Lactoferrin Concentration In Milk Research Articles

Effect of low milking frequency on the concentration of antimicrobial proteins in goat milk

This study examined the effects of low frequency milking on the concentrations of antimicrobial components in goat milk. Sixteen goats were divided into two groups of eight each: milking once every 2 d three times (for six days, three times group) or five times (for 10 days, five times group). On other days, milking was performed once daily. Milk was collected, and milk yield, somatic cell count (SCC), and the concentrations of some antimicrobial proteins such as lactoferrin (LF), S100A7, IgA, and sodium ions (Na+) in milk were measured. Milk yield significantly decreased in both the groups during the low-milking frequency period, followed by an increase above the low frequency milking period in both groups. In contrast, SCC and LF concentrations in milk increased in both groups during the low frequency milking period. The concentration of S100A7 in milk temporarily decreased after the low frequency milking period, followed by a significant increase. The S100A7 concentration during this period was higher in the five times group than in the three times group. These results indicated that low frequency milking induced a gradual decrease in milk yield and a concomitant increase in antimicrobial components, such as LF and S100A7, in milk. This increase in the antimicrobial components may be useful in preventing mastitis.

A common regulatory haplotype doubles lactoferrin concentration in milk

BackgroundBovine lactoferrin (Lf) is an iron absorbing whey protein with antibacterial, antiviral, and antifungal activity. Lactoferrin is economically valuable and has an extremely variable concentration in milk, partly driven by environmental influences such as milking frequency, involution, or mastitis. A significant genetic influence has also been previously observed to regulate lactoferrin content in milk. Here, we conducted genetic mapping of lactoferrin protein concentration in conjunction with RNA-seq, ChIP-seq, and ATAC-seq data to pinpoint candidate causative variants that regulate lactoferrin concentrations in milk.ResultsWe identified a highly-significant lactoferrin protein quantitative trait locus (pQTL), as well as a cislactotransferrin (LTF) expression QTL (cis-eQTL) mapping to the LTF locus. Using ChIP-seq and ATAC-seq datasets representing lactating mammary tissue samples, we also report a number of regions where the openness of chromatin is under genetic influence. Several of these also show highly significant QTL with genetic signatures similar to those highlighted through pQTL and eQTL analysis. By performing correlation analysis between these QTL, we revealed an ATAC-seq peak in the putative promotor region of LTF, that highlights a set of 115 high-frequency variants that are potentially responsible for these effects. One of the 115 variants (rs110000337), which maps within the ATAC-seq peak, was predicted to alter binding sites of transcription factors known to be involved in lactation-related pathways.ConclusionsHere, we report a regulatory haplotype of 115 variants with conspicuously large impacts on milk lactoferrin concentration. These findings could enable the selection of animals for high-producing specialist herds.

Concentrations of antimicrobial components in milk at dry off and postpartum and their relationships to new high somatic cell counts at quarter level in dairy cows.

We investigated the antimicrobial components in cow milk at dry off and postpartum and their contribution in preventing new high SCC at quarter level. Milk samples from 72 quarters of 19 lactating cows were collected at last milking before dry off and at 7 d after parturition. Milk yield of each cow was recorded and SCC, IgG, IgA, lactoferrin, lingual antimicrobial peptide (LAP), and S100A7 concentrations in each quarter milk sample were measured. The postpartum milk yield was significantly higher than that at dry off. The IgG, IgA and lactoferrin concentrations in milk at dry off were significantly higher than those at postpartum, whereas the LAP concentration was lower. Quarters with SCC < 300 000 cells/ml at both dry off and postpartum were classified as persistent low SCC (PL) whereas those that rose above that same threshold postpartum were classified as new high SCC (NH). At dry off, IgG and LAP concentrations in milk were significantly higher in PL than in NH. These results suggest that high LAP concentrations during the dry period may contribute toward the prevention of new high SCC.

Effects of twice a day teat bucket feeding compared to twice a day mother suckling on behaviour, health traits and blood immune parameters in dairy calves and immune parameters in cow's milk

Early separation of cow and calf is still common practice in dairy production systems, but interest in calf rearing with cow contact has been constantly increasing in recent years. We tested the hypothesis that calves suckling their mothers twice a day would benefit with regard to behaviour and health traits until four months of age, when compared to twice daily teat bucket fed calves, fed with comparable milk amounts. Moreover, the effect of calf contact beyond the colostrum period on immunoglobulin G (IgG) and lactoferrin content in cow’s milk was investigated in weeks 3, 8, 12 and 16 post partum. We conducted on-farm trials on two organic farms from 2018 to 2020, where we randomly assigned the new born calves by sex and parity status of the mother (primi- or multiparous) to either teat bucket feeding (BF, n = 30 cow-calf couples, 2 ×3–5 L/d warmed bulk milk) or mother suckling (MS, n = 28 cow-calf couples, 2 ×30 min contact after milking). MS calves performed less cross-sucking, but manipulations of objects did not differ between feeding treatments nor did avoidance distance towards humans. Clinical scores on vitality, body condition, indicators for diarrhoea and respiratory disorders, and number of medical treatments differed between farms, but not between feeding treatments. Lactate level (stress indicator) revealed contradictory results between farms (farm 1: BF>MS, farm 2: BF≤MS). Glucose content (indicator of energy level) and packed cell volume (low values indicating anaemia) were higher in MS compared to BF calves. No difference between feeding treatments was found with regard to the immune status indicators mean total protein and IgG content in calf serum. Consistently, average IgG content in cow’s milk did not differ between cows with or without calf contact and showed a clear decrease with time. The variability of lactoferrin content in milk was higher in cows with calf contact, but its average did not differ between feeding treatments nor did it change with time. We conclude that even part-time mother contact twice daily is a means to reduce abnormal behaviour, i.e. cross-sucking, as it satisfies sucking needs of calves better than teat bucket feeding. Although health traits did mostly not differ between feeding treatments, differences between farms underline the influence of management factors on calf health. Cow-calf contact twice daily had no effect on the build-up of the active immune defence until the age of four months.

Impact of maternal nutrition during early pregnancy and diet during lactation on lactoferrin in mature breast milk

ObjectiveLactoferrin supplementation is a promising strategy to prevent infections in neonates. Exploring whether maternal nutritional status in early pregnancy and maternal diet during lactation are associated with lactoferrin concentrations in mature human milk can provide early warning and allow timely adjustment. MethodsIn this follow-up cohort study, 206 participants were recruited at Peking University People's Hospital from June 2018 to June 2019. The levels of albumin and thyroid-stimulating hormones (TSH) were determined as nutritional indicators during early pregnancy. Information on maternal diet during lactation was collected with a semiquantitative food frequency questionnaire, and the lactoferrin concentrations in breast milk were examined at around 42 d postpartum. ResultsThe median level (interquartile range) of lactoferrin in breast milk was 2844.2 (2568.1, 3103.1) μg/mL. Overall, 5.5% of participants had lower albumin (<40 g/L), and 21.6% had elevated TSH (>2.5 mIU/L), respectively. The concentration of lactoferrin was higher (216.8 [13.4, 420.2] μg/mL) in women with lower albumin levels than in those with normal levels, and elevated TSH had no effect. A 1 g increase in egg intake led to a 0.3 (0.0, 0.6) μg/mL increase in lactoferrin concentration. Lactoferrin levels were also affected by intake of energy, protein, cholesterol, and vitamin A. ConclusionsWomen with lower albumin levels in early pregnancy had higher levels of lactoferrin in mature breast milk. TSH was not related to lactoferrin levels. Intake of energy, protein, cholesterol, and vitamin A may have contributed to lactoferrin concentrations in milk, and egg intake was positively associated with lactoferrin.

A comparison of 4 different machine learning algorithms to predict lactoferrin content in bovine milk from mid-infrared spectra

Lactoferrin (LF) is a glycoprotein naturally present in milk. Its content varies throughout lactation, but also with mastitis; therefore it is a potential additional indicator of udder health beyond somatic cell count. Condequently, there is an interest in quantifying this biomolecule routinely. First prediction equations proposed in the literature to predict the content in milk using milk mid-infrared spectrometry were built using partial least square regression (PLSR) due to the limited size of the data set. Thanks to a large data set, the current study aimed to test 4 different machine learning algorithms using a large data set comprising 6,619 records collected across different herds, breeds, and countries. The first algorithm was a PLSR, as used in past investigations. The second and third algorithms used partial least square (PLS) factors combined with a linear and polynomial support vector regression (PLS + SVR). The fourth algorithm also used PLS factors, but included in an artificial neural network with 1 hidden layer (PLS + ANN). The training and validation sets comprised 5,541 and 836 records, respectively. Even if the calibration prediction performances were the best for PLS + polynomial SVR, their validation prediction performances were the worst. The 3 other algorithms had similar validation performances. Indeed, the validation root mean squared error (RMSE) ranged between 162.17 and 166.75 mg/L of milk. However, the lower standard deviation of cross-validation RMSE and the better normality of the residual distribution observed for PLS + ANN suggest that this modeling was more suitable to predict the LF content in milk from milk mid-infrared spectra (R2v = 0.60 and validation RMSE = 162.17 mg/L of milk). This PLS +ANN model was then applied to almost 6 million spectral records. The predicted LF showed the expected relationships with milk yield, somatic cell score, somatic cell count, and stage of lactation. The model tended to underestimate high LF values (higher than 600 mg/L of milk). However, if the prediction threshold was set to 500 mg/L, 82% of samples from the validation having a content of LF higher than 600 mg/L were detected. Future research should aim to increase the number of those extremely high LF records in the calibration set.

Milk lactoferrin concentration of primiparous and multiparous sows during lactation

Lactoferrin (LF), a sialylated iron-binding glycoprotein, has numerous vital physiological functions including immunomodulation and protection against a large group of microorganisms, improving neurodevelopment, health, growth performance, and milk production. Lactoferrin occurs in human milk at a higher concentration compared with bovine milk, but little information is available on LF concentrations in porcine milk and the effects of sow parity on milk LF concentration. The objective of this study was to quantify the LF concentration in porcine milk and to compare that concentration between gilts and sows during lactation. We also investigated the effect of genetic background and litter size of the female pig on the LF concentration of porcine milk. The milk from 30 gilts and 35 sows was collected at 3 stages of lactation, namely colostrum, transition, and mature milk. Standard and experimental samples were analyzed by ultra-high performance liquid chromatography using a diode array UV detector. The following findings were reported: (1) porcine milk contained significant levels of LF with the highest concentration in colostrum, which decreased by ∼62% and ∼67% in transitional and mature milk, respectively; (2) mature gilt milk contained a 22% higher concentration of LF compared with sow milk, which was statistically significant; (3) breed line had an overall significant effect on the LF content of porcine milk; however, when the breed was considered, no significant difference was observed; and (4) LF concentration of porcine milk was not significantly influenced by the litter size. The presence of LF in a higher concentration in porcine milk suggests that LF is an important constituent of pig milk that might contribute to the optimum growth and development of piglets.

Breast Milk and Saliva Lactoferrin Levels and Postnatal Cytomegalovirus Infection.

Very low birth weight preterm infants are at risk for life-threatening infections in the NICU. Breast milk protects against infections but carries the risk of infection by cytomegalovirus (CMV) shed in mother's milk. Lactoferrin is a breast milk and saliva protein with potent neutralizing activity against CMV. VLBW, maternal breast milk fed infants in the NICU and their lactating mothers were enrolled and followed for 3 months/discharge. Breast milk and infant saliva samples were collected biweekly. Maternal CMV status was determined on breast milk. CMV was measured using quantitative polymerase chain reaction and lactoferrin by enzyme-linked immunosorbent assay. In an in vitro neutralization assay, the IC90 of purified human lactoferrin against CMV was 2.08 ng/mL. Bovine lactoferrins were more potent, IC90s > 10-fold higher. Lactoferrin was detected in all breast milk (median: 3.3 × 106 ng/mL) and saliva (median: 84.4 ng/swab) samples. Median CMV load in breast milk was 893 copies/mL. There was no correlation between breast milk lactoferrin concentration and CMV load. Five infants acquired postnatal CMV. There was no difference in saliva or breast milk lactoferrin concentration for mother-infant pairs and postnatal CMV acquisition. Lactoferrin neutralizes CMV in vitro, but concentrations in breast milk and saliva are likely too low for effective neutralization in vivo.

Lactoferrin concentration and expression in New Zealand cows milked once or twice a day.

This study evaluated the concentration and expression of lactoferrin (LF) in cows selected for once a day (OAD) milking compared to twice a day (TAD) milking. Milk samples were collected from the Massey University TAD and OAD herds. Milk traits and expression of LF and insulin-like growth factor 1 (IGF-1) were analyzed with a general linear model that included the fixed effects of milking frequency, lactation number, interaction between milking frequency and lactation number, and as covariates proportion of F, heterosis F×J and deviation from the herd median calving date. Cows milked OAD produced milk with higher (p<.01) concentrations of protein and lactose than TAD milked cows. Compared to TAD cows, cows milked OAD had higher expression of the LF gene (1.40 vs. 1.29 folds, p=.03) and the IGF-1 gene (1.69 vs. 1.48 folds, p=.007). The correlation between the expression of LF gene and the concentration of LF in milk was strong (r=.66 p<.001), but the correlation between the expression of the IGF-1 gene and LF concentration was stronger (r=.94, p<.001). These results suggest that milking frequency affects the milk composition and expression of milk composition genes at early lactation.

Milk lactoferrin and milk constituents in dairy Gyr heifers

The objective of this study was to describe the variation in bovine lactoferrin (LF) content in milk from dairy Gyr heifers during lactation and to evaluate its relationship with milk components. Milk samples were collected between August to October 2013 for performance test 1 (25 first lactation cows) and August to October 2014 for performance test 2 (20 first lactation cows), coordinated by the Brazilian Association of dairy Gyr Breeders (ABCGIL), in Uberaba, MG, Brazil. Milk yield (MY) of 45 primiparous cows was measured daily. The milk samples for analysis of LF concentration and of the other milk components such as somatic cell count and percentage of fat (F%), protein (P%), lactose (L%) and total solids (TS) were collected monthly. The somatic cell count (SCC) was transformed to a somatic cell score (SCS). Pearson correlation coefficients between LF and the other milk components were calculated. Analyses of variance were performed by mixed models including the fixed effects of test (1 = calving 2013 and 2 = calving 2014) year-season of sampling, lactation month and age of cow at calving as classificatory effects, and the effects of SCS and MY as covariates (linear effect). The effect of cow was analyzed as a random effect considering repeated measures over time. The mean LF concentration was 16.80 ± 12.41 μg/mL milk and the values showed variation across lactation indicating that the lactation month influences milk LF concentration. Lactoferrin content was correlated with SCS (0.64, P < 0.01). The correlation between LF and MY was −0.47 (P < 0.01) and between LF and P% was 0.47 (P < 0.01). The trends of the LF and SCS mean curves were similar, while opposite trends were observed between LF and MY mean curves. The concentration of LF in milk from primiparous dairy Gyr cows showed a strong association with MY level and udder health.

Phenotypic investigation of fine milk components in bovine milk and their prediction using mid-infrared spectroscopy

This study aimed to examine the phenotypic variation observed in fine milk components, the use of mid-infrared (MIR) spectroscopy to predict these components, and the correlations with other milk production traits. A total of 4116 milk samples were collected from 414 dairy cows from 44 Canadian herds. Overall 3117 samples were analyzed for casein micelle size, 986 and 937 for total and soluble calcium, respectively, and 2054 for lactoferrin. Individual milk MIR spectra were obtained. Linear mixed models with repeated records were used to test the effects of herd nested within breed, breed, days in milk class, parity, and season on milk components. Lactoferrin content in milk increased with stage of lactation and parity number. Prediction equations were developed by partial least squares regression to predict each milk component using MIR spectroscopy, with the greatest coefficient of determination achieved for lactoferrin (0.55). Pearson correlation coefficients for lactoferrin with somatic cell score and protein percentage were 0.49% and 0.40%, respectively. The estimated correlation increased to 0.53% for predicted lactoferrin and protein percentage. The examined fine milk components exhibited variation, but low accuracies for their MIR prediction may limit the utility of this technology for their phenotyping.

Variation in lactoferrin gene affects milk lactoferrin content and somatic cell count in murrah buffaloes

Lactoferrin plays an important role in antimicrobial defence and is a potential candidate gene for mastitis resistance. In the present investigation, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) studies were carried out in Murrah (Bubalus bubalis) buffaloes to detect single nucleotide polymorphisms (SNPs) of lactoferrin gene and to analyse the association between the observed polymorphisms with milk lactoferrin content and Somatic cell count (SCC). PCR-SSCP analysis revealed a total of 07 different variants in the partial coding region of the lactoferrin gene. PCR-SSCP analysis of exon 10 of lactoferrin gene revealed three and that of exons 4 and 5 revealed two unique patterns each, while all other exons exhibited monomorphic pattern. Comparison of nucleotide sequences of lactoferrin gene of the Murrah buffaloes with taurine reference sequence revealed a total of 14 point mutations, 09 of which were found to be in coding region. Conceptualized translation of nucleotide sequence revealed 07 amino acid changes. SSCP variants of exon 10 (P less than 0.01) had significant effect on milk SCC. The SSCP variants of exon 4 and exon 5 had significant (P less than 0.05) effect on lactoferrin content. The SCC and lactoferrin content in Murrah buffaloes was highest in 4th and above parity group. Stage of lactation had highly significant (P less than 0.01) effect on both milk SCC and lactoferrin content. There was a high and significant (p less than 0.01) correlation (0.741) between SCC and lactoferrin content in milk. The observed association between SSCP variants in lactoferrin gene with milk SCC and milk lactoferrin content can be used as prognostic markers for selection of animals for high lactoferrin content and low somatic cell count, as well as a marker of susceptibility/resistance to mastitis in Murrah buffaloes

Evaluation of microbiological, cellular and risk factors associated with subclinical mastitis in female buffaloes

ObjectiveThis study aimed to evaluate the microbiological and cellular milk profile for the diagnosis of subclinical mastitis in female buffaloes and to assess risk factors for predisposition of the disease.MethodsAnalyses were carried out by standard plate count (SPC), identification of species and antibiotic resistance, somatic cell count (SCC), electrical electrical conductivity of milk (ECM), and lactoferrin content in milk. Teat cups were swabbed to evaluate risk factors, observing hyperkeratosis, milking vacuum pressure and cleanliness of the site. Hence, 30 female buffaloes were randomly selected (15 from a group in early lactation and 15 in late lactation).ResultsThe most common bacteria in the microbiological examination were Staphylococcus spp., Streptococcus spp. and Corynebacterium sp. In the antibiotic sensitivity test, 10 (58.82%) of the 17 antibiotics tested were sensitive to all isolates, and resistant bacteria were Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus haemolyticus, and Escherichia coli. It was observed that positive samples in the microbiological examination showed total bacterial count between 9.10×103 to 6.94×106 colony forming units/mL, SCC between 42,000 to 4,320,000 cells/mL and ECM ranging from 1.85 to 7.40 mS/cm. It was also found that the teat cups had high microbial counts indicating poor hygiene, and even faults in the cleanliness of the animals’ waiting room were observed. It is concluded that values of SCC above 537,000 cells/mL and ECM above 3.0 mS/mL are indications of mammary gland infection for this herd; however, the association of these values with a microbiological analysis is necessary to more accurately evaluate the health status of mammary glands with subclinical mastitis.ConclusionThrough phenotypic characterization of bacteria involved in the samples, the genera Staphylococcus spp., Streptococcus spp., and Corynebacterimum bovis were the most prevalent in this study. Faults in environment and equipment hygienization are factors that are directly associated with mastitis.

High Levels of Chemokine C-C Motif Ligand 20 in Human Milk and Its Production by Oral Keratinocytes.

Chemokine C-C motif ligand 20 (CCL20) is implicated in the formation and function of mucosal lymphoid tissues. Although CCL20 is secreted by many normal human tissues, no studies have evaluated the presence of CCL20 in human milk or its production by oral keratinocytes stimulated by human milk. To evaluate the presence of CCL20 in breast milk and verify CCL20 secretion in vitro by oral keratinocytes stimulated with human and bovine milk, as well as its possible association with breast milk lactoferrin levels. The levels of CCL20 and lactoferrin were measured by enzyme-linked immunosorbent assay in human milk at three different stages of maturation from 74 healthy breastfeeding mothers. In vitro, oral keratinocytes were stimulated with human and bovine milk, and CCL20 was measured in their supernatant. High concentrations of CCL20 were detected in the human breast milk samples obtained during the first week (1,777.07 pg/mL) and second week postpartum (1,523.44 pg/mL), with a significantly low concentration in samples at 3-6 weeks postpartum (238.42 pg/mL; p < 0.0001). Human breast milk at different weeks postpartum stimulated higher CCL20 secretion by oral keratinocytes compared with bovine milk (p < 0.05). Such stimulation had no association with breast milk lactoferrin concentration. CCl20 is present at high levels in human milk, predominantly in the first and second week postpartum, but at significantly lower levels at 3-6 weeks postpartum. Human milk is capable of stimulating CCL20 secretion by oral keratinocytes, and this induction had no association with breast milk lactoferrin concentration.

Lactoferrin and Immunoglobulin G Concentration in Bovine Milk from Cows with Subclinical Mastitis during the Late Lactation Period

Background: Lactoferrin and immunoglobulin G in milk have an important role in udder resistance to infection in the involution period. Both proteins express antimicrobial activity- lactoferrin by the binding and sequestration of iron ion; and immunoglobulin G by complement activation, bacterial opsonization and agglutination. Many factors affect lactoferrin and immunoglobulin G concentrations in bovine milk, such as the stage of lactation, milk production, and intramammary infections. The aim of this study was to determine concentrations of lactoferrin and immunoglobulin G in milk from healthy cows and subclinical mastitic cows during the late lactation period, and to evaluate the relationship between them.Materials, Methods &amp; Results: A total of 150 quarter milk samples from 41 cows (Holstein-Friesian breed) in late lactation period were reviewed in this study. Milk samples were collected during morning milking, using aseptic techniques in sterile test tubes. From each sample, 0.1 mL of milk was plated on Columbia blood agar base with 5% defibrinated ovine blood, MacConkey agar and Sabouraud dextrose agar and incubated for 24 h - 48 h (bacteria) and 5 days (yeasts, mould) at 37oC. Milk samples for detection lactoferrin and immunoglobulin G concentration were skimmed at 1,400 g for 45 min and stored at -20°C until analysis. Lactoferrin concentration in bovine milk was determined using the Bovine Lactoferrin ELISA Quantitation Set. Milk samples were diluted at a ratio of 1:10,000. Plates were read at 450 nm absorbence values. Immunoglobulin G concentration was determined by the immunodiffusion method using radial immunodiffusion (RID) plates. Milk samples were diluted in a ratio of 1:30. Reading of results was done after incubation for 48 h by measuring the diameter of the precipitation ring. The highest mean lactoferrin concentration was observed in udder quarters infected with contagious pathogens (Streptococcus agalactiae and Staphylococcus aureus), while the highest mean immunoglobulin G concentration was detected in milk samples where minor mastitis pathogens (coagulase-negative staphylococci and Corynebacterium spp.) were isolated. Milk samples where Staphylococcus aureus was isolated had the lowest immunoglobulin G concentration, and the lowest lactoferrin concentration was observed in samples infected with enviromental pathogens (Streptococcus dysgalactiae).Discussion: This study showed that lactoferrin and immunoglobulin G concentrations are higher in milk samples from subclinical mastitic cows than in milk from normal lactating cows. Lactoferrin concentrations in milk samples from udder quarters infected with major mastitis pathogens were significantly higher than in milk infected with minor mastitis pathogens. The lowest concentration of immunoglobulin G was detected in milk samples where Staphylococcus aureus was isolated, while the highest immunoglobulin G concentration was observed in milk samples from quarters infected with minor mastitis pathogens. Lactoferrin and immunoglobulin G concentrations were significantly and positively correlated in all milk samples. This means that cows with high lactoferrin concentrations have high immunoglobulin G concentrations. In quarter milk samples infected with Staphylococcus aureus, lactoferrin and immunoglobulin G concentrations were negatively correlated. The cause of these findings could be the suppression of local immune response of mammary gland.

Change in viable bacterial count during preservation of milk derived from dairy cows with subclinical mastitis and its relationship with antimicrobial components in milk.

The objectives of the present study were to investigate the change in the number of viable pathogens during preservation of milk obtained from cows with subclinical mastitis and the association between the decreasing ratio of viable bacteria during preservation and the somatic cell count (SCC) and the values of lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO). After preservation of milk at room temperature for 0, 0.5, 1, 2, 3, 4 and 5 hr, the bacterial colonies in the milk were counted to determine the number of colony forming units (CFUs). Fresh skim milk was used to determine the values of LAP, LPO and LF. Bacteria were not detected in 19.4% of milk samples, and this percentage increased up to 30% after 5 hr of preservation. The number of Staphylococcus aureus and Streptococcus uberis in milk did not change significantly during the 5-hr incubation, whereas significant decreases were observed in the number of coliforms, coagulase-negative staphylococci, yeasts and Corynebacterium bovis. High SCC significantly decreased CFUs of S. aureus and yeast after preservation of milk for 4 to 5 hr. High LF concentration in milk was associated with decrease in CFU of S. aureus during 4-hr preservation. These results suggest that the viable counts of some pathogens in milk decreased during preservation at room temperature after collection, which may be attributed to the leukocytes and antimicrobial components present in milk.

Suspension of milking in dairy cows produces a transient increase in milk lactoferrin concentration and yield after resumption of milking

Lactoferrin is a multifunctional glycoprotein with a range of antimicrobial and immune-related properties that is found at >10-fold higher concentration in human milk (~1.7g/L) relative to bovine milk (~0.15g/L). Consumer demand is increasing for bovine lactoferrin through a wide range of nutritional and cosmetic consumer products. Increasing lactoferrin yield and concentration in bovine milk could assist in satisfying this increasing demand and may also help in increasing resistance to bovine mammary infection. Two experiments with cows in mid and late lactation were carried out to examine milking strategies to increase milk lactoferrin concentration and yield. Milking was suspended in cows normally milked twice daily, for periods of 2, 4, or 7d (mid lactation) or 2 or 4d (late lactation) after which cows were milked out and twice-daily milking resumed for 4d. In all groups, lactoferrin concentration was significantly increased during the remilking period, approaching concentrations similar to those found in human milk (~1g/L). Lactoferrin yields were significantly higher in all treatment groups, although increasing the nonmilking period beyond 2d offered no advantage. Milk yield was lower initially after resumption of milking but recovered to preexperimental values by the fourth day of remilking in all groups, except the 4-d nonmilking group in late lactation. Milk somatic cell count was significantly elevated in all groups at the start of remilking but had substantially reduced by d 4 and reached a preexperimental level in the 2-d nonmilking group of mid-lactation cows. In summary, extended milking intervals can be used as a tool to produce a short-term increase in the concentration and yield of lactoferrin from bovine milk during established lactation, without any apparent long-term effects on milk yield and quality.

Association of polymorphism within LTF gene promoter with lactoferrin concentration in milk of Holstein cows.

This study analyzed the association between single nucleotide polymorphism (A/C) in position -28 located in the TATA box of LTF gene and the lactoferrin concentration in bovine milk secreted by healthy and infected udders. Out of 241, 69 cows were selected into the experimental group and were divided into 3 groups according to mean value of somatic cell count (SCC): I < 180,000 cells/mL, II: 180,000-350,000 cells/mL and III > 350,000 cells/mL. In each SCC group, three LTF genotypes: AA, AC and CC were identified by PCR-SSCP method. A total of 604 milk samples were collected monthly and lactoferrin concentration was measured by ELISA. The 1-way ANOVA within SCC groups was performed to estimate association of -28 A/C genotypes with mean lactoferrin concentration per lactation. In the group of healthy cows (< 180,000 cells/mL) LTF concentration in milk cows with the AA genotype (107.58 ± 17.92 μg/mL) was significantly higher than in homozygotes CC (52.09 ± 19.01 μg/mL). Unexpectedly, in cows with elevated SCC (> 350,000 cells/mL) we observed a significant opposite relationship (207.21 ± 28.50 in CC vs 115.0 ± 28.6 μg/mL in AA). We hypothesized that a promoter with allele C, which cannot be recognized as a TATA sequence is becoming more accessible for other transcription factors, which may induce alternative LTF gene expression. We assume that our results demonstrate a very interesting effect of differential gene expression depending on polymorphism in a key regulatory motif (TATA box) and also on the health status of mammary tissues.

Structural heterogeneity and multifunctionality of lactoferrin.

Lactoferrin or lactotransferrin is a multifunctional glycoprotein found in blood circulation, mucosal surfaces, neutrophils, and in various secretory fluids, such as milk, bile, tears, nasal secretion, pancreatic juice, and saliva. The lactoferrin content in milk varies between different mammalian species and, within one species, between lactation periods. Although lactoferrin is known to be involved with immunoprotection, its functions are not limited to the regulation of innate immunity, but extend to iron transfer to cells, control of the level of free iron in blood and external secretions, interaction with DNA, RNA, heparin, and polysaccharides, and pronounced antimicrobial and antiviral activities. This multifunctionality is determined by the fact that lactoferrin belongs to the class of hybrid proteins possessing both ordered domains and functionally important intrinsically disordered regions. Structurally, lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa consisting of two homologous domains known as N-terminal and C-terminal lobes. These lobes are unevenly glycosylated (with the C-lobe typically containing more N-linked glycosylation sites). Each lobe can bind a single ferric ion concomitantly with one bicarbonate anion. Lactoferrin and its lobes have a wide spectrum of antimicrobial and antiviral activities, with the antimicrobial and antiviral potentials dependent on the type of microbes and viruses. Often, the N-lobe possesses the majority of antimicrobial activities. In addition, lactoferrin and its lobes possess clear anti-cancer, wound healing, anti-inflammatory, and immunomodulation activities.

Lactoferrin gene variants, their expression in the udder and mastitis susceptibility in dairy cattle

Lactoferrin gene (LF) is regarded as one of the potential markers of mastitis susceptibility/resistance in dairy cattle. The study’s aim was therefore, to investigate the feasibility of two single nucleotide polymorphisms (SNP), placed in the 5′-flanking region and 3′-untranslated region of the LF gene, to serve as mastitis markers. The associations between these SNP and the expression of LF, both on mRNA and protein level, were estimated in the milk of Polish Holstein-Friesian cows. The relationships between polymorphisms and cows’ estimated breeding values (EBV) for somatic cell count were also calculated. It was shown that both polymorphisms have a significant impact on lactoferrin content in milk, and that LF+32 SNP is associated with the cow’s EBV for somatic cell count. No association between SNP chosen for the study and lactoferrin mRNA abundance in milk somatic cells was observed. We propose LF+32 SNP for a molecular marker of mastitis resistance in dairy cows.