A simple, rapid method for differential cell counts in porcine mammary secretions.

The use of the fluorescent dye acridine orange for making differential cell counts in mammary secretions from sows was investigated, and the variations in cell type during the lactation period were also studied. In untreated samples of mammary secretions polymorphonuclear leucocytes, mononuclear phagocytes, lymphocytes and epithelial cells could be identified with certainty, and the methodological error was small (1.80 to 2.22 per cent). The mammary secretions could be stored at 4 degrees C for six hours without any effect on the differential count. Washing the secretions decreased the percentage of polymorphonuclear leucocytes and increased the percentage of epithelial cells. The polymorphonuclear leucocytes predominated in colostrum (58.0 to 65.5 per cent) and epithelial cells predominated in milk (60 to 89 per cent). Polymorphonuclear leucocytes were the predominant phagocytes in all mammary secretions (7.6 to 65.5 per cent).     

A method for preparation of chromosomes from bovine zygotes and blastocysts

A simple technique for making chromosome preparations from zygotes and early blastocysts is described. The morphological features of blastocysts and total number of cells greatly influence the quality of the preparation.     

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.     

Secretory component and IgA expression by epithelial cells in sow mammary gland and mammary secretions

Secretory component (SC) and IgA expression of epithelial cells were studied in the mammary tissue and mammary secretions of sows. In mammary tissue, SC was not detected until day 105 of gestation. From the time of delivery (day 115) to the time of established lactation, the proportion of epithelial cells containing sc rose from 20 per cent to nearly 100 per cent. There was no IgA in alveolar epithelial cells until day 105 of gestation; on day 115, IgA positive epithelial cells were present in 10 per cent of the alveoli, which increased to 80 per cent during lactation. Epithelial cells represented more than 20 per cent of the total cells in colostrum, and predominated over leucocytes in milk. In colostrum, these epithelial cells (9 to 15 μm) showed weakly positive membrane, sc, contained cytoplasmic SC and had a limited capacity for in vitro proliferation. Ten per cent of epithelial cells contained intracytroplasmic IgA. In milk, the epithelial cells were larger (15 to 40 μm) with a higher expression of both membrane and intracytoplasmic sc; 66 per cent of these cells expressed intracytoplasmic IgA. These data showed that the capacity of mammary epithelium to process IgA to secretory IgA was complete at the end of mammary gland organisation, and established that the epithelial cells of milk contribute to the transfer of IgA to neonates.     

A study of bovine T-cell subsets in the blood and mammary gland secretions during the dry period

Blood and mammary secretions were obtained from cows throughout the dry period. Quantitative and qualitative assays were performed to determine the cell types and cell distributions at weekly intervals from day of dry off until parturition. The total cell counts in secretions increased during involution and remained at high levels until a few weeks prepartum. The macrophages were the predominant cell type in mammary secretions whereas the numbers of lymphocytes were always less than neutrophils or macrophages. Enriched mononuclear cell populations derived from blood and mammary secretions were also evaluated using "T-cell rosette" assays. Changes observed in the relative distribution of three T-cell subsets in secretions did not reflect the dynamics of the cells in the peripheral blood. T-cell subsets that predominated in mammary secretions were the EN+ EAET+ and EN-EAET+ phenotypes. Distinct patterns of migration or differentiation of T-cell subsets were suggested by the changes of subsets observed in mammary secretions collected throughout the dry period.     

Persistence of antibiotics in bovine mammary secretions following intramammary infusion at cessation of milking

A study was conducted to determine the persistence of antibiotic preparations for use in nonlactating cows in bovine mammary secretions following intramammary infusion at cessation of milking. Five commercially available antibiotic formulations were evaluated using 311 cows. All quarters of each cow were sampled once only during the nonlactating period and most cows were sampled at or near parturition. Antibiotic residues were detected qualitatively by the Bacillus stearothermophilus disc assay. Great variation between different antibiotics in persistence in mammary secretion was observed. In general, mammary secretions from most mammary glands infused with cloxacillin or penicillin-dihydrostreptomycin were positive at 28–35 days after infusion and some were positive at 42–49 days after infusion. On the other hand, <13% of mammary secretions at 7 days after infusion of novobiocin and 50% of mammary secretions at 14 days after infusion of penicillin-novobiocin were positive for antibiotics. Cephapirin benzathine persisted for about 21 days after infusion. Some samples that were positive for antibiotics after initial testing were negative following heating of samples, suggesting that component(s) of dry secretion can inhibit growth of B. stearothermophilus and influence the interpretation of results. Colostrum samples from all quarters except one were negative for antibiotics. These data suggest that nonlactating-cow antibiotic formulations persist primarily during the early to mid-nonlactating period. Based upon present methods of formulation, it would appear that antibiotic preparations for use in nonlactating cows most likely provide little protection during the periparturient period, at a time when mammary glands are highly susceptible to new intramammary infections.     

A rapid method for the large scale preparation of bovine leukemia virus antigen

Bovine leukemia virus (BLV) is the etiologic agent responsible for enzootic bovine leukosis. Detection of cattle seropositive for BLV and laboratory studies of BLV require the preparation of large quantities of BLV antigen. A convenient and economical method for concentrating virus from large volumes of cell culture medium involves the precipitation of the virus in the cold by polyethylene glycol (PEG). The present work demonstrates the feasibility of rapidly precipitating BLV with 10% PEG and subsequently separating the resuspended virus from the residual PEG on a disposable desalting column.     

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10⁷ cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.     

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.     

Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected.Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells.In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.     

Immunofluorescent immunoglobulin differentiation of vibrio fetus antibodies from bovine cervico-vaginal secretions

Reports on discrepancies between local and systemic immunity started to appear about 50 years ago (cf. Tomasi & Bienenstock 1968). Protection against infections has been shown in many cases to be closely related to the antibody content of external secretions and more or less independent from the serum antibody level.     

Restriction endonuclease patterns of bovine herpesvirus type 1 isolated from bovine mammary glands

Restriction endonuclease analysis was used, in conjunction with viral neutralization and growth-curve experiments, to compare a bovine herpesvirus type 1 (BHV-1) isolate, originally obtained from bovine mammary gland lesions, with a standard BHV-1 strain, infectious bovine rhinotracheitis virus. Although differences were not detected by viral neutralization or growth-curve experiments, restriction fragment patterns generated by Bam HI, Eco RI, Hind III, and Hpa I, revealed definite differences between the isolate and the prototype strain. Additionally, Eco RI, Hind III, and Hpa I patterns revealed that the mammary gland isolate had DNA-fragment patterns characteristic of infectious pustular vulvovaginitis strain of BHV-1, type 2b. Seemingly, type-2b isolates, similar to types 1 and 2a, may be capable of causing divergent types of infection of variable severity in cattle.     

An evaluation of the mononuclear cells derived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties and non-specific esterase staining.

The distribution of mononuclear cells isolated from the bovine mammary gland during the nonlactating (dry) period was examined using monoclonal antibodies against leukocyte cell surface antigens, cellular light scattering properties, and the presence of nonspecific esterase. Most of the mononuclear cells isolated during the dry period were lymphocytes. T cells predominated until about 1 week prior to parturition. During the week prior to calving, the percentage of B cells increased until it approximated T cells. The ratio of CD4:CD8 cells was 2-3:1 for mammary gland T cells. This was similar to the ratio found in peripheral blood. At dry-off, about 12% of mammary mononuclear cells were macrophages. The macrophage percentage increased (to about 30%) at mid-dry and remained at this levels until parturition. PMN's were isolated with the mononuclear cells during the first 2 weeks dry and the week prior to calving. Three methods were used to identify mammary macrophages. Esterase staining (as an enzymatic method), forward angle/90 degrees light scatter (based on size and internal complexity), and MHC class II/forward angle light scatter (based on size and surface markers) were compared. Each method yielded similar specificity for macrophage identification. Non-adherent cell fractions, obtained by passage of the cells over Sephadex G-10 columns, were enriched in CD4 positive T cells, somewhat depleted of B cells, and depleted of macrophages and PMN's. Cells eluted from G-10 columns, with lidocaine, were mostly lymphocytes, but reflected the cells loaded onto the column.     

Examination of chemotactic properties of bovine mammary macrophages.

An in vitro model system in which polymorphonuclear leukocytes (PMN) migration under agarose was employed to examine the ability of mammary macrophages to release chemoattractants for PMN. Mammary macrophages were incubated in Hanks' balanced salt solution for up to 12 h in the presence of Staphylococcus aureus. The possibility that the chemotactic activity is mediated through release of interleukin 1 (IL-1) and prostaglandins (PGs) by mammary macrophages was investigated. The data showed that release of chemotactic activity peaked 6 h following addition of S. aureus in the culture medium of mammary macrophages. Very low levels of IL-1 were detected in the same culture medium. Addition of indomethacin, a PGs synthesis inhibitor, was ineffective in altering the chemotactic activity detected in the culture medium of macrophages. These data suggest that it is highly unlikely that the chemotactic activity is mediated through the production of IL-1 and PGs by the mammary macrophages.