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.     

Assessment of bovine mammary gland macrophage oxidative burst activity in a chemiluminescence assay

A major bactericidal mechanism of neutrophils and macrophages is the generation of toxic oxygen-free radicals upon phagocytosis of microbes. Studies were conducted to assess the oxidative metabolism of bovine mammary gland macrophages. Bovine mammary gland macrophages were challenge exposed with a variety of phagocytic stimuli in an in vitro, luminol-assisted chemiluminescence assay. A measurable oxidative burst was observed when macrophages were challenge exposed with heat-aggregated bovine immunoglobulin, opsonified zymosan, and nonosponified zymosan. Addition of superoxide dismutase decreased mammary gland macrophage chemiluminescence in a dose-dependent manner. Brucella abortus, when opsonified with antiserum, lacteal antibody, or normal serum, produced an oxidative event, whereas nonopsonified B abortus did not. When challenge exposed with phagocytic stimuli, mammary gland macrophages produced an oxidative burst similar to that produced by other phagocytes for which an oxidative event is known to be bactericidal.     

Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages

Chronic bovine brucellosis is characterized by persistent infection of the mammary gland. The interaction of live Brucella abortus with bovine mammary gland macrophages was studied in vitro. Opsonization of smooth B abortus strain 2308 and rough strain 45/20 was required for phagocytosis by mammary gland macrophages. When opsonized with specific antiserum, strains 2308 and 45/20 stimulated a considerable oxidative burst when phagocytized by mammary gland macrophages. Intracellular survival rates for strain 2308 were significantly higher than those for strain 45/20. After being phagocytized, B abortus localized in phagosomes and phagolysosomes of mammary gland macrophages.     

Resistance of preimplantation bovine embryos to infection with Brucella abortus

Preimplantation bovine embryos were exposed in vitro to Brucella abortus to determine if the bacteria would adhere to zona pellucida (ZP)-intact embryos or adhere to or infect ZP-free embryos. Brucella abortus was not isolated from ZP-intact or ZP-free groups of embryos after 10 sequential antibiotic-free washings. Brucella abortus was isolated from all groups containing ZP-defective embryos after the exposure period and washing. Detrimental effects on healthy in vitro development of embryos were not observed.     

Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.     

Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.     

Use of hydrophobic cloths as antibody adsorbents for enzyme immunoassay: detection of Brucella antigens

A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Bovine ileal dome lymphoepithelial cells: endocytosis and transport of Brucella abortus strain 19

Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitium and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propria contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.     

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.     

Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunoglobulins.     

Further evidence that bovine IgM does not fix guinea pig complement

In a study of sera from cattle vaccinated with 3 X 10(10) cfu of Brucella abortus strain 19, it was found that IgG1 antibody measured by an indirect ELISA was the only isotype to correlate with standard complement fixing antibody titers using heated serum samples and guinea pig serum as a source of complement. A supplement of normal unheated bovine serum resulted in IgM fixing guinea pig complement, giving data similar to those obtained with unheated serum in the complement fixation test.     

Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components?

Purified bovine isotypes IgM, IgG1, IgG2, and IgA (secretory), affinity purified with Brucella abortus, were tested in a complement fixation test (CFT) for their ability to activate guinea pig complement directly or in the presence of 'normal' bovine serum. Only IgG1 fixed guinea pig complement in the direct test and approximately 250 ng of antibody was required to activate 50% of 3 CH50 units with a standard amount of antigen. Addition of 'normal' bovine serum as an additional source of complement resulted in activation of guinea pig complement by IgM, IgG2 and secretory IgA at levels of approximately 1200, 700 and 2250 ng, respectively for 50% of 3 CH50 units. Addition of 'normal' bovine serum did not enhance complement activation by bovine IgG1.     

Persistent plaque formation in experimental murine brucellosis

Primary plaque forming cells (PFC) are present in spleens of mice 150 days or more following an infection with Brucella abortus. The development of primary plaques in mice long after antigenic challenge is an uncommon phenomenon, unlike the plaque formation (PF) induced by a non-living antigen. The mechanism of this persistent PF has been now investigated in light of a prolonged persistence of the corresponding antigen in tissues. Living E. coli, inoculated in massive dose into mice, survived in their organs for a brief time, while concomitantly PFC disappeared by day sixteen. Infection with B. abortus, in contrast, induced persistent presence of bacteria in the organs of inoculated mice and stimulated long lasting plaque formation. Only direct plaques were found during all stages of infection. Repeated inoculations of dead B. abortus also induced continuous production of primary plaques, whereas an interval in supply of the antigen resulted in disappearance of PFC. Rifampin (40 mg/kg) eliminated bacteria from the treated mice, which resulted in the disappearance of primary PFC. It seems likely that long lasting PF in B. abortus infected mice is connected with a constant antigenic stimulus operating in the carrier state.     

Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.     

Influence of arachidonic acid metabolites and steroids on function of bovine polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMN) from 4 ovariectomized healthy cows were incubated with 0 (control), 10(-8), 10(-7), and 10(-6) M arachidonic acid metabolites of the cyclo- and lipoxygenase pathways for 30 minutes, and with steroids for 2 hours. Immediately after incubation, PMN were subjected to the following function assays: chemotaxis against zymosan-activated serum, chemotaxis against arachidonic acid metabolite or steroid at the doses given (only control PMN were tested), random migration, ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, antibody-independent and -dependent cell-mediated cytotoxicity (AICC and ADCC). Prostaglandin F2 alpha was chemoattractant and stimulated ingestion of 125I-IdUR-S aureus. Prostaglandin E2 stimulated cytochrome C reduction, whereas prostacyclin inhibited iodination of proteins. Thromboxane B2 stimulated ADCC. Leukotriene B4 was chemoattractant for bovine PMN and stimulated random migration and AICC. 5-Hydroxyeicosatetraenoic acid was also chemoattractant, but inhibited ingestion of 125I-IdUR-S aureus. 15-Hydroxyeicosatetraenoic acid was chemoattractant and decreased ADCC. Lipoxin A4 stimulated random migration, whereas lipoxin B4 inhibited chemotaxis against zymosan-activated serum, but was chemoattractant and stimulated cytochrome C reduction. 12-Hydroxyhepadecatrienoic acid and 12-hydroxyeicosatetraenoic acid did not influence any of the PMN functions tested. Of the steroids tested, cortisol increased ADCC, and progesterone stimulated cytochrome C reduction, but decreased ADCC. 17 beta-Estradiol and estrone were chemoattractant and stimulated cytochrome C reduction. In addition, estrone also stimulated random migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of vitamins A and E on superoxide production and intracellular signaling of neutrophils in Holstein calves.

The effects of vitamin A and vitamin E treatment on superoxide (O2-) production of neutrophils and their intracellular signaling, including intracellular Ca2+, protein kinase C (PKC), and tyrosine kinase (TK), were examined in Holstein calves. After treatment with vitamin A, heat-aggregated IgG (H-agg.IgG)-induced O2- production in neutrophils ranged from 3.7+/-0.4 to 4.3+/-0.9 nmol (mean +/- SD), and it was significantly (P<0.05) higher than that in the control calves. Opsonized zymosan (OPZ)-induced O2- production was similar with that of control calves. Intracellular signaling of neutrophils in vitamin A-treated calves was enhanced compared with that of control calves when stimulated with H-agg. IgG, but not with OPZ. After treatment with vitamin E, H-agg.IgG- and OPZ-induced O2- production of neutrophils ranged from 4.2+/-0.8 nmol to 5.4+/-0.5 nmol and from 7.8+/-0.6 nmol to 8.1+/-0.4 nmol (mean +/- SD), respectively, and they were significantly (P<0.05) higher than those in control calves. Intracellular signaling was also enhanced compared with that of control calves. These results suggested that the effects of vitamin A and E treatment on O2- production were correlated to changes in intracellular signaling of neutrophils in Holstein calves.     

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inhibition of equine neutrophil function by phosphodiesterase inhibitors

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge, which may contribute to inflammation and lung damage. Inhibition of phosphodiesterase isoenzymes (PDEs) has been shown to attenuate human neutrophil functions including superoxide production, leukotriene (LT)B4 biosynthesis, enzyme and chemokine release. As equine neutrophils contain predominantly the isoenzyme, PDE4, the present study was undertaken to investigate the effects of rolipram, a PDE4 inhibitor, on equine neutrophil function. For comparison, the effects of the nonselective PDE inhibitor, theophylline, were examined. Cells from both normal horses and COPD horses in remission were used. Superoxide production was significantly inhibited by both rolipram [32.2 +/- 2.6 vs. 10.1 +/- 1.1 nmol/10(6) cells and 49.8 +/- 6.8 vs. 22.7 +/- 2.2 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M human recombinant (hr) C5a] and theophylline (19.0 +/- 0.6 vs. 10.2 +/- 0.6 nmol/10(6) cells and 24.3 +/- 2.1 vs. 10.7 +/- 0.9 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M C5a). However, superoxide production induced by serum treated zymosan was inhibited only by theophylline (10(-3) M). Neither hrC5a- nor platelet activating factor (PAF)-induced neutrophil adherence to fibronectin coated plastic was reduced by rolipram (10(-5) M). These results demonstrate that the effects of PDE inhibitors on equine neutrophils are both stimulus and function dependent. The PDE4 inhibitors may reduce neutrophil activation in vivo in horses with COPD.