Kerst Stelwagen

Immune components of bovine colostrum and milk

Colostrum and milk provide a complete diet for the neonate. In ruminants, colostrum is also the sole source of initial acquired immunity for the offspring. Milk therefore plays an important role in mammalian host defense. In colostrum, the concentration of immunoglobulins is particularly high, with IgG being the major immunoglobulin class present in ruminant milk, in contrast to IgA being the major immunoglobulin present in human milk. Immunoglobulins are transported into mammary secretions via specialized receptors. In addition to immunoglobulins, both colostrum and milk contain viable cells, including neutrophils and macrophages, which secrete a range of immune-related components into milk. These include cytokines and antimicrobial proteins and peptides, such as lactoferrin, defensins, and cathelicidins. Mammary epithelial cells themselves also contribute to the host defense by secreting a range of innate immune effector molecules. A detailed understanding of these proteins and peptides offers great potential to add value to the dairy industry. This is demonstrated by the wide-ranging commercial applications of lactoferrin derived from bovine milk. Knowledge of the immune function of milk, in particular, how the gland responds to pathogens, can be used to boost the concentrations of immune factors in milk through farm management practices and vaccination protocols. The latter approach is currently being used to maximize yields of bovine milk-derived IgA directed at specific antigens for therapeutic and prophylactic use. Increasingly sophisticated proteomics technologies are being applied to identify and characterize the functions of the minor components of milk. An overview is presented of the immune factors in colostrum and milk as well as the results of research aimed at realizing this untapped value in milk.

Regulation of yield loss and milk composition during once-daily milking: a review

Abstract Once-daily milking (ODM) of ruminants results in loss of milk production. In cows this loss is very variable between individuals but in recent short-term trials, on average, the loss was 13%. Full lactation studies indicate losses of 35–50% but documentation from some commercial farms using ODM suggests that losses are lower. ODM also results in changes in mammary permeability leading to changes in milk composition through increased influx of serum proteins and ions and increased efflux of lactose and potassium. Somatic cell count is also increased by ODM. Yield loss occurs through a combination of acute and chronic changes in mammary function. Anatomical and physiological characteristics which are likely to minimise production losses on ODM include production of a concentrated milk, possession of a large cistern, good drainage from the alveolar compartment and a low initial somatic cell count. Efficient removal of strippings, use of oxytocin and/or injection of bovine somatotropin all act to restore yield during ODM. It is proposed that the mechanism of the yield loss is through changes in mammary function initiated by changes in cell shape during alveolar filling. Central to these changes is an increased permeability of the udder through impairment of tight junctions between the alveolar cells. This effect appears to be linked to a mild inflammatory response which occurs in the last 12 h of a 24-h period of milk accumulation. The ability to further reduce production losses on ODM could lead to much wider adoption of ODM. Key to this is the development of methods to identify high-producing animals tolerant of extended milking intervals.

Expression of a β-Defensin mRNA, Lingual Antimicrobial Peptide, in Bovine Mammary Epithelial Tissue Is Induced by Mastitis

ABSTRACT The expression of a β-defensin, the lingual antimicrobial peptide (LAP), in response to mastitis was investigated by real-time PCR of RNA from mastitic and control udder quarters. There was a positive relationship between somatic cell count in milk and LAP expression. In situ hybridization showed that LAP mRNA was expressed in epithelial cells of mastitic tissue. These results suggest that LAP plays a role in the innate immune response to mastitis.

Transcriptome profiling of Streptococcus uberis-induced mastitis reveals fundamental differences between immune gene expression in the mammary gland and in a primary cell culture model.

Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. A bovine cDNA microarray comprising approximately 22,000 expressed sequence tags was used to evaluate the transcriptional changes that occur in the mammary gland after the onset of clinical Strep. uberis mastitis. Five lactating Friesian heifers were intramammary infused in an uninfected quarter with approximately 1,000 to 1,500 cfu of a wild-type strain of Strep. uberis. Microarray results showed that Strep. uberis mastitis led to the differential expression of more than 2,200 genes by greater than 1.5-fold compared with noninfected control quarters. The most highly upregulated genes were associated with the immune response, programmed cell death, and oxidative stress. Quantitative real-time reverse transcription PCR analysis confirmed the increase in mRNA expression of immune-related genes complement component 3, clusterin, IL-8, calgranulin C, IFN-gamma , IL-10, IL-1beta, IL-6, toll-like receptor-2, tumor necrosis factor-alpha, serum amyloid A3, lactoferrin, LPS-bonding protein, and oxidative stress-related genes metallothionein 1A and superoxide dimutase 2. In contrast, a decrease of mRNA levels was observed for the major milk protein genes. Bovine mammary epithelial cells in culture challenged with the same Strep. uberis strain used to induce clinical mastitis in the in vivo animal experiment did not cause a change in the mRNA levels of the immune-related genes. This suggests that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not Strep. uberis. However, challenging epithelial cells with different Strep. uberis strains and Staphylococcus aureus resulted in an increase in the mRNA expression of a subset of the immune-related genes measured. In comparison, an Escherichia coli challenge caused an increase in the majority of immune-related genes measured. Results demonstrate the complexity of the bovine mammary gland immune response to an infecting pathogen and indicate that a coordinated response exists between the resident, recruited, and inducible immune factors.

Partitioning of milk accumulation between cisternal and alveolar compartments of the bovine udder: relationship to production loss during once daily milking

Experiments were undertaken to validate a method (using adrenaline injection) for determination of the size of cisternal and alveolar compartments in the udder, to use this method to determine the pattern of milk accumulation in the udder over time and to determine the relationship between the size of the alveolar and cisternal compartments and tolerance of once daily milking. Cows received intrajugular injections of adrenaline (3 mg) immediately before milking, to block milk ejection and allow harvesting of the cisternal milk fraction. This was followed by removal of the alveolar fraction 30 min later after intrajugular oxytocin (5 i.u.) injection. Results obtained were similar to those obtained by catheter drainage. The alveolar compartment was 90% full at 16 h post milking while the cisternal compartment filled more slowly and was only 70% full at 24 h post milking. At full capacity (measured at 40 h), the volumes of milk contained in the cisternal and alveolar compartments were similar. In a further experiment involving identical twin cows, it was shown that the greater the degree of filling of the cisternal compartment at 24 h, the lower was the production loss on once daily milking. This suggests that the freedom of the alveoli to drain was an important factor in the production loss on once daily milking. Although there were significant correlations within twin sets for milk yield and the size of udder compartments, the relationship within twin sets for yield loss on once daily milking was not statistically significant.

Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells.

Tight junctions (TJ) between adjacent epithelial cells play an important role in maintaining mammary function in the differentiated mammary gland. Mouse mammary cell lines (HC11 and Comma-1D) were used to investigate the effect of the lactogenic hormones prolactin (PRL) and glucocorticoids on the formation of mammary TJ. TJ formation was assessed by an increase in transepithelial electrical resistance and a decrease in paracellular flux of radiolabeled inulin. Both PRL and the synthetic glucocorticoid dexamethasone (DEX) stimulated TJ formation. The biggest effect on TJ formation was observed when both hormones were used in combination, but only when cells were pretreated with DEX. The effects of PRL and DEX are mediated, at least in part, via expression of the transmembrane TJ protein occludin. In summary, these data are the first to show an effect of PRL on mammary TJ formation and the expression of TJ proteins, and confirm the TJ-stimulating effects of glucocorticoids that have been reported previously.

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.

Epigenetic Regulation of Milk Production in Dairy Cows

It is well established that milk production of the dairy cow is a function of mammary epithelial cell (MEC) number and activity and that these factors can be influenced by diverse environmental influences and management practises (nutrition, milk frequency, photoperiod, udder health, hormonal and local effectors). Thus, understanding how the mammary gland is able to respond to these environmental cues provides a huge potential to enhance milk production of the dairy cow. In recent years our understanding of molecular events within the MEC underlying bovine lactation has been advanced through mammary microarray studies and will be further advanced through the recent availability of the bovine genome sequence. In addition, the potential of epigenetic regulation (non-sequence inheritable chemical changes in chromatin, such as DNA methylation and histone modifications, which affect gene expression) to manipulate mammary function is emerging. We propose that a substantial proportion of unexplained phenotypic variation in the dairy cow is due to epigenetic regulation. Heritability of epigenetic marks also highlights the potential to modify lactation performance of offspring. Understanding the response of the MEC (cell signaling pathways and epigenetic mechanisms) to external stimuli will be an important prerequisite to devising new technologies for maximising their activity and, hence, milk production in the dairy cow.

Effect of unilateral once or twice daily milking of cows on milk yield and udder characteristics in early and late lactation.

Twelve multiparous British Friesian cows in early (40 +/- 23 d in milk; n = 6) or late (216 +/- 17 d in milk; n = 6) lactation were used to study the effects of milking frequency on yield, udder volume and milk storage within the udder. After a 2 week control period of twice daily milking, diagonally opposed udder halves within a cow were milked once or twice daily for 3 weeks. Milk yield was 28-38% lower from the halves that were milked once daily than from halves that were milked twice daily. The loss of milk yield, expressed as a decrease in the relative milk yield quotient (an index that accounts for pretreatment differences), was greater for cows in early than in late lactation (0.59 v. 0.68). Empty udder-half volume was not decreased by once daily milking, suggesting that no cell loss occurred. Instead, once daily milking reduced the secretion efficiency (units of milk per unit of empty udder-half volume) by 46 and 27% respectively in early and late lactation; thus, at least part of the loss was due to reduced metabolic activity of the mammary epithelium. There were positive correlations between the relative milk yield quotient and the proportion (r = 0.804) or volume (r = 0.644) of cisternal milk in the glands that were milked once daily. These results confirm that, during extended milking intervals, milk loss was smallest for cows that stored a larger proportion of milk in the gland cistern.

cDNA microarray analysis reveals that antioxidant and immune genes are upregulated during involution of the bovine mammary gland.

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.