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.     

Disassociation of bactericidal and fungistatic activities from the oxidative burst of avian macrophages

Avian peritoneal exudate macrophages, when exposed to phagocytic stimuli, produced an appreciable oxidative burst as measured by production of chemiluminescence, superoxide anion, and hydrogen peroxide. Metabolic inhibitors of the oxidative burst and scavengers of oxygen radicals clearly inhibited macrophage chemiluminescence, but had no significant effect on macrophage bactericidal activity against Escherichia coli or fungistatic activity against Candida tropicalis. Therefore, avian macrophages were capable of oxygen-independent bactericidal and fungistatic activities.     

Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus

Nonvaccinated pregnant cows were segregated retrospectively into 2 groups following inoculation with Brucella abortus strain 2308. One group resisted infection (resistant cows) and the other group developed active infections (susceptible cows) and subsequently aborted. Mammary gland macrophages collected from the 2 groups of cows were compared, using in vitro functional assays. In a chemiluminescence assay, mammary gland macrophages from resistant cows produced significantly (P = 0.014) higher oxidative burst activity than did macrophages from susceptible cows. Macrophages from resistant cows had significantly (P = 0.038) greater bacteriostatic activity against B abortus than did macrophages from susceptible cows. Differences in lysosomal enzymatic activity or Fc receptor expression were not observed for macrophages from the 2 groups of cows. Differences in macrophage function may be one factor responsible for natural resistance to Brucella infection in cattle.     

Comparison of phagocytosis and chemiluminescence by blood and mammary gland neutrophils from multiparous and nulliparous cows

Neutrophils were isolated from the blood and mammary gland of 3 multiparous lactating cows and 3 nulliparous heifers. Neutrophil function was evaluated by phagocytosis and luminol-dependent chemiluminescence. Peroxidase activity was detected by use of transmission electron microscopy. Compared with that for blood neutrophils, percentage of phagocytosis was 9.6% lower for neutrophils isolated from the mammary gland of lactating cows, but this difference was not observed between neutrophils isolated from the mammary gland and from the blood heifers. Similarly, after subtraction of chemiluminescence values in the absence of zymosan, phagocytosing neutrophils from the mammary gland of lactating cows had lower chemiluminescence than did those from the blood of such cows. For heifers, however, chemiluminescent activity by phagocytosing neutrophils obtained from the mammary gland was similar to that of blood neutrophils. Chemiluminescent activity of resting neutrophils from the mammary gland of lactating cows pretreated with cytochalasin B was not inhibited, compared with that of nontreated resting neutrophils (controls). This was attributed to xanthine oxidase activity. Transmission electron microscopy of mammary gland neutrophils from lactating cows revealed peroxidase-positive material associated with milk-fat globule membranes and with phagosomes containing zymosan. Results indicated that ingestion of fat and casein by neutrophils isolated from milk caused a decrease in phagocytic and chemiluminescent activity. Also, luminol-dependent chemiluminescence was not a reliable measure of milk neutrophil function, because of interference by xanthine oxidase.     

Endotoxin lipopolysaccharide from Escherichia coli and its effects on the phagocytic function of systemic and pulmonary macrophages in turkeys.

The effect of Escherichia coli lipopolysaccharide (LPS) on the competence of pulmonary macrophages and phagocytic cells from the systemic circulation of turkeys was examined using luminol-enhanced zymosan-stimulated chemiluminescence. The results showed a rapid and accelerated oxidative burst in both systemic and pulmonary macrophages in LPS-treated turkeys that was significantly greater than in untreated controls. However, this increased oxidative metabolism induced by LPS was not associated with enhanced intracellular bacterial killing by pulmonary macrophages. Turkeys treated with LPS showed a highly significant decrease in pulmonary bactericidal activity against Staphylococcus aureus challenge, indicating a defect in pulmonary macrophage function induced by LPS.     

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.     

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.     

Recombinant bovine interferon-gamma, but not interferon-alpha, potentiates bovine neutrophil oxidative responses in vitro

In this study we have addressed the in vitro effects of recombinant bovine interferon-gamma (rBoIFN-gamma) and interferon-alpha (rBoIFN-alpha 1) on oxidative functions of bovine neutrophils. Treatment with rBoIFN-gamma, but not rBoIFN-alpha 1, enhanced the luminol-dependent chemiluminescence (LDCL) response of bovine neutrophils to both opsonized zymosan particles and phorbol myristate acetate. Pre-incubation of neutrophils for 2 h at 39 degrees C with rBoIFN-gamma resulted in a 40% increase in both LDCL and release of hydrogen peroxide by neutrophils stimulated with opsonized zymosan. This enhancement was observed at doses ranging from 0.2 to 2000 units of rBoIFN-gamma per ml. In contrast to the results observed in the LDCL and hydrogen peroxide assays, preincubation of neutrophils with rBoIFN-gamma had no effect on the levels of superoxide anion released in response to opsonized zymosan. Pre-incubation with rBoIFN-gamma increased phorbol myristate acetate (PMA)-stimulated LDCL by 30%, although it had no effect on either superoxide anion or hydrogen peroxide release in response to PMA stimulation. Neither recombinant interferon directly elicited an oxidative burst from neutrophils in the absence of zymosan or PMA stimulation.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

In utero infection with PRRS virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets

The putative immunosuppressive effect of PRRS virus (PRRSV) on innate immune responses was studied in piglets infected in utero with PRRSV. Phagocytosis and oxidative burst capacities in 2-, 4- and 6-week-old in utero infected piglets were investigated and compared with age-matched control piglets. Phagocytic capacity of blood monocytes against Salmonella bacteria was investigated by flow cytometry. Oxidative burst in blood monocytes and in alveolar lung macrophages was investigated by luminol- and lucigenin-enhanced chemiluminescence, respectively. Decreased phagocytosis against Salmonella was found in blood monocytes from 4- and 6-week-old infected piglets compared to controls. In contrast, 2-week-old infected piglets showed phagocytic responses comparable to age matched control piglets. While oxidative burst capacity was increased in blood (PBMC) from in utero PRRSV infected piglets, the oxidative burst capacity of alveolar lung macrophages was decreased, especially in 2- and 4-week-old piglets, compared to age-matched control piglets. The present results indicate that in utero infection with PRRSV inhibits phagocytosis against Salmonella in blood monocytes as well as the oxidative burst capacity of alveolar macrophages. These observations indicate that PRRSV in utero infection induces at state of immunosuppression in piglets paving the way for enhanced secondary infections.     

Effect of strenuous exercise stress on chemiluminescence response of equine alveolar macrophages

Bronchoalveolar lavage samples were collected using a fibreoptic endoscope from horses at specified times before and after single bouts of exercise. Lucigenin-dependent phagocytic chemiluminescence was used to assess the effect of exercise on the alveolar macrophage metabolic activity in response to stimulation by opsonised zymosan. A profound suppressive effect on the chemiluminescence production was present throughout the first three days after exercise. However, the cellular composition of lavage fluids was not altered by the exercise. It is suggested that strenuous exercise may jeopardize the antimicrobial function of alveolar macrophages which may lead to an increase in susceptibility to infection.     

Effect of milk stasis on Brucella abortus infection of the mammary gland in goats

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.     

Oxidative responses in ferret macrophages

Although the basic function of T and B lymphocytes in ferrets has been known for some time, the function of mononuclear phagocytes has not been described in this species. The present study has characterised basic oxidative responses in ferret macrophages, and has investigated the effects of endogenous and exogenous modulators of macrophage function on oxidative capacity in vitro. Macrophages derived from the blood or lungs of ferrets were shown capable of generating the reactive oxygen intermediate (ROI) molecules superoxide and hydrogen peroxide, and secreting a lysosomal enzyme (acid phosphatase), in response to appropriate stimuli. A T cell supernatant (derived from mitogen-stimulated peripheral blood lymphocytes) was able to activate both blood- and lung-derived macrophages for enhanced ROI production, while specific ROI inhibitors (superoxide dismutase and catalase) were able to partially ablate ROI activity. The accumulation of nitrite in culture supernatants, as an indicator for the production of reactive nitrogen intermediates, could not be demonstrated by ferret macrophages derived from either tissue source. In contrast to the enhancing effects of TCS on the oxidative function of blood-derived macrophages, exposure to bacterial LPS caused marked suppression of ROI and lysosomal enzyme production by these cells. Finally, the generation of superoxide anion, following phagocytosis of live or heat-killed Mycobacterium bovis or zymosan, indicated that ROI production in response to phagocytic stimulation was relatively weak in ferret blood-derived macrophages. These results are discussed in relation to the study of immune function in a novel species, and with particular reference to research into tuberculosis (Tb), since ferrets are important wildlife vectors of bovine Tb in New Zealand.     

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.     

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.     

Neutrophil apoptosis during the resolution of bovine mammary gland injury

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.     

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.     

Humoral and cellular factors affecting the neutrophil response of the locally immunised mammary gland to staphylococcal infection

Locally immunising the non-lactating ovine mammary gland by infusing killed Staphylococcus aureus enhances neutrophil accumulation in mammary secretion during subsequent staphylococcal infection. Immunological factors influencing this increased neutrophil response were studied in the present experiments. Glands locally immunised with killed Brucella abortus supported a greater neutrophil response to staphylococcal infection than did glands immunised with killed S. aureus. An enhanced neutrophil response to staphylococcal infection was recorded in 3 of 7 ewes locally immunised with zymosan. Passive immunisation by intramammary infusion of secretions from immunised glands conferred an enhanced neutrophil response during staphylococcal infection. Absence of haemolytic complement in secretions of immunised glands suggested complement was not implicated in the response. Secretions of immunised glands contained elevated concentrations of mononuclear cells. When exudates rich in mononuclear cells were established by infusion of endotoxin into non-immunised glands there was no increase in the neutrophil response to subsequent staphylococcal infection. However, when the mononuclear cell concentration was elevated by intramammary infusion of staphylococcal antigens in systemically immunised ewes there was an increase in the neutrophil response to subsequent infection. Thus humoral and cellular characteristics of the locally immunised mammary gland influence the kinetics of the neutrophil influx during staphylococcal infection.     

The production and characterization of anti-bovine CD14 monoclonal antibodies

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.