Pharmacologic enhancement or suppression of phagocytosis by bovine neutrophils

Sixty-three drugs, belonging to 10 chemical classes, were tested in vitro to determine effects on phagocytosis of 32P-labeled Staphylococcus aureus by neutrophils isolated from milk. Within each class, the number of antibiotics tested were: nonsteroidal anti-inflammatory drugs (NSAID; 8), peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), beta-lactam antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and antibacterial quinolones (8). Percentage of phagocytosis was determined after incubating (2 hours at 37 C) 12.5 x 10(6) viable neutrophils, 200 x 10(6) 32P-labeled S aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics tested were 1,000, 500, and 10 micrograms/ml of incubation media. When compared with nonantibiotic controls at the highest drug concentration, the NSAID acetylsalicylic acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively, and benzydamine, indomethacin, phenylbutazone, ibuprofen, and acetominophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0, and 18.2%, respectively. The peptolids novobiocin and pristinamycin decreased phagocytosis 24.5 and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and gentamicin decreased phagocytosis 21.1, 15.4, and 19.2%, respectively. For the tetracyclines and fusidic acid, minocycline and doxycycline decreased phagocytosis 39.8 and 54.2%, respectively. The beta-lactam antibiotics carfecillin, cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8, and 23.8%, respectively. The secretolytic agent, bromhexin, increased phagocytosis 10.8%. These data indicate that the potential for enhanced phagocytosis exists through use of some NSAID, and for depressed phagocytosis through use of aminoglycosides, peptolids, tetracyclines, and beta-lactams, as well as certain other NSAID.     

The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli

Encapsulated strains of Escherichia coli were found to be more resistant to phagocytosis and killing by bovine neutrophils; requiring in the order of 100 times more serum opsonins than non-encapsulated strains. Mid-lactation pooled whey from cows with no history of mastitis was opsonic for non-encapsulated strains, but had no effect on encapsulated organisms. In contrast, early lactation pooled whey (5-10 days post-partum) was opsonic for all strains of E. coli. It is concluded that since early lactation milk contains sufficient opsonins, severe E. coli mastitis at this stage of lactation is not due to opsonic deficiency.     

Specific antibody in milk whey and phagocytosis of Actinomyces pyogenes by neutrophils in vitro

Milk samples were collected from 21 non-pregnant cows to study the ability of milk whey to support in vitro bactericidal activity of neutrophils against Actinomyces pyogenes. Significant differences (P less than 0.01) existed in opsonising ability of milk whey samples from individual cows. Antibody titres to A pyogenes in milk whey were determined using an enzyme linked immunosorbent assay. Bactericidal activity of neutrophils incubated with milk whey was positively correlated (P less than 0.05) with titres of IgG2 and IgM antibodies but not with IgG1 or IgA antibodies.     

Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk

Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.     

Relationship between the chemiluminescent response of bovine neutrophils and changes in intracellular free calcium concentration.

The relationship between luminol dependent chemiluminescent (LDCL) response and changes in intracellular free Ca2+ concentrations in the bovine neutrophils was evaluated. LDCL responses and changes in intracellular Ca2+ concentrations of neutrophils were clearly detected by the stimulation with opsonized zymosan (OPZ), concanavalin A(ConA), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA). Patterns of LDCL responses and intracellular Ca2+ of neutrophils showed characteristic features for each stimulant. PMA was a weak stimulant of the intracellular Ca2+ concentration, whereas it was a strong stimulant of LDCL response. Con A strongly stimulated an increase in the intracellular Ca2+ concentration, but was a weak stimulant of LDCL response. LDCL response of intracellular Ca(2+)-depleted neutrophils treated with ionomycin, stimulated with each stimulant was inhibited markedly without extracellular Ca2+. The sustained phase of intracellular Ca2+ concentrations stimulated with OPZ was inhibited significantly (P < 0.05) by the preincubation with anti-CD18 antibody, whereas the transient phase of intracellular Ca2+ concentrations was not inhibited. These results indicate that LDCL response is regulated at least in part by the elevation of the intracellular Ca2+, and a rise in intracellular Ca2+ concentration, which may be mediated by specific receptors appears to be essential in the LDCL response of bovine neutrophils.     

Serum immunoglobulin concentrations in cattle naturally infected with bovine leukemia virus

In order to elucidate whether natural infection of BLV in cattle might induce humoral immunological responses, changes in IgG1, IgG2, and IgM concentrations in the sera of infected cattle were determined. Twelve BLV-infected cattle were used. Cattle of different breeds were classified serologically and haematologically into BLV + PL+, BLV + PL- and BLV-free groups. Ig concentrations in the sera of the three groups were quantitated using a commercial single radial immunodiffusion assay. The findings were compared to those of BLV-free cattle. The serum IgM concentrations were significantly lower in cattle with PL (P less than 0.001) than in BVL + PL- and BLV-free cattle. The IgM concentrations tended to be lower in BLV+ PL- than those of BVL-free cattle. There were no significant differences in IgG1 and IgG2 serum concentrations between the three experimental cattle groups. IgG1 was the predominant subtype in the sera of all cattle groups.     

Effect of milk or colostrum on phagocytosis of glass- or plastic-adherent Streptococcus agalactiae by bovine granulocytes

In the presence of milk, bovine polymorphonuclear cells (PMN) assumed a polarized shape, a feature of motile cells, and their adherence to plastic was augmented. Milk enhanced the phagocytosis of glass- or plastic-adherent Streptococcus agalactiae. In contrast, PMN were not stimulated by colostrum.     

Relationship between mastitis pathogen numbers in bulk tank milk and bovine udder infections in California dairy herds

Samples of bulk tank milk and cow-composite milk from 23,138 dairy cows from 50 California dairies were examined by use of microbiologic procedures. The number of colonies of mastitis pathogens isolated per milliliter of bulk tank milk (used as a predictor of the percentage of infected cows in the herd) was evaluated, using simple regression analysis and Spearman's rank correlation. Correlations between the pathogens and the percentage of cows in each herd shedding the pathogens were found for Streptococcus agalactiae (r = 0.71) and mycoplasma (r = 0.59), but were considerably lower for other pathogens. When greater than or equal to 4,000 colonies of Streptococcus agalactiae were found per milliliter of bulk tank milk, at least 7% of the cows in the herd was shedding this organism. However, a pattern was not found between the number of mycoplasma colonies per milliliter of bulk tank milk and the percentage of infected cows in the herd.     

Phagocytosis of serum-resistant and serum-sensitive coliform bacteria (Klebsiella) by bovine neutrophils from blood and mastitic milk

Phagocytic activity of neutrophil leukocytes from bovine blood and mastitic milk was determined for 2 strains of Klebsiella, 1 resistant and the other sensitive to serum bactericidal activity. Both strains were easily phagocytized in the presence of an opsonic agent, but milk neutrophils seemed to be less efficient than blood neutrophils in this respect. Phagocytosis was maximal after incubation for 60 minutes at 37 C and decreased markedly with reduction in incubation temperature. The opsonic activity of mastitic milk was considerably higher than that of normal milk and approached that of fresh bovine serum. Precolostral calf serum was deficient in opsonic activity and anti-bovine leukocyte serum was antiphagocytic.     

Relationship between milk production and plasma concentrations of oxidative stress markers during hot season in primiparous cows

Oxidative stress markers, ascorbic acid, and sulfhydryl (SH) residue concentration in primiparous cows' plasma and their relationship to milking performance during hot seasons were investigated. The rectal temperature of cows correlated negatively with SH residue (r = −0.38, n  = 38, P  < 0.05) and ascorbic acid (r = −0.34, P  < 0.05) concentrations in the cows' plasma. The group with a higher concentration of ascorbic acid over the mean value produced significantly more milk ( P  < 0.05) than did the group with a lower ascorbic acid concentration. Although the cows' milk production showed a positive correlation with ascorbic acid concentration in plasma (r = 0.47, P  < 0.05), the relation of SH residue concentration to milk yield was not constant. The plasma SH residue concentration during the hot season correlated positively with milk protein % (r = 0.38, P  < 0.05), lactose % (r = 0.35, P  < 0.05), and solid-non-fat (SNF) % (r = 0.47, P  < 0.05), respectively, but not with fat %. On the other hand, ascorbic acid concentration in plasma showed negative correlations with milk fat % (r = −0.34, P  < 0.05) and protein % (r = −0.49, P  < 0.05), but correlated positively with lactose % (r = 0.52, P  < 0.05). The produced amount (g/day) of milk protein (r = 0.42, P  < 0.05), lactose (r = 0.61, P  < 0.05), and SNF (r = 0.56, P  < 0.05) showed positive correlations with ascorbic acid concentration in plasma.     

特异性IgY抗体对中性粒细胞吞噬功能的影响

鸡卵黄免疫球蛋白 (Yolk Immunoglobulins,Ig Y)是一种存在于鸡蛋黄中的 Ig G。Ig Y比哺乳动物Ig G多一个 CH4区 ,为 7S免疫球蛋白 ,分子量约为1 80 k Da,含 67～ 70 k Da的重链和 2 2～ 30 k Da的轻链 [1 ]。抗绿脓杆菌、葡萄球菌及沙门氏菌的特异性Ig Y抗体能够抑制绿脓杆菌生长 ,抑制葡萄球菌产生肠毒素以及抑制沙门氏杆菌结合人肠细胞 (Caco2 ) [2 ] 。抗绿脓杆菌 Ig Y含漱预防胆囊纤维化患者绿脓杆菌感染 [3]。虽然 Ig Y不激活补体 ,不与哺乳动物细胞的 Fc受体结合 ,但 Ig Y在抗细菌性感染方面能发挥抗菌和防止炎症发生的作…     

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.     

Activation of bovine neutrophils by recombinant bovine tumor necrosis factor-alpha

The in vitro effect of bovine recombinant tumor necrosis factor-alpha (rbTNF-alpha) on bovine neutrophil function and the possibility that rbTNF-alpha and recombinant bovine interferon-gamma (rbIFN-gamma) act synergistically were investigated. Treatment of neutrophils with rbTNF-alpha (0.05 micrograms/ml; approximately 50 U/ml) at 37 degrees C for 2.5 h resulted in enhancement of antibody independent neutrophil-mediated cytotoxicity (AINC) and inhibition of random migration and chemotaxis. The same treatment resulted in a slight decrease in iodination and cytochrome C reduction, but did not affect Staphylococcus aureus ingestion, or antibody dependent cell-mediated cytotoxicity. Kinetic and inhibitor studies indicated that the action of rbTNF-alpha was rapid and was independent of protein and RNA synthesis by neutrophils. Evaluation of the synergistic activities of rbTNF-alpha and rbIFN-gamma indicated that treatment of neutrophils with these two cytokines simultaneously resulted in additive enhancement of AINC and inhibition of random migration and chemotaxis. There was no additive effect of the two cytokines on inhibition of iodination or cytochrome C reduction.     

Effect of milk fraction on concentrations of cephapirin and desacetylcephapirin in bovine milk after intramammary infusion of cephapirin sodium

Clinical mastitis in dairy cows is commonly treated with intramammary (IMM) antimicrobial agents. Pharmacokinetic data are used to design treatment regimens and determine withholding times. In some pharmacokinetic studies, investigators measure antimicrobial concentrations in foremilk, whereas in others, they use bucket milk or do not specify the milk fraction sampled. Our objective was to compare antimicrobial concentrations in foremilk, bucket milk, and strippings after IMM treatment of six healthy Holsteins. One mammary gland/cow was infused with 200 mg of cephapirin (CEPH) after each of the two milkings, using different milking frequencies and treatment intervals in a randomized crossover design. Treated glands were sampled at the first milking following each infusion. Antimicrobial concentrations in milk were measured using HPLC/MS/MS. CEPH concentration was higher in foremilk (geometric mean 44.2 μg/mL) than in bucket milk (15.7 μg/mL) or strippings (18.5 μg/mL), as it was true for desacetylcephapirin (DAC) (59.5, 23.0, and 30.2 μg/mL, respectively). This finding, which was based on milk samples collected at the first milking after IMM infusion, suggests that pharmacokinetic data based on drug concentrations in foremilk may be misleading. Strippings were more representative of bucket milk than foremilk. The relationship between milk fraction and antimicrobial concentration should be investigated for other IMM antimicrobial agents. Meanwhile, it is essential that pharmacokinetic and residue studies report the fraction of milk that was analyzed.     

Relationship between blood selenium concentration or glutathione peroxidase activity, and milk selenium concentrations in New Zealand dairy cows

AIM: To determine the relationships between blood selenium (Se) concentrations or glutathione peroxidase activity (GSH-Px), and milk Se concentrations in dairy cows. METHODS: Seventy-two Friesian dairy cows were either untreated or injected with 0.5, 1.0 or 2.0 mg Se/kg liveweight as barium selenate (BaSeO4) formulations, resulting in 6 groups of animals with mean blood Se concentrations that varied from 212 to 2272 nmol/l. Milk samples were collected on Days 104 and 188, and blood samples were collected prior to treatment and on Days 41, 76, 104, 188, 244, and 292 after Se injection. RESULTS: Significant quadratic relationships between blood Se and milk Se concentrations, as well as blood GSH-Px activity and milk Se concentrations, were evident at Days 104 and 188. Using combined data, these were represented by the equations: milk Se = 27.3 + 0.073 blood Se -0.00001 (blood Se)2; R2=0.79, p<0.005, and; milk Se = 34.8 + 4.99 GSH-Px -0.068 (GSHPx)2; R2=0.79, p<0.005. CONCLUSIONS: The Se status of dairy cows can be assessed from milk Se concentrations. CLINICAL SIGNIFICANCE: Bulk-tank milk Se concentrations could be evaluated as a method to assess the Se status of dairy herds.