Functional maturation during bovine granulopoiesis

Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow. These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process. The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species. Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation. Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells. Functions and receptor expressions assessed in the three maturation stages were: Fc-IgG(2) receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity. Immature cells expressed already Fc-IgG(2) receptor and CD11b on their cytoplasma membrane. Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis. Promyelocytes, myelo-cytes and metamyelocytes showed no respiratory burst activity. Only band and segmented cells produced reactive oxygen species. Myeloperoxidase was present at all stages of maturity. Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors. Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows.

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis

Influence of 17 beta-estradiol, progesterone, and dexamethasone on diapedesis and viability of bovine blood polymorphonuclear leukocytes

The aim of the current study was to investigate whether polymorphonuclear leukocyte (PMN) diapedesis and viability are influenced by steroid hormones. Using an in vitro model with different types of cell layers ( bovine mammary epithelial cells and fibroblasts), we investigate whether steroid hormone treatments (17beta-estradiol, progesterone, and dexamethasone) have an influence on the diapedesis capacity and viability of PMN. In addition, we studied apoptosis of PMN in the in vitro model and evaluated the influence of different types of cell layers and steroid hormone treatments on this process. A significant decrease in the number of viable PMN in the lower compartment of the in vitro model (i.e., number of migrated PMN x viability after migration) was found after 17beta-estradiol treatment, whereas no influence was detected after progesterone or dexamethasone treatment. The effect of 17beta-estradiol was not due to a lower viability before migration as none of the treatments caused a significant effect on the viability before diapedesis. This treatment effect was not influenced by endogenous 17beta-estradiol or progesterone levels before isolation because there was no correlation between these plasma levels and PMN diapedesis capacity or viability. Furthermore, migration through epithelial cells caused a significant decrease in viability of PMN due to increased apoptosis but not necrosis

Differential effects of steroids and retinoids on bovine myelopoiesis in vitro

Pregnancy and parturition are associated with physiological changes caused by steroid hormones. Alterations in number, maturity, and function of polymorphonuclear leukocytes observed in dairy cows at parturition suggest a common causative relationship with steroid hormones. This study was designed to investigate the effects of progesterone, 17-beta-estradiol, and hydrocortisone on the proliferation of bovine progenitor cells. An in vitro culturing system was used, and colonies were scored after 7 d of incubation. At low concentrations, 17-beta-estradiol inhibited proliferation of granulocyte progenitor cells. Hydrocortisone reduced growth of granulocyte and monocyte colonies, whereas myelopoiesis was not altered by progesterone. Furthermore, we studied the effect of retinoids on colony formation of bovine bone marrow cells. All-trans- and 9-cis-retinoic acid stimulated growth of granulocyte colonies and inhibited proliferation of the monocyte lineage. The addition of the 13-cis-isomer also increased numbers of granulocyte colony-forming units. This study indicates that steroid hormones may be responsible for alterations in the bovine hematopoietic profiles observed in circulation during the postpartum period. White blood cells, especially polymorphonuclear leukocytes, which are derived from bone marrow, are an important first line defense against mastitis. Therefore, these effects of steroids might contribute to the increased susceptibility of dairy cows to Escherichia coli mastitis. We furthermore hypothesize that an important role might be attributed to retinoic acid in its regulation of bovine myelopoiesis. Modulation of myelopoiesis in favor of the granulocyte lineage during the acute-phase reaction may be an adaptive mechanism designed to increase the capacity of first-line defense to intramammary infections

L-selectin and beta(2)-integrin expression on circulating bovine polymorphonuclear leukocytes during endotoxin mastitis

The aim of this in vivo study was to examine the effect of intramammarily administered endotoxin (lipopolysaccharide, LPS) on the expression of L-selectin (CD62L) and the beta(2)-integrin subunits CD11b and CD18 on circulating bovine PMN. Six early lactating cows were infused with Escherichia coli LPS. The adhesion molecules under study were stained at the cell surface and analyzed flow cytometrically. In addition, some of the clinical parameters associated with adhesion molecule mobilization such as fever, blood cortisol levels, somatic cell count (SCC), and total and differential blood leukocyte count were measured. In analogy with observations during clinical coliform mastitis, a progressive decrease of CD62L expression levels was observed early after LPS infusion, concomitantly with a continuous rise of CD11b and CD18 density. However, no correlation was found between the kinetics of CD11b and CD18 density. The initial changes in adhesion molecule expression paralleled the decrease in blood PMN numbers, together with the increase in rectal temperature, cortisol levels, SCC, and number of circulating immature PMN. In conclusion, intramammarily administered LPS seems to play an important role in modulating adhesion receptor expression on circulating bovine PMN. Interestingly, in contrast to coliform mastitis, the net CD18 variation is not principally influenced by CD11b upregulation during endotoxin administration. The knowledge of adhesion molecule kinetics in relation to the different parameters evaluated in the present study contributes to an improved understanding of the inflammatory reaction

Effect of sampling method and storage conditions on albumin, retinol-binding protein, and N-acetyl-β-D-glucosaminidase concentrations in canine urine samples

Urinary markers for renal dysfunction are gaining interest but effects of sampling method, storage conditions, and urinary tract inflammation or infection on these markers are unclear Therefore, the objectives of the current study were to determine the difference in urinary albumin (uALB), urinary retinol-binding protein (uRBP), and urinary N-acetyl-beta D-glucosaminidase (uNAG) concentrations in cystocentesis and voided samples and to investigate concentration changes after storage at -20 degrees C and at -80 degrees C Effects of a protease inhibitor were also assessed in samples stored at -80 degrees C for 12 months In a pilot experiment, influence of in vitro hematuria, pyuria, and bacteriuria on the urinary markers was evaluated A mixed model was used to calculate mean differences and 95% confidence intervals Urinary ALB, uNAG, and uRBP concentrations were similar in voided and cystocentesis samples After storage for 4 months at -20 degrees C, uALB concentration was not affected and uRBP concentration showed a mild and clinically irrelevant decrease, whereas uNAG activity was significantly lower compared with fresh samples After storage for 12 months at -80 degrees C, uALB and uRBP concentrations did not differ from fresh samples but uNAG activity was severely decreased Protease inhibitor addition did not preserve uNAG activity Experimental hematuria, pyuria, and bacteriuria did not seem to affect urinary markers although further research is neede

Influence of sex steroids on the viability and CD11b, CD18 and CD47 expression of blood neutrophils from dairy cows in the last month of gestation

In the period around parturition, cows experience an increased susceptibility for the development of Escherichia coli mastitis. This increased susceptibility has been correlated with a decreased functionality of neutrophils. In the current study, it is suggested that the decreased neutrophil functionality may be induced by the extensive alterations in sex steroid levels occurring around parturition. It was first hypothesized that 17 beta-estradiol and progesterone influence the viability, apoptosis and necrosis of blood neutrophils from cows in their last month of gestation. Subsequently, it was hypothesized that 17 beta-estradiol modulates the expression of CD11b, CD18 or CD47 thereby explaining its influence on the migration of bovine neutrophils. Neither 17 beta-estradiol nor progesterone significantly influenced viability, apoptosis or necrosis in spontaneous apoptosis conditions. However, when apoptosis was induced with TNF-alpha and gliotoxin, progesterone exerted a survival effect ( P < 0.05). In addition, 17 beta-estradiol treatment of bovine blood neutrophils significantly decreased the expression of CD47 ( P < 0.05) but not of CD11b or CD18. It can be concluded that 17 beta-estradiol and progesterone do not affect spontaneous apoptosis of bovine blood neutrophils while a survival effect was observed for progesterone on induced neutrophils apoptosis. Moreover, our results concerning the influence of 17 beta-estradiol on the CD11b, CD18 and CD47 expression extend previous demonstrations of the suppressive effect of 17 beta-estradiol on neutrophils migration and indicate that the altered expression of CD47 may contribute to this phenomenon

Potential therapeutic application of bacteriophages and phage-derived endolysins as alternative treatment of bovine mastitis

The increase in bacterial drug resistance causes major difficulties in the clinical treatment of a growing number of bacterial infections worldwide. Consequently, there is an urgent need to develop novel anti-bacterial agents to control these resistant pathogens and to complement the currently used antibiotics. Mastitis is the most prevalent disease impacting dairy cattle, and therefore one of the costliest diseases in the global dairy industry. The excessive use of curative as well as preventive antibiotics in this sector entails a real risk for the emergence of antimicrobial resistance. Moreover, these traditional antimicrobial agents are often ineffective and lead to residues in the milk, which can affect dairy product consumers. As an alternative therapeutic approach, bacteriophages and phage-encoded endolysins have been proposed and are currently (re)investigated as potential antibacterial agents against mastitis

Limited association between disinfectant use and either antibiotic or disinfectant susceptibility of Escherichia coli in both poultry and pig husbandry

Background Farm disinfectants are widely used in primary production, but questions have been raised if their use can select for antimicrobial resistance. The present study examined the use of disinfectants in poultry and pig husbandry and its contribution to the antibiotic and disinfectant susceptibility of Escherichia coli (E. coli) strains obtained after cleaning and disinfection. On those field isolates antibiotic susceptibility was monitored and susceptibility to commonly used active components of farm disinfectants (i.e. glutaraldehyde, benzalkoniumchloride, formaldehyde, and a formulation of peracetic acid and hydrogen peroxide) was tested. Results This study showed a high resistance prevalence (> 50%) for ampicillin, sulfamethoxazole, trimethoprim and tetracycline for both production animal categories, while for ciprofloxacin only a high resistance prevalence was found in broiler houses. Disinfectant susceptibility results were homogenously distributed within a very small concentration range. Furthermore, all E. coli strains were susceptible to in-use concentrations of formaldehyde, benzalkoniumchloride and a formulation of peracetic acid and hydrogen peroxide, indicating that the practical use of disinfectants did not select for disinfectant resistance. Moreover, the results showed no indications for the selection of antibiotic resistant bacteria through the use of disinfectants in agricultural environments. Conclusion Our study suggests that the proper use of disinfectants in agricultural environments does not promote antibiotic resistance nor reduce E. coli disinfectant susceptibility