Comparison between lactoferrin and subepithelial matrix protein binding in Staphylococcus aureus associated with bovine mastitis.

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.     

Response of Staphylococcus aureus isolates from bovine mastitis to exogenous iron sources

Staphylococcus aureus can survive in conditions of extremely low iron concentration. The ability of S. aureus to use two exogenous hydroxamate types of siderophores (desferrioxamine and ferrichrome) and four iron-containing proteins found in cattle (hemin, hemoglobin, ferritin, and lactoferrin) were tested on 16 reference and clinical isolates. For all strains tested, ferrichrome and desferrioxamine showed strong growth-promoting activities in a disk diffusion assay and in liquid medium. The heme proteins hemin and hemoglobin were also found to support growth in culture media lacking other iron sources, while lactoferrin failed to do so. On media containing the iron chelator dipyridyl, ferritin induced a growth inhibition effect that was further enhanced in the presence of lactoferrin in seven of the 13 tested strains. Staphylococcus aureus was able to bind hemin and the level of binding activity was not increased after growth in iron-rich or -poor media. Dot-blot competition tests showed that biotin-labeled lactoferrin binds to S. aureus, and this binding can be inhibited by unlabeled lactoferrin. Expression of lactoferrin-binding activity was independent of the level of iron in the medium and the iron saturation status of lactoferrin. For each strain tested, ligand blots showed lactoferrin-binding proteins of molecular weights ranging from 32 to 92 kDa. Possible functions of these lactoferrin-binding proteins could not be related to iron acquisition mechanism in S. aureus.     

Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis

The objective of the present study was to evaluate the therapeutic potential of bovine lactoferrin or lactoferricin in combination with penicillin G against Staphylococcus aureus. Minimal inhibitory concentrations of lactoferrin, lactoferricin, penicillin, and combinations of lactoferrin or lactoferricin with penicillin were determined for 15 S. aureus strains including several strains resistant to beta-lactam antibiotics. The fractional inhibitory concentration index indicated a synergistic effect between lactoferrin and penicillin. Combination of lactoferrin with penicillin increased the inhibitory activity of penicillin by two- to fourfold and reduced the growth rate in S. aureus strains tested, whereas the increase in the inhibitory activity of lactoferrin by penicillin was 16- to 64-fold. The addition of iron to the medium containing a combination of penicillin and lactoferrin had no effect on growth inhibition. Electron microscopy revealed that concentration below the minimal inhibitory concentrations of penicillin induced important ultrastructure alterations, which were further enhanced by the presence of lactoferrin. When S. aureus cells were grown in the presence of a combination of penicillin and lactoferrin, changes in the protein profile of the bacteria, including the disappearance of several protein bands due to the presence of lactoferrin, were observed. These data suggest that bovine lactoferrin or lactoferricin in combination with beta-lactam antibiotics can increase the antibacterial activity of these antibiotics against S. aureus resistant to antibiotics.     

Antibacterial and antiviral activity of camel milk protective proteins.

Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.     

Binding of zinc to bovine and human milk proteins

Zn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine alpha s1-casein had the greatest Zn-binding capacity (approximately 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and the protein precipitated at a free Zn concentration of 1.7 mM. Zn binding increased with increasing pH in the range 5.4-7.0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to alpha s1-casein indicating common binding sites for these two metals. Bovine beta-casein bound up to 8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.5 mM, while kappa-casein bound 1-2 atoms Zn/mol. Whole bovine and human casein bound 5-8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (Kapp) for all caseins were similar (log Kapp 3.0-3.2). Enzymic dephosphorylation of alpha s1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin, which bound over 8 atoms Zn/mol, the bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and lactotransferrin, had little capacity for Zn binding.     

The effect of bovine milk lactoferrin on human breast cancer cell lines

The evidence that biologically active food components are key environmental factors affecting the incidence of many chronic diseases is overwhelming. However, the full extent of such components in our diet is unknown, as is our understanding of their mechanisms of action. Beyond the interaction of these food components with the gut and intestinal immune functions, whey proteins such as lactoferrin are being tested as anticancer agents. Lactoferrin is an iron-binding protein that has been reported to inhibit several types of cancer. In the present work, the effects of bovine milk lactoferrin on human breast cancer HS578T and T47D cells were studied. The cells were either untreated or treated with lactoferrin concentrations ranging from 0.125 to 125 μM. Lactoferrin decreased the cell viability of HS578T and T47D by 47 and 54%, respectively, and increased apoptosis about 2-fold for both cell lines. Proliferation rates decreased by 40.3 and 63.9% for HS578T and T47D, respectively. For the T47D line, cell migration decreased in the presence of the protein. Although the mechanisms of action are not fully known, the results gathered in this work suggest that lactoferrin interferes with some of the most important steps involved in cancer development.     

A probe for capture and Fe3+-induced conformational change of lactoferrin selected from phage displayed peptide libraries

Linear pentadecamer and cyclic hexamer peptide phage libraries were used to isolate phage clones with binding affinity toward lactoferrins purified from human and bovine milk. Phage clones with high specificity toward lactoferrin were selected with different binding strengths depending on the sequence of the peptide displayed by the phage. Phages coated to a microtiterplate were able to capture lactoferrin from crude milk samples without prior treatment. One of the selected sequences, EGKQRR, failed to bind to lactoferrin. In contrast, a branched tree-peptide bearing 4 EGKQRR sequences did bind to lactoferrin (Kd approximately 29 microM) and was also capable of inhibiting the binding of the phage to lactoferrin (IC(50) approximately 17 microM), indicating that avidity was important. Unexpectedly, the affinity of the phage for lactoferrin was influenced by the amount of bound Fe(3+), with a much lower affinity when lactoferrin was saturated with Fe(3+) as compared with the iron-depleted or partially saturated (natural) lactoferrin. As the phage does not bind to the Fe(3+)-binding site, the difference in binding affinity is due to differences in conformation of lactoferrin induced by Fe(3+). These results demonstrate that avidity or multipoint attachment and Fe(3+)-induced conformational changes play an important role in the binding of the selected phage to lactoferrin. Thus, we could demonstrate that, by the use of selected phage clones, we are able not only to detect lactoferrin, but also to capture lactoferrin from crude milk samples. Furthermore, the extent of phage binding provides additional information about the iron content and the concomitant conformation of lactoferrin.     

Enhancement of the activity of novobiocin against Escherichia coli by lactoferrin

The activity of novobiocin against Escherichia coli ATCC 25922 and three E. coli strains that were isolated from cases of bovine mastitis was determined in timekill studies in the presence of bovine lactoferrin. Lactoferrin alone did not affect the growth of any of the strains of E. coli. A combination of 1.0 mg/ml of lactoferrin and novobiocin at 1/16x minimum inhibitory concentration (MIC) was bactericidal for E. coli ATCC 25922. When the concentration was increased to 3.0 mg/ml of lactoferrin, novobiocin was bactericidal at 1/64x MIC. Among the mastitis strains tested, 6789 and 6806 were more susceptible to killing by novobiocin than was strain 6800. Strains 6789 and 6806 were killed when treated with novobiocin concentrations of 2, 1/2, and 1/4x MIC. When these strains were also treated with lactoferrin at 3.0 mg/ml, there was a bacteriostatic effect at novobiocin concentrations of 1/8 and 1/16x MIC for strains 6789 and 6800. Strain 6806 appeared to be more susceptible to the combination of lactoferrin and novobiocin as was evidenced by a bactericidal effect over the 24-h testing period. The combination treatment with cephapirin and lactoferrin showed that there was a synergistic bactericidal effect against all of the E. coli strains tested. These studies indicate that lactoferrin can potentiate the activity of antibiotics against Gram-negative bacteria.     

Screening of Bifidobacterium spp. based on in vitro growth responses to bovine lactoferrin

In the present study, we investigated the in vitro growth responses of fourteen strains of four Bifidobacterium spp. (Bifidobacterium infantis, B. breve, B. bifidum, B. longum) against bovine lactoferrin (bLf) at various concentrations. Bacterial strains were grown in deMan, Rogosa and Sharpe (MRS) broth with or without bLf, and growth was monitored by measuring absorbance at 660 nm. A dose‐dependent and strain‐dependent growth response was observed. Bifidobacterium spp. were ranked into high, medium and low according to their calculated relative growth response levels against lactoferrin (Lf). Strains showed better growth responses against holo‐type Lf. However, no inhibitory effects at high concentrations (4 mg mL^?1) or with apo‐type Lf were observed. These results strongly suggest that the growth response of Bifidobacterium spp. against bLf may be a selection criterion for their use in fermented products. In addition, use of holo‐type Lf in fermented food products may be more effective for probiotic growth, and could also be used as a source of iron to the host.     

High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin

We have studied the inactivation of a panel of eight test bacteria (two Escherichia coli strains, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Shigella flexneri, Pseudomonas fluorescens and Staphylococcus aureus) by high pressure in the presence of bovine lactoferrin (500 microg/ml), pepsin hydrolysate of lactoferrin (500 microg/ml), lactoferricin (20 microg/ml) and nisin (100 IU/ml). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria, however, the former had a narrower bactericidal spectrum than the two latter compounds. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The sensitisation of the test bacteria to these antimicrobials under pressure was transient, since no bactericidal activity was observed when bacteria were pressure treated before exposure to the compounds. We propose a mechanism of pressure-promoted uptake of these antimicrobial proteins and peptides in gram-negative bacteria to explain this sensitisation.     

Separation of lactoferrin-a and -b from bovine colostrum

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.     

Lactoferrin research,technology and applications

Lactoferrin is an iron-binding glycoprotein present in milk as well as other exocrine secretions and neutrophil granules in mammals. Lactoferrin is considered to be an important host defense molecule and has a diverse range of physiological functions such as antimicrobial/antiviral activities, immunomodulatory activity, and antioxidant activity. During the past decade, it has become evident that oral administration of lactoferrin exerts several beneficial effects on the health of humans and animals, including anti-infective, anticancer, and anti-inflammatory effects. This has enlarged the application potential of lactoferrin as a food additive. The technology of producing bovine lactoferrin on a factory scale was established over 20 years ago. Bovine lactoferrin is purified by cation-exchange chromatography from bovine skim milk or whey, and is commercially available from several suppliers. Recombinant human lactoferrin is produced by Aspergillus niger, transgenic cows, and rice, and its efficacy is being evaluated. In this article, we review basic research and technological aspects of the application of lactoferrin.     

Comparative sequence analysis of spa gene of Staphylococcus aureus isolated from bovine mastitis: characterization of an unusual spa gene variant

The protein A encoding gene spa of four Staphylococcus aureus strains isolated from bovine clinical mastitis was amplified by PCR and sequenced. The four strains were selected after an initial screening of spa gene of 41 strains isolated from mastitic cows and were subjected to detailed investigations. According to the sequencing results the spa gene of three strains (M1, M2, M3) appeared with gene segments encoding five (E, D, A, B and C) and four (E, A, B and C) IgG binding domains for two (M1, M3) and one (M2) strain, respectively and with gene segments encoding four, two and two repeats of the octapeptide Xr-repeats for the strains M1, M2 and M3, respectively. For the remaining Staph. aureus strain (M4) gene segments encoding IgG binding domains E, D and A and a new domain BC with a size of 219 bp could be observed. The BC domain appears, with a deletion of a 123 bp segment from the border region between both domains, as fused domain of both previously characterized domains. The Xr-region of this strain had 11 octapeptide repeats.     

Two apparent molecular forms of bovine lactoferrin

Bovine lactoferrin was prepared by CM-Sephadex column chromatography from defatted colostrum. When partially purified lactoferrin was analyzed by SDS-PAGE apparently two polypeptides of different size appeared. The polypeptides were transferred to a nitrocellulose sheet and visualized using antirabbit serum raised against the small polypeptide. Two polypeptides appeared clearly when stained by an immunological method. The color intensity of the two polypeptides was similar when the polypeptides were stained with Coomassie Brilliant Blue R-250. A high similarity of cyanogen bromide cleavage patterns between the two polypeptides was also observed in those stained with dye and immunologically visualized. Therefore, it is suggested that these polypeptides are lactoferrins. Comparison of the peptide patterns of the two lactoferrin molecules being deglycosylated suggested that sugar moieties may be one, but not all, of the causes of the size heterogeneity in lactoferrin. These results suggest that bovine colostrum contains two lactoferrin molecules of different size, although the physiological significance of the heterogeneity is not yet known.     

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk,rectal feces,and teat apices

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates (“IM” and “TA”) were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen–derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.     

Bovine Staphylococcus aureus mastitis: strain recognition and dynamics of infection.

Restriction enzyme fragmentation pattern (REFP) analysis was used to recognise Staphylococcus aureus strain variation in naturally occurring bovine subclinical mastitis. Multiple colony REFP analysis identified eight distinct strains of S. aureus in addition to the original strains A and B that were infused via the intramammary route, indicating that individual quarters of the udder may be colonised simultaneously by more than one strain of S. aureus. Examination of multiple colonies per milk sample may benefit bacterial strain recognition as an epidemiological tool in mastitis investigations. The dynamics of intramammary infection were determined using a novel double crossover experimental challenge. Quarters remained persistently infected for several weeks following challenge in all four cows, irrespective of the challenge strain. This indicated that no alteration of the original subclinical infection, including the possible induction of clearance of the quarters infected with S. aureus, or replacement of the original strain by the infused strain was induced by challenge. The persistent subclinical infection in all four animals supports previous reports on the chronicity of S. aureus intramammary infection in dairy cows.     

乳铁蛋白的分离及纯化

乳铁蛋白 (Lactoferrin ,简称Lf)作为一种糖蛋白 ,主要存在于哺乳动物的各种外分泌物中 ,如乳汁、眼泪等 ,乳汁尤其是初乳中Lf的含量很高[1] 。Lf是一种多功能蛋白质 ,不仅具有广谱的抗菌作用 ,而且还能够增强机体的抗病毒、抗感染、抗肿瘤的能力和免疫能力。因此 ,从来源丰富的牛初乳中提取出Lf,把它作为功能性成分应用于食品行业 ,具有广阔的应用和开发前景。本文通过盐析法和层析法从牛初乳中分离出乳铁蛋白 ,并应用酶联免疫法 (ELISA)检测不同分离纯化方法所获得的乳铁蛋白的含量 ,并用电泳分析其纯度。所获得的乳铁蛋白的分子质量为 780 0 0u ,其纯度大约为 92 %。     

Determination of lactoferrin in bovine milk,colostrum and infant formulas by optical biosensor analysis

An automated, rapid, sensitive and label-free biosensor-based immunoassay for lactoferrin in bovine milk utilising surface plasmon resonance (SPR) optical detection is described. Lactoferrin content is estimated from its specific interaction with an anti-bovine lactoferrin antibody immobilised on the sensor surface in a direct- binding assay format. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilisation, flow-rate, contact time and regeneration have been defined and non-specific binding considerations evaluated. Performance parameters include a working range of 0–1000 ng mL⁻¹, a method-detection limit of 19.9 μg mL⁻¹ in undiluted milk, overall instrument response RSD_R of 3.50%, a mean inter-assay RSD_R of 10.8% for milk and surface stability over ca 500 samples. The technique was applied to the measurement of lactoferrin content of consumer milks, colostrum and infant formulas, and temporal change during early bovine lactation.     

牛乳铁蛋白抗菌活性及其影响因素的研究

乳铁蛋白具有广谱的抗菌性 ,它通过直接性的“铁剥夺”和间接性的“膜渗透”2种机制发挥抗菌作用。文中开展了牛乳铁蛋白抗菌活性的研究 ,研究表明 ,牛乳铁蛋白对大肠杆菌的最小抑菌浓度为 2mg/mL ,对金黄色葡萄球菌的最小抑菌浓度为 1 6mg/mL。葡萄糖、蔗糖、麦芽糖、牛血清白蛋白 (BSA)、尿素、硫酸铵在 0～ 1 0mg/mL ,乳糖、可溶性淀粉在 0～ 4mg/mL ,酪蛋白在 0～ 1 2mg/mL的质量浓度范围内 ,对牛乳铁蛋白的抗菌活性影响不大。当NaCl或KCl的浓度为2 5～ 1 0 0mmol/L ,MgCl2 或CaCl2 的浓度为 1 0～ 5 0mmol/L时 ,牛乳铁蛋白的抗菌活性就会大大降低。随着缓冲盐浓度和有机酸浓度 (2 5～ 1 0 0mmol/L)的增大 ,牛乳铁蛋白的抗菌活性呈下降趋势 ,且在微碱性pH环境下牛乳铁蛋白表现出较强的抗菌活力。     

Inhibitory effects of human and bovine milk constituents on rotavirus infections

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.