Modulation of bovine mononuclear cell proliferation during physiological transitions of the mammary gland

Mammary secretions and blood were collected from five primiparous Holstein cows 14 d following cessation of milking and 14 d prior to parturition for preparation of serum and mammary secretion skim fractions. Mammary secretions and blood were collected from the same animals 15 to 18 d following cessation of milking and 2 to 13 d prior to parturition for isolation of mononuclear cells. Effects of serum on mammary gland mononuclear cell proliferation and skim fractions from mammary secretions on blood mononuclear cell proliferation were evaluated. Mononuclear cell proliferation was evaluated in a mitogen-induced lymphocyte proliferation assay and in a mixed leukocyte assay. Proliferative responses of blood and mammary gland mononuclear cells did not vary significantly between the two time periods evaluated. Mammary secretion skim fractions obtained at both time periods significantly suppressed blood mononuclear cell proliferation. In contrast, exogenous serum enhanced mammary gland mononuclear cell proliferation in response to mitogens and allogeneic cells. Ability to enhance in vitro proliferation of mammary mononuclear cells isolated during physiological transitions of the mammary gland may suggest the potential for enhancing mammary mononuclear cell proliferation in vivo to reduce incidence of new intramammary infections at times when the mammary gland is highly susceptible.     

Observations on Corynebacterium bovis infection of the bovine mammary gland. II. Experimental infection

Experiments are described in which lactating cows were exposed to Corynebacterium bovis either by dipping the teats in a suspension of the bacteria or by inoculating the bacteria into the teat duct or the teat sinus. All three methods readily led to 'infection' being established. The effect of these infections on somatic cell count was minor and no clinical mastitis resulted during the course of the experiments although some cases occurred subsequently. There was evidence that 44% of these infections were confined to the teat duct. Quarters excreting C. bovis in milk continued to do so during endotoxin-induced inflammation and showed a similar cellular response to that of uninfected quarters.     

Observations on Corynebacterium bovis infection of the bovine mammary gland. I. Natural infection

Data from experiments in 55 commercial herds have been examined to study the patterns of Corynebacterium bovis infection. Such infections are most common in herds that are not subjected to regular control methods but their commercial significance appears to be small. In herds using teat disinfection and dry cow therapy levels of infection with this organism are low. There is no evidence from these data to suggest that C. bovis infections protect the udder against invasion by a major pathogen. On the contrary these infections do not persist when major pathogens invade.     

Phylogenetic studies on Corynebacterium bovis isolated from bovine mammary glands

Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.     

Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-γ and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-γ positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.     

Leukocytic infiltration of bovine mammary parenchymal tissue in response to Corynebacterium bovis colonization

Morphological changes and local leukocyte response to prolonged Corynebacterium bovis colonization were studied in lactating bovine mammary glands. Morphometric analysis of parenchymal tissue demonstrated no adverse effects of colonization on the synthetic and secretory activity of mammary epithelium. Numbers of macrophages, lymphocytes, and plasma cells were higher in tissue from C. bovis-colonized quarters. However, there were no differences in numbers of neutrophils between colonized and uninfected quarters. Results suggest persistent C. bovis colonization may elicit effector cell populations in lactating mammary tissue where leukocyte concentrations tend to be lower.     

Changes in the lipid composition of the secretions of the bovine mammary gland during the dry period.

To study initiation of milk fat synthesis, lipid composition of mammary secretions at -60, -40, and -10 d prepartum was studied in lactating and nonlactating Holstein cows. Eleven cows were dried off, and 13 cows were milked twice per day throughout the normal dry period. Total neutral lipid was similar in late lactation milk (-60 d) from lactating cows, 2.1 g/dl, and in milk from the dry group, 2.2 g/dl. Neutral lipids decreased to 1.3 and .9 g/dl in quarters from dry cows at -40 and -10 d prepartum. In secretions from dry quarters, triglycerides were 97% of total lipids at -60 d and decreased to 85 and 91% at -40 and -10 d, respectively. Conversely, FFA and monoglycerides increased during the dry period. Lipids associated with fat globule membrane components increased during the dry period. These increases were 10 times for cholesterol, 20 times for cholesteryl esters, and twice for phospholipids. In general, the content of fat globule core lipids (triglycerides) exhibited a pattern opposite that of membrane lipids (cholesterol, cholesteryl esters, phospholipids) during the prepartum period. Proportions of core lipids tended to decrease, whereas proportions of membrane lipids increased in prepartum mammary secretions. Lipid composition of prepartum secretions may be influenced by blood lipids, somatic cells, and alterations in mammary lipid synthesis.     

Lactoferrin concentration during involution of the bovine mammary gland.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.     

Tolerability of N-chlorotaurine in the bovine mammary gland

N-Chlorotaurine (NCT) is a promising endogenous agent for topical treatment of infections. We tested the tolerability and pharmakokinetics of NCT in the bovine mammary glands in a phase 1 study. Three concentrations of NCT in water (0.1%, 1.0%, 2.0%) were administered intramammarily in each of two cows. Into two quarters of the udder 100 ml NCT was injected into each twice daily for 5 d, while 0.9% NaCl was injected into the other two quarters in a randomized and blinded manner. Samples of milk were taken to determine the number of leucocytes and the activity of NCT, and samples of urine and blood to determine the taurine and chloride concentration. Chloride concentrations in serum samples were determined by an ISE-Unit of a Modular-System of the Roche Diagnostics company. The udder was monitored clinically for signs of inflammation. Oxidative activity could be detected in the milk after single irrigations for 15 min (0.1% NCT) and for maximally 5 h (1% and 2% NCT), respectively. On day 2, leucocytes increased to 4 x 10(6)/ml in the NCT group, while they remained 1 x 10(6)/ml in the saline group. However, on days 3-5 they increased to (5-7) x 10(6) in both the NCT and control group without any statistical difference. One day after the end of dosing the number decreased significantly and reached the baseline (<1 x 10(6)/ml) on day 10. The decrease was similar in both groups. Except for sporadic slight induration of single quarters in both groups and slight reduction of milk performance no disorders occurred. Taurine levels in blood and urine did not change. Irrigation of the bovine mammary gland with both NCT and saline caused a transient increase of leucocytes in the milk, but no severe side effects. The absence of residues and decay products may be a great advantage of NCT over other antimicrobial agents.     

Bovine mononuclear leukocyte subpopulations in peripheral blood and mammary gland secretions during lactation.

Florescence flow cytometry and monoclonal antibodies were used to analyze the composition of leukocytes from peripheral blood and mammary gland secretions. Samples were obtained from Holstein dairy cows, free of IMI, at six time points during the lactation cycle. The percentage of monocytes in peripheral blood mononuclear cells varied from 15 to 31%; the lowest percentage occurred at the early nonlactation period. The percentage of T lymphocytes from mammary gland secretions varied from 16% during the periparturient period to 62% at late lactation. The B lymphocytes varied from 7% at late lactation to 25% during the periparturient period. Macrophages varied from 21 to 69%; the highest percentage occurred during the periparturient period. The mean ratio of CD4+:CD8+ T lymphocytes in the peripheral blood and mammary gland secretions was 1.53 and .85, respectively. A subpopulation of activated CD8+ T lymphocytes present in mammary gland secretions throughout the lactation cycle coexpressed a new activation molecule, ACT2. Increases in the proportion of CD8+ T lymphocytes were associated with an increase in the percentage of CD8+, ACT2+ T lymphocytes. Activated CD8+ T lymphocytes may play an important role in the regulation and expression of the local immune response to pathogens.     

In vitro growth of mastitis-associated streptococci in bovine mammary secretions

Cell-free, fat-free mammary secretions were tested in vitro for ability to support growth of streptococci associated with mastitis. Secretions were obtained prior to drying off, during the dry period, at calving, and during lactation from four cow treatment groups. Treatment groups were dry cow therapy, dry cow therapy and mammary glands subjected to induced inflammation 7 d post-drying-off, no dry cow therapy and no induced inflammation, no dry cow therapy but mammary glands subjected to induced inflammation. Growth of Streptococcus uberis, Streptococcus faecalis, and Streptococcus agalactiae in secretions from nonlactating glands was unaffected by induced inflammation. Growth of Streptococcus bovis was significantly inhibited in secretion obtained 14 d after induced inflammation. Dry cow therapy had no effect on streptococcal growth in secretion obtained 7 d after therapy. Streptococcal growth was greatest in secretions from involuted glands, and there was little or no evidence for growth inhibitory factors in cell-free, fat-free secretions obtained during the dry period. Milk from lactating glands inhibited streptococcal growth, and the inhibitory factor was presumptively identified as lactoperoxidase. Apolactoferrin, immunoglobulin, or both had little effect on streptococcal growth.     

Differential distribution of T lymphocyte subpopulations in the bovine mammary gland during lactation.

The existence and distribution of T lymphocyte subpopulations in the mammary parenchymal tissue of cows were immunohistochemically detailed during early lactation. CD2+, CD4+, and CD8+ T lymphocytes were localized primarily in the mammary parenchymal tissue. CD8+ T lymphocytes were predominant over CD4+ T lymphocytes and occurred in close contact with the alveolar epithelium and between epithelial cells in the central area of the upper mammary gland. CD4+ T lymphocytes were present in equal numbers in the epithelial and connective tissue area. Occasionally, both CD4+ and CD8+ T lymphocytes formed cell clusters in the interalveolar connective tissue. The ratio of CD4+ T lymphocytes to CD8+ T lymphocytes was less than 1.0 and was lower in the epithelial area than in the connective tissue. The distribution of CD4+ and CD8+ T lymphocytes was similar in the parenchymal tissue of the gland cistern. The observation that there is a preferential presence of CD8+ T lymphocytes in the epithelial area of the bovine mammary gland during early lactation might indicate that these cells participate in the maintenance of the integrity of the epithelium.     

Intramammary challenge of the bovine mammary gland with coliform bacteria during early involution

Isolates of Escherichia coli (n = 12), Klebsiella pneumoniae (n = 20), and Klebsiella oxytoca (n = 10) were used to challenge involuting mammary glands at 7 d of the dry period. Bacteria were selected for challenge on the basis of their ability to grow in a pooled source of dry cow secretion obtained at 21 d of involution. Challenge bacteria were classified as highly adapted (in vitro growth greater than 7 cfu log10/ml) or poorly adapted (growth less than 2 cfu log10/ml) for growth in dry cow secretion. Intramammary infusion of Escherichia coli, K. pneumoniae, and K. oxytoca resulted in 0, 40, and 30%, respectively, of quarters infected. Isolates highly adapted for growth in dry cow secretion caused 75% of K. pneumoniae and 67% of K. oxytoca experimental intramammary infections. Results indicated that the ability to overcome inhibitory properties of dry cow secretion was related to the establishment of K. pneumoniae and K. oxytoca intramammary infections in the dry gland. There was no evidence that growth of E. coli in dry cow secretion related to pathogenicity in the dry gland. Experimental challenge using multiple isolates did confirm the resistance of the involuting mammary gland to E. coli infection.     

Enzyme-linked immunosorbent assays of bovine lactoferrin and a 39-kilodalton protein found in mammary secretions during involution

Enzyme-linked immunosorbent assays were developed for two proteins found in mammary secretions during the non-lactating period: lactoferrin and a 39-kdal protein. Minimum detectable concentrations were .12 ng/ml for lactoferrin and .0037 ng/ml for the 39-kdal protein. Standard curves were linear in the ranges of .4 to 100 ng/ml for lactoferrin and 2.4 to 30 ng/ml for the 39-kdal protein. The immunoassays were used to measure concentrations of the proteins in mammary secretions during involution. From the last day of milking to d 30 of involution, mean concentrations of lactoferrin and the 39-kdal protein increased from .82 +/- .35 to 78.5 +/- 12.43 mg/ml and from 11.8 +/- 3.8 to 84.4 +/- 21.1 micrograms/ml, respectively. The immunoassays described will be valuable for further studies on the synthesis and secretion of these proteins during mammary involution.     

Expression and regulation of glucose transporters in the bovine mammary gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.     

Electrolytes in bovine prepartum mammary secretions and their usefulness for predicting parturition

Accurately predicting parturition in cows can be difficult because of the variability in gestation length and uncertainty of the time of parturition. The goal of the present study was to determine the electrolyte concentrations of prepartum mammary secretions of gravid cows and to investigate retrospectively the possible predictive relationships between these concentrations and the time of parturition. Twenty-three cows were sampled once daily for 3 to 12 d before they calved. The concentrations of calcium, inorganic phosphorus, chloride, magnesium, sodium, and potassium were determined by photometric methods. In addition, the concentrations of calcium, magnesium, and inorganic phosphorus were determined using rapid test kits. The correlation between the photometrically measured electrolyte concentrations and the time of parturition was highest for inorganic phosphorus (r = 0.74). The inorganic phosphorus concentration was 11.8 to 26.5 mmol/L in cows that calved within 24 h of sample collection. When 11.8 mmol/L was used as a cutoff concentration for inorganic phosphorus, 21.7, 47.8, and 87.0% of cows calved within 24, 48, and 72 h, respectively. Within 12 d prepartum, the time of parturition can be estimated, with an error of ± 32.4 h, by using the results of simultaneous determination of inorganic phosphorus and sodium concentrations in mammary secretions and a formula derived from a multiple regression analysis. The results of a phosphate field test positively and significantly correlated with those of the photometric analysis (r = 0.69) and the time of parturition (r = 0.41).