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.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

Cryopreservation of bovine peripheral lymphocytes and its effect on the $\text{in vitro}$ response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to ?30°C at 1.3°C/minute and further to ?80°C at 6°C/minute and then rapidly to ?196°C by dropping in liquid nitrogen. The cells were recovered by rapid thawing in water at 30–35°C and washed twice before use in the stimulation test. Ten percent DMSO had a much better protective effect than 5%; addition of 25% fetal bovine serum to the freezing had no favourable effect. In most of the 16 animals used in the experiments the frozen lymphocytes gave the same or a higher response to Con A than those kept in the DMSO containing medium at 4°C for two hours.The responses of the frozen cells were comparable to those of fresh lymphocytes (kept at 4°C for two hours in medium without DMSO).     

Monocyte-induced potentiation of bovine fetal thymocyte mitogenic responses to concanavalin A

Peripheral blood monocytes significantly potentiated the mitogenic response of bovine fetal thymocytes to Concanavalin A as measured by incorporation of [3H] thymidine into cellular DNA. Mononuclear cells obtained from either normal or Mycobacterium bovis sensitized cattle were cultured with or without purified protein derivative (PPD) for 24 hours at which time bovine fetal thymocytes and concanavalin A were added. After 3 days of culture, both activated or non-activated monocytes significantly potentiated Con A-induced blastogenic responses. of monocytes from thymocyte cultures completely abrogated thymocyte responses to Concanavalin A.     

Physiological and morphological changes in bovine mammary glands following intramammary infusion of recombinant interferon-gamma.

Eleven lactating dairy cows were used to evaluate the response of bovine mammary glands to increasing doses of recombinant bovine interferon (rBoIFN)-gamma. Right front and rear quarters were intramammarily infused with eight different doses (10(2) U to 2 x 10(8) U/quarter) of rBoIFN-gamma; each dose was tested in at least two quarters. Left udder halves served as within animal controls in which quarters were injected with a saline placebo or were not infused at all. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 6, 24, 36 and 48 h following infusion. Animals were slaughtered immediately following the 48 h sampling period and mammary tissue was obtained for morphometric analyses. Milk composition was similar between control quarters and those quarters infused with up to 10(5) U of rBo-IFN-gamma during the entire sampling period. Quarters infused with 10(6) U and 10(7) U of rBoIFN-gamma had higher milk somatic cell counts (SCC) following treatment compared with preinfusion values. Changes in the composition of mammary secretion were most dramatic in quarters infused with greater than or equal to 10(8) U of rBoIFN-gamma as indicated by the significant increase in SCC and milk pH with a concomitant decrease in lactose concentration when compared with pre-infusion values or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in luminal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of rBoIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of bovine lymphocyte response to phytohemagglutinin by a specific 5-lipoxygenase inhibitor

AA861, a specific 5-lipoxygenase inhibitor, inhibited bovine lymphocyte response to phytohemagglutinin (PHA). Mitogen-stimulated cultures of mononuclear cells produced leukotriene B4 (LTB4) in 24 hours. The production of LTB4 was completely inhibited by concentrations of AA861 that inhibited mitogen-induced 3H-thymidine incorporation. The inhibition of lymphocyte proliferation was totally reversed by LTB4 but not by leukotriene C4 or leukotriene D4. The inhibition of interleukin-2 (IL-2) production by AA861 was also completely reversed by addition of exogenous LTB4 to lymphocyte cultures. Thus, endogenous LTB4 production appeared to be necessary for PHA-induced IL-2 production and lymphocyte proliferation.     

Commercial serum inhibition of phytohemagglutinin induced blastformation

Growth supporting capability of commercial versus autologous, homologous and millipored autologous sera, obtained from animals maintained in our laboratory were studied using phytohemagglutinin (PHA) stimulated sheep lymphocyte cultures. A significant reduction in proliferation of PHA-stimulated lymphocytes was observed in cultures containing commercial sera as compared to cultures containing autologous or homologous sera. Moreover, commercial sera containing cultures both with and without PHA, demonstrated significantly reduced culture cellularity. The cause of reduced growth supporting capability of commercial sera remains to be determined but was not due to the presence of cells, activated complement components or preservatives in commercial sera.     

Mycoplasma bovis suppression of bovine lymphocyte response to phytohemagglutinin

The effect of viable Mycoplasma bovis on the in vitro bovine peripheral blood lymphocyte response to phytohemagglutinin (PHA) was studied. Results showed that M. bovis did not act as a mitogen for bovine lymphocytes. Viable M. bovis produced a dose and time dependent suppression of the PHA stimulated lymphocyte response. Suppression was not a result of differences in the viability of infected or control lymphocyte cultures. The suppressive effect of M. bovis was found to be independent of the concentration of PHA used in the test and the lymphocyte response could not be restored by supplementation of the culture medium with arginine. Delay for 48 h after PHA stimulation before adding M. bovis to the lymphocyte cultures diminished, but did not prevent, the suppression of the lymphocyte response. These results show that suppression of the lymphocyte response does not require the presence of M. bovis during the period of PHA stimulation, and that M. bovis was capable of interrupting [3H]-thymidine incorporation in lymphocytes which were actively synthesizing DNA.     

Generation of neutrophil chemoattractants by phagocytosing bovine mammary macrophages

Macrophages from bovine mammary gland were cultured in vitro and the growth medium collected at intervals. Using an in vitro system in which neutrophils migrated under agarose, both chemotactic and chemokinetic activity for bovine neutrophils was detected in supernatants of macrophage cultures to which heat killed preopsonised Staphylococcus aureus had been added. The suspensions of killed bacteria were not themselves chemotactic for neutrophils and no chemotactic activity was present either in supernatants from unstimulated macrophage cultures or in sonicated macrophages. The chemotaxin(s) was generated within two hours of the addition of staphylococci to the cultures and was largely stable to heating at 56 degrees C for 45 minutes, although its activity was reduced by boiling for 15 minutes. Traces of proteolytic activity were also detected in some supernatants. Substantial proteolytic activity was found in lysates of neutrophils. Unlike chemotaxis, proteolytic activity was suppressed by addition of milk from early lactation and containing high natural levels of protease inhibitors. Proteolytic activity was destroyed by boiling for 15 minutes.     

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。     

Morphometric analysis of involuting bovine mammary tissue after 21 or 42 days on non-suckling

Mammary gland involution was morphologically evaluated 21 or 42 d after prevention of suckling of one udder half in 10 crossbred beef cows. Parenchymal tissue was taken from lower, middle and upper zones of each quarter from the teat to the ventral body wall. Udder halves, trimmed of extraparenchymal tissue, were weighed and used for DNA determination. DNA content was reduced 50 and 64% after 21 and 42 d of involution. However, the percentage of tissue occupied by epithelium was similar in suckled and nonsuckled glands. Well-differentiated cells, typical of suckled glands, were rarely observed in nonsuckled glands. Alveolar structure was evident in nonsuckled glands, but the number of cells per alveolar cross-section was reduced (30 vs 22). Unlike in suckled glands, there was a marked gradation in classification of epithelial cells across zones in involuting glands. For example, nearly 10% of the epithelium was well-differentiated in the tissue from the upper zone, whereas no well-differentiated cells were found in the lower zones. Regression of the mammary parenchyma does not occur uniformly through the udder, so use of single biopsy to study involution should be avoided. Presence of alveoli after 42 d indicates that redevelopment of the udder with subsequent lactations is less dramatic than suggested from study of other species.     

Secretion of alpha-hemolysin by bovine mammary isolates of Staphylococcus aureus.

A total of 262 strains of Staphylococcus aureus isolated from the mammary gland of dairy cows were examined for the production of alpha-hemolysin. Strains were cultured in a liquid medium of casein hydrolysate and yeast extract in an atmosphere of 7% (v/v) CO2 in air. The assay consisted of a dot immunoblotting technique employing bacterial culture supernatants and a mouse monoclonal antibody specific for alpha-hemolysin. Ninety-four percent (247) of 262 strains were positive for alpha-hemolysin by this method, when cultured in the laboratory. This figure is compared with those obtained in previous studies which typically based their results on the hemolytic patterns of isolates on blood agar plates.     

Effect of intramammary devices on the outcome of induced Escherichia coli infection of bovine mammary quarters

Eighteen Holstein cows, free of intramammary infection, were fitted with smooth (n = 9) or abraded (n = 9) intramammary devices (IMD) in 2 diagonally opposed quarters within 4 weeks after calving. The 2 other quarters of each cow were used as controls. Three to 6 weeks after IMD insertion, depending on when milk somatic cell counts returned to a base-line value of less than 4 X 10(5)/ml, all cows were subjected to bacterial challenge exposure in the front or rear quarters by intracisternal injection of about 30 colony-forming units of Escherichia coli/quarter. Challenge exposure was done immediately after milking. Three weeks after the initial bacterial exposure, the other quarter pairs were similarly challenge exposed. Quarter bacteriologic status, concentration of milk somatic cells, and clinical observations (rectal temperature, milk appearance, udder palpation, and general condition of the cow) were monitored. Infection developed in 14 of 16 (88%) quarters with smooth IMD vs 16 of 16 (100%) control quarters and in 7 of 17 (41%) quarters with abraded IMD vs 17 of 17 (100%) control quarters. The difference in infection frequency between quarters with smooth IMD and quarters with abraded IMD was significant (P less than 0.05). Protection against establishment of infection was associated with somatic cell counts greater than 8.0 X 10(5)/ml in milk collected immediately after milking (7 of 12 quarters) or 4 hours later (11 of 12 quarters). In 10 quarters (59%) of cows fitted with abraded IMD, secretory abnormalities appeared before bacterial challenge inoculation. Abnormal milk or visible blood was observed over periods varying from 2 weeks after insertion through the entire lactation.     

A method for preparation of IgA from bovine mammary secretions.

A method for preparing purified IgA from bovine mammary secretions is described. Whey was initially fractionated by gel filtration and fractions containing IgA were pooled, concentrated and digested with pepsin. The digest was rechromatographed on the same type of gel twice and the resulting IgA preparation tested for purity by an enzyme immunoassay procedure. Five different preparations tested were found to contain no measureable IgM or IgG2 and 0.8% to 1.1% IgG1 on a weight basis. If colostral whey was digested with pepsin prior to chromatography, the IgA preparations contained 1.1% to 2% IgG1 and no measureable IgM and IgG2. The procedure provides a reasonably easy method of eliminating most of the contaminating IgG1 (dimeric) and allows preparation of quantities of IgA for immunochemical studies and standardization of serological techniques.     

Secretion of hydrogen peroxide by phagocytic cells from bovine non-lactating mammary glands

Phagocytic cells from non-lactating bovine mammary glands have the capacity to secrete hydrogen peroxide when exposed to the soluble membrane stimulant phorbol myristate acetate (PMA). Unfractionated cell suspensions, containing mainly neutrophils and macrophages, and cell monolayers enriched for macrophages secreted hydrogen peroxide. A correlation was observed between the amount of hydrogen peroxide secreted, the antibacterial activity of the cells and the number of neutrophils present in the cell suspensions. Pre-exposure of cells to PMA significantly impaired their antibacterial activity against Staphylococcus aureus suggesting the importance of oxygen metabolism in the bactericidal capacity of these cells.