In vitro function of bovine neutrophils against Actinomyces pyogenes

Factors that influenced the in vitro bactericidal activity of bovine neutrophils against Actinomyces pyogenes were investigated. Neutrophils and serum from 2 clinically normal donor cows were incubated with bacteria for 2 hours. To determine bactericidal activity, colony-forming units were counted after a 48-hour incubation on blood agar plates. Microscopic examination indicated that in the presence of serum, bacteria were cell associated after incubation, whereas when serum was replaced by medium, bacteria were not cell associated. Bactericidal activity of neutrophils was similar whether the sera were heat-treated at 56 C for 30 minutes or were not heated. Heating the serum at 65 C for 30 minutes significantly (P less than 0.001) reduced bactericidal activity. Bactericidal activity decreased (P less than 0.001) as serum concentration (less than 10%) decreased. More than 80% of the bacteria were killed within the 40 minutes of incubation. The opsonizing capacity of serum varied significantly (P less than 0.01) among 12 cows. Similarly, neutrophil bactericidal activity (by cow) was affected significantly (P less than 0.001). Preincubation of serum with A pyogenes significantly (P less than 0.001) reduced the opsonizing ability of the serum. Culture filtrate of A pyogenes was not chemotactic for neutrophils in vitro.     

Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.     

Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and mammary gland neutrophil function in vitro.

Modulation of peripheral blood and mammary gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 microgram/mL rBoGM-CSF. Neutrophils treated with rBoGM-CSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections.     

Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.     

In vitro and in vivo effects of recombinant bovine interferon-tau on bovine leukemia virus

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.     

Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.     

The effects of acute exposure to deoxynivalenol on some inflammatory parameters in miniature pigs

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.     

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.     

Effects of in vitro and in vivo administration of recombinant bovine interferon-gamma on bovine neutrophil responses to Brucella abortus

The effects of in vitro and in vivo treatment of bovine polymorphonuclear leukocytes with recombinant bovine interferon-gamma on in vitro bovine polymorphonuclear leukocyte functions and the survival of Brucella abortus were determined. Activation of neutrophils in vitro with interferon-gamma resulted in enhanced production of O2- and myelopeoroxidase-H2O2-halide activity by neutrophils in the presence of B. abortus. The improved iodination responses were correlated with an enhanced ability to perform iodination in the presence of 5'-guanosine monophosphate and adenine which have previously been shown to contribute to inhibition of neutrophil myeloperoxidase-H2O2-halide activity by B. abortus. The ability of opsonized B. abortus to survive in the presence of neutrophils activated in vitro or in vivo was partially decreased by approximately 10% of control when compared to survival rates within control phagocytes. These results suggest that activation of neutrophils with recombinant interferon-gamma partially enhances their oxidative metabolic responses, resulting in a slightly enhanced ability to kill virulent B. abortus.     

Total glutathione and glutathione reductase in bovine erythrocytes and liver biopsy

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.     

A colorimetric assay for quantitating bovine neutrophil bactericidal activity

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.     

Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant

Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species. In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein. The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection. Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils. These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo.     

精氨酸在体内和体外试验中对鲤鱼免疫力的影响

本试验采用体外和体内2种方法来研究精氨酸(Arg)对鲤鱼免疫力的影响。体外试验中,在培养基中分别添加0、0.5、1.0、1.5和2.0mmol/L的Arg,将肾脏白细胞培养不同的时间段后测定增殖指数、呼吸爆发活力、吞噬活力和杀菌率。体内试验中,将平均体重37g的鲤鱼随机分为5组,每组3个重复,每个重复10条,分别体内注射0、25、50、100和200 mg/kg鱼体重的Arg,商业饲料投喂2周,进行肾脏白细胞增殖指数、呼吸爆发活力和吞噬活力的测定,以及血清和肝胰脏一氧化氮(NO)含量、一氧化氮合成酶(NOS)活性及血清白蛋白(ALB)含量的测定。体外试验结果表明:添加Arg能够提高肾脏白细胞的增殖指数、呼吸爆发活力、吞噬活力和杀菌率。培养12和24h后,1.0、1.5和2.0mmol/L Arg组肾脏白细胞增殖指数显著高于0mmol/L Arg组(P0.05);培养6、12和24h后,1.0mmol/L Arg组肾脏白细胞呼吸爆发活力显著高于0mmol/L Arg组(P0.05);培养12和24h后,1.0、1.5和2.0mmol/L Arg组肾脏白细胞吞噬活力显著高于0mmol/L Arg组(P0.05);培养18h后,1.0、1.5和2.0 mmol/L Arg组肾脏白细胞杀菌率显著高于0 mmol/L Arg组(P0.05)。体内试验结果表明:50、100和200 mg/kg Arg组肾脏白细胞呼吸爆发活力、吞噬活力和增殖指数显著高于0mg/kg Arg组(P0.05);100和200mg/kg Arg组血清ALB和NO含量显著高于0 mg/kg Arg组(P0.05),50、100和200mg/kg Arg组血清NOS活性显著高于0mg/kg Arg组(P0.05);50、100和200mg/kg Arg组肝胰脏NO含量显著高于0mg/kg Arg组(P0.05),25和50mg/kg Arg组肝胰脏NOS活性显著高于0mg/kg Arg组(P0.05)。由此可见,Arg提高了鲤鱼肾脏白细胞的免疫力,体外试验表明Arg的适宜浓度为1.0mmol/L。正常摄食情况下,Arg在鲤鱼体内注射适宜浓度为50~100mg/kg。     

Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation

To examine the effect of parity on polymorphonuclear neutrophils (PMN) function, phagocytic and bactericidal activity of the PMN isolated from blood and milk against Staphylococcus aureus was compared between groups of 6 primiparous and 6 multiparous healthy dairy cows during early lactation using bacteriological and PMN-pathogen interaction assays. Latex-stimulated luminol-amplified chemiluminescence (CL) and viability of these PMN were also investigated. The phagocytosis and killing of S. aureus by blood were remarkably higher than those of milk PMN. Similarly, the CL and viability in blood PMN were markedly higher than in milk PMN. Both in blood and in milk the phagocytosis of S. aureus by PMN in primiparous cows was substantially higher than in multiparous cows. The killing activity of blood PMN against S. aureus was 42.3+/-3.4% and 23.2+/-1.7% in primiparous and multiparous, respectively. Milk PMN killed only 20.7+/-2% S. aureus in primiparous and 10.2+/-1.3% in multiparous cows. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows. The pronounced reduction in phagocytic and bactericidal activity in blood and milk-resident PMN from multiparous cows, in part, resulted from the pronounced decrease of PMN viability and free radicals production capacity; this suggests that heifers' udders could be more protected against S. aureus, which remains to be tested in the field.     

Effects of human IL-8 isoforms on bovine neutrophil function in vitro

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.     

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.     

Comparative in vitro activity of three cephalosporins against Staphylococcus aureus isolated from bovine mastitis

This report compares the in vitro activity of three cephalosporins (cephalothin, cefoxitin and ceftriaxone) against 57 Staphylococcus aureus strains isolated from cows with clinical mastitis on the basis of the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). The majority of the S aureus strains showed resistance to cefoxitin and ceftriaxone and sensitivity to cephalothin. The highest MICs and MBCs were found for cefoxitin and ceftriaxone. Antimicrobial tolerance (MBC/MIC greater than or equal to 32:1) was observed in relation to cephalothin and ceftriaxone. The data suggest that these cephalosporins may not be effective for the treatment of staphylococcal bovine mastitis. The precise definition of their antimicrobial efficacies requires more detailed in vitro and in vivo studies.     

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.     

Serum amyloid A and haptoglobin levels in bovine amyloidosis

Serum amyloid A (SAA) and haptoglobin (Hp) levels were determined in 25 cows suffering from amyloidosis. SAA levels in cows with amyloidosis ranged between < 0.3 and 225.8 microg/ml, with a median level of 105.1 microg/ml, and Hp levels ranged between < 20 and 1860 microg/ml, with a median level of 950 microg/ml. These levels were significantly higher than the levels observed in healthy cows (SAA levels ranged from < 0.3 to 13.5 microg/ml, with median of 1.4 microg/ml, and Hp levels were undetectable in all cases), but were not significantly different from the levels observed in control cows with chronic inflammation. There was a significant correlation between SAA and Hp levels in cows with chronic inflammation , but not in cows with amyloidosis. It was concluded that the serum SAA levels in cows with amyloidosis might be changed by some factor other than inflammation.