11 to 20% range. Non-5/8 strains significantly differed from type 5 in lactoferrin, fibronectin, fibrinogen, and IgG and also from type 8 in fibrinogen and IgG binding. The differences in protein binding between type 5 and type 8 were nonsignificant. The degree of lactoferrin binding in all types positively correlated with laminin binding. Lactoferrin and fibrinogen bindings were correlated in type 5 and type 8 strains. Lactoferrin and fibronectin bindings were correlated only in type 5 strains. These data suggest that bovine lactoferrin binding is common and associated with subepithelial matrix protein interactions in certain serotypes of S. aureus.

Topics: Animals; Cattle; Extracellular Matrix Proteins; Female; Fibrinogen; Fibronectins; Immunoglobulin G; Lactoferrin; Laminin; Mastitis, Bovine; Protein Binding; Serotyping; Staphylococcal Infections; Staphylococcus aureus

Bovine lactoferrin (BLf), an acute-phase iron-binding secretory protein present in secretions of the bovine udder, was demonstrated to bind to the following staphylococcal species associated with bovine intramammary infections: S. epidermidis, S. warneri, S. hominis, S. xylosus, S. hyicus, and S. chromogenes. The degree of 125I-labeled BLf uptake significantly varied among the blood agar-grown cells of all six species of coagulase-negative staphylococci tested. Isolates identified as S. xylosus demonstrated the highest (mean, 35.1 x 10(6) +/- 13.3 x 10(6) nmol) and S. hyicus the lowest (mean, 10.7 x 10(6) +/- 5.9 x 10(6) nmol) binding to 125I-BLf. BLf binding was optimum at an acidic pH, with time-dependent binding saturation ranging from 70 min for S. warneri to 240 min for S. hominis. The BLf-binding mechanism was specific, with affinity constants (Ka values) ranging between 0.96 x 10(6) and 11.90 x 10(6) liters/mol. The numbers of BLf-binding sites per cell, as determined by using Scatchard analysis, were as follows: S. epidermidis, 3,600; S. warneri, 1,900; S. hominis, 4,100; S. xylosus, 4,400; S. hyicus, 6,100; and S. chromogenes, 4,700. 125I-BLf binding to all species was inhibited by unlabled BLf and unlabeled human lactoferrin, whereas none of the various plasma, connective tissue, or mucosal secretory proteins or carbohydrates tested caused significant interference. BLf-binding receptors of the six coagulase-negative staphylococcal species demonstrated marked differences in patterns of susceptibility to proteolytic or glycolytic enzyme digestion and to heat or periodate treatment. These data suggest that the BLf-binding components in S. epidermidis and S. warneri are proteins containing glycosidyl residues. In the remaining four species, the proteinaceous nature of the BLf-binding component was evident, but the involvement of glycosidyl residues was not clear. Results of this study establish the presence of specific binding components for BLf on coagulase-negative staphylococci isolated from bovine intramammary infections.

Topics: Animals; Bacterial Proteins; Binding Sites; Carrier Proteins; Cattle; Cell Membrane; Coagulase; Female; Kinetics; Lactoferrin; Mastitis, Bovine; Species Specificity; Staphylococcal Infections; Staphylococcus

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.

Topics: Aerosols; Albumins; Animals; Escherichia coli Infections; Female; Iron; Lactoferrin; Lactoglobulins; Lung; Mice; Neutrophils; Staphylococcal Infections; Transferrin

Topics: Animals; Cattle; Citrates; Female; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Pregnancy; Staphylococcal Infections; Staphylococcus aureus

Ten Holstein-Friesian cows were distributed according to their lactoferrin and lysozyme concentrations in milk into groups with high and low concentrations. In each cow, a front and a rear mammary quarter was infected by inoculation of 10(8) colony forming units of Staphylococcus aureus while the other two quarters were infused with 2 ml of sterile milk. The reaction was observed during the following nine days. After 10 hours the cell count, the lactoferrin and lysozyme concentrations were increased in the infected and control quarters. In milk samples with a high initial lactoferrin concentration the colony forming units of S. aureus were higher than in those with a low concentration. In milk samples with a high lysozyme concentration with colony forming units of S. Aureus were significantly lower than in those with low concentrations. These results show, that the lysozyme concentration in milk of healthy udders could indicate the preparedness for defense against infectious diseases.

Topics: Animals; Cattle; Cattle Diseases; Female; Lactoferrin; Lactoglobulins; Mammary Glands, Animal; Milk; Muramidase; Staphylococcal Infections; Staphylococcus aureus

The mean lactoferrin (Lf) concentration determined by electroimmunodiffusion (EID) assay of whey preparations from 80 quarters of 20 normal lactating cows was 0.35 mg/ml. The mean alpha-lactalbumin (alpha-LAC) and bovine serum albumin (BSA) concentrations were 2.01 mg/ml and 0.29 mg/ml, respectively. The mean was significantly related to cell count (P smaller than 0.01), BSA (P smaller than 0.05), stage of lactation (P smaller than 0.05), and milk production (P smaller than 0.05). The Lf-milk production relationship was the only negative correlation. In 11 cows with mastitis, there was a significant (P smaller than 0.01) increase in mean Lf concentration in infected quarters from 0.55 mg/ml on day 1 of the infection to 1.89 mg/ml by day 3. By day 15 clinical signs had subsided and mean Lf concentrations had decreased to near day 1 values. On day 3 quarters infected with coliform bacteria (clinical mastitis generally more severe) had mean Lf values more than twofold greater than those quarters infected with species of Staphylococcus or Streptococcus (milder clinical signs). Noninfected (control) quarters of cows having coliform bacteria-infected quarters had slightly increased mean Lf concentrations, where Lf concentration in contral quarters of cows having quarters infected with gram-positive organisms remained unchanged.

Topics: Animals; Cattle; Electrophoresis, Polyacrylamide Gel; Escherichia coli Infections; Female; Lactalbumin; Lactation; Lactoferrin; Lactoglobulins; Mastitis, Bovine; Milk; Pregnancy; Serum Albumin, Bovine; Staphylococcal Infections; Streptococcal Infections