lactoferrin and Mastitis

As a step toward prevention of bovine mastitis, a plasmid-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete bovine lactoferricin and bovine tracheal antibacterial peptides. For this purpose, a series of mammary tissue-specific expression vectors, harboring the antibacterial peptide gene, the 5'-flanking regulation sequence of goat beta-casein, and the bovine growth hormone polyadenylation signal sequence, were constructed using a eukaryotic expression vector pIRES1-neo. The mammary gland tissue-specific expression vector carrying the antimicrobial peptide genes dissolved in physiologic saline was injected directly into the lactating mammary glands of goats. The milk samples after injection were checked by Tricine-SDS-PAGE and bacterium inhibition zone assay. The results of these tests showed that the mammary gland tissue-specific expression vector driven by the goat beta-casein gene promoter could efficiently direct the expression of antibacterial peptides in goat milk; the expression of antibacterial proteins lasted for 3 to 6 d. All of the milk samples collected from the mammary glands that had been injected with different vectors harboring the antibacterial peptide gene(s) exhibited bacteriostatic activity against different bacterial pathogens. These results demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antibacterial peptide gene into the goat mammary gland, enabling secretion of a bioactive form of antibacterial peptide in the milk. This successful expression of antibacterial peptides in goat mammary glands provided a possible method to prevent mastitis in ruminants.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Escherichia coli K12; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Vectors; Goat Diseases; Goats; Lactoferrin; Mammary Glands, Animal; Mastitis; Milk; Recombinant Proteins; Staphylococcus aureus; Time Factors

This study aimed to determine if intrauterine-infused lipopolysaccharides (LPS) can be translocated to the mammary glands and induce an inflammatory response. Thirty-seven goats were divided into two experiments. Nineteen goats (control group, n = 9; LPS group, n = 10) were subjected to intravenous injection of LPS, and eighteen goats (control group, n = 8; LPS group, n = 10) were subjected to intrauterine infusion of LPS. Milk and blood samples were collected before and after the LPS challenge, to measure the blood leukocyte count (BLC), plasma LPS-binding protein (LBP), milk yield, milk somatic cell count (SCC), lactoferrin (LF), milk lactoperoxidase (LPO) activity, and pro- and anti-inflammatory cytokines in plasma and milk. Mammary gland tissues were collected from the parenchyma before and after the LPS challenge, for immunohistochemistry of LPS. In the intravenous injection experiment, the BLC (P < 0.001) and milk yield (P = 0.009) were lower, whereas the LF concentration (P < 0.001) and milk LPO activity (P < 0.001) were higher in the LPS group compared to that in the control group. LPS was detected in the mammary gland 3 and 24 h after intravenous injection of LPS. In the intrauterine infusion experiment, the mean concentrations of IL-1β and IL-6 in milk were higher in the LPS group compared to that in the control group (P = 0.004 and P = 0.017, respectively), whereas there were no changes in milk yield or SCC. LPS was detected in the connective tissues and interepithelial spaces of the alveoli of the mammary glands 24 h after intrauterine infusion of LPS. We conclude that intrauterine-infused LPS can be translocated to the mammary glands from the uterus, however, the amount of translocated LPS might not be enough to induce symptoms of clinical or subclinical mastitis.

Topics: Acute-Phase Proteins; Administration, Intravenous; Animals; Carrier Proteins; Female; Goats; Lactation; Lactoferrin; Lipopolysaccharides; Mammary Glands, Animal; Mastitis; Membrane Glycoproteins; Milk; Uterus

Topics: Adult; Body Mass Index; Breast Diseases; Breast Feeding; Calcium; Candidiasis; Female; Humans; Infant; Infant, Newborn; Lactoferrin; Male; Mastitis; Milk, Human; Mothers; Neuropeptides; Plasma; Postpartum Period; Prospective Studies; Surveys and Questionnaires; Sweden; Young Adult

Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.

Topics: Amino Acid Sequence; Animals; Antibodies, Bacterial; Bacterial Adhesion; Bacterial Proteins; Bacterial Vaccines; Base Sequence; Cattle; DNA, Bacterial; Epithelial Cells; Female; Immunoglobulin G; Lactoferrin; Mastitis; Mice; Models, Animal; Recombinant Proteins; Streptococcal Infections; Streptococcus; Vaccines, Subunit; Virulence Factors

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

Lactoferrin gene is one of the important candidate genes for mastitis resistance. The gene is located on chromosome BTA 22 and consists of 17 exons spanning over 34.5 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exons 7 and 12 of lactoferrin gene and to analyze association between its genetic variants and incidence of clinical mastitis in Murrah buffalo. The amplification of exons 7 and 12 of lactoferrin gene yielded amplicons of 232- and 461-bp sizes. PCR-restriction fragment length polymorphism (RFLP) analysis of 232-bp amplicon using BccI restriction enzyme revealed three genotypes (AA, AB, and BB) with frequencies of 0.62, 0.22, and 0.16, respectively. The frequencies of two alleles, A and B, were estimated as 0.73 and 0.27. Hpy188I-RFLP for 461-bp amplicon revealed polymorphism with three genotypes, CC, CD, and DD, with respective frequencies of 0.06, 0.39, and 0.56, whereas frequencies for C and D alleles were 0.25 and 0.75. The chi-square (χ(2)) analysis revealed a significant association between incidence of clinical mastitis and genetic variants of exon 7, and animals of AA genotype of exon 7 were found to be least susceptible to mastitis. The findings indicate potential scope for incorporation of lactoferrin gene in selection and breeding of Murrah buffaloes for improved genetic resistance to mastitis.

Topics: Animals; Buffaloes; Dairying; Female; Genetic Predisposition to Disease; Genetic Variation; Incidence; India; Lactoferrin; Mastitis; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length

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

Abstract Secretory immunoglobulin A (sIgA) is the dominant immunoglobulin in human milk, and apart from the obvious contribution it makes towards the protection of the infant, sIgA may also form an important part of the defense of the mammary gland. This report involves a mother (M8) who participated in a research study investigating the relationships between symptoms and changes in the physiology of the lactating breast during mastitis. Breastmilk samples were collected on Days 14, 30, 60, and 90 postpartum, to establish the normal reference range of biochemical markers, and during periods of breast inflammation. M8 experienced seven episodes of blocked duct(s) during the first 19 weeks, five of which occurred within the 90-day reference sample collection period. On analysis, it was found there was no detectable sIgA present in her milk samples. Medical referral and further testing resulted in a diagnosis of selective IgA deficiency, of which the mother had not been previously aware. M8 showed little variation in her milk composition even when suffering with blocked duct(s), although there was an increase in the concentration of lactoferrin in both breasts at reference collection days 14-90. Lactoferrin concentration was also unusually high at Day 14 (15 g/L) in the left breast and continued to be increased in this breast until Day 60. The absence of sIgA in this mother's breastmilk may have been a contributing factor in her experiencing recurrent blocked ducts.

Topics: Adult; Biomarkers; Breast; Female; Humans; IgA Deficiency; Immunoglobulin A, Secretory; Lactation; Lactoferrin; Mastitis; Milk, Human

The objective was to investigate changes in milk composition that reflect variations in breast permeability, milk synthesis, and immune response in women before, during, and after mastitis.. Mothers (n = 26) were followed prospectively from day 5 postpartum to the end of their lactation. Milk from each breast, blood, 24-hour urine samples, and data on breast and systemic pathologies were collected at reference intervals during the first 3 months postpartum, daily during the occurrence of any breast inflammation, and 7 days after resolution of symptoms, and was analyzed using mixed-model analysis (repeated measures).. There was a significant difference in sodium (p < 0.001), chloride (p < 0.001), serum albumin (p < 0.02) and lactose (p < 0.003) in the breast with mastitis when compared with both the contralateral asymptomatic breast and "healthy" breasts. Inflammation of the whole breast was a significant predictor for a decreased glucose (p < 0.01) and hyperacute systemic symptoms predicted a decrease in milk glucose (p < 0.03) and an increased lactoferrin (p < 0.05) and sIgA (p < 0.03).. There is an increased breast permeability, reduced milk synthesis, and increased concentration of the immune components sIgA and lactoferrin with increasing severity of breast and systemic symptoms. The changes observed in milk composition during periods of increased breast permeability cannot be solely explained by the current theory of permeability of the paracellular pathway and further research in this area is required.

Topics: Adult; Biomarkers; Breast; Cell Membrane Permeability; Chlorides; Female; Humans; Immunoglobulin A, Secretory; Lactation; Lactoferrin; Lactose; Mastitis; Milk, Human; Prospective Studies; Serum Albumin; Severity of Illness Index; Sodium

Lactoferrin concentration (LFC) in normal and mastitic milk of dairy goats were examined. LFC in bulk milk collected from 70 dairy goat farms and individual milk samples from 10 goats with mastitis were measured by enzyme-linked immunosorbent assay (ELISA) and their reaction time in methylene blue reduction test (MBRT) monitored. Bulk milk samples were categorized into three grades, such as high, normal and low, based on the reaction time in MBRT. The mean LFC in milk that was considered high quality (167 microg/ml) was significantly lower than that of those graded as normal (218 microg/ml) and low quality (304 microg/ml), while mean LFC in mastitic milk was 587 microg/m l. The correlation coefficient between milk LFC and MBRT time was found to be -0.7. Three goats were inoculated with Staphylococcus aureus into one of their udder halves. The mean milk LFC was found to be significantly higher (1500 microg/ml) than the control (30 microg/ml). These findings suggest that milk LFC may be useful as an index for intramammary infection in goats.

Topics: Analysis of Variance; Animals; Blotting, Western; Dairying; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Goat Diseases; Goats; Lactoferrin; Mastitis; Methylene Blue; Milk; Staphylococcus aureus

Mastitis is a common complication of human lactation. We examined milk specimens from eight women with clinical mastitis to determine their content of anti-inflammatory components. Antioxidant activity (spontaneous cytochrome c reducing activity), selected pro-inflammatory cytokines (IL-6, IL-1beta), selected endogenous cytokine control molecules (sIL-6R, sIL-1RII, and sTNFRI), lactoferrin, Na(+):K(+) ratios, and milk bioactivities that cause shedding of sIL-1RII from human polymorphonuclear leukocytes (PMN), suppress PMN aggregation, and suppress PMN adherence responses were not increased compared to normal milks. Neither the bioactivities that deplete PMN intracellular Ca(2+) stores nor those that block Ca(2+) influx into fMLP-stimulated PMN were significantly increased in mastitis milks. In contrast, levels of TNFalpha, sTNFRII, and IL-1RA and bioactivities that cause shedding of sTNFRI from human PMN were significantly increased compared to normal milks. Mastitis milk has the same anti-inflammatory components and characteristics of normal milk, with elevations in selected components/activities that may help protect the nursing infant from developing clinical illness due to feeding on mastitis milk.

Topics: Adult; Antioxidants; Bacterial Infections; Breast Feeding; Calcium Signaling; Cell Adhesion; Cell Aggregation; Cytochrome c Group; Cytokines; Female; Humans; Infant; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Lactoferrin; Mastitis; Milk Proteins; Milk, Human; Neutrophils; Opsonin Proteins; Oxidation-Reduction; Potassium; Receptors, Interleukin-1; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Sodium; Staphylococcus aureus; Tumor Necrosis Factor-alpha

Although an elevated sodium concentration in human milk is suggested to be an indicator of mastitis, it is unclear whether elevated sodium concentrations are associated with immunological and inflammatory mediators in human milk. We conducted a cross-sectional study to evaluate the relationships between elevated breast milk sodium concentrations and levels of lactoferrin, lysozyme, secretory leukocyte protease inhibitor (SLPI), interleukin-8 (IL-8), and RANTES (regulated on activation normal T cell expressed and secreted) in human milk at 6 weeks postpartum in 96 lactating women in Blantyre, Malawi. Mastitis, as indicated by an elevated breast milk sodium concentration, was present in 15.6% of the women. Women with and without mastitis had respective median levels of other factors as follows: lactoferrin, 1,230 versus 565 mg/liter (P < 0. 0007); lysozyme, 266 versus 274 mg/liter (P = 0.55); SLPI, 76 versus 15 microg/liter, (P < 0.0002); IL-8, 339 versus 25 ng/liter (P < 0. 0001); and RANTES, 82 versus 3 ng/liter (P < 0.0001). Elevated sodium concentrations in breast milk are associated with an increase in levels of some immunological and inflammatory factors in breast milk.

Topics: Adolescent; Adult; Chemokine CCL5; Cross-Sectional Studies; Female; Humans; Interleukin-8; Lactoferrin; Malawi; Mastitis; Milk, Human; Muramidase; Potassium; Proteinase Inhibitory Proteins, Secretory; Proteins; Secretory Leukocyte Peptidase Inhibitor; Serine Proteinase Inhibitors; Sodium

The bacteriostatic activity of bovine lactoferrin (Lf) against mastitic bacteria was assessed with an in vitro microassay. The most susceptible species was Escherichia coli; all of the 35 isolates tested were susceptible to bacteriostasis by apo-Lf (0.1 mg ml-1), although a few strains showed a lower degree of inhibition. Heterogeneity among strains was more pronounced among 10 isolates of Staphylococcus aureus, four of which were apparently unaffected by apo-Lf (1 mg ml-1). Under the same conditions, Streptococcus agalactiae (six isolates) and Str. uberis (five isolates) resisted the bacteriostatic action of apo-Lf.

Topics: Animals; Cattle; Escherichia coli; Lactoferrin; Lactoglobulins; Mastitis; Milk; Staphylococcus aureus; Streptococcus; Streptococcus agalactiae

Mastitis was found to be a sizeable clinical problem in a group of lactating Gambian mothers. The mean monthly incidence was 2.6% and repeated episodes of mastitis were common. The role of milk antimicrobial factors in the local defence of the breast against mastitis was investigated by analysis of IgA, IgG, IgM, C3, C4, lactoferrin and lysozyme in the breast milk of 10 mastitis patients. Acute inflammation of the breast was accompanied by the rapid appearance of high concentrations of serum-derived immunoproteins in mastitic milk. Changes in the milk levels of lactose, sodium and transferrin indicated that this was due to a temporary opening of the paracellular pathway. Concentrations of secretory immunoproteins (IgA, lactoferrin and lysozyme) exhibited a delayed response, being elevated one week after the attack of mastitis. The normal milk of mastitis sufferers was significantly deficient in IgA, C3 and lactoferrin when compared with other lactating women suggesting that the former were predisposed to mastitis.

Topics: Complement C3; Complement C4; Female; Humans; Immunoglobulin A; Immunoglobulin G; Lactoferrin; Lactose; Mastitis; Milk, Human; Muramidase; Pregnancy; Secretory Component