Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response.     

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca²⁺) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ?1 μM 2-APB significantly reduced ROS production, whereas 50 and 100 μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ?10 μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca²⁺ channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

Linoleic acid increases adhesion,chemotaxis, granule release,intracellular calcium mobilisation,MAPK phosphorylation and gene expression in bovine neutrophils

Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 μM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 μM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2–5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2 h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.     

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils

2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.     

D-Lactic acidosis in calves as a consequence of experimentally induced ruminal acidosis

In order to test the hypothesis that ruminal drinking in calves can lead to D-lactic metabolic acidosis, ruminal acidosis was induced in nine calves by intraruminal application of untreated whole milk via a stomach tube. The amount of the daily force-fed liquid was 3 x 1 l. The experimental design called for an end of intraruminal applications if two or more of the following signs were observed: severe depression, estimated degree of dehydration >10%, absence of sucking reflex, lack of appetite for two consecutive feedings, severe metabolic acidosis with calculated Actual Base Excess (ABE) <-15 mmol/l. The procedure was scheduled to be discontinued on the 17th day of experiment. The onset of ruminal acidification occurred rapidly, and mean pH value fell from 6.70 (+/-0.48) to 4.90 (+/-0.38) after the first application. The following days the pH values varied between 4 and 5. Rumen acidity was characterized biochemically by a significant increase in both isomers of lactic acid. The effects of the intraruminal administration on the calves were detrimental; eight of nine calves showed an acute disease process. According to the pre-established clinical standard, seven of nine calves were removed from the intraruminal feeding schedule. All but one of the calves developed severe systemic acidosis. The increase in anion gap demonstrated the net acid load. In all the calves D-lactate levels were found to show a significant and rapid increase. On the contrary, L-lactate never deviated from physiological levels. These observations confirm that, in young calves as in adult cattle, ruminal acidosis may lead to a clinically manifested D-lactic metabolic acidosis.     

Role of platelet-activating factor in alveolar septal injury associated with experimentally induced pneumonic pasteurellosis in calves

OBJECTIVE: To determine whether platelet-activating factor (PAF) is involved in acute lung microvascular injury associated with pneumonic pasteurellosis in calves. ANIMALS: 15 healthy 2- to 4-week-old male Holstein calves. PROCEDURE: Calves were anesthetized and inoculated intrabronchially with saline (0.9% NaCl) solution (n = 5) or 1x10(9) Pasteurella haemolytica organisms (n = 10). Of the 10 calves inoculated with P haemolytica, 5 also were treated with WEB 2086, a potent inhibitor of PAF, and 5 were treated with vehicle. Blood and bronchoalveolar lavage samples were collected before and 1, 2, 4, and 6 hours after inoculation of P. haemolytica. Blood samples were analyzed to evaluate total number and differential counts of leukocytes, dilute whole-blood leukocyte deformability, size of neutrophils, and neutrophil CD11b expression. Bronchoalveolar lavage samples were analyzed for total number and differential counts of nucleated cells, total protein concentration, and hemoglobin concentration. Size and gross and histologic appearance of lung lesions also was determined. RESULTS: Treatment of calves with WEB 2086 reduced size of lung lesions, attenuated the increase in microvascular permeability, and reduced neutrophil infiltration in the first 4 hours after inoculation. Treatment with WEB 2086 also attenuated a decrease in leukocyte deformability, increase in size of neutrophils, and CD11b expression by circulating neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: It appears that PAF is a major mediator for altered lung microvascular permeability and activation of circulating neutrophils in the first 4 hours after onset of pneumonic pasteurellosis in calves.     

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.     

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.     

Comparison of L-selectin and Mac-1 expression on blood and milk neutrophils during experimental Escherichia coli-induced mastitis in cows

OBJECTIVE: To evaluate L-selectin (CD62L) and Mac-1 (CD11b) expression at the surface of blood and milk neutrophils during the early inflammatory response to Escherichia coli-induced mastitis in cows. ANIMALS: 6 healthy Holstein heifers in early lactation. PROCEDURE: Blood and milk samples were collected before and after intramammary administration of 10(4) CFU's of E coli in the left mammary gland quarters. Bacterial counts and electrolyte concentrations in milk, rectal temperature, differential blood leukocyte counts, milk somatic cell counts, neutrophil viability, and the expression of CD62L and CD11b on blood and milk neutrophils were determined longitudinally. RESULTS: Bacteria grew during the first 6 hours after inoculation with a pronounced leukocytic influx. Coincident with neutrophil influx was an increase in CD62L+ and CD11b+ milk neutrophils, as well as an improved viability of milk neutrophils. The peak of the inflammatory reaction was reached approximately 12 hours after E coli inoculation. From that time forward, changes in CD62L and CD11b expression were opposed to each other, with a decrease in CD62L expression and an increase in CD11b expression on blood and milk neutrophils; the magnitude of the differences in CD62L and CD11b expression between blood and milk neutrophils decreased. Percentages of CD62L+ and CD11b+ milk neutrophils increased to percentages that were similar to blood neutrophils (ie, approx 92%). CONCLUSIONS AND CLINICAL RELEVANCE: The presence of adhesion molecules on a large percentage of milk neutrophils during the acute inflammatory response, together with the changes in receptor density, suggest a major role for CD62L and CD11b in neutrophil function during coliform mastitis.     

In vitro evaluation of the role of platelet-activating factor and interleukin-8 in Mannheimia haemolytica-induced bovine pulmonary endothelial cell injury

OBJECTIVE: To develop an in vitro model of the bovine alveolar-capillary interface and to evaluate the roles of interleukin-8 (IL-8) and platelet-activating factor (PAF) in neutrophil-mediated endothelial injury induced by infection with Mannheimia haemolytica. SAMPLE POPULATION: Cultured bovine pulmonary microvascular endothelial cells, freshly isolated bovine neutrophils, and monocyte-derived bovine macrophages. PROCEDURE: A coculture system was developed in which endothelial cells were grown to confluence in tissue culture inserts, neutrophils were added to the inserts, and macrophages were added to tissue culture wells. Mannheimia haemolytica-derived lipopolysaccharide (LPS) or supernatant was added to activate macrophages, and inhibitors of PAF or IL-8 were added to the insert. Endothelial cell cytotoxicity and permeability (ie, albumin leakage) and neutrophil activation (ie, adhesion, degranulation [lactoferrin expression], and superoxide production) were assessed. RESULTS: The addition of M haemolytica-derived LPS to bovine macrophages in the coculture system resulted in significant increases in endothelial cell cytotoxicity and permeability and neutrophil degranulation and adhesion. Inhibition of IL-8 reduced endothelial cell permeability and neutrophil degranulation induced by exposure to M haemolytica-derived supernatant, whereas inhibition of PAF decreased superoxide release by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of bovine macrophages by M haemolytica-derived LPS resulted in neutrophil activation and neutrophil-mediated endothelial damage. Neutrophil-mediated endothelial injury and neutrophil degranulation were, at least in part, mediated by IL8, whereas PAF promoted superoxide release by neutrophils in this in vitro system designed to mimic the in vivo events that occur during the early stages of bovine pneumonic pasteurellosis.     

Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

Evaluation of lipopolysaccharide-induced activation of equine neutrophils

OBJECTIVE: To evaluate lipopolysaccharide (LPS)-induced activation of equine neutrophils in blood. SAMPLE POPULATION: Blood samples from 5 healthy adult Thoroughbreds. PROCEDURES: Neutrophil integrin (CD11/CD18) expression, size variation, degranulation, and deformability were measured with and without incubation with LPS. Time and concentration studies were done. The mechanism of endotoxin-induced neutrophil activation was investigated by inactivating complement or preincubating neutrophils with inhibitors of tumor necrosis factor-alpha (TNF-alpha) synthesis, prostaglandin-leukotriene synthesis, or platelet-activating factor. RESULTS: Incubation of equine neutrophils with LPS increased cell surface expression of CD11/CD18, decreased neutrophil deformability, increased and decreased neutrophil size, and induced neutrophil degranulation. The LPS-induced neutrophil activation was attenuated by addition of inhibitors of TNF-alpha and prostaglandin-leukotriene synthesis. CONCLUSIONS AND CLINICAL RELEVANCE: Equine neutrophils are readily activated in vitro by LPS, resulting in increased expression of integrin adhesion molecules, decreased deformability, variation in neutrophil size, and degranulation. The tests used to detect activated neutrophils in this study may be useful in detecting in vivo neutrophil activation in horses with sepsis and endotoxemia.     

Influence of sex steroids on the viability and CD11b, CD18 and CD47 expression of blood neutrophils from dairy cows in the last month of gestation

In the period around parturition, cows experience an increased susceptibility for the development of Escherichia coli mastitis. This increased susceptibility has been correlated with a decreased functionality of neutrophils. In the current study, it is suggested that the decreased neutrophil functionality may be induced by the extensive alterations in sex steroid levels occurring around parturition. It was first hypothesized that 17beta-estradiol and progesterone influence the viability, apoptosis and necrosis of blood neutrophils from cows in their last month of gestation. Subsequently, it was hypothesized that 17beta-estradiol modulates the expression of CD11b, CD18 or CD47 thereby explaining its influence on the migration of bovine neutrophils. Neither 17beta-estradiol nor progesterone significantly influenced viability, apoptosis or necrosis in spontaneous apoptosis conditions. However, when apoptosis was induced with TNF-alpha and gliotoxin, progesterone exerted a survival effect (P<0.05). In addition, 17beta-estradiol treatment of bovine blood neutrophils significantly decreased the expression of CD47 (P<0.05) but not of CD11b or CD18. It can be concluded that 17beta-estradiol and progesterone do not affect spontaneous apoptosis of bovine blood neutrophils while a survival effect was observed for progesterone on induced neutrophils apoptosis. Moreover, our results concerning the influence of 17beta-estradiol on the CD11b, CD18 and CD47 expression extend previous demonstrations of the suppressive effect of 17beta-estradiol on neutrophils migration and indicate that the altered expression of CD47 may contribute to this phenomenon.     

Anti-lipopolysaccharide antibody administration mitigates ruminal lipopolysaccharide release and depression of ruminal pH during subacute ruminal acidosis challenge in Holstein bull cattle

The effects of anti-lipopolysaccharide (LPS) antibody on rumen fermentation and LPS activity were investigated during subacute ruminal acidosis (SARA) challenge. Eleven Holstein cattle (164 ± 14 kg) were used in a 3 × 3 Latin square design. Cattle were fed a roughage diet on days −11 to −1 (pre-challenge) and day 2 (post-challenge), and a high-grain diet on days 0 and 1 (SARA challenge). For 14 days, 0-, 2-, or 4-g of anti-LPS antibody was administered once daily through a rumen fistula. Ruminal pH was measured continuously, and rumen fluid and blood samples were collected on days −1, 0, 1, and 2. Significantly lower ruminal LPS activity on day 1 was observed in the 2- and 4-g groups than those in the 0-g group. In addition, significantly higher 1-hr mean ruminal pH on SARA challenge period (days 0 and 1) was identified in the 4-g group than in the 0-g group. However, rumen fermentation measurements (total volatile fatty acid [VFA], VFA components, NH₃-N and lactic acid) and peripheral blood metabolites (glucose, free fatty acid, beta-hydroxybutyrate, total cholesterol, blood urea nitrogen, aspartate aminotransferase and gamma-glutamyl transferase) were not different among the groups during the experimental periods. Therefore, anti-LPS antibody administration mitigates LPS release and pH depression without the depression of rumen fermentation and peripheral blood metabolites during SARA challenge in Holstein cattle.     

Ruminal acidosis and the rapid onset of ruminal parakeratosis in a mature dairy cow: a case report

A mature dairy cow was transitioned from a high forage (100% forage) to a high-grain (79% grain) diet over seven days. Continuous ruminal pH recordings were utilized to diagnose the severity of ruminal acidosis. Additionally, blood and rumen papillae biopsies were collected to describe the structural and functional adaptations of the rumen epithelium. On the final day of the grain challenge, the daily mean ruminal pH was 5.41 ± 0.09 with a minimum of 4.89 and a maximum of 6.31. Ruminal pH was under 5.0 for 130 minutes (2.17 hours) which is characterized as the acute form of ruminal acidosis in cattle. The grain challenge increased blood beta-hydroxybutyrate by 1.8 times and rumen papillae mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A synthase by 1.6 times. Ultrastructural and histological adaptations of the rumen epithelium were imaged by scanning electron and light microscopy. Rumen papillae from the high grain diet displayed extensive sloughing of the stratum corneum and compromised cell adhesion as large gaps were apparent between cells throughout the strata. This case report represents a rare documentation of how the rumen epithelium alters its function and structure during the initial stage of acute acidosis.     

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.     

Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance

We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.