Intramammary Infusion Of Endotoxin Research Articles

Supplemental fish oil does not alter immune competence or the pathophysiological response to an intramammary infusion of endotoxin in peri-partum multiparous Holstein cows

The objective was to determine the effects of supplementing the diet with fish oil during the peri-partum period on the immune competence and the pathophysiological response to a lipopolysaccharide-induced mastitis challenge. Multiparous Holstein cows (n=30) were completely randomized to one of two treatments at 3 weeks pre-partum. Treatments differed only in the source of supplemental lipid and included either Energy Booster or fish oil. Treatment diets were fed from -21 d relative to expected date of parturition until 10 d post partum. Treatments were fed as a bolus prior to the a.m. feeding. The dose of lipid during the pre-partum period was 250 g/d, whereas the amount of lipid supplemented post partum was adjusted to the level of intake, approximately 0.92% of the previous day's dry matter intake. Ex-vivo analyses of immune competence were measured including the antimicrobial activity of whole blood against Escherichia coli, Salmonella typhimurium and Candida albicans as well as the production of interferon-gamma by peripheral blood mononuclear cultures. At 7 days in milk cows were infused with 100 microg of Esch. coli lipopolysaccharide into one rear quarter. Supplementing fish oil increased plasma concentrations of eicosapentaenoic and docosahexaenoic acids, but had no affect on the proportions of arachidonic acid at calving. Fish oil did not influence the production of interferon-gamma or the antimicrobial activity of whole blood against any of the microorganisms. Furthermore, fish oil had no ameliorative effect on either the local or the systemic acute phase response following an intramammary lipopolysaccharide challenge in early lactating Holstein cows. Supplementing fish oil in the diet of peri-partum cows will not protect them from deleterious effects of an excessive acute phase response.

Mild Dietary Copper Insufficiency Depresses Blood Neutrophil Function in Dairy Cattle

Abstract Effects of dietary copper insufficiency on bovine blood neutrophil function were evaluated. Dairy heifers were maintained on a basal (6-7 mg Cu/kg diet) or copper-supplemented (20 mg Cu/kg diet) diet beginning 84 d prepartum. At approximately 35 d postpartum, heifers were bled for neutrophil isolation 1 d prior to and 2 d following intramammary infusion of endotoxin in 2 mammary quarters. Neutrophils were evaluated for in vitro production of superoxide anion, reduction of nitroblue tetrazolium, and bactericidal activity. Copper status did not affect neutrophil reduction of nitro-blue tetrazolium or production of superoxide anion. Neutrophil bacterial phagocytosis and number of bacteria ingested per neutrophil were similarly unaffected by diet. Prior to endotoxin infusion neutro-phils isolated from copper-supplemented heifers were significantly more effective at killing ingested bacteria than neutrophils from unsupplemented heifers. Endotoxin infusion eliminated diet-related differences in bacteri...

Total and Differential Leucocyte Counts and Lymphocyte Subpopulations in Lymph, Afferent and Efferent to the Supramammary Lymph Node, During Endotoxin‐Induced Bovine Mastitis

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.

Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4 h after infusion (p.i.). Neutrophils appeared in afferent lymph 2 h p.i., and in efferent lymph and milk 4 h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4 h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-α (TNF-α) concentration tended to increase in afferent lymph at 2 h p.i., and increased in milk at 4 h p.i. The level of IL-1β increased at 4 h p.i. in milk, but was not detected in lymph. Interferon-γ was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1β and TNF-α are not necessary for IL-8 production and release in response to endotoxin.

The Effect of Ascorbic Acid and l-Histidine Therapy on Acute Mammary Inflammation in Dairy Cattle

Ascorbic acid and L-histidine were investigated as antioxidant therapies for acute mammary inflammation. Mastitis was induced in eight nonpregnant Holstein cows by intramammary infusion of endotoxin. Treatments were administered in a 4 × 4 Latin square crossover design with 1-wk periods between challenges with endotoxin. Four individual treatments, control, ascorbic acid only, L-histidine only, and ascorbic acid plus L-histidine, were applied. Two doses of 25g of ascorbic acid administered intravenously at 3- and 5-h postendotoxin challenge increased milk production recovery (9% higher, P<0.02) and tended to reduce the extent of rumen stasis. Two doses of 25g of L-histidine similarly administered decreased plasma antioxidant activities 5.5% (P<0.05). However, ascorbic acid and L-histidine had no effects on rectal temperature, heart rate, respiratory rate, and dry matter intake. The data suggested that ascorbic acid provided some potential benefit for recovery from acute mammary inflammation in dairy cattle.

Hemostasis in cows with endotoxin-induced mastitis

Summary Hemostasis was evaluated in cows with experimentally induced endotoxemia and mastitis, caused by intramammary infusion of endotoxin (1 mg) derived from Escherichia coli. Hemostatic tests included prothrombin time; activated partial thromboplastin time; thrombin time; fibrinogen, fibrin(ogen) degradation products, and platelet concentrations; and antithrombin-III and plasminogen activities. Significant alterations were observed in the mean values of most analytes (prothrombin time was increased; thrombin time was increased with subsequent decrease; activated partial thromboplastin time, fibrinogen concentration, plasminogen activity, and platelet concentration were decreased; and antithrombin-III activity and fibrin(ogen) degradation products concentration were unchanged) at 1 or more postchallenge sample collection times (3, 12, or 24 hours) after endotoxin administration, compared with mean values obtained from samples prior to endotoxin administration. These data indicated activation of hemostatic mechanisms, initiated either directly by endotoxin or by inflammatory mediators released or produced in response to endotoxin infusion.

Milk antimicrobial drug residue assay results in cattle with experimental, endotoxin-induced mastitis

Summary Antimicrobial drug residue testing was performed on milk samples obtained from 8 cows with experimental endotoxin-induced mastitis, using 4 commercially available assay kits. Although none of the cows in the study received antimicrobials, only 1 of the 4 assay procedures, assay C, had consistently negative results (specificity = 1.00). The proportion of positive assay results varied from 0 to 1.00 among combinations of sampling time, sample status (endotoxin-infused quarter vs composite noninfused sample). The proportion of positive results found when assay C was used (0) differed significantly from the proportion found when the 3 other assays were used. The proportion of positive results did not differ significantly between assay A (0.45) and assay B (0.48); however, both assays had a significantly lower proportion of positive assays than did assay D (0.86). Logistic regression models were developed predicting positive milk antimicrobial drug residue assay results as a function of assay kit, sample status, and time interval following experimental challenge exposure. Using assay A as a baseline risk, assay B and assay D were more likely to have positive assay results, and assay C had a decreased risk of positive assay results. Milk samples from endotoxin-infused quarters were at increased risk for positive assay results, compared with noninfused composite samples. Samples collected from endotoxin-infused quarters or control quarters were at increased risk for positive assay results following the intramammary infusion of endotoxin. Our findings suggest that specificity of milk antimicrobial drug residue assays varies greatly among assay kits and that intramammary inflammation may increase the proportion of false-positive assay results.

Phmacodynamics and pharmacokinetics of carprofen, a non‐steroidal anti‐inflammatory drug, in healthy cows and cows with Escherichia coli endotoxin‐induced mastitis

The pharmacodynamics of carprofen and its pharmacokinetics in plasma and milk of healthy cows and cows with endotoxin-induced mastitis were studied after a single intravenous dose of 0.7 mg/kg body weight. Carprofen was administered to five clinically healthy cows and to the same cows 3 weeks later, 2 h after intramammary infusion of endotoxin. Mastitis developed in all endotoxin-infused quarters. The pharmacokinetic characteristics of carprofen in healthy cows were a small volume of distribution (0.09 l/kg), a relatively low systemic clearance (2.4 ml/h kg), and a long elimination half-life (30.7 h). In the mastitic cows, systemic clearance (1.4 ml/h kg) was significantly lower (P less than 0.01), and elimination half-life (43.0 h) was significantly longer (P less than 0.01) than in the normal animals. Concentrations of carprofen in milk from healthy quarters were below the limit of detection for the assay (0.022 micrograms/ml). In milk from mastitic quarters, concentrations of carprofen increased up to 0.164 micrograms/ml during the first 12 h after induction of mastitis, but were less than 0.022 micrograms/ml at 24 to 48 h. Compared with the untreated mastitic controls, carprofen treatment significantly reduced heart rate (P less than 0.01), rectal temperature (P less than 0.001), quarter swelling (P less than 0.01) and other parameters measured. Local and systemic adverse reactions to carprofen were not observed.

Sequestration of N-acetyl-β-D-glucosaminidase in somatic cells during experimental bovine mastitis induced by endotoxin, Staphylococcus aureus or Streptococcus agalactiae

Experimental mastitis was induced in cows by intramammary infusion of endotoxin, Staphylococcus aureus or Streptococcus agalactiae. The inflammatory response was monitored by somatic cell counting and N-acetyl-beta-D-glucosaminidase (NAGase). NAGase activity was analysed in fresh milk samples in parallel with samples treated by a cycle of freezing and thawing combined with detergent treatment to release the cell-bound NAGase. Before the udder reacted by inflammation, the total NAGase activity consisted of free extracellular activity. Later on when the inflammation was established, much of the milk NAGase remained sequestered intracellularly. S agalactiae was linked with a high degree of cellular NAGase sequestration indicating a blockage of the lysosomal release function from the phagocytes. S aureus delayed the inflammatory response.

Effect of Steroidal Anti-Inflammatory Drugs on Escherichia coli Endotoxin-Induced Mastitis in the Cow

Effects of intramammary infusion of prednisolone (40mg) or intramuscular injection of dexamethasone (30mg) or flumethasone (5mg) on local and systemic signs in Escherichia coli endotoxin-induced mastitis were studied. The effect of varying intervals (0, 2, and 4h) between intramammary infusion of endotoxin and prednisolone in the same quarter was determined.Intramammary infusion of endotoxin (.01mg lipopolysaccharide of E. coli) produced inflammation of the infused quarter, fever, tachycardia, and leukopenia followed by a neutrophilic leukocytosis in the blood and a decrease in plasma zinc and iron concentrations. All corticosteroid treatments, except intramammary administration of prednisolone 4h after endotoxin infusion, enhanced leukocytosis and diminished local signs of inflammation. Intramuscular injection of dexamethasone or flumethasone together with intramammary infusion of endotoxin and intramammary administration of prednisolone 2h after lipopolysaccharide infusion completely abolished the febrile response. Abolishment of fever and attenuation of several hematologic and blood biochemical changes may be explained by diminished synthesis of endogenous mediators within the inflamed quarters due to glucocorticosteroid action.

Induction of inflammatory responses by endotoxin in the non‐lactating ovine mammary gland

In non-lactating ewes, intramammary infusion of endotoxin provokes an intense but transient inflammatory response as assessed by mammary lavage. The variability of the method for sampling the leucocytes in mammary secretions was examined. Initial lavage with 20 ml pyrogen-free saline recovered 83.0 +/- 2.8 and 84.4 +/- 3.0% of the total leucocyte numbers harvested by three serial washes of quiescent and inflamed glands, respectively. Lavage of glands prior to infusion of endotoxin did not affect the magnitude of the subsequent inflammatory response. Three treatments at 3.5 day intervals with the glucocorticoid, dexamethasone, which inhibits the inflammatory response to intramammary infusion of antigen in systemically immunized ewes, did not affect the inflammatory response to endotoxin. However, the variability of the inflammatory response to activated complement was greatly increased in dexamethasone-treated ewes; this effect was attributed to the induction of lactation which results in loss of sensitivity of the gland to the inflammatory activity of activated complement. The results indicated that the macrophages and lymphocytes which are present in mammary secretions in large numbers, and dexamethasone-sensitive lymphocytes in the gland, played a trivial role in the induction of inflammatory responses to endotoxin.

Effects of Endotoxin on Mammary Secretion of Lactating Cows

The objectives were to describe the magnitude and time course of changes in milk pH, Na, K, lactose, and somatic cells and to determine if paracellular pathways were altered after infusion of Escherichia coli endotoxin (serotype #O128:AB12) to produce inflammation in one-half of the udder of the goat. Intramammary infusion of endotoxin increased pH, number of somatic cells, and Na and decreased K and lactose in milk. Sodium and number of somatic cells were increased by as little as .1μg of endotoxin; .25μg produced changes in most of the other parameters; maximal effect was elicited by 1μg of endotoxin. The gland response peaked from 5 to 7h after infusion of endotoxin with an increase in milk cellularity as the only significant effect noted in the control gland. Infusion of [14C]lactose into the gland and [99mTc]albumin into the blood demonstrated that large molecules were more able to cross into and out of udder halves after endotoxin treatment. It is suggested that ion interchange rather than bulk flow across paracellular paths is responsible for changes. In addition, endotoxin appeared to reduce lactose secretion and synthesis.

Endotoxin Induced Migration of Leukocytes from Blood to Milk

Guinea pigs were separated from young on days 5 to 7 of lactation. They were anesthetized with ether and were infused intramammary via the teat canal with either sterile saline (.5ml) or Escherichia coli endotoxin (026:B6:500μg/.5ml). Each animal served as its own control by having sterile saline in one gland and endotoxin in the other. Animals were sacrificed 4, 6, 8, and 12h later to determine the time of maximum migration of polymorphonuclear leukocytes from blood to milk. A control group of animals having had no intramammary infusion was sacrificed. Tissues were prepared for observation by light and electron microscopy. Five fields per slide, five slides per animal, and three animals per period were examined by light microscopy, and numbers of polymorphonuclear leukocytes per field were categorized for presence a) in the capillary, b) in interstitial space, c) touching the basal lamina, d) between epithelial cells of an alveolus, and e) in the lumen of an alveolus. Polymorphonuclear leukocytes were in small numbers in the lumen by 4h and peaked at about 8h after intramammary infusion of endotoxin. By 12h numbers had decreased from 8h. Polymorphonuclear leukocytes were observed by electron microscopy in the intercellular spaces of the alveolar epithelium. A time sequence model using the guinea pig to study mechanisms of leukocyte migration into milk based upon tissue studies may be used in research aimed at controlling mastitis.

The effect of an intramammary infusion of endotoxin on experimentally induced mycoplasmal mastitis

The infusion of 10 microgram of endotoxin lipopolysaccharide from Escherichia coli into the mammary gland of four cows 16 h before inoculation with ureaplasmas did not prevent, or even diminish, the subsequent ureaplasmal mastitis. There was no reduction in the severity or duration of the inflammatory cell response in milk or in the clinical appearance of the resulting mastitis. Also, the excretion of ureaplasmas was not reduced. A similar experiment with Mycoplasma dispar in two cows demonstrated that endotoxin was again ineffective in preventing the mastitis. Furthermore, there was some indication that the proliferation and excretion of this mycoplasma was enhanced in endotoxin-treated quarters.

The effect of an intramammary infusion of endotoxin on the establishment of experimental mastitis by Streptococcus agalactiae inthe cow

An infusion of E. coli endotoxin (10 microgram) into the mammary gland of the cow 16 h before experimental infection with St. agalactiae prevented the establishment of mastitis. A clinical examination of the affected gland did not reveal signs of inflammation, all organisms were eliminated from the milk by 36 h after infection. Mammary glands not pretreated with endotoxin, and injected with the same number of viable streptococci, showed signs of severe inflammation the milk yield was reduced and the injected pathogen could be isolated from the gland for up to 14 days when sampling was discontinued. The injection of endotoxin alone produced a rapid increase in the cell count of the milk, there were some signs of inflammation and 80% of the cells in the milk were neutrophils. The cell counts in the milk remained above normal for 7-10 days. Untreated control quarters appeared to be unaffected by the injection of E. coli endotoxin into an adjacent quarter, on the other hand the injection of St. agalactiae into the adjacent quarter on the same side of the udder produced a cell response which reached a peak after 2 days and returned to preinoculation levels by the fourth day. No inflammation was observed in these control quarters and no streptococci were isolated from the milk.