Morphologic changes in the bovine mammary gland during involution and lactogenesis

Morphologic changes developing during bovine mammary involution were examined. Quarter biopsy specimens were obtained weekly from 5 cows beginning the day milking was discontinued through parturition. Light and electron microscopic examination of mammary tissue indicated a gradual reduction in synthetic and secretory activity of alveolar epithelium as involution progressed. Light microscopic morphologic analysis revealed increases in stroma and nonactive secretory epithelium, with concomitant decreases in epithelium, lumen, and fully active secretory epithelium during the first 2 weeks of involution. Electron microscopic analysis of alveolar epithelium revealed decreased number of organelles associated with milk synthesis and secretion during this time. These changes reversed gradually beginning 2 weeks before parturition, and by the time of calving, cell structure was typical of lactating glands. Tissue from infected quarters had less synthetic and secretory ability as indicated by significantly higher percentages of stroma and nonactive cells, but lower percentages of lumen and moderately active cells, compared with uninfected quarters. Infected quarters also had more leukocytes infiltrating the epithelium, lumen, and stroma, compared with uninfected quarters. Microscopic examination of macrophages and neutrophils suggested these cells removed milk components and cellular debris during involution. Large numbers of plasma cells, with distended cisternae of rough endoplasmic reticulum, suggested local antibody production during the periparturient period.     

Growth inhibition of environmental mastitis pathogens during physiologic transitions of the bovine mammary gland

Ten dairy cows were infused intramammarily near drying off with concanavalin A (conA) or phytohemagglutinin (PHA). Mammary secretions were collected during physiologic transitions of the udder and were used in an in vitro microbiological assay to determine growth inhibition of mastitis pathogens. As mammary involution progressed, in vitro growth inhibition of Klebsiella pneumoniae, Escherichia coli, and Streptococcus uberis increased. Mammary secretions from conA- and PHA-treated glands had significantly increased bacterial growth inhibition. Secretions contained significantly increased concentrations of lactoferrin and a decreased citrate:lactoferrin molar ratio earlier in the dry period than did control mammary secretions. Greatest bacterial growth inhibition was observed in mammary secretions obtained 7 days before parturition. However, differences in secretion composition or bacterial growth inhibition were not found between conA- or PHA-treated and control udder halves during the prepartum period. Bacterial growth inhibition by mammary secretion decreased markedly during early lactation. A highly significant positive correlation was found between bacterial growth inhibition and concentrations of lactoferrin, serum albumin, and immunoglobulin G. A highly significant negative correlation was found in the citrate:lactoferrin molar ratio during early involution and the peripartum period.     

The effect of Escherichia coli endotoxin and culture filtrate on the lactating bovine mammary gland

The pathogenesis of coliform mastitis was studied by observing pathological changes in lactating glands after infusion of either endotoxin or the sterile culture filtrate (CCF) of the medium in which Escherichia coli strain B117 had been grown. Both infusions produced a rapid and intense inflammatory response by 4 h with a marked increase of serum proteins in the milk. Before dispersing into the milk, neutrophils were attached to the ductular epithelium; highest cell counts in the milk were recorded when the tissue reaction had waned. Oedema of the ductular epithelium occurred, particularly where neutrophils were actively migrating. The infusion of CCF produced, in addition to inflammation, degeneration and necrosis of ductular cells. The smallest lesions healed very rapidly. There was evidence of differing cell susceptibility to the necrotising toxin as well as uneven distribution over the epithelial surface. All changes observed were confined to the regions of the teat and lactiferous sinuses with little effect on the secreting tissue. The role of the necrotising toxin in the natural disease remains undetermined.     

The survival of serum resistant Escherichia coli in the bovine mammary gland following experimental infection.

Serum resistant strains of Escherichia coli were injected into one or two quarters of the udders of eight healthy dairy cows. Animals receiving infection into two quarters showed variation in their ability to eliminate the bacteria. This variation extended from elimination from both glands to complete failure to remove the organisms from either gland. In most cases, the organisms were removed from one gland before the clinical signs of infection were observed, but persisted in the other gland for three to four days. Following a single infection most animals eliminated the organisms before the appearance of clinical signs, but one retained the bacteria for four days. The retention of bacteria within the gland for a period longer than the initial inflammatory response resulted in their survival within neutrophils; and in some of these glands spasmodic clinical signs of mastitis reappeared for up to at least 40 days after the initial infection. These signs were associated with the reappearance of the same serological strain of E coli. Bovine serum albumin levels in the milk were found not to constitute the most effective marker of the serum components involved in the bactericidal activity.     

Changes in the microscopic anatomy of the bovine teat canal during mammary involution

Changes in the microscopic anatomy of the bovine teat canal were examined during mammary involution. Morphometric analyses revealed a significant (P less than 0.05), temporary dilatation of the teat canal lumen on day 7 of the nonlactating period. Additionally, the teat canal epithelium physiologically atrophied as evidenced by decreased cross-sectional area and thickness during the first 30 days of the nonlactating period, significantly so (P less than 0.05) between days 0 and 7. This physiologic atrophy was due mainly to a reduction in area and thickness of the stratum granulosum and may have resulted from continuing keratinization, a process that led to increased thickness of the keratin layer and formation of a functional plug during later stages of involution. Changes in cells of the stratum granulosum indicated a decrease in the rate of epithelial cell maturation during involution. The mitotic index (percentage of basal cells in mitosis) of the teat canal epithelium decreased significantly (P less than 0.05) between days 0 and 7 of the nonlactating period. Bacteria, observed in histologic sections, appeared to colonize only certain regions of the keratin layer. Seemingly, changes in the teat canal during mammary involution may be important factors in changing susceptibility to new intramammary infection during the early and mid-nonlactating periods.     

Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses

The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.     

Neutrophil apoptosis during the resolution of bovine mammary gland injury

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.     

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats

Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 μg kg⁻¹ body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 × 10⁶ colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 × 10⁶ CFU ml⁻¹ E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.     

Growth inhibition of Escherichia coli by nitrate reductase

The metabolite of a not nearer identified Pseudomonas strain inhibited or inactivated Escherichia coli in presence of nitrate.     

Biopsy of the bovine mammary gland.

A technique is described for biopsy of the bovine udder, employing sedation and local anaesthesia. Tissue samples of approximately 5 g were obtained by electrocautery from two quarters of the udder of a cow laterally recumbent. Care was taken to ensure complete haemostasis which was achieved by electrocoagulation and ligation. Postoperative recovery was rapid, and loss of yield was no greater in biopsied glands than in control glands of the same cow. Yield from all quarters returned to preoperative levels within 48 h.     

The role of IGFBP-5 in mammary gland development and involution

Insulin-like growth factor-I (IGF-I) plays an important role as a survival factor during mammary gland development and remodelling during involution of the mature/lactating mammary gland, and elevated concentrations have been associated with increased risk of breast cancer. The actions of IGF-I are modulated by a family of binding proteins (IGFBPs) and we have shown that IGFBP-5 is associated with cell death in the mammary gland and more recently provided the first evidence that it is causally related to apoptosis of the mammary gland. A transgenic mouse expressing IGFBP-5 on a mammary-specific promoter led to impaired mammary development involving inhibition of IGF-signalling and involving members of the Bcl-2 family. Subsequent studies in vitro and in vivo using exogenous IGFBP-5 treatment have added support to this concept. Although the effects of IGFBP-5 did appear to involve inhibition of IGF action, a role for IGF-independent effects cannot be ruled out. Such IGF-independent effects involve potential interactions with components of the extracellular matrix involved in tissue remodelling including plasminogen activator inhibitor-1 (PAI-1). In addition, intracellular events involving nuclear localisation of IGFBP-5 have been shown to have the ability to inhibit cell proliferation. Thus, IGFBP-5 seems important for regulating both apoptosis and cell proliferation in the mammary gland during development and post-lactation involution.     

退化期的乳腺凋亡及其调节机理

在动物哺乳周期中,乳腺经历发育、分化和退化的循环过程。动物的妊娠期、哺乳期和退化期与乳腺细胞数量的变化有密切关系,乳腺细胞的数量变化决定了哺乳期间的产奶量,退化期的幸存细胞数量可能影响哺乳期的生产力。近来研究表明:乳腺退化期主要通过细胞凋亡途径减少细胞数量。本文对退化期的乳腺凋亡及其调节机理进行了综述。     

Characterization of immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution

The aim of this study was to characterize the immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus intramammary infections (IMI) were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of toll-like receptor 2 (TLR2) and TLR4 was significantly higher in S. aureus-infected quarters than in uninfected controls at the three involution stages studied. Protein expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α and IL-17 was significantly affected by IMI; being higher in S. aureus-infected than uninfected quarters during all evaluated stages. In S. aureus-infected and uninfected quarters protein expression of lactoferrin increased from day 7–14 of involution, decreasing significantly to day 21 in mammary quarters with chronic infections. The number of monocytes-macrophages was significantly higher in S. aureus-infected than in uninfected control quarters at 7 and 21 d of involution. The number of T lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters at 7 and 14 d of involution while the number of B lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters during all evaluated stages, showing a progressive increase as involution advanced. These results demonstrated a sustained and exacerbated innate and adaptive immune response during chronic S. aureus IMI, playing a critical role in the infection control during active involution.     

Localization of fibroblast growth factor I (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis, lactation and involution

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.     

Cytokine activity in bovine mammary gland secretions during the periparturient period.

The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples.