Christian Burvenich

Defense of the Bovine Mammary Gland by Polymorphonuclear Neutrophil Leukocytes

The primary phagocytic cells of the bovine mammary gland, polymorphonuclear neutrophil leukocytes (PMN), and macrophages, comprise the first line of defense against invading pathogens. In the normal healthy mammary gland, macrophages predominate and act as sentinels to invading mastitis-causing pathogens. Once invaders are detected, macrophages, and possibly mammary epithelial cells, release chemoattractants that direct migration of PMN into the area. In the mammary gland, protection is only effective if rapid influx of PMN from the circulation and subsequent phagocytosis and killing of bacteria occur. The second line of defense against infection consists of a network of memory cells and immunoglobulins that interact with the first line of defense. To minimize mammary tissue damage caused by bacterial toxins and oxidative products released by PMN, elimination of invading bacteria must proceed quickly. Therefore, the inflammatory response needs to be regulated. Hormones, metabolites, and acute phase proteins act to influence the outcome of mastitis, especially around parturition. The number of circulating PMN in cows during early lactation is highly heritable and closely related to susceptibility to clinical mastitis at this time. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host–pathogen interactions. For example, the cloning and expression of proteins such as recombinant bovine soluble (rbos) CD (cluster of differentiation) 14 antigens, may provide ways of minimizing damaging effects of endotoxin during acute coliform mastitis. Soluble CD14 binds and neutralizes lipopolysacharide (LPS) and causes local recruitment of PMN after binding of CD14-LPS complexes to mammary epithelial cells. Development of transgenic animals that express rbosCD14 in their milk could prevent infection by Gram-negative pathogens.

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.

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.

Immune surveillance of mammary tissue by phagocytic cells

The leukocytes in milk consist of lymphocytes, neutrophil polymorphonuclear leukocytes (PMN) and macrophages. Lymphocytes together with antigen-presenting cells function in the generation of an effective immune response. Lymphocytes can be divided into two distinct subsets, T- and B-lymphocytes, that differ in function and protein products. The professional phagocytic cells of the bovine mammary gland are PMN and macrophages. In the normal mammary gland macrophages are the predominate cells which act as sentinels to invading mastitis causing pathogens. Once the invaders are detected, macrophages release chemical messengers called chemoattractants that cause the directed migration of PMN into the infection. Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal. However, their presence is like a double-edged sword. While the PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemicals which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity. Permanent scarring will result in a loss of milk production. Resident and newly migrated macrophages help reduce the damage to the epithelium by phagocytosing PMN that undergo programmed cell death through a process called apoptosis. Specific ligands on the neutrophil surface are required for directed migration and phagocytosis. In response to infection, freshly migrated leukocytes express greater numbers of cell surface receptors for immunoglobulins and complement and are more phagocytic than their counterparts in blood. However, phagocytic activity rapidly decreases with continued exposure to inhibitory factors such as milk fat globules and casein in mammary secretions. Compensatory hypertrophy in non-mastitic quarters partially compensates for lost milk production in diseased quarters. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-parasite interactions. For example the cloning and expression of proteins that bind endotoxin may provide ways of reducing damaging effects of endotoxin during acute coliform mastitis. The successful formation of bifunctional monoclonal antibodies for the targeted lysis of mastitis causing bacteria represents a new line of therapeutics for the control of mastitis in dairy cows.

Pathophysiological effects of endotoxins in ruminants. 1. Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration.

Data from the literature on the clinical effects of bacterial endotoxins in ruminants are reviewed. Special attention is paid to the effects on body temperature and reticulo-rumen motility. Furthermore, the effects of repeated intravenous injection of endotoxin are summarised. Pathophysiological disturbances after intramammary infusion of endotoxins proved to be identical to those found after intravenous injection of non-lethal doses. Strikingly, however, no marked inhibitory effect on rumen motility nor abortion was observed after intramammary infusion of endotoxins. Moreover, in cows that were made tolerant to endotoxin by daily intravenous injections, intramammary infusion of one-fifth of this daily dose produced a maximum effect on body temperature and plasma Zn concentrations. This suggests that inflammatory endogenous mediators were released in the udder and then absorbed into the blood circulation, rather than the absorption of endotoxin.

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.

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.

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.