Dagmar Hoeben

Tumor necrosis factor-α and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and of NO(x) (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:O37 bacteria or endotoxin O111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the E. coli-infected cows were treated with a bactericidal antibiotic (Enrofloxacin; Bayer AG, Leverkusen, Germany) i.v. at 10 hr and subcutaneously (sc) at 30 hr after infection. NO(x) concentrations transiently increased maximally 10- to 11-fold in milk of E. coli-infected quarters with or without antibiotic treatment at 24 hr and after endotoxin administration. NO(x) concentrations did not change in milk of unchallenged quarters and in blood plasma. Increases of NO(x) were proceeded by a transient (96- to 149-fold) rise of milk TNF-alpha concentrations, which in endotoxin-administered quarters was maximal at 6 hr and in infected quarters without or with Enrofloxacin treatment at 10 and 14 hr. In blood plasma TNF-alpha concentrations only moderately increased to peaks in endotoxin-administered cows at 6 hr and in E. coli-infected cows at 14 hr postchallenge. In one severely sick, nontreated E. coli-infected cow milk, TNF-alpha response at 14 hr was excessive and followed by a spectacular rise of NO(x) concentration in milk between 48 and 72 hr. In conclusion, a possible clinical relevance of nitric oxide production associated with a rise of intramammary and systemic TNF-alpha during acute mastitis by E. coli infection and endotoxin in lactating dairy cows is indicated, but could not be inhibited by antibiotic treatment.

Role of endotoxin and TNF-α in the pathogenesis of experimentally induced coliform mastitis in periparturient cows

Twelve cows were experimentally infected in two quarters with 1 x 10(4) cfu Escherichia coli per quarter and six cows were infused with 500 microg endotoxin into two quarters. Six cows infected intramammarily with Esch. coli were treated intravenously with a bactericidal antibiotic 10 h after infection and subcutaneously 20 h later. Blood and milk samples were collected from all cows at regular time intervals. Milk production decreased more rapidly, but was less pronounced, after endotoxin infusion than (during Esch. coli mastitis. The milk production losses in the noninflamed quarters were negligible in endotoxin mastitis, but were substantial during Esch. coli mastitis, probably due to more pronounced systemic effects. Reticulorumen motility was inhibited only during Esch. coli mastitis. Changes in plasma haptoglobin were more pronounced during Esch. coli mastitis, although they occurred sooner during endotoxin mastitis. No changes in plasma activities of enzymes such as lactate dehydrogenase, glutamic-oxaloacetic transaminase and gamma-glutamyl transpeptidase were observed. Concentrations of tumour necrosis factor-alpha increased in both types of mastitis. Absorption of these cytokines into the circulation was highest during Esch. coli mastitis, especially in the untreated control group. We found only minor differences between the treated and untreated Esch. coli groups, but there were larger differences between the Esch. coli groups and the endotoxin group. These differences were probably due to differences in kinetics, composition and amounts of different cytokines released in the mammary gland and subsequently absorption into the circulation. Endotoxin is probably not directly responsible for the systemic changes during coliform mastitis.

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.

Chemiluminescence of bovine polymorphonuclear leucocytes during the periparturient period and relation with metabolic markers and bovine pregnancy-associated glycoprotein.

The respiratory burst activity of polymorphonuclear leucocytes (PMN) was evaluated in eight Holstein cows from 8 weeks before until 6 weeks after calving by chemiluminescence (CL). The CL response started to decrease 1 week before parturition, reaching a minimum during the first 2 weeks after calving. From week 3 of lactation, CL increased again and returned to original levels by week 6 of lactation. Plasma concentrations of 3-hydroxybutyric acid, total bilirubin and bovine pregnancy-associated glycoprotein started to increase before parturition to reach a maximum during the first or second week of lactation. The concentrations of glutamic-oxaloacetic transaminase, lactate dehydrogenase, non-esterified fatty acids and bilirubin increased after calving, reaching a maximum during the second week. A small decrease in plasma cholesterol during the week before and after calving was followed by an increase. The CL response of the PMN showed significant temporal relationships with the plasma concentrations of 3-hydroxybutyric acid, bovine pregnancy-associated glycoprotein, bilirubin, glutamic-oxaloacetic transaminase, non-esterified fatty acids; that with cholesterol was nearly significant. This means that the change in the CL response with time coincided with the changes in plasma concentrations of these substances with time and that these changes were significantly related with each other. The results of this study show that the decreased respiratory burst activity of bovine PMN around parturition may be related to the extent of the metabolic and hormonal changes. Although the causative relationships are not proven, these results support earlier results suggesting that 3-hydroxybutyric acid and bovine pregnancy-associated glycoprotein may directly affect neutrophil function, whereas non-esterified fatty acids, cholesterol, bilirubin, and liver enzymes may have potential as diagnostic markers of impaired neutrophil function and consequently increased disease risk around parturition.

Pregnancy-associated glycoprotein and decreased polymorphonuclear leukocyte function in early post-partum dairy cows

Phagocytosis and oxidative burst activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and pregnancy-associated glycoprotein (bPAG) concentrations in plasma were evaluated in two longitudinal studies in dairy cows from 3 weeks before until 5 weeks after calving, carried out in the United States and in Europe. Ingestion of Staphylococcus aureus by blood PMN increased during the first week after calving and normalised 3 weeks post-partum. Phagocytosis of Escherichia coli did not change in the early post-partum period. In both studies, a significant decrease in oxidative burst activity of PMN was observed between 1 and 3 weeks after calving. In all cows, a very significant increase in plasma bPAG concentration was found between 1 week before and 2 weeks after calving. The peak of bPAG concentration in plasma immediately preceded the alterations of blood PMN functions. These results suggest that bPAG may be associated with inhibition of PMN function of dairy cows during the early post-partum period.

Modulation of the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland by growth hormone

This review is focused on the possible interactions of prolactin and somatotrope hormone in the modulation of inflammation of the mammary gland. Several different models are examined: Escherichia coli, Streptococcus uberis, and endotoxin mastitis. Subsequently, the release of growth hormone and insulin-like growth factor during fever and mastitis, the immunophysiological effects of GH on E. coli mastitis, S. uberis and endotoxin mastitis, the galactopoietic action of rBST on healthy and mastitis cows as well as the immunologic effects of GH on leukocytes in healthy and diseased cows are discussed. It can be concluded that the underlying regulation of the neuro-endocrine network is fundamental in the normal function of the immune system.

In vitro effect of ketone bodies, glucocorticosteroids and bovine pregnancy-associated glycoprotein on cultures of bone marrow progenitor cells of cows and calves

Changes in the number, maturity and function of neutrophils, concomitant changes in plasma concentrations of hormones and metabolites, and the increased susceptibility of cows to infectious diseases around parturition, led us to investigate the effect of beta-hydroxybutyric acid (BHBA), acetoacetic acid (AcAc), hydrocortisone-21-acetate (HCAc) and bovine pregnancy-associated glycoprotein (bPAG) on the proliferation of bovine bone marrow progenitor cells in methylcellulose in vitro cultures. Myeloid progenitors were stimulated with concanavalin A-stimulated leukocyte conditioned medium (LCM) and erythroid progenitors with erythropoietin in the presence of hemin. Erythroid and myeloid colonies were scored after five and seven days, respectively. BHBA and AcAc induced inhibitory effects on the proliferation of bovine bone marrow cells at concentrations of 1.0, 2.5, and 5.0 mM. HCAc significantly inhibited growth of progenitors at concentrations of 10, 20, 50, and 100 ng/ml, and bPAG at concentrations of 2400 and 3000 ng/ml. The results of this study suggest that in the cow high concentrations of BHBA, AcAc, HCAc and bPAG, which can be reached in the circulation around calving, could alter the number of circulating neutrophils after parturition. This phenomenon might contribute to the increased susceptibility of dairy cows to environmental mastitis.

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.

Treatment of acute Escherichia coli mastitis in cows with enrofloxacin: effect on clinical signs and chemiluminescence of circulating neutrophils.

We have studied the effect of treatment with enrofloxacin on local and general clinical signs and chemiluminescence of circulating polymorphonuclear leucocytes during experimentally induced Escherichia coli mastitis in cows immediately afer parturition. Twelve cows were infected with 10(4) cfu Esch. coli P4:032 into both left quarters. Six cows received an intravenous injection of 5 mg enrofloxacin/kg at 10 h after infection and a second enrofloxacin treatment administered subcutaneously at 30 h post infection. The other six cows were controls that received no treatment. General clinical signs (fever, tachycardia, loss of appetite, reduced rumen motility and depression) were similar in both groups. Local clinical signs, such as swelling, pain and firmness of the inflamed mammary quarters, were less severe in the treated cows. We saw no difference in the appearance of the milk: flecks and watery or purulent milk were observed in both groups. The beneficial effects of treatment with enrofloxacin were mainly on milk production and composition. The decline in milk production and the changes in milk concentrations of lactose, Na+ and bovine serum albumin were less pronounced in the treated cows. Treatment with enrofloxacin accelerated the clearance of bacteria from the infected quarters, but had no effect on the chemiluminescence response of isolated polymorphonuclear leucocytes. The changes in the number of circulating leucocytes and the appearance of immature neutrophils in the circulation of the treated cows indicated possible beneficial effects on migration of neutrophils into the inflamed glands. Higher milk somatic cell counts in the treated cows supported this hypothesis. The results of this study indicated that treating cows that have been experimentally infected with Esch. coli mastitis after parturition with enrofloxacin reduced the severity of the disease, especially the decline in milk production and the changes in milk composition.