Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines.     

Morphometric analysis of proinflammatory cytokines in mammary glands of sows suggests an association between clinical mastitis and local production of IL-1beta, IL-6 and TNF-alpha

Twelve healthy primiparous sows received intramammary inoculation with Escherichia coli (serotype O127) during the 24-h period preceding parturition. Mammary gland biopsy samples were taken immediately before inoculation (0 h) and from the inoculated and the contralateral non-inoculated glands 24 h after inoculation. The analyses of interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) by immunohistochemistry revealed that the production of these proinflammatory cytokines significantly increased in the inoculated mammary glands of sows that developed clinical signs of mastitis (affected group, n=4) 24 h after inoculation. This was also true for IL-8 in the inoculated mammary glands of sows that did not develop clinical signs of mastitis (nonaffected group, n=8). Sows that developed clinical signs of mastitis displayed significantly lower constitutive production of IL-1beta than did sows that remained clinically healthy. The data indicate that the development of clinical symptoms of coliform mastitis in the sow is associated with a locally increased proinflammatory cytokine production in response to intramammary E. coli infection.     

Enhanced cutaneous hypersensitivity reactions are associated with ovine high and low cortisol responsiveness to acute endotoxin challenge

Inbred rodent studies have demonstrated that cutaneous hypersensitivity reactions are exacerbated in stress-susceptible, and attenuated in stress-resistant strains of mice. This physiological response was, in part, mediated by activation of the hypothalamic-pituitary-adrenal axis during the acute restraint stress. A study was conducted to examine whether or not cutaneous hypersensitivity reactions are also associated with variable cortisol responsiveness to inflammatory stress in an outbred ovine population. High (H), medium (M), and low (L) cortisol responsive sheep were identified from a population of 110 females based on their estimated breeding values for cortisol concentration measured 4 h post-systemic challenge with Escherichia coli endotoxin (400 ng kg(-1)). Cutaneous hypersensitivity reactions to phytohaemagglutinin (PHA), 1-chloro-2, 4-dinitrobenzene (DNCB), and Candida albicans cellular antigen (CAA) were measured in these variable cortisol-responding sheep, in addition to serum interleukin (IL)-6, interferon (IFN)-gamma, and ovalbumin (OVA)-specific IgG concentrations. When compared to the M cortisol responders, both H and L cortisol responders had significantly greater cutaneous swelling during the elicitation phase in response to DNCB (P < 0.05) and CAA (P < 0.05); a similar but not significant trend was observed during the PHA challenge. The primary, but not the secondary, IgG response to OVA was significantly lower in the H and L cortisol responders when compared to the M cortisol responders. Differences in serum IL-6 or IFN-gamma concentration were not observed across variable cortisol-responsive groups. Together, these results demonstrate that cutaneous hypersensitivity reactions are enhanced in outbred H and L cortisol-responding sheep, independent of systemic modulation by IL-6 and IFN-gamma.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.     

Effect of magnesium deficiency in the hen on egg production and hatchability of eggs

The influence of the level of magnesium (Mg) in the ration on reproduction by the hen was studied in 2 experiments. In experiment 1, it was found that feeding a semi‐purified ration, containing 56 p.p.m. of Mg, resulted in decreased egg production and reduced serum Mg concentration within 10 to 14 days. Although fertility of eggs was not affected noticeably by Mg deficiency, hatchability of fertile eggs was decreased markedly. The influence of inadequate dietary Mg was partially alleviated by adding 250 p.p.m. Mg to the ration. In experiment 2, a decline in hatchability preceded decreases in egg production and food consumption. A decrease in Mg concentration in egg contents accompanied the decline in hatchability. Hens fed rations containing 50 or 150 p.p.m. added Mg produced eggs of relatively low hatchability by day 14 of the trial. Subsequent to being fed a ration containing 550 p.p.m. added Mg, these hens increased in rate of egg production while hatchability of fertile eggs also increased. The results indicate that the dietary Mg requirement of the laying hen, sufficient to maintain a high rate of production of eggs which hatch satisfactorily, is 400 or more p.p.m.     

The xMAP technique can be used for detection of the inflammatory cytokines IL-1beta, IL-6 and TNF-alpha in bovine samples

Infectious diseases can cause large health problems in cattle. The infections cause an acute inflammatory response, mediated by pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. By mapping the pattern of cytokines during inflammations, valuable information about the course of an infection is gained. The aim of the present study was to evaluate a particle-based flow cytometric method, the xMAP technique, using ovine/bovine reagents, for quantification of IL-1beta, IL-6 and TNF-alpha, for application in studies on ruminant infectious diseases with emphasis on bovine milk and plasma samples. Singleplex, duplex and triplex xMAP assays were evaluated, and limits of detection (LODs) as well as intra- and inter-assay variabilities were determined for each assay. Cross-reactivity between reagents in multiplex assays was also tested. In addition, presence of cytokines in milk and plasma samples from healthy and mastitic cows was studied. The LODs were significantly lower for singleplex xMAP assays than for duplex and triplex assays. In singleplex assays, the LODs were 0.08, 0.2 and 0.5 ng/ml, for IL-1beta, IL-6 and TNF-alpha, respectively. Corresponding LODs in triplex assays were 2.0, 6.5 and 3.5 ng/ml. Data indicate that the linear ranges of the multiplex assays were narrower than in singleplex assays. The intra-assay coefficients of variation were < or =10.7% for singleplex assays, while they ranged from 6.2 to 23.2% in the triplex assay. The inter-assay variance ranged from 5.1 to 35.8% in singleplex assays, and from 8.8 to 78.4% in triplex assays. Cross-reactivity between reagents was not observed, and all three cytokines were detected in bovine milk and plasma samples collected from cows with clinical mastitis. In conclusion, our results show that the xMAP technique can be used for quantification of IL-1beta, IL-6 and TNF-alpha in bovine samples, and that further work is required to optimize the multiplex assays.     

Enhanced IL-6 transcriptional response to adenosine receptor ligands in horses with lower airway inflammation

Reasons for performing study: Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objectives: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL‐6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR‐subtypes in each BAL sample was determined by quantitative RT‐PCR and compared to that in 13 other tissues. Bronchoalveolar lavage cells were stimulated either with the adenosine analogue NECA, CGS‐21680 (A_2AAR selective agonist) or with a combination of NECA and SCH‐58261 (A_2AAR antagonist) and IL‐6 expression assessed. Results: Bronchoalveolar lavage cells predominantly expressed A_2BAR, with lower A_2AAR levels and marginal A₃AR expression; A₁AR was not detected. This pattern was similar to that of PBMCs but different from the other tissues tested. No significant differences in AR expression in BAL cells from both groups were detected, although a trend for decreased A_2BAR in airway‐compromised horses was observed. Treatment of BAL cells with the nonselective agonist NECA upregulated IL‐6 expression in cells from airway‐compromised horses, but levels remained unchanged in control animals. Furthermore, blockage of A_2AAR with SCH‐58261 enhanced IL‐6 mRNA induction by NECA in both groups, with higher levels in airway‐compromised horses; the amplitude of this response correlated with neutrophil count. Conclusions: These results demonstrate the presence of an adenosine/IL‐6 inflammatory axis in the bronchoalveolar milieu of airway‐compromised horses. While A_2BAR is the predominant proinflammatory AR subtype expressed, A_2AAR appears to modulate inflammatory signalling (IL‐6 expression) by adenosine. Potential relevance: This study supports selective AR targeting as a potential therapeutic approach for the modulation of inflammation in the equine lower respiratory tract.     

Liver mitochondrial oxygen consumption and proton leak kinetics in broilers supplemented with dietary copper or zinc following coccidiosis challenge

Mitochondrial respiration was assessed in sixteen 7‐day‐old broilers as a subset of a larger study assessing the effects of Cu and Zn supplementation above requirements with a coccidiosis challenge on gain/feed ratio. The birds were selected from four treatments (four birds/treatment): a control diet (Cu 15 mg/kg and Zn 60 mg/kg) + coccidiosis challenge (CC), a Cu diet with 245 mg/kg Cu from tribasic copper chloride (TBCC) + CC, a negative control diet (Cu 15 mg/kg and Zn 60 mg/kg) ‐ CC and a Zn diet with 2000 mg/kg Zn from ZnO. The diets were composed of 49% corn, 40% soybean meal, 6.2% vegetable oil (diet dry matter = 90.62%, crude protein = 21.37%, fat = 7.7%, metabolizable energy = 12.1 MJ/day) and were fed for 14 days. Birds were dissected, and approximately one gram of liver tissue was used for mitochondrial oxygen consumption and proton leak kinetics assays. Respiratory control ratio and mitochondrial proton leak assessed by calculating rates of oxygen consumption at 175mV membrane potential were greater for the negative control group, but there were no differences in average gain/feed among treatments. In summary, broilers that did not undergo coccidiosis challenge had lower proton leak and higher respiratory control ratio. However, the impact of supplementation of Cu and Zn above requirements did not appear to prevent changes in respiratory control ratio and proton leak kinetics with coccidiosis challenge.     

Influence of dietary carboxymethylcellulose on the apparent absorption of calcium, magnesium and phosphorus in rats

Introduction   Dietary pectin vs cellulose has been shown to enhance apparent magnesium absorption in rats (unpublished data). This observation supports that of V ahouny et al. (1987), indicating that pectin feeding produced a more positive balance of magnesium in young, growing rats than did cellulose feeding. Pectin is a soluble, viscous and readily fermentable non-starch polysaccharide, but is not digested by animal enzymes. Pectin feeding raises the viscosity of small intestinal digesta in rats (J udd and T ruswell 1985). The increase in magnesium absorption seen after pectin feeding could relate to its fermentation, which may lower the pH of the small intestinal digesta, which in turn raises the solubility of minerals, rendering them more available for absorption. Both dietary lactulose and lactose, which are not digestible but highly fermentable, lower the pH of small intestinal digesta and increase mineral absorption in rats (H eijnen et al. 1993). The influence of viscosity per se on mineral absorption is unknown. In contrast to the above-mentioned mechanism, the observed stimulatory effect of pectin feeding on magnesium absorption (unpublished data) could relate to viscosity of small intestinal contents. To provide clues as to this possibility, the model compound carboxymethylcellulose (CMC) may be used. This compound is not digested by animal enzymes, is non-fermentable, but is highly viscous and has been shown to raise the viscosity of small intestinal contents in broiler chickens (S mits et al. 1997). Thus we fed rats on diets with different levels of CMC to study the influence of digesta viscosity on apparent absorption of calcium, magnesium and phosphorus.     

Regulation and role of intrapituitary IL-6 production by folliculostellate cells

Interleukin-6, mainly produced by monocytes and macrophages is known to influence the secretion of anterior pituitary hormones and is, therefore, considered to play an important role in the interaction between the immune system and the endocrine system. However, IL-6 represents not only a lymphocyte message but is also produced within the anterior pituitary. Folliculostellate (FS) cells have been identified as the source of the intrapituitary IL-6 production in the normal pituitary, whereas in pituitary adenomas IL-6 is produced by the tumor cells themselves. The present review summarizes the knowledge about the regulation of the intrapituitary IL-6 synthesis and release in FS cells. Moreover, the possible roles of the intrinsic IL-6 production for function and growth of normal and adenomatous endocrine pituitary cells are discussed.     

Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-gamma production

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRgammadelta+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRalphabeta+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-gamma, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses.     

Mycoplasma synoviae lipoprotein MSPB, the N-terminal part of VlhA haemagglutinin, induces secretion of nitric oxide, IL-6 and IL-1beta in chicken macrophages

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing respiratory disease and infectious synovitis. M. synoviae haemagglutinin VlhA is an abundant surface-exposed lipoprotein and immunodominant antigen. Post-translational cleavage of VlhA generates two proteins, MSPB and MSPA. MSPB, the amino-terminal end of VlhA, is a lipoprotein of about 40-50 kDa but can appear in truncated forms (tMSPB) of about 20-30 kDa. The aim of this study was to determine whether MSPB and tMSPB can stimulate chicken macrophages to secrete NO and cytokines. Macrophages derived from chicken monocytes (MDM) or the MQ-NCSU macrophage cell line were stimulated with M. synoviae protein extracts containing MSPB or tMSPB, as well as with purified MSPB and tMSPB. Proteins from detergent extractions induced IL-6 secretion in MDM and NO secretion in MQ-NCSU. Both MSPB and tMSPB were capable of inducing NO secretion in MQ-NCSU, as well as IL-6 and IL-1beta in MDM. The activity of IL-6 induced by purified tMSPB was similar to the effect of 60 pg/ml of recombinant chicken IL-6. The effect of IL-1beta induced by tMSPB was comparable to the effect of 10 ng/ml of recombinant IL-1beta. Whereas all samples containing MSPB were able to induce NO, IL-6 and IL-1beta, it seemed that the purified tMSPB of about 20 kDa was the most potent in its ability to induce IL-6 and IL-1beta in MDM. Compared to MSPB, tMSPB lacks about 200 amino acids in its carboxyl-terminal part. Therefore, our results suggest that the major part of the stimulating activity is associated with the amino-terminal part of MSPB, most likely with its lipid moiety.     

Blunted pathogen-associated molecular pattern motif induced TNF, IL-6 and IL-10 production from whole blood in dogs with lymphoma

Lymphoma is associated with a higher risk of sepsis as compared to other forms of neoplasia in people and dogs which might be due to alterations in cytokine production. The objective of this study was to compare bacterial pathogen associated molecular pattern (PAMP) motif-induced TNF, IL-6, and IL-10 response of whole blood from dogs with na?ve lymphoma and healthy dogs. We hypothesized that whole blood from dogs with lymphoma would exhibit an impaired cytokine response to LPS, lipoteichoic acid (LTA), and peptidoglycan (PG) stimulation compared to whole blood from healthy dogs. Whole blood was collected from dogs with lymphoma (n=20) and healthy dogs (n=15) and stimulated with PAMPs or phosphate buffered saline. Whole blood production of TNF, IL-6 and IL-10 was measured. Whole blood from dogs with lymphoma had reduced TNF, IL-6 and IL-10 production capacity after LPS, LTA and PG stimulation compared to whole blood from healthy dogs. These data could partially explain why dogs with lymphoma have a higher risk for infection compared to dogs with other forms of neoplasia.     

Effects of dietary ruminally protected L-carnitine on plasma metabolites in sheep following a sub-lethal ammonia challenge

In Experiment 1, lambs were randomly assigned to 0.25, 1.00, 2.50, 5.00 and 10.00 g/day of dietary ruminally protected L-carnitine (RPLC) and were allowed to adapt for 20 days. Plasma samples were obtained at 0, 120 and 240 min after RPLC feeding. Plasma L-carnitine (LC) concentrations increased (p<0.01) for all levels of RPLC treatment, however, no differences were observed due to level of RPLC or time. Plasma LC concentrations were 27.05 and 57.83 micromol/l for baseline and pooled RPLC treated sheep, respectively. In Experiment 2, lambs were randomly assigned to 0, 0.125, 1.06 and 2.0 g/day of RPLC and were adapted as in Experiment 1. Plasma was collected at 0, 15, 30, 60, 90, 180, 240 and 360 min after oral ammonia challenge (300 mg/kg BW urea). Plasma LC concentrations increased with treatment relative to control (p<0.01). Plasma LC concentrations were 35.7, 44.2, 60.5 and 65.7 micromol/l for the 0, 0.125, 1.06 and 2.0 g/day treatments, respectively. RPLC tended to decrease plasma ammonia at some time points (time x treatment; p=0.10). We conclude that RPLC increased plasma LC concentrations, but had only modest effects on plasma ammonia concentrations and had no effect on plasma urea or glucose concentrations.     

Pharmacokinetics of ivermectin in sheep following pretreatment with Escherichia coli endotoxin

The comparative pharmacokinetics of ivermectin (IVM), between healthy and in Escherichia coli lipopolysaccharides (LPS) injected sheep, was investigated after an intravenous (IV) administration of a single dose of 0.2 mg/kg. Ten Suffolk Down sheep, 55 ± 3.3 kg, were distributed in two experimental groups: Group 1 (LPS): treated with three doses of 1 μg LPS/kg bw at ?24, ?16, and ?0.75 hr before IVM; group 2 (Control): treated with saline solution (SS). An IV dose of 0.2 mg IVM/kg was administered 45 min after the last injection of LPS or SS. Plasma concentrations of IVM were determined by liquid chromatography. Pharmacokinetic parameters were calculated based on non‐compartmental modeling. In healthy sheep, the values of the pharmacokinetic parameters were as follows: elimination half‐life (2.85 days), mean residence time (MRT) (2.27 days), area under the plasma concentration curve over time (AUC, 117.4 ng day^?1 ml^?1), volume of distribution (875.6 ml/kg), and clearance (187.1 ml/day). No statistically significant differences were observed when compared with the results obtained from the group of sheep treated with LPS. It is concluded that the acute inflammatory response (AIR) induced by the intravenous administration of E. coli LPS in adult sheep produced no changes in plasma concentrations or in the pharmacokinetic behavior of IVM, when it is administered intravenously at therapeutic doses.