Ultrastructural features of bovine mammary epithelial cells grown on collagen gels.

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.

Culture of bovine mammary epithelial cells on collagen gels.

A method for the isolation of lobules of acini from bovine mammary gland and their storage in liquid nitrogen is described. After further dissociation of freshly prepared or frozen lobules, clumps of cells are obtained which attach to collagen gels and give rise to colonies which, on morphological criteria, appear predominantly epithelial. Storage for up to 6 months did not adversely affect viability. Increase in colony area involved cell division, was more rapid in air than in 95% oxygen and was enhanced by fetal calf serum.

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

The mammary glands of eight non-lactating dairy cows were infused with either endotoxin or the sterile culture filtrate in which Escherichia coli had been grown (CCF). Animals were necropsied at intervals up to 24 h after infusion. Only mild inflammation of the teat and lactiferous sinuses occurred after 4 h, subsiding gradually to 24 h. After mixing dry cow secretion with either endotoxin or CCF, an intense inflammatory response was produced on inoculation of quarters of a lactating cow; CCF also produced necrosis of the epithelium. Thus, dry gland secretions do not inhibit the effect of filtrate or endotoxin. It was concluded that the refractory nature of the gland to these toxins in the non-lactating animal is due to the changes which occur in the ductular epithelium during involution.

Incidence of rotavirus in artificially reared pigs and some effects of diarrhoea on the physiology and histology of the gastrointestinal tract.

Pigs reared on a milk substitute from two days old often developed diarrhoea, but suckled littermates remained healthy. Only in a few pigs was diarrhoea associated with the presence of rotavirus. Rotavirus was also present in some healthy pigs, and was associated with a reduction in villus length. Pigs with diarrhoea usually had an increased amount of digesta in the stomach and a reduction in lactase activity in the small intestine but villus length was unchanged. There was no evidence of lactose malabsorption.

Inhibition of the bactericidal activity of bovine polymorphonuclear leucocytes and related systems by casein.

Polymorphonuclear leucocytes (PMN) obtained from lactating cows' udders were deficient in their ability to kill Staphylococcus aureus compared with PMN isolated from blood. However, blood PMN suspended in separated milk or in the presence of casein were similarly impaired. Casein was found to inhibit in vitro the bactericidal activities of histone, the lactoperoxidase-H2O2-KI system and PMN lysates. Electron microscopy showed that casein was ingested by PMN with the formation of phagocytic vacuoles. These observations provide the basis of a hypothesis explaining the bactericidal deficiency of milk PMN and the consequent susceptibility of the udder to infection.

Progressive pathology of severe Escherichia coli mastitis in dairy cows.

An investigation was made using light and electron microscopy of the progressive pathological changes in nine experimental and two natural cases of severe Escherichia coli mastitis in dairy cows. The duration of infection varied from 18 hours to 13 days. Epithelial lesions were not found in glands which had been infected for more than 24 hours. However, the epithelia of the sinuses and large ducts became hyperplastic after 60 hours of infection and by six days hyperplasia was extensive on the crests of folds. The leucocyte response in the lumina of the glands and subepithelial tissue showed a progressive change from an acute neutrophil reaction to a chronic mononuclear cell infiltration within the first 36 hours of infection. The only changes affecting the secretory tissue occurred after six days of infection and were typical of mammary gland involution which was probably a direct consequence of anorexia.

Pathogenesis of experimental bovine mastitis following a small inoculum of Escherichia coli.

Mastitis was produced in four quarters of two lactating cows by the inoculation of 50 or 200 viable Escherichia coli. Changes were investigated by light microscopy and scanning electron microscopy. After 10 hours the changes were confined to the superficial layer of the epithelium of the teat and lactiferous sinuses; single cells or small groups of cells were damaged and were being extruded from the tissue. By 14 hours there was extensive necrosis and sloughing of the epithelial cells which did not extend beyond the basement membrane, and an intense inflammatory response associated with the epithelial damage. The somatic cells in the milk, mainly polymorphonuclear leucocytes, increased 40 to 250 times and strongly inhibited the survival of E coli. Epithelial damage appeared first in the ventral portions of the gland and then extended through the lactiferous sinus to the large ducts and secreting tissue. This pattern of damage together with the failure to observe bacteria attached to the epithelia was consistent with the production of diffusible toxins by the organisms.

The aetiology of diarrhoea in pigs weaned at two days of age.

When pigs are weaned at two days of age large numbers of Excherichia coli appear in the anterior gut and the incidence of diarrhoea rises. The two phenomena do not appear to be directly related because the strains of E coli isolated are not serotypes previously found to be associated with neonatal pig scouring. Representative strains of the non-enteropathogenic serotypes did not produce enterotoxin and did not adhere to small intestine brush borders. Moreover when antibiotics were fed to eliminate E coli from the gut, the pigs still scoured. Rotavirus was detected in the gut contents and gut epithelium of scouring pigs and a bacteria-free filtrate of gut contents produced diarrhoea when administered to germ-free pigs. It is suggested that rotavirus may be one of the causes of the scouring seen shortly after weaning pigs at two days of age.

Hyperacute Escherichia coli mastitis of cattle in the immediate post-partum period.

The pathology of hyperacute coliform mastitis was studied in 5 post-parturient cows. In all infected quarters infiltration of neutrophils was negligible. In all except one case there was severe damage to the ductular and secretory system, involving most areas of the gland. Bacteria were dense in infected alveoli, and there was evidence of substantial phagocytosis of bacteria by the secretory epithelium. The exception showed a large lesion in the middle of the gland from which the spread was ductular; other infections were consistent with spread via the teat canal. The organisms were largely confined to the ductular/secretary lumen and there was little invasion of the parenchyma. The severity of the disease was considered due to the absence of the inflammatory response seen in mid lactation.

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.

Enzyme catalysis in multiphase systems: direct determination of water distribution.

The distribution of water and the formation of microphases during the synthesis of octyl octanoate and the hydrolysis of triolein catalyzed by immobilized lipase have been directly monitored in real time using confocal laser microscopy.

An ultrastructural study of the sinus epithelium in the mammary gland of the lactating ewe.

The teat and lactiferous sinus epithelium from the mammary glands of 23 lactating ewes was examined by light and electron microscopy. Most of the sinus epithelium consisted of two layers of non-secretory cells but, in the lactiferous sinus, cells with the same ultrastructural features as alveolar secretory cells were also found. Secretory cells sometimes occupied more than 50% of the total area of the sinus. Many non-secretory cells in the lactiferous sinus possessed a single cilium but they were less common in the teat sinus. 'Accessory glands', which opened directly into the lumen of the gland, were found beneath the epithelium in both the teat and the lactiferous sinuses. From their ultrastructure it was clear that these glands consisted of normal secretory alveoli and that they produced normal milk components. It is suggested that the mixed population of secretory and non-secretory cells in the lactiferous sinus provides unique material for the experimental study of many aspects of mammary gland physiology.

The epithelial cells and cell fragments in human milk.

The cell fragments and epithelial cells in human milk were examined in samples obtained from 30 women: 3 of these provided sequential samples at weekly intervals for 110 days. Membrane-bound cytoplasmic fragments in the sedimentation pellet greatly outnumbered the population of intact cells in all samples. Most of the fragments were derived from secretory cells and contained numerous cisternae of rough endoplasmic reticulum, lipid droplets and Golgi vesicles containing casein micelles. Secretory epithelial cells were present in small numbers in all samples and after the 2nd month of lactation replaced the macrophage as the predominant cell type. Ductal epithelial cells represented less than 1% of the total cell population up to 8 days post-partum, but thereafter they were very rarely found. They occurred in aggregates of 2--4 cells and possessed tight junctions that circumscribed the area of cell-cell contact. All samples of milk contained squamous epithelial cells derived from the galactophores and/or the skin of the nipple. Bacteria were often attached to the surface of the squamous cells. The possible relationship between the presence of secretory epithelial cells in milk and the occurrence of milk proteins in the blood of lactating women is discussed.

The origin, structure and occurrence of corpora amylacea in the bovine mammary gland and in milk.

Corpora amylacea were present in variable numbers in the mammary glands of 8 lactating cows but they were not observed in the glands of pregnant or virgin heifers. In primiparous cows they were relatively simple bodies composed of 2--3 concentric layers but in multiparous animals, more complex multilayered structures were also noted. Corpora amylacea were formed in alveoli lined with actively secreting cells. The initial stage of their formation involved the aggregation, fusion and compaction of casein micelles in the alveolar milk. The resulting structure acted as a nucleus for further growth which occurred by the addition of material of micellar origin to its surface. Bodies containing a large proportion of crystalline material were common. Contact of corpora amylacea with the alveolar epithelium often caused flattening or irreversible damage to the secretory cells. The corpora amylacea recovered from milk corresponded in appearance to various stages in their growth seen in the mammary gland. They were usually present in the colostrum of multiparous cows but never in that from primiparous animals. In only 1 cow was the colostrum and milk found to be consistently free of corpora amylacea.

Ultrastructural surface changes associated with dextran synthesis by Leuconostoc mesenteroides.

When Leuconostoc mesenteroides NCDO 1875 was grown in MRS broth and fixed for electron microscopy in the presence of ruthenium red, the cell wall appeared as a triple-layered structure similar to other, gram-positive bacteria. When such logarithmic-phase cultures were exposed to sucrose, the appearance and growth of a uniform layer of electron-dense material was evident on the surface of the cell wall. After 2 h in the presence of sucrose, the formation of this surface coat (110 to 130 nm thick) was complete. For 85 to 90% of the cells, continued exposure to sucrose did not produce any further change in their appearance, but the rest of the population began to accumulate insoluble capsular dextran at the surface of their coat material. Within 18 h, these cells had produced a large capsule (maximum diameter, 6 micrometer) composed mainly of an extensive reticulum of fine filaments. Periodate-reactive carbohydrate was localized cytochemically in the capsular dextran and in the surface coat of all cells. It is suggested that the surface coat of sucrose-grown cells represents a cell-bound dextran-dextransucrase complex and that the acapsulate cells produce the relatively soluble S dextran reported by previous workers.