Phagocytosis of Staphylococcus aureus by somatic cells in dry cow secretion of mammary gland

Independently of the stage of the dry period, phagocytosis and killing of Staphylococcus aureus by somatic cells isolated from dry cow secretion were significantly higher in a medium of diluted secretion than in 1% serum and in PBS. Intramammary introduction of vaccine from killed Staphylococcus aureus cells caused, in the steady state dry period, the significant increase of phagocytic and bactericidal activity of somatic cells, examined in the medium of diluted secretion of the mammary gland. The isolated secretion deriving both from vaccinated and non-vaccinated cows causes--in the steady state dry period--the disappearance of the significance of differences between the somatic cells coming from various cows. Phagocytosis is highly significantly correlated with the total and intracellular survival of bacteria; similarly, highly significant correlation exists between the total and intracellular survival of S. aureus.     

Genetic Variation Affecting the Expression of Catalase in DROSOPHILA MELANOGASTER: Correlations with Rates of Enzyme Synthesis and Degradation

Both second and third chromosome substitution lines isolated from natural populations of Drosophila melanogaster affect the expression of catalase (EC 1.11.1.6) at both the larval and adult stages of development. In each case, the level of catalase activity is strongly related to the level of catalase-specific cross-reacting material. Turnover studies employing the catalase inhibitor 3-amino-1,2,4-triazole were conducted on a selected number of lines. Although the variation in steady state levels of catalase protein was highly significant among lines, variation in intracellular degradation rate was not. These results suggest that the different steady state levels observed among lines largely reflect different rates of catalase synthesis.     

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.     

Selenium Concentration and Glutathione Peroxidase (GSH-Px) Activity in Serum of Cows at Different Stages of Lactation

The aim of the study was to evaluate the activity of glutathione peroxidase (GSH-Px) and the concentration of selenium in Holstein-Friesian cows at different stages of lactation. Selenium was determined spectrofluorimetrically and GSH-Px activity using a Sigma CGP1 Glutathione Peroxidase Cellular Activity Assay kit. Mean serum selenium concentration was highest in early-lactation multiparous cows (0.18 μg/ml) and the lowest in dry cows (0.111 μg/ml). In early lactation, serum selenium concentration was significantly (P ≤ 0.01) higher in multiparous cows than in cows from the other groups. Mean GSH-Px activity in the serum of dry cows was over twice lower than in late-lactation cows (P ≤ 0.01) and over four times lower than in first-calving heifers and multiparous cows in early lactation (P ≤ 0.01). The coefficients of Spearman's rank correlation between GSH-Px activity and selenium concentration in the cows at different stages of lactation were not significant. A significant (P ≤ 0.01) mean positive correlation (0.46) was found between GSH-Px activity and serum selenium concentration for all the cows analysed together. The highest Se concentration and GSH-Px activity found in the serum of cows during the first stage of lactation may suggest that the generation of reactive oxygen species and their derivatives was higher during this period compared to the other stages, thus placing the cows at a greater risk of oxidative stress. It is therefore essential to give particular attention during this period to meeting the cows' requirement for selenium and other feed components that increase, directly or indirectly, the capacity of the body's antioxidant system.     

Sex steroid hormones do not influence the oxidative burst activity of polymorphonuclear leukocytes from ovariectomized cows in vitro

During the periparturient period, dairy cows are subjected to physiological changes that may induce immunosuppression and an increased susceptibility of the animal to bacterial infections such as mastitis. The incidence of clinical environmental mastitis is high during the last period of gestation, at parturition and during the first month of lactation, suggesting a potential influence of sex steroid hormones. Efficient functioning of polymorphonuclear leukocytes (PMN) is necessary during the early phase of infection to clear the mammary gland from invading pathogens. The purpose of this study was to investigate the effect of sex steroid hormones on the oxidative burst activity of isolated PMN from ovariectomized cows. Ovariectomy was performed to minimize the interference of endogenous estrogen and progesterone levels, which are known to vary extensively during the estrus cycle. Isolated PMN were incubated with different concentrations of 17beta-estradiol, estrone or progesterone. A flow cytometric technique was used to quantify the oxidation of intracellular 2',7'-dichlorofluorescin by the oxidative burst system of PMN following stimulation with phorbol myristate acetate. Staurosporine was used as a positive control for our in vitro model. No statistically significant changes in PMN oxidative burst activity were observed at physiological or pharmacological levels of the three sex steroid hormones. A large variation existed in the oxidative burst activity among cows. In an additional experiment, the expression of estrogen receptor alpha and of progesterone receptor in PMN was evaluated immunohistochemically. No specific staining was detected for both receptors in isolated PMN following incubation with different concentrations of sex steroid hormones.     

Role of serum in the intracellular killing of staphylococci in rabbit monocytes

Shayegani, Mehdi G. (U.S. Veterans Administration Hospital, Philadelphia, Pa.), and Stuart Mudd. Role of serum in the intracellular killing of staphylococci in rabbit monocytes. J. Bacteriol. 91:1393-1398. 1966.-Although some intracellular killing occurs in rabbit monocytes with heated normal serum or even in monocytes washed three times with Hanks' solution and with staphylococci not exposed to serum, efficient killing of coagulase-positive Staphylococcus aureus cells in the mononuclear phagocytes of rabbits is shown to require heat-labile components of serum. The effect of serum in promoting phagocytosis and intracellular killing may be exhibited either by presensitization of the staphylococcal cells before contact with leukocytes or by the presence of serum in the phagocytic system. Under any conditions studied the rate of intracellular killing of S. aureus is very slow.     

Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca²⁺ release from intracellular Ca²⁺ stores and anti-PGN-IgGdependent Ca²⁺ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41]     

Effects of continuous lactation and short dry periods on mammary function and animal health

The dry period is required to facilitate cell turnover in the bovine mammary gland in order to optimize milk yield in the next lactation. Traditionally, an 8-week dry period has been a standard management practice for dairy cows based on retrospective analyses of milk yields following various dry period lengths. However, as milk production per cow has increased, transitioning cows from the nonlactating state to peak milk yield has grown more problematic. This has prompted new studies on dry period requirements for dairy cows. These studies indicate a clear parity effect on dry period requirement. First parity animals require a 60-day dry period, whereas lactations following later parities demonstrate no negative impact with 30-day dry period or even eliminating the dry period when somatotropin (ST) is also used to maintain milk yields. Shortened dry periods in first parity animals were associated with reduced mammary cell turnover during the dry period and early lactation and increased numbers of senescent cells and reduced functionality of lactating alveolar mammary cells postpartum. Use of ST and increased milking frequency postpartum reduced the impact of shortened dry periods. The majority of new intramammary infections occur during the dry period and persist into the following lactation. There is therefore the possibility of altering mastitis incidence by modifying or eliminating the dry period in older parity animals. As the composition of mammary secretions including immunoglobulins may be reduced when the dry period is reduced or eliminated, there is the possibility that the immune status of cows during the peripartum period is influenced by the length of the dry period.     

Prediction of Vitamin A,Vitamin E,Selenium and Zinc Status of Periparturient Dairy Cows Using Blood Sampling During the Mid Dry Period

Vitamins A and E, and the trace elements selenium (Se) and zinc (Zn) are essential for the health and performance of dairy cows. Their concentrations often decrease around calving and extra supplementation is sometimes recommended at that time. However, the need for this varies, for example depending on quantity and quality of feedstuffs in the diet. The aim of this study was to measure the concentrations of serum vitamin A (S-vit A) and vitamin E (S-vit E), plasma Se (P-Se) and serum Zn (S-Zn) in blood samples taken at several time points from one month before to one month after calving, and to evaluate if a blood sample taken during the mid dry period can accurately predict the blood concentration at calving and early lactation. Dairy cows on 3 different feeding regimens during the dry period were included in the study. A significant decrease in the concentrations of S-vit A and S-vit E, and S-Zn, was observed at calving, and P-Se was significantly lower during the dry period and at calving than in early lactation. The blood concentrations of S-vit E and P-Se in the mid dry period significantly predicted the occurrence of values considered marginal or deficient at the time of calving. The data indicate that a mid dry period concentration of ≥5.4 mg/l of S-vit E and ≥0.09 mg/l of P-Se will result in a 90% chance that the cow stays above marginal levels at calving given that a feed of the same quality is offered.     

金葡菌荚膜多糖血清型的研究进展

金葡菌是引起奶牛乳腺炎的一种主要的致病菌,致病性金葡菌多数含有荚膜成分,金葡菌荚膜多糖有11种血清型。从不同血清型荚膜多糖的结构,基因构成,抗吞噬作用,致病性等方面作了介绍,并简要介绍了金葡菌荚膜多糖血清型的国内外研究现状。     

Quantification of phagocytosis in human neutrophils by flow cytometry.

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

Effects of forage NDF content and body condition score on forage intake by Holstein-Friesian dairy cows in the dry period

This study used individual weekly results for 160 non-lactating Holstein-Friesian dairy cows in the last 5 weeks of gestation to develop regression equations based on forage NDF content and individual body condition score (BCS) for predicting dry matter (DM) intake. Results were used from treatments in which cows received the same forage and no concentrates throughout the dry period. Ten different conserved forages, either grass silages or mixtures of grass silage and barley straw, were fed in six different experiments and forage NDF ranged from 452 to 689 g/kg DM. On average cows gained 390 g live weight per day, which is less than conceptus growth at this stage - suggesting some mobilisation of maternal tissues to support conceptus growth. BCS remained unchanged at 2.5 over the dry period. DM intake declined from 10.79 kg/day 5 weeks before calving to 9.32 kg/day in the week before calving, with half of this decline occurring in the final week before calving. Intake as a percentage of live weight was moderately predicted (R2 = 0.61 for the entire period) from measures of diet composition (NDF) and cow state (BCS). There were highly significant negative effects of forage NDF and increased BCS on DM intake. The effect of BCS on DM intake was greatly reduced in the week before calving, possibly as a result of a change in metabolic priorities from gaining to losing body reserves.     

Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and secretion of serum albumin

A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones.The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.     

Expression of mRNA for follicle-stimulating hormone suppressing protein in ovarian tissues of cows.

The expression of bovine follicle-stimulating hormone (FSH)-suppressing protein (FSP) mRNA was investigated in different ovarian tissues of cows. Northern blot analysis, using a cDNA probe to bovine FSP, demonstrated that the FSP gene in the bovine ovary is highly expressed in a pool of isolated granulosa cells. Two bands (2.8 and 1.8 kb) were observed in all tissues expressing the mRNA. FSP mRNA was low in small antral follicles and increased in growing follicles to reach a maximum in preovulatory follicles. Low amounts of mRNA of steady state FSP were observed in all stages of the corpus luteum as well as in the corpus luteum of pregnant cows, in the corpus albicans and theca tissue, whereas this mRNA could not be detected in the liver. These results are consistent with the hypothesis that, in cows, FSP functions as an autocrine regulator in developing follicles to facilitate luteinization of granulosa cells.     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Linking exocytosis and endocytosis during phagocytosis

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome-related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome-related organelle secretion could also be implicated in phagosome formation.     

Influence of season and estradiol on secretion of luteinizing hormone in ovariectomized cows

The hypotheses that secretion of luteinizing hormone (LH) varies with season and that estradiol may modulate the seasonal fluctuation in secretion of LH in cows were investigated. Seven mature cows were ovariectomized approximately 30 days before initiation of the experiment. Three of the ovariectomized cows (OVX-E2) were administered a subcutaneous estradiol implant that provided low circulating levels of 17 beta-estradiol. The remaining 4 cows (OVX) were not implanted. From December 21, 1982, to September 20, 1984, blood samples were collected sequentially (at 10-min intervals for 6 h) at each summer and winter solstice, and each spring and autumn equinox. In addition, from March 17, 1983, to March 17, 1984, sequential samples were collected midway between each solstice and equinox. Concentration of LH was measured in all samples, and concentration of estradiol was measured in pools of samples. An annual cycle in mean serum concentration of LH and amplitude of LH pulses was detected in both groups of cows. The seasonal pattern did not differ in the two treatment groups. Serum concentration of LH and amplitude of LH pulses were highest around the spring equinox, decreased gradually to the autumn equinox, and then increased and peaked again during the following spring equinox. Frequency of LH pulses and concentration of estradiol in serum did not vary with season. Circulating concentrations of LH and amplitude of pulses tended to be higher in OVX-E2 than OVX cows throughout the experimental period. Frequency of pulses of LH was lower in OVX-E2 than OVX cows throughout the experiment. Concentrations of estradiol were higher in the implanted cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of oxidative stress and metabolic adaptation on PON1 activity and MDA level in transition dairy cows

Serum PON1 is a HDL-associated enzyme that protects lipoproteins, both LDL and HDL, against oxidation and it is considered as an antioxidative/anti-inflammatory component of HDL. Dairy cows are highly susceptible to oxidative stress which commonly occurs in late pregnancy and early lactation. During the transition period, increased production of reactive oxygen species is associated to processes of metabolic adaptation to a low-energy balance. We investigated serum paraoxonase-1 (PON1) activity and malondialdehyde (MDA) concentration to assess the antioxidative/prooxidative status during pregnancy and the postpartum period. In order to evaluate metabolic homeostasis, common metabolic parameters (glucose, triglyceride, total cholesterol, HDL-C and albumin concentrations) were determined as well. A significantly lower PON1 activity was found in late pregnancy and early postpartum (P<0.05) compared to the first and the second trimester of pregnancy and the mid-lactation. MDA level was significantly higher (P<0.05) in the dry period compared to pregnant lactating and postpartum cows. Serum glucose concentration (P<0.001) was lower in the early and late puerperium indicating low-energy balance in the early lactation. Serum triglyceride and albumin concentrations were lower in late puerperium (P<0.001), while total cholesterol and HDL-C were lower during the dry period (P<0.05) as well as in early postpartum (P<0.001). Significant correlations of PON1 activity with glucose (P<0.05), albumin (P<0.05), total cholesterol (P<0.001) and HDL-C (P<0.001) were also found. The observed lower serum PON1 activity and higher MDA level in late pregnancy and early postpartum could indicate a prooxidants/antioxidants imbalance influenced by reproductive stress and metabolic adaptation in the transition period of dairy cows.     

Albumin synthesis and secretion by isolated morphologically characterized avian hepatic parenchymal cells

Cell suspensions were prepared from rooster liver by sequential perfusion with EGTA (ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid) and collagenase. Tissue morphology during disaggregation showed the appearance of discrete intercellular spaces in response to EGTA perfusion. This effect apparently permits the collagenase greater access to the liver cells and accounts for the greater cell yields obtained with the sequential procedure. The cell suspensions contained 95% parenchymal cells, and the enrichment for parenchymal cells was found to occur at two stages in the disaggregation procedure. Parenchymal cells remain viable in suspension culture for several hours and continue to synthesize and secrete serum albumin. Analysis of albumin secretion shows a minimum processing time of 30 min. between synthesis and secretion while intracellular albumin attains an apparent steady state after approximately 100 min. of labeling with [³H]-leucine. These studies indicate that rooster parenchymal cells can be used to examine the processing and secretion of proteins by a cell committed to the constitutive secretion of multiple protein products.