muramidase and Mastitis

Staphylococcus aureus causes mastitis as a result of community-acquired or nosocomial infections. Lysozyme (LYSO) is an enzyme that is upregulated in many organisms during the innate immune response against infection by bacterial pathogens. Baicalin is a bioactive flavonoid that can bind to enzymes, often to potentiate their effect. Here we tested the effects of baicalin on the activity of LYSO using the S. aureus mastitis mouse model and neutrophilic granulocyte model of S. aureus infection. In our experiments, S. aureus counts decreased with increasing baicalin concentration. Furthermore, qPCR and western blot analyses showed that LYSO expression was unaffected by baicalin, while fluorescence quenching and UV fluorescence spectral analyses showed that baicalin binds to LYSO. To test whether this binding increased LYSO activity, we assessed LYSO-induced bacteriostasis in the presence of baicalin. Our results showed that LYSO-induced S. aureus bacteriostasis increased with increasing concentrations of baicalin, and that baicalin binding to LYSO synergistically increased the antibacterial activity of LYSO. These results demonstrate that baicalin enhances LYSO-induced bacteriostasis during the innate immune response to S. aureus. They suggest baicalin is a potentially useful therapeutic agent for the treatment of bacterial infections.

Topics: Animals; Anti-Infective Agents; Blotting, Western; Cells, Cultured; Female; Flavonoids; Host-Pathogen Interactions; Mammary Glands, Animal; Mastitis; Mice, Inbred BALB C; Microbial Viability; Muramidase; Neutrophils; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Fluorescence; Staphylococcal Infections; Staphylococcus aureus

To explore the pathogenesis of caprine contagious agalactia (CA), we assessed the ability of Mycoplasma agalactiae (Ma) to modulate the immune system in host tissues by immunohistochemically and chronologically characterizing the main cell subsets present during the mammary immunoinflammatory response. Fifteen lactating goats were inoculated with 10(10) colony-forming units (cfu) of Ma and killed 5, 15 or 45 days post-inoculation (dpi). Blood was taken before necropsy to determine antibodies and milk to determine mycoplasma number. Cells in mammary tissue expressing lysozyme, myeloid-histiocyte antigen (Mac387), major histocompatibility complex class II antigen, immunoglobulin G (IgG), IgA, and CD3, CD4 and CD8 lymphocytes were determined by immunohistochemistry. Results indicate an innate immune response in animals sacrificed at 5dpi, characterized by an abundance of Mac387+ and lysozyme+ cells, that was unable to block or control Ma infection. Elevated numbers of all the cell subsets of the specific immune response (MHC-II+, IgG+, IgA+, CD3+, CD4+ and CD8+ cells) were observed during the subacute stage of the inflammatory process, represented by the 15dpi group. However, these findings could not be correlated with an intense antibody response in blood. The chronic stage of the inflammatory process observed in the goats killed at 45dpi was mainly characterized by expansion of the CD8 compartment at the expense of the CD4 subset leading to a reduced CD4/CD8 ratio. These results contribute to establishing the basic morphological and immunohistochemical characterization of the local immune response against Ma in the goat's mammary gland.

Topics: Animals; Antigens, Bacterial; Antigens, CD; Female; Goat Diseases; Goats; Histocompatibility Antigens Class II; Immunoglobulin A; Immunoglobulin G; Immunohistochemistry; Mammary Glands, Animal; Mastitis; Muramidase; Mycoplasma agalactiae; Mycoplasma Infections

The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P < 0.02) and Staphylococcus aureus (P < 0.05) as well as the cold-spoilage organism Pseudomonas fragi (P < 0.02). The growth of an organism involved in cheese-making, Lactococcus lactis, was not affected by the presence of lysozyme in milk. The supplementation of control milk with purified lysozyme did not achieve the same inhibitory effect as milk from transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.

Topics: Animals; Animals, Genetically Modified; Anti-Bacterial Agents; Bacteria; Consumer Product Safety; Escherichia coli; Female; Goats; Humans; Lactococcus lactis; Mastitis; Milk; Muramidase; Pseudomonas; Staphylococcus aureus

The effect of parenteral administration of two subcutaneous injections of vitamin E and Se (5 mg and 0.1 mg/kg of body weight, respectively) during the dry period on the mammary health and milk somatic cell counts of 25 dairy ewes was investigated. Supplementation reduced somatic cell counts (5.4 vs. 6.0 log10) during the subsequent lactation but had no effect on the incidence of clinical mastitis (4% vs. 6%) and intramammary infections (9.0% vs. 11.3%). Furthermore, the administration of vitamin E and Se was associated with differences in differential cell counts of milk samples (macrophages, 48.8% vs. 38.4%; polymorphonuclear neutrophils, 40.1% vs. 50.7%; and eosinophils, 0.7% vs. 1.4% for control ewes and ewes receiving supplements, respectively). The administration of these supplements also increased erythrocyte glutathione peroxidase activity (139.5 vs. 86.3 U/ml of packed cell volume) and the percentage of blood neutrophils that reduced nitroblue tetrazolium after bacterial extract stimulation (48.6% vs. 38.7%). Parenteral administration of vitamin E and Se to ewes during the dry period appeared to have influenced mammary gland status during the subsequent lactation and particularly total and differential milk cell counts.

Topics: Animals; Cell Count; Dietary Supplements; Female; Glutathione Peroxidase; Leukocyte Count; Lymphocytes; Macrophages; Mammary Glands, Animal; Mastitis; Milk; Muramidase; Neutrophils; Selenium; Sheep; Sheep Diseases; Staphylococcal Infections; Streptococcal Infections; Vitamin E

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

Human milk contains important immunological factors that protect the breast from infection and are thought to protect infants from infection, including human immunodeficiency virus (HIV) infection. Human milk immunological factors have not been well characterized in HIV-infected lactating women. Lysozyme, secretory leukocyte protease inhibitor (SLPI), sodium (an indicator of mastitis), and HIV were measured in breast milk of 334 HIV-infected women at 6 weeks postpartum. Women with mastitis, as indicated by elevated breast milk sodium concentrations, had higher median levels lysozyme (290 vs 221 mg/L, p < 0.04), SLPI (38 vs 19 mg/L, p < 0.0001) and HIV (920 copies/mL vs undetectable, p < 0.0001) compared with women without mastitis. Lower total plasma carotenoid levels (p < 0.02) and higher maternal HIV load (p < 0.006) by quartile were risk factors for mastitis. Mastitis, as indicated by elevated breast milk sodium levels, is associated with high concentrations of immunological factors and higher HIV load in breast milk.

Topics: Adult; Carotenoids; Female; HIV Infections; Humans; Longitudinal Studies; Malawi; Mastitis; Milk, Human; Muramidase; Pregnancy; Proteinase Inhibitory Proteins, Secretory; Proteins; Puerperal Disorders; Risk Factors; Secretory Leukocyte Peptidase Inhibitor; Sodium; Viral Load

A highly presumptive identification of Nocardia farcinica was made of 47 bacterial isolates. Fifteen isolates from Alberta, 9 from Ontario, and 2 each from New Brunswick, Newfoundland, and Nova Scotia were from clinical cases involved in the Canadian mastitis epizootic. Seventeen additional isolates from Alberta were recovered from farm milk bulk tanks from herds found to have cows involved in the epizootic. All isolates were shown by high-performance liquid chromatography to possess mycolic acids of a size consistent with the genus Nocardia. All isolates were resistant to a concentration of 5 micrograms/mL of mitomycin C. Forty-five isolates grew well and 2 showed reduced growth in the presence of 50 micrograms/mL of kanamycin acid sulfate. Forty-six isolates were resistant to 5-fluorouracil at a concentration of 20 micrograms/mL. All isolates were resistant to lysozyme. Resistance to these compounds supported the placement of the isolates in the genus Nocardia. Thirty-five isolates produced strong beta-galactosidase reactions and 12 showed weak reactions. The demonstration of beta-galactosidase activity further supports the identification of the isolates as nocardiae. Attempts to identify the bacteria to species by high-performance liquid chromatography of mycolic acid esters were frustrated, since two species of Nocardia were found to have indistinguishable mycolic acid patterns. The physiological and growth characteristics of the isolates were consistent with Nocardia farcinica.

Topics: Animals; Bacterial Typing Techniques; beta-Galactosidase; Canada; Cattle; Dairying; Disease Outbreaks; Drug Resistance, Microbial; Mastitis; Microbial Sensitivity Tests; Milk; Mitomycin; Muramidase; Mycolic Acids; Nocardia

The morbidity rates, ++features of lactation formation, and serum and breast milk lysozyme levels were studied in 91 parturients who showed various breast functional activity in the early postpartum period. The highest frequency of postpartum ++pyo-septic diseases was found in females with hyperlactation. There was earlier appearance of foremilk and milk in these females. Healthy mothers with higher breast functional activity exhibited the greatest levels of lysozyme in the milk and marked decreases in its levels in the blood within the first 3 days as compared to those observed in females who had normal or insufficient quantities of milk.

Topics: Adolescent; Adult; Breast; Female; Humans; Lactation; Mastitis; Milk, Human; Muramidase; Postpartum Period; Reference Values; Time Factors

The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.

Topics: Animals; Anti-Bacterial Agents; Colony Count, Microbial; Escherichia coli; Mastitis; Microbial Sensitivity Tests; Muramidase; Salmonidae; Staphylococcus aureus; Streptococcus; Trout

Topics: Electric Stimulation Therapy; Female; Humans; Lactation Disorders; Mastitis; Milk, Human; Muramidase; Pregnancy; Risk; Time Factors; Transcutaneous Electric Nerve Stimulation

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

Topics: Animals; Clinical Enzyme Tests; Female; Mastitis; Milk; Muramidase; Pregnancy; Sheep; Sheep Diseases

Topics: Blood Proteins; C-Reactive Protein; Complement System Proteins; Female; Humans; Immunoglobulins; Mastitis; Muramidase; Pregnancy; Puerperal Infection

Topics: Antibody Formation; Breast Feeding; Complement System Proteins; Female; Humans; Immunity, Active; Infant; Infant, Newborn; Mastitis; Methods; Muramidase; Pregnancy; Properdin