Effect of a biological response modifier on cellular death mechanisms at drying off

Agents that increase natural protective mechanisms have been proposed for prevention and treatment of intramammary infections. The objective of this study was to describe the effects of a single intramammary infusion of a lipopolysaccharide (LPS)-based biological response modifier (BRM) on cellular death mechanism in uninfected and Staphylococcus aureus-infected bovine mammary glands during involution. Three groups of 12 cows, each one including 6 Staph. aureus-infected and 6 uninfected, were infused in two mammary quarters with BRM or placebo and slaughtered at 7, 14 and 21 d of involution. In infected quarters, BRM treatment produced a significant increase in percent of stained epithelial cells for the apoptosis-promoting protein Bax at every observation period. In addition, BRM produced a significant increase of immunostained stromal cells for Bax compared with placebo-treated quarters. BRM treatment produced an increase in percentages of epithelial cells staining with active caspase-3 at 7 d and 14 d of involution compared with placebo-treated quarters and a significant decrease in percentages of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive epithelial cells at 7 d and 21 d of involution. In addition, BRM treatment caused an increase in percentage of stromal cells immunostaining for active caspase-3 and TUNEL. An increase of active caspase-3 and TUNEL epithelial and stromal cell immunostaining was observed in Staph. aureus-infected compared with uninfected quarters. Cellular proliferation, determined by Ki-67 immunostaining, was increased in epithelial and stromal cells from Staph. aureus-infected compared with uninfected quarters at every observation period. These results provide new insights into the mechanism of mammary cell death in uninfected and Staph. aureus-infected bovine mammary gland during involution and illustrate the effects of LPS-based BRM on apoptosis and cell proliferation during mammary involution.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

Histological response of the bovine mammary gland to intramammary devices.

Histologic response of the bovine mammary gland to presence of three intramammary device models (abraded, star, or grooved) was studied in 12 lactating cows. Uninfected quarters fitted with devices exhibited greater leukocyte infiltration into teat and gland cistem linings as well as into mammary parenchyma adjacent to the gland cistem compared with unfitted control quarters. Cytologic alterations to cistem linings in device-fitted quarters ranged from degeneration and sloughing of surface cells of the double-layered epithelium to hyperplasia, stratification, and keratin formation. In uninfected quarters, quantification of mammary parenchymal components showed no differences among models for percentages of epithelium, but percentage of lumen was lowest and stroma highest for the star intramammary device model, suggesting reduced secretory activity or mammary involution in response to these intramammary devices. Presence of bacterial infection amplified the histologic responses to all devices. Leukocyte infiltration remained greater in device-fitted quarters compared with controls but was elevated over uninfected quarters for all treatments. Likewise, hyperplasia, stratification, and keratin formation of the cistem epithelial lining were more frequently observed in infected quarters. Among models in infected quarters, percentage of lumen was lowest and stroma highest in quarters fitted with abraded devices. In most cases, presence of infection masked any effect of devices on mammary parenchymal components. Plaque formation was observed on all models and tended to be thicker on those retrieved from infected quarters. Electron microscopic examination showed that plaque was composed of leukocytes, cell debris, keratin, and amorphous material. Results demonstrated that most intramammary device models were successful in stimulating leukocytosis into the gland, and tissues from fitted quarters displayed alterations to cisternal linings; however, quarters fitted with these devices exhibited reduced secretory activity.     

Effect of recombinant cytokines on leucocytes and physiological changes in bovine mammary glands during early involution

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rbolL-2) (2 x 10(5) units, n = 6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 microg, n = 4) and recombinant bovine interleukin-1beta (rbolL-1beta) (10 microg, n = 10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rbolL-2 and rboGM-CSF were produced in a yeast expression system, while rbolL-1beta was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rbolL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rbolL-1beta induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rbolL-1beta compared with control PBS quarters. In addition, infusion of glands with rbolL-1beta lowered the citrate:lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rbolL-1beta at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rbolL-1beta, also increased the rate of mammary gland involution during the early dry period.     

Mammary serum amyloid A3 activates involution of the mammary gland in dairy cows

The dry period is a nonlactating phase in which senescent mammary cells are regenerated, which is thought to optimize milk production in the subsequent lactation. In bovines, the dry period normally coexists with pregnancy and the lactogenic hormones delay mammary gland involution and impair the activation of immune system to fight the risk of intramammary infections. Conventionally, long dry periods of up to 60 d are required to allow sufficient mammary regeneration for full milk yield in the next lactation. The aim of this study was to evaluate the potential of mammary serum amyloid A3 (M-SAA3) as an activator of the involution of the mammary gland. One milligram of recombinant M-SAA3 and the corresponding negative controls (saline solution and lipopolysaccharide) were infused into the mammary gland via the teat canal, and mammary secretion samples were taken during the first 3 d after drying off to analyze metalloproteinase activity, somatic cell count, protein, and fat contents. Primary mammary gland epithelial cell cultures and bovine dendritic cells, obtained from necropsy tissue and blood, respectively, were incubated with and without M-SAA3 and cytokine expression was quantified. Last, the protective role of the M-SAA3 against infections was evaluated after a Staphylococcus aureus challenge. Matrix metalloproteinase 9 activity, a key protein that directly participates in the onset of the involution process, was greater in quarters treated with the M-SAA3. Protein content was increased in mammary secretions compared with control quarters. M-SAA3 increased cytokines directly related to innate immunity in both epithelial and dendritic cells and reduced the infection by Staphylococcus aureus.     

Histopathology of staphylococcal mastitis in unbred dairy heifers

Histologic observations of mammary tissue samples from unbred heifers revealed that secretory parenchyma from uninfected quarters was undeveloped, exhibiting small alveoli with a limited luminal area and a large interalveolar stromal area. Tissues from quarters infected with Staphylococcus aureus were less developed, exhibiting less alveolar epithelial and luminal areas and more interalveolar stroma compared with tissues from uninfected quarters. Such quarters also demonstrated minimal secretory activity. Macroscopic and microscopic abscesses were observed in one quarter with S. aureus intramammary infection. Staphylococcus aureus-infected quarters showed greater leukocyte infiltration into mammary parenchymal components and cistern lining compared with uninfected quarters. Quarters infected with non-aureus staphylococci also exhibited greater leukocyte infiltration and greater percentages of interalveolar stroma compared with uninfected controls. Results demonstrated that presence of infection increased leukocytosis into the mammary gland and reduced secretory activity in heifers, suggesting a deleterious effect on future milk production.     

Effects of intramammary infusions of casein hydrolysate,ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70 mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7 g), lactose (5.1 g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na⁺ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K⁺ concentrations decreased. As involution advanced, the Na⁺:K⁺ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na⁺ concentration and in the Na⁺:K⁺ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.     

Intramammary infections in heifers during early lactation following intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin at the first milking after parturition

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73.1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61.8% (21/34) were cured following treatment with penicillin-novobiocin and 39.6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52.8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41.4% (12/29) were cured following treatment with penicillin-novobiocin and 23.1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.     

Determination of milk and mammary tissue concentrations of ceftiofur after intramammary and intramuscular therapy

Twenty-five Staphylococcus aureus strains isolated from bovine mastitis were tested for their susceptibility to ceftiofur. Zone diameter for 30 micrograms disks averaged 39 mm, and minimum inhibitory concentrations ranged from .5 to 1 microgram/ml. Tissue and milk concentrations were determined from biopsy and quarter milk samples collected from eight cows treated with either intramammary infusion of 100 or 200 mg of ceftiofur, one or two intramuscular injections of 500 mg of ceftiofur, or combination therapy of intramammary infusion coupled with intramuscular injection. Three additional cows received two intramammary infusions of 200 mg of cephapirin at 24-h intervals. Intramuscular injections of ceftiofur resulted in tissue and milk concentrations below detectable limits. Staphylococcus aureus was not eliminated from infected mammary glands by infusion of 100 mg of ceftiofur or by injection of 500 mg of ceftiofur by 48 h after treatment. Combination therapy of 100 mg of ceftiofur infused and 500 mg injected reduced S. aureus numbers in milk and tissue markedly, as did infusion of 200 mg of ceftiofur. Cows receiving intramammary infusion of 200 mg of ceftiofur (two doses at 24-h intervals) had highest concentrations in milk (450 micrograms/ml at 4 and 6 h) and in tissue (.08 microgram/mg at 30 h). These concentrations are similar to those obtained with two 200-mg doses of cephapirin at 24-h intervals. Histologic analysis of mammary parenchymal tissues showed that combination therapy resulted in higher percentages of alveolar luminal area and lower percentages of interalveolar stroma compared with infusion or injection alone. Histology of quarters receiving combination therapy was not different from that of quarters receiving cephapirin infusion alone.     

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 μg/quarter per d with or without 10 μg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered ∼2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.     

Examination of intramammary devices from infected and uninfected mammary quarters by scanning electron microscopy

Polyethylene intramammary devices were removed from six infected and four uninfected mammary quarters of seven lactating cows and examined by scanning electron microscopy. Infecting organisms included Corynebacterium bovis, coagulase-negative staphylococci, and an unidentified fungus. Intramammary devices from infected quarters had amorphous material adhering to large areas of the polyethylene. Large numbers of inflammatory cells and microorganisms were found concentrated within the material. Devices from uninfected quarters had less amorphous material with few adhering inflammatory cells. The amorphous material appeared to be restricted to abraded surfaces of the polyethylene. Milk somatic cell counts in stripping milk of quarters infected with Corynebacterium bovis and coagulase-negative staphylococci before and 3 wk after removal of intramammary device averaged 1.2 X 10(6) and .2 X 10(6)/ml, respectively. Results suggest that increased cell counts of infected quarters containing intramammary devices were associated with microbial colonization of the amorphous material.     

Effect of alpha-tocopherol deprivation on the involution of mammary gland in sheep

The objective of this study was to investigate the effect of alpha-tocopherol deprivation on mammary gland involution and apoptosis in sheep. Two groups of four single lamb ewes were used. The control group received 100 mg/d of RRR-alpha-tocopherol supplementation and the experimental group received no vitamin E supplementation. After 3 mo of suckling, ewes were dried off, and blood samples from the jugular vein and tissue biopsies from the mammary gland were collected at d 1, 3, 5, and 8 after dry-off. The experimental group had lower plasma concentrations of alpha-tocopherol (1.8 vs. 4.2 micromol/L), lower glutathione peroxidase activity in erythrocytes, and higher concentration of malondialdehyde in plasma than the control group. Immunohistochemical analysis of tissue samples resulted in marked differences of bcl-2 and bax protein expressions during involution and between groups. The bax expression was decreased by alpha-tocopherol deprivation at 1, 3, and 5 d, but not at 8 d, while the bcl-2 score was higher only at 8 d (1.5 vs. 0.0 for experimental and control groups, respectively). As a result, the bcl-2 to bax ratios were increased for the experimental group at 1 and 8 d. During involution, apoptotic counts increased (from 0.12 to 4.06%), but no effects were detected in relation to bcl-2 to bax ratio and alpha-tocopherol. These results indicate that alpha-tocopherol can control bcl-2 expression, but not apoptosis in cells of the mammary gland during involution.     

Comparison of tilmicosin and cephapirin as therapeutics for Staphylococcus aureus mastitis at dry-off

Forty-four cows (26 Jerseys and 18 Holsteins) that had at least 1 mammary quarter that was naturally (n = 12) or experimentally (n = 84) infected with Staphylococcus aureus were allotted to three treatment groups of approximately equal number at the end of lactation. Cows were dried off by abrupt cessation of milking, and dry cow therapy was administered as an intramammary infusion of cephapirin benzathine at 10 ml per quarter, an intramammary infusion of tilmicosin (solution containing 300 mg/ml) at 5 ml per quarter, or a subcutaneous injection of tilmicosin at 5 mg/kg of body weight on the day of drying off and another injection 4 d later. Mammary secretions were monitored during the dry period and postpartum for antimicrobial residues, intramammary infection (IMI) status, and somatic cell counts. Results demonstrated the following percentage cures for IMI caused by Staph. aureus at 28 d postcalving based on individual mammary quarters: cephapirin benzathine, 78.1%; tilmicosin infused, 74.2%; and tilmicosin injected, 9.1%. During the first 4 wk after drying off, the mean concentration of tilmicosin in mammary secretions from cows infused with the antibiotic remained approximately 10-fold higher than that in secretions from cows injected with the antibiotic (3.43 vs. 0.32 ppm), and, by the time of calving, concentrations for cows treated with both methods were below the dilution limit of the assay (< 0.1 ppm). Results demonstrated that intramammary infusion of tilmicosin was equally as effective as cephapirin benzathine in curing IMI caused by Staph. aureus at drying off; however, the subcutaneous injection of tilmicosin at the dose used was not effective as a dry cow therapeutic against Staph. aureus.     

Effect of intramammary device on new infection rate, milk yield, and milk somatic cell counts in maryland dairy herds

Effectiveness of a polyethylene intramammary device against naturally occurring infections was evaluated in three Maryland herds over 2 yr. Treated cows [62] were fitted with intramammary devices in all quarters of udders. Control cows [62] were sham treated. Rates of new intramammary infection over single lactation in treated and control quarters of primiparous cows averaged 18 and 27%. Reduction of infection rate was due primarily to fewer Corynebacterium bovis infections. Infection rate between multiparous cow treatments were similar. In uninfected quarters cell counts in strippings averaged .11 to .13 X 10(6)/ml and in quarters fitted with intramammary devices concentrations were only .22 to .31 X 10(6) cells/ml. But in infected quarters with intramammary devices, cell counts of strippings were 1.38 to 1.48 X 10(6)/ml. Concentrations of somatic cells of strippings in infected quarters without devices averaged .48 to .63 X 10(6)/ml. Dairy herd improvement cell counts for primiparous and multiparous cows with and without intramammary devices were similar and averaged .2 X 10(6)/ml. Neither milk nor fat production differed. The intramammary device as currently designed is incapable of stimulating a leukocytosis sufficient in stripping milk to prevent intramammary infection.     

Quantification and immunoglobulin classification of plasma cells in nonlactating bovine mammary tissue

Plasma cell populations in bovine mammary tissue were examined during involution using electron microscopic and immunohistochemical techniques. Biopsies were taken from each quarter of five Jersey cows at weekly intervals beginning at drying off through parturition. Ultrastructural examination of stromal plasma cells revealed rough endoplasmic reticulum cisternae engorged with flocculent material, indicative of antibody synthesis. Plasma cells were observed proximal to alveolar epithelial cells. This association may facilitate transport of antibody through epithelium and into milk. Immunoglobulin-producing plasma cell numbers increased gradually from drying off, reached peak concentrations 2 wk prepartum, and dropped significantly during the last 2 wk of gestation. Plasma cells producing immunoglobulin G1 and G2 were the most numerous types observed during the nonlactating period followed by cells producing immunoglobulin M and immunoglobulin A. Plasma cells producing immunoglobulin were more numerous during the last 2 wk of gestation and in tissue infected with minor pathogens than in uninfected quarters. Exposure to minor pathogens may have enhanced sensitized B-lymphocyte proliferation into antibody producing plasma cells through antigenic stimulation. Results of plasma cell distribution during the nonlactating period in bovine mammary tissue indicate times when local immunostimulation of B-lymphocytes may be most effective in enhancing immunity to intramammary infection.     

Apoptosis and proliferation in Staphylococcus aureus-challenged,nonlactating mammary glands stimulated to grow rapidly and develop with estradiol and progesterone

Bovine mastitis is a common and costly disease in the dairy industry and is known to negatively affect the amount of epithelium in nonlactating mammary glands. Despite this recognition, an understanding of the mechanisms contributing to reductions in epithelium is lacking. The objective of this study was to evaluate cellular apoptosis and proliferation in uninfected and Staphylococcus aureus-infected mammary glands that were stimulated to rapidly grow and develop. Estradiol and progesterone injections were administered to 18 nonlactating dairy cows to induce mammary growth, and 2 quarters from each animal were infused with saline or Staph. aureus. Mammary tissues were collected at 5 (n = 9) and 10 d (n = 9) postinfusion and examined using quantitative bright field and florescent immunohistochemistry. Staphylococcus aureus mammary glands tended to have a greater number of mammary epithelial cells undergoing apoptosis than saline quarters. In the stromal compartment, challenged quarters contained a lower proportion of cells undergoing apoptosis than saline quarters overall; however, cell types undergoing apoptosis were differentially affected. Staphylococcus aureus quarters contained a lesser percentage of apoptotic fibroblasts while also containing more nonapoptotic immune cells than saline quarters in the intralobular stroma compartment. A similar number of proliferating epithelial cells were present in Staph. aureus and saline mammary tissues, but more proliferating cells were present in the intralobular stroma compartment of Staph. aureus-infused quarters than those infused with saline. When these cellular responses are considered together, it indicates that changes in cellular apoptosis and proliferation contribute to changes in the gland structure by potentiating the expansion of the intralobular stromal compartment, via cellular accumulation, and limiting the amount of epithelium due to increases in cellular apoptosis in affected glands. Reductions in mammary epithelium are expected to reduce future milk yields and productive herd life.     

Cellular proliferation and apoptosis in Staphylococcus aureus–infected heifer mammary glands experiencing rapid mammary gland growth

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus–infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.     

Intramammary response to modified intramammary devices.

Twenty-five cows in three experiments were used to evaluate the following intramammary devices: abraded surfaces; abraded and sulfur hexafluoride-coated; abraded, sulfur hexafluoride-coated, and weighted; and smooth surfaces and weighted. The objectives of the experiments were 1) to determine whether coating the abraded intramammary devices prevented amorphous deposits and bacterial adherence on the devices, 2) to determine whether addition of weight to devices increased the concentration of somatic cells in milk, and 3) to evaluate milk production and response of mammary epithelial cells to the devices. Milk SCC and NAGase (EC 3.2.1.30) from quarters fitted with devices were increased in quarter bucket and stripping milk, but increases were similar among all devices. Macrophages were the predominate cell type in quarter milk before insertion and neutrophils after insertion. Moderate to heavy deposits of amorphous material were observed on all devices, regardless of time residing in the gland. Approximately 50% of the devices were colonized after intramammary inoculation with Corynebacterium bovis. Milk production in control quarters and in quarters with devices were similar. Neither coating with sulfur hexafluoride nor addition of weight to devices reduced amorphous deposits or bacterial adherence or enhanced milk somatic cell response.     

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.