Concentration of lactoferrin and transferrin throughout lactation in cow's colostrum and milk

The evolution of the concentration of lactoferrin and transferrin was studied in cow's colostrum and milk throughout lactation. The highest concentrations of both proteins were found in the first milking (0.83 mg/ml for lactoferrin and 1.07 mg/ml for transferrin), decreasing sharply during the first days of lactation (colostral period). Thereafter, the concentrations of these proteins decreased slowly, reaching their definitive values during the third week post-partum (0.09 mg/ml for lactoferrin and 0.02 mg/ml for transferrin). The ratio transferrin/albumin in colostrum (first milking), mature milk, milk from mastitic cows and serum was determined, and found to be four times greater in colostrum than in mature or mastitic milk, suggesting a specific transport of transferrin from blood to milk.     

Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.     

The release of bradykinin in bovine mastitis.

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain and oedema. Despite early reports of the presence of kinins in milk, no previous study has investigated the role of the kinin system in bovine mastitis. The present study indicated that mastitis was accompanied by raised levels of bradykinin (BK) in milk and the increased levels of BK correlated with the severity of mastitis. Raised BK levels in mastitic milk were not dependent on the presence of inflammatory cells, nor were they secondary to changes in blood levels of BK. In milk from sub-clinically inflamed quarters, BK was raised in those milks where Staphylococcus aureus (S. aureus) was isolated but not in those milks where no pathogen was isolated. Increasing S. aureus artificially, also caused an increase in the milk BK. Increases in milk BK were not restricted only to the mastitic quarters of the udder. In udders in which mastitis was detected in one or more quarters, BK increases were also detected in the apparently uninvolved quarters.     

Presence of CTAK/CCL27, MCP-3/CCL7 and LIF in human colostrum and breast milk

Human colostrum and breast milk are known to contain high levels of cytokines and chemokines, which are thought to contribute to the development of the newborn. The aim of this study was to investigate the difference in the presence and levels of 21 soluble cytokines and chemokines in paired samples of human colostrum (day 2 after delivery) and breast milk (day 4–5 after delivery) by using the multiplex technology. Of the 21 cytokine investigated in 10 pairs of samples, only β-NGF was absent in both colostrum and milk, while INF-α2, SCF and TNF-β were present in colostrum but not in human milk. As a general rule, colostrum contained higher concentrations of cytokines and chemokines with respect to breast milk. The majority of cytokines, detected in colostrum alone or in colostrum and human milk (IL-1α, IL-2Rα, IL-3, IL-16, IL-18, GRO-α, HGF, IFN-α2, M-CSF, MIF, MIG, TNF-β, SDF-1α, TRAIL) have been described in previous studies, while for the first time we describe the presence of additional cytokines either in colostrum alone (SCF) or in both colostrum and breast milk (CTAK/CCL27, MCP-3/CCL7, LIF). Our data confirm and expand previous studies showing that some cytokines/chemokines, which might contribute to the development of the gastro-intestinal and nervous systems, are overexpressed in human colostrum and breast milk, and might contribute to the development of these systems.     

The serum-free growth of cultured cells in bovine colostrum and in milk obtained later in the lactation period

Seven established cell lines, including both epithelial cells and fibroblasts (MDCK, Vero, CV-1, NRK, 3T3, F2408, and NIL8) and four early passage cell strains (bovine articular chondrocytes, bovine smooth muscle cells, human foreskin fibroblasts, and rat embryo cells) were cultured in serum-free medium supplemented with milk obtained 1 day after birth (colostrum) or 80 days after birth (older milk). MDCK, Vero, CV-1, NRK, and 3T3 grew readily in colostrum and attained saturation densities ranging from 22% to 63% of that in serum. There was no growth of F2408, NIL8, or the early passage strains in bovine colostrum. None of the 11 cell cultures grew in older milk. The temporal dependence of growth in milk was examined in detail using MDCK cells. Growth equivalent to that in serum occurred in 3% colostrum and in 15% milk obtained 2 days after birth. Milk obtained 3 days and 10 days after birth was not effective as a growth supplement for MDCK cells at any concentration. Those cells, unable to grow in colostrum or in older milk, could be induced to grow if culture dishes were precoated with fibronectin. In addition to fibronectin, it was necessary in some cultures to supplement colostrum or older milk with insulin and/or transferrin in order to achieve growth. In the presence of fibronectin and appropriate factors, the final saturation density attained in colostrum or older milk ranged from 25% to 100% of that in serum. The fibronectin contents of bovine colostrum and milk were determined. The fibronectin level of colostrum was found to be approximately 5% of bovine serum. There was no detectable fibronectin in the 80-day-old milk.     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

Activation of Plasmin in Mastitic Milk

Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Bactericidal effect of bovine normal and immune serum, colostrum and milk against Helicobacter pylori

Serum and colostrum but not post-colostral milk from non-immunized Friesian cows was found highly bactericidal for Helicobacter pylori NCTC 11637. This bactericidal activity was destroyed by heating at 56°C for 30 min and restroed by the addition of fetal calf serum as a source of complement, indicating that the bactericidal effect was probably dependent on an antibody-complement system. Systemic, serial immunization of non-lactating, pregnant cows with H. pylori resulted in high specific antibody titres in serum and colostrum. No titres were found in post-colostral milk, even after booster-immunization during lactation. Immunization did not enhance the bactericidal activity of serum and colostrum, but increased it in post-colostral milk. The bactericidal activity was not correlated with titres of specific antibody or with IgG concentrations.     

Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.     

Bacteriological and haematological studies of clinical mastitis in goats in Ibadan, OYO State, Nigeria

An investigation was undertaken to identify the bacteria from clinical cases of caprine mastitis in Ibadan and to compare the haematological values of mastitic and non-mastitic does. Bacteriological examination of milk samples from 35 clinically affected udders was performed. A total of 53 bacterial isolates belonging to seven bacteriological genera, were recovered from the milk of the mastitic does examined. The coagulase-negative staphylococci (Staphylococcus epidermidis) were the most common pathogen in this study with an incidence of (50.9%); followed by Escherichia coli with (15.1%) incidence, while Streptococcus species and Bacillus cereus had an incidence of 9.4 and 7.5%, respectively. However, Mannhiemia (P.) haemolytica, Corynebacterium species and Klebsiella pneumoniae showed an incidence of 5.7%, respectively. The profile of bacteria encountered in this investigation are among the pathogen that have been incriminated as sources of contamination of fresh milk meant for human consumption and are therefore of public health importance.

The haematological values of mastitic and non-mastitic does were compared statistically. The erythrocytic values of mastitic does were significantly (P < 0.05) lower than that of the non-mastitic does. However, the total and differential leucocyte counts of the mastitic does were significant (P < 0.05) higher than that of the non-mastitic does. The full haematological counts are true reflection of the health status of an animal. Total and differential leucocyte counts in lactating does are true reflection of mastitis and will even assist the clinicians in predicting the prognosis of mastitic animals.     

Leptin in bovine colostrum and milk.

We studied leptin content in bovine colostrum, milk and plasma during the first month of lactation, and investigated relationships between selected milk components and milk leptin in five multiparous dairy cows. Colostrum/milk yield and composition were measured on days 0, 10, 20, and 30 of lactation. Leptin was assayed using a multi-species leptin RIA kit. Leptin concentration was 56 % lower in mature milk (day 10) than colostrum (13.90 vs. 6.14 microg/l; p < 0.001), but remained steady over the twenty days afterwards. Daily secretion of leptin into mature milk was 28 % lower than into colostrum (173.2 microg/d vs. 220.0 microg/d; p = 0.09) notwithstanding an 80 % increase in production. Colostrum and milk leptin levels correlated with fat (0.90; p < 0.001) and choline phospholipid (0.76; p < 0.05). Plasma and milk leptin decreased during the first month, but remained higher in milk, and highest in colostrum. Thus, leptin is present in large quantities in colostrum, less so and more variably in untreated milk, and is likely to be decreased in skimmed milk. These findings have implications for the use of untreated milk and colostrum-based (functional) food products.     

研究: 奶牛与水牛初乳中乳寡糖组分比较研究

乳寡糖是由乳汁中含量丰富的固体物质组成．研究结果表明,乳寡糖有提高免疫、益生元及抗感染等作用,已发现与婴儿肠道发育、神经智力发育等多方面关系密切．水牛奶是除牛奶外的第二大奶源,国际上公认其为营养含量高、口感好的优质乳制品,但目前针对水牛乳寡糖的研究多以美洲水牛为研究对象,尚无中国水牛的相关研究．本研究利用固相萃取对已脱脂和除去蛋白质的广西水牛初乳乳汁样品进行纯化,并采用苯胺 (aniline,Bn)衍生化试剂对其进行衍生化处理,通过UPLC-ESI-Q-TOF-MS液相质谱进行优化后,对水牛初乳中的寡糖组分进行测定并与牛乳进行了对比,最终测得奶牛初乳中19种及水牛初乳中的9种乳寡糖组分,并对二者的种类及含量进行比较,发现在两种初乳的乳寡糖中,中性糖二糖m/z 385.15和中性糖三糖m/z 547.21以及酸性糖m/z 635.23均为其主要寡糖成分,与其他乳寡糖相比含量相对较高．总体而言水牛初乳中的中性寡糖占比比奶牛初乳高,二者中性糖占乳寡糖总量的比例分别为88.88%和63.16%．     

Estimation of Mineral and Trace Element Profile in Bubaline Milk Affected with Subclinical Mastitis

The milk samples from buffaloes of Murrah breed at mid lactation stage, reared at an organised dairy farm, were screened for subclinical mastitis based on bacteriological examination and somatic cell count following International Dairy Federation criteria. Milk samples from subclinical mastitis infected and healthy buffaloes were analysed to evaluate physicochemical alterations in terms of protein, fat, pH, electrical conductivity, chloride, minerals (sodium, potassium and calcium) and trace elements (iron, zinc, copper and selenium). In the present study, protein, fat, zinc, iron, calcium and selenium content was significantly lower (P < 0.001), while pH and electrical conductivity were significantly higher in mastitic milk as compared to normal milk. Concentration of electrolytes mainly sodium and chloride significantly increased with higher somatic cell count in mastitic milk and to maintain osmolality; potassium levels decreased proportionately. Correlation matrix revealed significantly positive interdependences of somatic cell count with pH, electrical conductivity, sodium and chloride. However, protein, fat, calcium and potassium were correlated negatively with elevated somatic cell count in mastitic milk. It is concluded that udder infections resulting in elevated somatic cells may alter the mineral and trace element profile of milk, and magnitude of changes may have diagnostic and prognostic value.     

Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.     

Lipid composition and vitamin E content in human colostrum and mature milk

Lipidic components, as well as fatty acid composition and vitamin E content were determined in colostrum (days 3-5 of postpartum) and mature milk (day 21) in 8 women from Murcia (Spain). Triglycerides concentration was higher and cholesterol and esterified cholesterol were lower in mature milk than in colostrum, whereas phospholipid content was similar. These differences indicate that the diameter of milk fat globules increases in mature milk. The percentage of medium-chain fatty acids (12:0 and 14:0) increased in mature milk as compared to colostrum, reflecting de novo synthesis of fatty acids. With the only exception of stearic acid which was lower in mature milk than in colostrum, the remaining long-chain fatty acids was similar. The proportion of both linoleic (18:2) and eicosapentaenoic (20:5) acids found in mature milk and colostrum is higher than in studies from other countries and may reflect the intake of high proportions of polyunsaturated fat from vegetable oils and fish in the studied women. Both vitamin E content and vitamin E/linoleic acid ratio in mature milk are lower than in colostrum, evidencing the efficient mechanism of mammary gland vitamin E uptake around parturition.     

Nutrient composition and concentrations of immunoglobulins in milk of sows supplemented with L-carnitine

Recent studies have shown that L-carnitine supplementation of sows increases growth of their piglets during the suckling period. In this study, the composition of the milk of sows supplemented with L-carnitine was determined to find out whether an altered milk composition could account for the increased growth rates of the piglets. Milk of 13 control sows and 14 sows supplemented with L-carnitine (125 mg/d during pregnancy, 250 mg/d during lactation) was collected 5-8 h after birth (colostrum) and on days 10 and 20 of lactation. Concentrations of fat and lactose and the energy content in milk at day 10 and 20 did not differ between both groups of sows. Sows supplemented with L-carnitine had a higher concentration of protein in colostrum (p < 0.05) while concentrations of fat, lactose, immunoglobulins G, M and A as well as the energy content in colostrum did not differ between both groups of sows. These findings show that milk composition does not play a major role for the increased postnatal growth of piglets from sows supplemented with L-carnitine observed in recent studies.     

Human colostrum: identification of minor proteins in the aqueous phase by proteomics

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.     

Histochemical Observations on Mycoplasma after Staining with Acridine Orange

Mycoplasma colonies and Mycoplasma cells in preparations from infected milk and lymph nodes were observed for their fluorescent qualities after treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or red-orange. When treated after exposure to ribonuclease, the colonies fluoresced lime-green. There was no fluorescence when both ribonucleic acid and deoxyribonucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears of mastitic milk or smears of infected lymph nodes was not definitive because of the large amount of nonspecific ribonucleic acid-rich material present during inflammatory reactions.