Hilde Dosogne

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.

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.

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.

Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis

Abstract. Objective and design: To investigate the effect of enrofloxacin on endotoxin resorption during bovine Escherichia coli mastitis.¶Animals: 12 healthy early post partum Holstein cows.¶Treatment: Mastitis was induced by intramammary infusion of 104 cfu E. coli P4:O32. Six cows were treated twice according to the usual enrofloxacin therapy: 5 mg/kg enrofloxacin 1) intravenously at 10 h and 2) subcutaneously at 30 h after challenge. The other 6 cows served as non-treated controls.¶Methods: Blood and milk samples were collected at several time points after challenge. LPS in plasma was quantified using the limulus amœbocyte lysate (LAL) assay. The somatic cell count (SCC) and cfu of milk samples were also analysed.¶Results: Occasional LPS peaks were detected in the plasma of 2 control cows at 6 h post-challenge and of 1 enrofloxacin-treated cow at 10 h post-challenge (P < 0.01 and P < 0.05, respectively, in comparison with time 0), just before enrofloxacin treatment. After enrofloxacin treatment, no significant LPS amounts were detected in the plasma of treated cows, but neither in the control cows.¶Conclusion: During induced coliform mastitis, LPS resorption in plasma occured only sporadically and within 10 h post-challenge. Whereas enrofloxacin treatment clearly limited bacterial growth in milk, significant effects on LPS resorption could not be detected. This suggests that enrofloxacin treatment of E. coli mastitis is predominantly beneficial by its bactericidal activity and is not associated with enhanced resorption of endotoxins.

Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils

Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli. During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function. In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma. Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha. Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E. coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN. Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C. The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies. When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity. This is the first study reporting the effects of apoptosis on bovine PMN function. These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection. Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland.

Increased surface expression of CD11b receptors on polymorphonuclear leukocytes is not sufficient to sustain phagocytosis during Escherichia coli mastitis in early postpartum dairy cows.

Phagocytosis, CD11a and CD11b adhesion receptor expression, O2-production and maturity of circulating polymorphonuclear leukocytes (PMN) were studied during acute coliform mastitis in early postpartum dairy cows to obtain a better insight in the role of neutrophils in the pathology of this disease. The mammary gland of twelve newly calved high-yielding dairy cows was experimentally infected with Escherichia coli. Variability in clinical signs of mastitis and inhibition of milk production among cows was very high. There was a significant negative correlation between the number of circulating neutrophils immediately before infection and severity of mastitis represented by the decrease in milk production of non-infected quarters two days after infection. Pre-infection phagocytosis of E. coli, CD11a and CD11b receptor expression, phorbol myristate acetate (PMA)-induced O2-production and maturity of neutrophils on a per cell basis were not related to severity of mastitis. However, significant correlations between severity of mastitis and the total number of phagocytic PMN and mature PMN in blood immediately before infection were found. PMN characteristics responded differently to mastitis depending on the severity of the disease. Neutrophil functions from cows classified as severe (S) and moderate (M) responders to infection of the mammary gland were compared. Surface expression of CD11a receptors on PMN was decreased in all cows 24 h after infection, and this decrease was long-continued in S responders. A biphasic upregulation of the number of CD11b receptors on PMN was observed with a more pronounced response in S cows. PMN phagocytosis was decreased 12 h after infection in S cows and 18 h after infection in S and M cows and was normalized 24 h post-infection. The decrease of phagocytosis coincided with the first peak of CD11b receptor expression. Phorbol myristate acetate (PMA)-induced production of O2-by PMN was decreased for three days after infection in S responders compared to only one day in M responders and was followed by an upregulation. These data demonstrate a complexity in alterations of PMN functions during mastitis and suggest the involvement of differences in systemic factors dependent on severity of mastitis.

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.

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.

Decreased number and bactericidal activity against Staphylococcus aureus of the resident cells in milk of dairy cows during early lactation

Phagocytic and bactericidal activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and milk, against Staphylococcus aureus, was compared between groups of six healthy dairy cows in early, mid- and late lactation using a bacteriological assay. PMN were isolated from blood with a high degree of purity, but the cells isolated from milk contained variable amounts of macrophages (Mphi) and lymphocytes (L). The results were therefore calculated using the percentage PMN in order to evaluate phagocytosis and killing by PMN only. Blood PMN phagocytosed 82% Staph. aureus and milk PMN 43% on average and there was no significant difference between the different stages of lactation. The bactericidal activity of blood PMN against Staph. aureus was 36+/-8% in early lactation (significantly different from mid lactation, P < 0.05), 64+/-10% in mid lactation and 53+/-6% in late lactation. Milk PMN killed only 6+/-3% Staph. aureus in early lactation (significantly different from mid lactation, P < 0.01), 27+/-3% in mid lactation and 20+/-9% Staph. aureus in late lactation. The ratio of the bactericidal activity of milk to blood PMN was 0.08, 0.43 and 0.22 in early, mid- and late lactation, respectively. In addition to the decreased function. the number of cells in milk (somatic cell count, SCC) was also 60% lower in early lactation than in mid lactation cows (P < 0.01). Our results suggest an impairment of blood and milk-resident PMN bactericidal activity against Staph. aureus and a decreased number of milk-resident PMN in dairy cows at the onset of lactation.