Roger Heyneman

Role of the neutrophil leucocyte in the local and systemic reactions during experimentally induced e.coli mastitis in cows immediately after calving

Mammary leucocytes are the major contributors to natural defence against mastitis after a microorganism has entered the gland. This paper reviews the role of the neutrophil granulocyte during acute coliform mastitis in cows in the periparturient period. Qualitative and quantitative aspects of several neutrophil cell functions before and during experimentally induced infections are briefly discussed.

Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0.001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0.001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0.001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1.7, 2.6 and 2.4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN. the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

Elevated levels of β-hydroxybutyric acid in periparturient cows and in vitro effect on respiratory burst activity of bovine neutrophils

The in vitro effect of normal (0.01 to 1 mM) and subketotic (1 to 2.5 mM) doses of butyric acid on the respiratory burst activity of bovine polymorphonuclear leucocytes (PMNL) isolated from blood was studied by luminol-enhanced, PMA (phorbol-12-myristate-13-acetate)-induced chemiluminescence (CL). The subketotic concentrations of butyric acid induced a significant inhibitory effect on CL. In a cell free assay, consisting of sonicated cells and H2O2, no changes in activity could be observed. The activity of the myeloperoxidase was not significantly altered as shown by the ortho-dianisidine-oxidation assay. Also, the production of O(2)- measured by the cytochrome c reduction assay was not affected by different doses of butyric acid. Butyric acid had no scavenging effect on hypochlorite. The reason for the inhibitory effect on CL may be a decreased production of H2O2. Indeed, luminol-enhanced CL evaluates the production of H2O2. This could not be confirmed in the other assays mentioned above, because H2O2 was added externally in these assays. In conclusion, because of this inhibitory effect on the respiratory burst activity of PMNL, the elevated blood level of butyric acid after parturition in high yielding cows may be, in part, responsible for the higher susceptibility to local and systemic infections during the postpartum period and during subclinical and clinical ketosis.

Classification of newly calved cows into moderate and severe responders to experimentally induced Escherichia coli mastitis.

In the present study newly calved cows were tentatively classified as moderate and severe responders to experimentally induced Escherichia coli mastitis based upon the reactive oxygen species (ROS)-generating capacity of their blood neutrophils before infection. The groups differed in blood and milk composition prior to infection. This initial classification was supported by the corresponding variation in clinical symptoms and in the changes in milk production and composition measured during mastitis. Responses of newly calved cows to Esch. coli challenge varied from mild to severe symptoms of inflammation in infected glands and differed in the intensity of systemic disturbances and general illness. Losses in milk yield and compositional changes were most pronounced in inflamed glands and in severe responders. In inflamed glands milk yield and composition did not return to preinfection level in either moderate or severe responders. The yields of lactose, alpha-lactalbumin, casein and fat followed the same pattern as milk yield. It is concluded that the severe and long lasting systemic disturbances observed in severe responders can be ascribed to absorption of endotoxin from infected glands into circulation, indicating the important role of endotoxin in the pathology of coliform mastitis in periparturient cows. Evaluation of the ROS-generating capacity of blood neutrophils and blood and milk composition before infection might help to predict the cows sensitivity to Esch. coli mastitis.

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood.

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.

Influence of antimicrobial agents on bactericidal activity of bovine milk polymorphonuclear leukocytes

The influence of nine commonly used antibiotics on the respiratory burst activity of bovine milk polymorphonuclear leukocytes (PMNL) of high yielding cows was studied in vitro. Cellular oxidative activity was quantitated after preincubation with drugs at different concentrations and assayed by a PMA (12,13-phorbol myristate acetate)-induced luminol-enhanced chemiluminescence (CL) technique. All antibiotics except sulphadiazine and enrofloxacin decreased CL at the highest concentration. Enrofloxacin significantly increased CL. Oxytetracycline inhibited CL even at low doses. The decreased CL with danofloxacin and oxytetracycline was mainly induced by their color, which caused absorption of the blue light emitted by luminol. Production of superoxide radicals measured by the cytochrome c reduction assay was lowered by danofloxacin, penicillin and chloramphenicol. The decreased CL with ceftiofur was due to inhibition of myeloperoxidase (MPO) activity and to scavenging of reactive oxygen species. Interference with the MPO-H2O2-halide system was also observed with spiramycin, erythromycin and oxytetracycline, while the latter was also observed with penicillin. The stimulatory effect of enrofloxacin might be due to an improvement of the penetration of luminol into the PMNL or to a stimulation of the production of H2O2. Potentiation of the action of PMA by changing the ratio between bound and free intracellular Ca2+ might also be involved. Our results suggest that many antibiotics may affect neutrophil function at concentrations that may be found in milk immediately after intramammary treatment or at concentrations higher than those found in milk after intramammary treatment.

Effect of antibiotics on the phagocytotic and respiratory burst activity of bovine granulocytes.

The influence of antibiotics on respiratory burst (phorbol-12-myristate-13-acetate (PMA)-stimulated luminol-enhanced chemiluminescence) and phagocytosis (flow cytometry) by bovine granulocytes was studied in vitro. Phagocytosis was impaired by 1000 micrograms/ml of oxytetracycline, chloramphenicol, erythromycin and spiramycin. All antibiotics, except sulphadiazine, decreased chemiluminescence at 1000 micrograms/ml or lower concentrations. Enrofloxacin increased chemiluminescence. The inhibition by oxytetracycline and danofloxacin was due to absorption of the light emitted by luminol at 425 nm. Oxytetracycline, ceftiofur, spiramycin and erythromycin affected the myeloperoxidase-H2O2-halide system. Ceftiofur, penicillin and danofloxacin showed scavenging effects on H2O2 and OCI. Penicillin and ceftiofur might interfere with luminol. Chloramphenicol, penicillin and ceftiofur affected the production of superoxide radicals. In summary, the observed effects of antibiotics might be of importance during treatment of infectious diseases in normal and immunocompromised animals. However, before classifying a drug as immunosuppressive, attention has to be paid to possible interference with the chemiluminescence assay.