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Abstract

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 × 109 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. (Am J Vet Res 1999;60:1307–1311)

Free access
in American Journal of Veterinary Research

Abstract

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-α (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 F-actin content and CD11b 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 CD11b 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. (Am J Vet Res 2000;61:380–386)

Full access
in American Journal of Veterinary Research

Abstract

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 haemolyticaderived LPS resulted in neutrophil activation and neutrophil- mediated endothelial damage. Neutrophilmediated endothelial injury and neutrophil degranulation were, at least in part, mediated by IL-8, 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. (Am J Vet Res 2002; 63:394–401)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis.

Animals

12 adult ponies.

Procedure

Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Results

19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (α1-antitrypsin I), 65,000 (α1-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies.

Conclusion

Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies.

Clinical Relevance

Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies. (Am J Vet Res 1998;59:1234–1237)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To determine whether platelets become activated and form platelet-neutrophil aggregates during near-maximal treadmill exercise in horses.

Animals

4 Thoroughbreds.

Procedure

Horses were subjected to 4 standardized exercise tests on a treadmill, and blood samples were collected before exercise, at treadmill speed of 12 m/s, and 5 minutes after exercise. Flow cytometric techniques were used to identify activated platelets, and flow cytometric and microscopic techniques were used to identify platelet-neutrophil aggregates.

Results

Platelet-neutrophil aggregates increased from 2.8 ± 0.4% at rest to 17.2 ± 1.1% and 14.7 ± 1.6% during and after exercise, respectively. Platelet activation was not detected during or after exercise.

Conclusions

Platelet-neutrophil aggregates consistently form during strenuous exercise in horses. (Am J Vet Res 1998;59:393–396)

Free access
in American Journal of Veterinary Research

SUMMARY

Objectives

To determine whether platelets are hyperaggregable or form platelet-neutrophil aggregates during the prodromal stages of acute laminitis of ponies.

Animals

Healthy adult ponies: 8 experimental and 6 control.

Procedures

Acute laminitis was induced by oral administration of corn starch and wood flour to 8 ponies, and indices of platelet activation were evaluated. Blood samples were collected before and at 4, 8, 12, 24, 28, and 32 hours after carbohydrate administration, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, and platelet-neutrophil aggregates were determined. When lameness was first detected, ponies were euthanatized and arteriography and histologic examination of hooves were performed.

Results

Carbohydrate overload was associated with hyperaggregability of platelets throughout the prodromal stages of laminitis and increased numbers of platelet-neutrophil aggregates. Reduction of blood supply to affected hooves was variable, and blood clots were found in 6 of 11 laminitis-affected hooves.

Conclusions

Platelets were hyperaggregable throughout the prodromal stages of carbohydrate-induced laminitis and formed platelet-neutrophil aggregates. Platelet-neutrophil aggregates may initiate or contribute to development of acute laminitis.

Clinical Relevance

Anti-platelet therapy may be useful for treatment of acute alimentary laminitis in horses. (Am J Vet Res 1997;58:1376–1380)

Free access
in American Journal of Veterinary Research

Objective

To determine whether cats with inflammatory hepatic disease had concurrent inflammatory bowel disease (IBD), pancreatitis, or chronic interstitial nephritis.

Design

Prospective case series.

Sample Population

78 tissue sections of liver, intestine, pancreas, and kidney from cats that had previous necropsy examinations at the teaching hospital.

Procedure

We reviewed histologic sections of liver, intestine, pancreas, and kidney from cats that had previous necropsy examinations and determined the prevalence of lymphocytic portal hepatitis, cholangiohepatitis, IBD, pancreatitis, and chronic interstitial nephritis, and the relationship among them.

Results

36 cats had lymphocytic portal hepatitis, 18 had cholangiohepatitis, and 24 did not have inflammatory hepatic disease. The prevalence of IBD (10/36; 28%) and pancreatitis (5/36; 14%) in cats with lymphocytic portal hepatitis was not significantly different from cats without inflammatory hepatic disease. The prevalence of IBD (15/18; 83%) and pancreatitis (9/18; 50%) was greater (P < 0.05) for cats with cholangiohepatitis, compared with cats without inflammatory hepatic disease. Thirty-nine percent of cats (7/18) with cholangiohepatitis had IBD and pancreat Evidence of IBD in association with cholangiohepatitis was characterized by infiltration of lymphocytes and plasma cells into the lamina propria; however, neutrophilic infiltrates also were found in 6 of 15 (40%) cats with cholangiohepatitis. Pancreatitis was mild in all cats.

Clinical Implications

Cats with a diagnosis of cholangiohepatitis should be evaluated for IBD and pancreatitis, (J Am Vet Med Assoc 1996;209:1114-1116)

Free access
in Journal of the American Veterinary Medical Association

Summary

Hemorheologic alterations induced by incremental treadmill exercise were examined in 5 Thoroughbreds. Blood viscosity; pcv; rbc filterability, density gradient profile, and shape; serum and rbc electrolyte concentrations; and plasma total solids and lactate concentrations were measured before exercise, at treadmill speeds of 9 and 13 m/s, and 10 minutes after exercise. Exercise was associated with significant (P < 0.05) increases in pcv, blood viscosity, and plasma total solids concentration. After adjustment of pcv to 40% by adding or removing each horse's own plasma, blood viscosity remained significantly greater in the sample obtained at 13 m/s, compared with that in samples taken at rest. Filterability of rbc was significantly decreased at 13 m/s, compared with values from other sampling times. During exercise, a significantly greater proportion of the rbc were less dense and were found in the upper layers of the rbc density gradient profile, compared with resting values. This change was associated with a significant increase in rbc mean cell volume. Rapid increases in serum sodium and potassium concentrations during exercise were accompanied by significant increases in rbc potassium and chloride concentrations. This study revealed a consistent pattern of hemorheologic alterations associated with exercise in Thoroughbreds, suggesting that multiple hemorheologic tests are needed to adequately define these complex alterations during exercise in horses.

Free access
in American Journal of Veterinary Research

Summary

Effects of echinocytosis on blood rheology and exercise performance were evaluated for 5 Thoroughbreds. Echinocytosis was induced by administration of furosemide (1 mg/kg of body weight, im, q 12 h) for 4 days. Furosemide treatment resulted in decreases in serum sodium and serum chloride concentrations and in RBC chloride and potassium concentrations. Echinocytosis was associated with increased rbc density as determined by rbc density gradient centrifugation. However, samples containing echinocytes were more filterable than control samples, indicating that echinocytes were not rigid cells. Erythrocyte sedimentation rate was decreased in blood samples containing echinocytes, indicating that cell-to-cell interaction was reduced. Whole blood viscosity was not altered by presence of echinocytes. Echinocytes did not impair the capacity of horses to complete treadmill exercise tests, nor did they alter heart rate or blood gas variables. However, plasma lactate concentration was higher in samples obtained during exercise at a treadmill speed of 9 m/s. Echinocytosis was associated with higher postrace creatine kinase activity. These data indicate that echinocytes may be dense, but not rigid cells, which have decreased tendency to aggregate and do not increase whole blood viscosity. Therefore, echinocytes are unlikely to inhibit or obstruct microvascular blood flow.

Free access
in American Journal of Veterinary Research

Summary

The hypothesis that equine laminitis is caused by thrombosis of vessels in the laminar corium (dermis) was investigated. Hemostatic alterations were evaluated by determining platelet count, platelet survival, platelet adhesiveness to vascular subendothelium, activated clotting time, and whole blood recalcification time. Thrombosis of vessels in the hoof wall was evaluated by scintigraphic studies of the hoof wall after administration of indium-111 (111In)-labeled platelets, contrast arteriography, and histologic examination. Platelet count remained constant before and at the onset of lameness; however, survival of 111In-labeled platelets was shortened. Scintigraphy of affected feet revealed accumulation of 111In-labeled platelets distal to the coronary band. Arteriography of disarticulated saline-perfused feet revealed marked reduction in blood supply to affected hooves. Histologic examination of the laminar dermis disclosed variable numbers of microthrombi in dermal veins of affected feet from 3 of 4 ponies with laminitis. Whole blood recalcification time was shortened at 8 hours after administration of carbohydrate and was prolonged at the onset of laminitis. Activated clotting time was prolonged at 32 hours after carbohydrate administration and at the onset of lameness. Plasma endotoxin-like activity was detected in 1 of 4 affected ponies. These data confirm that microvascular thrombosis existed at the onset of lameness in ponies with carbohydrate-induced laminitis and indicate that systemic coagulopathy may have preceded development of thrombosis.

Free access
in American Journal of Veterinary Research