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Abstract

OBJECTIVE To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS).

ANIMALS 8 healthy adult horses.

PROCEDURES Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays.

RESULTS Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone.

CONCLUSIONS AND CLINICAL RELEVANCE Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine plasma endotoxin concentration in horses competing in a 48-, 83-, or 159-km endurance race and its importance with regard to physical, hematologic, or serum and plasma biochemical variables.

Animals—83 horses.

Procedure—Weight and rectal temperature measurements and blood samples were obtained before, during, and after exercise. Blood samples were analyzed for plasma endotoxin concentration; serum antiendotoxin antibody titers; thromboxane B2 (TxB2) and 6- keto-prostaglandin F (PGF) concentrations; tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) activities; WBC, plasma protein, lactate, serum electrolyte, and calcium concentrations; PCV; and creatine kinase activity.

Results—Detection of plasma endotoxin increased during exercise for horses competing at all distances but occurred more frequently in the 48- and 83-km groups. Plasma lactate concentration was significantly greater when endotoxin was concurrently detected. Endotoxin in plasma was not significantly associated with success of race completion. Plasma TxB2 and PGF concentrations and serum IL-6 activity significantly increased with exercise. Horses that had an excellent fitness level (as perceived by their owners) had greater decreases in serum antiendotoxin antibody titers during exercise than did horses perceived as less fit. In horses with better finish times, TxB2 and PGF concentrations were significantly greater and TNFα activity was significantly less than that of slower horses.

Conclusions and Clinical Relevance—Endotoxemia developed during endurance racing, but was significantly correlated with increased plasma lactate concentration and not with other variables indicative of endotoxemia. Plasma TxB2 and PGF concentrations and serum TNFα activity may be associated with performance success. (Am J Vet Res 2003;64:754–761)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To compare physiologic, hematologic, and selected serum and plasma biochemical variables obtained from horses competing in 48-, 83-, or 159- km endurance rides before competition and at the same cumulative distance points.

Animals—83 horses.

Procedure—Weight and rectal temperature measurements and blood samples were obtained from horses before, during, and after 1 of 3 rides conducted on the same day. Plasma protein (PP), lactate, WBC, serum electrolyte, and calcium concentrations; PCV; and creatine kinase (CK) activity were determined. Assessments were made to determine whether any differences among groups, with respect to total distance competed, could be explained by differences in lap speed or conditioning and to investigate the effect of time in transit or on-site prior to competition on results of blood analyses or competition outcome.

Results—Horses in the 159-km distance group had the lowest preride serum sodium, chloride, bicarbonate, and calcium concentrations. As hours in transit increased, preride PP concentration was significantly greater; serum sodium, chloride, and bicarbonate concentrations were lower; CK activity at 159 km was greater; and horses were more likely to be eliminated. The preride sodium was significantly greater in horses that completed the ride, compared with those eliminated.

Conclusions and Clinical Relevance—Among distance groups, distance ridden, speed, level of fitness, and years of experience of horses had little effect on the variables examined. Electrolyte and water supplementation and earlier arrival at the event may be beneficial for horses that are transported long distances to endurance competition. (Am J Vet Res 2003;64:746–753)

Full access
in American Journal of Veterinary Research

Abstract

Objective— To evaluate effects of polymyxin B sulfate (PMB) on response of horses to endotoxin, using an ex vivo model.

Animals—8 healthy horses.

Procedure—In a crossover design, 3 doses of PMB (100, 1,000, and 10,000 U/kg of body weight) and physiologic saline solution (control) were evaluated. Prior to and for 24 hours after administration of PMB, blood samples were collected into heparinized tubes for use in 2 assays. For the endotoxin-induced tumor necrosis factor (TNF) assay, blood samples were incubated (37 C for 4 h) with 1 ng of Escherichia coli or Salmonella Typhimurium endotoxin/ml of blood. Plasma was harvested and assayed. For the residual endotoxin activity assay, plasma was collected into sterile endotoxin-free borosilicate tubes, diluted 1:10 with pyrogen-free water, and incubated for 10 minutes at 70 C. Escherichia coli endotoxin (0.1 or 1 ng/ml of plasma) was added to the thawed samples prior to performing the limulus ameobocyte lysate assay. Serum creatinine concentrations were monitored for 1 week.

Results—Compared with baseline values, PMB caused a significant dose- and time- dependent decrease in endotoxin-induced TNF activity. Compared with baseline values, residual endotoxin activity was significantly reduced after administration of 10,000 U of PMB/kg. Compared with baseline values, 1,000 and 5,000 U of PMB/kg should inhibit 75% of endotoxin-induced TNF activity for 3 and 12 hours, respectively. Serum creatinine concentrations remained within the reference range.

Conclusion and Clinical Relevance—Results of the study suggest that PMB is a safe, effective inhibitor of endotoxin-induced inflammation in healthy horses. ( Am J Vet Res 2001; 62:72–76)

Full access
in American Journal of Veterinary Research

Abstract

Case Description—Primary hypoaldosteronism without concurrent hypoadrenocorticism was diagnosed in an 8-year-old female alpaca with acute onset of weakness progressing to recumbency within 6 hours after onset.

Clinical Findings—Hematologic testing at admission revealed profound hyponatremia, hypochloremia, and acidemia with a normal blood potassium concentration. Further diagnostic testing, including an ACTH stimulation test, led to a diagnosis of hypoaldosteronism in conjunction with normal cortisol production.

Treatment and Outcome—The hembra responded well to IV polyionic fluid therapy with sodium supplementation and was managed successfully long term with free access to saline (0.9% NaCl) solution in addition to water ad libitum.

Clinical Relevance—To our knowledge, this is the first reported case of hypoaldosteronism in an alpaca. Hypoaldosteronism should be considered in alpacas as a possible differential diagnosis for refractory hyponatremia or for hyponatremia in which an underlying etiology is not determined.

Restricted access
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine concentrations of 2 acute-phase proteins (serum amyloid A [SAA] and lipopolysaccharide-binding protein [LBP]) in serum samples obtained from horses with colic and identify relationships among these acute-phase proteins and clinical data.

Animals—765 horses with naturally developing gastrointestinal tract diseases characterized by colic (ie, clinical signs indicative of abdominal pain) and 79 healthy control horses; all horses were examined at 2 university teaching hospitals.

Procedure—Serum concentrations of SAA and LBP were determined by immunoturbidometric and dotblot assays, respectively.

Results—SAA and LBP concentrations were determined for 718 and 765 horses with colic, respectively. Concentrations of SAA were significantly higher in nonsurvivors than in survivors, and horses with enteritis or colitis and conditions characterized by chronic inflammation (eg, abdominal abscesses, peritonitis, or rectal tears) had SAA concentrations significantly greater than those for horses with other conditions. Serum concentrations of LBP did not correlate with outcome, disease process, or portion of the gastrointestinal tract affected.

Conclusions and Clinical Relevance—Circulating concentrations of SAA were significantly higher at admission in horses with colic attributable to conditions having a primary inflammatory cause (eg, enteritis, colitis, peritonitis, or abdominal abscesses) and were higher in horses that failed to survive the episode of colic, compared with concentrations in horses that survived. Serum concentrations of LBP did not correlate with survival. Analysis of these findings suggests that evaluation of SAA concentrations may be of use in identifying horses with colic attributable to diseases that have inflammation as a primary component of pathogenesis. (Am J Vet Res 2005;66:1509–1516)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine which antimicrobials that are used to treat neonatal foals with septicemia attributable to Escherichia coli will minimize endotoxin release from bacteria and subsequent activity of inflammatory mediators while maintaining bactericidal efficacy.

Sample Population—Blood samples from 10 healthy foals.

ProcedureEscherichia coli isolates A and B were isolated from 2 septicemic foals, and minimal inhibitory concentrations (MIC) were determined for 9 antimicrobials. Five of these antimicrobials were tested in vitro at 2 and 20 times their respective MIC. Whole blood or mononuclear cells grown in tissue- culture media were incubated with 105 colonyforming units of E coli and each antimicrobial or saline (0.9% NaCl) solution. After 6 hours, number of viable bacteria remaining was determined, and supernatant was tested for endotoxin and tumor necrosis activity.

Results—Testing in whole blood was compromised by bactericidal effects of the blood itself. In mononuclear cell suspensions, each antimicrobial significantly reduced the number of viable bacteria to low or undetectable amounts. Antimicrobials did not differ significantly in efficacy of bacterial killing. Amikacin used alone or in combination with ampicillin resulted in significantly less endotoxin activity than did ampicillin, imipenem, or ceftiofur alone. There was a correlation between TNF-α and endotoxin activity.

Conclusions and Clinical Relevance—Aminoglycosides appear less likely to induce endotoxemia and TNF-α synthesis during bactericidal treatment of E coli septicemia, compared with β-lactam antimicrobials. Use of ampicillin, imipenem, or ceftiofur in the treatment of septicemic neonatal foals should be accompanied by appropriate treatment for endotoxemia. (Am J Vet Res 2002;63:660–668)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To compare daily endogenous cortisol production rate and the pharmacokinetics of an IV bolus of hydrocortisone between neonatal foals and adult horses.

Animals—10 healthy full-term 2- to 4-day-old foals and 7 healthy adult horses.

Procedures—Blood samples were collected from each horse every 15 to 20 minutes for 24 hours for determination of 24-hour mean cortisol concentration. Afterward, dexamethasone (0.08 mg/kg) was administered IV to suppress endogenous cortisol production. Twelve hours afterward, hydrocortisone sodium succinate (1.0 mg/kg) was administered as a rapid IV bolus and serial blood samples were collected to determine hydrocortisone pharmacokinetics. Cortisol concentrations, daily cortisol production rate, and hydrocortisone pharmacokinetics were determined, and results were compared between adult horses and foals.

Results—The mean ± SD 24-hour cortisol concentration was significantly lower in foals (20 ± 4 ng/mL) than in horses (26 ± 6 ng/mL), but the daily cortisol production rate was significantly greater in foals (6,710 ± 320 ng/kg/d) than in horses (2,140 ± 400 ng/kg/d). For hydrocortisone, foals had a significantly greater volume of distribution at steady state (1.92 ± 1.11 L/kg) and total body clearance (1.39 ± 0.108 L/kg/h) and significantly lower peak plasma concentration (1,051 ± 343 ng/mL) than did horses (0.58 ± 0.15 L/kg, 0.349 ± 0.065 L/kg/h, and 8,934 ± 3,843 ng/mL, respectively).

Conclusions and Clinical Relevance—Important differences were detected in cortisol production and metabolism between neonatal foals and adult horses consistent with lower plasma protein binding of cortisol in foals. This decrease may contribute to cortisol insufficiency during prolonged critical illness in neonatal foals.

Full access
in American Journal of Veterinary Research