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Abstract

Objective—To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes.

Animals—30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old).

Procedure—Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion.

Results—Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to- CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age.

Conclusions and Clinical Relevance—In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency. ( Am J Vet Res 2001;62:1413–1417)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine an infusion rate of butorphanol tartrate in horses that would maintain therapeutic plasma drug concentrations while minimizing development of adverse behavioral and gastrointestinal tract effects.

Animals—10 healthy adult horses.

Procedure—Plasma butorphanol concentrations were determined by use of high-performance liquid chromatography following administration of butorphanol by single IV injection (0.1 to 0.13 mg/kg of body weight) or continuous IV infusion (loading dose, 17.8 µg/kg; infusion dosage, 23.7 µg/kg/h for 24 hours). Pharmacokinetic variables were calculated, and changes in physical examination data, gastrointestinal tract transit time, and behavior were determined over time.

Results—A single IV injection of butorphanol was associated with adverse behavioral and gastrointestinal tract effects including ataxia, decreased borborygmi, and decreased defecation. Elimination half-life of butorphanol was brief (44.37 minutes). Adverse gastrointestinal tract effects were less apparent during continuous 24-hour infusion of butorphanol at a dosage that resulted in a mean plasma concentration of 29 ng/ml, compared with effects after a single IV injection. No adverse behavioral effects were observed during or after continuous infusion.

Conclusions and Clinical Relevance—Continuous IV infusion of butorphanol for 24 hours maintained plasma butorphanol concentrations within a range associated with analgesia. Adverse behavioral and gastrointestinal tract effects were minimized during infusion, compared with a single injection of butorphanol. Continuous infusion of butorphanol may be a useful treatment to induce analgesia in horses. (Am J Vet Res 2001;62:183–189)

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in American Journal of Veterinary Research

Abstract

Objective

To determine blood protein concentration, immunoglobulin concentration, and lymphocyte profiles in equine infectious anemia virus (EIAV) seropositive, naturally infected horses without clinical signs of disease.

Animals

26 clinically normal seropositive horses, 6 febrile ponies with experimentally induced EIA, and 52 clinically normal seronegative horses and ponies.

Procedure

Serum and EDTA-anticoagulated blood were obtained from all horses and ponies, and total serum protein and albumin concentrations, immunoglobulin concentrations, and blood lymphocyte subset counts were determined.

Results

Compared with seronegative horses, EIAV seropositive inapparent carrier horses had no significant difference in serum reverse transcriptase activity, PCV, or platelet count. Inapparent carrier horses had increased plasma total solids and serum globulin concentrations and decreased serum albumin concentration and albumin-to-globulin ratio. Total serum immunoglobulin and serum IgM concentrations were increased. In-apparent carrier horses had significantly decreased percentages of CD5+ and CD4+ blood lymphocytes.

Conclusions

Serum protein and lymphocyte subset changes in EIAV-infected inapparent carrier horses are consistent with immune activation or chronic inflammation, both of which may, in part, be the result of virus-induced polyclonal B-cell activation.

Clinical Relevance

EIAV seropositive horses have immune-related abnormalities consistent with ongoing viral activity regardless of the duration they have been infected, even when the usual signs of disease (anemia, fever, weight loss) are not apparent. (Am J Vet Res 1998;59:1009–1015)

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in American Journal of Veterinary Research

SUMMARY

Objective

To evaluate the ability of nucleic acid amplification techniques to detect Rhodococcus equi in equine buffy coat, blood, and tracheal wash fluid and to differentiate between virulent and avirulent strains of the bacteria.

Sample Population

Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses.

Procedure

Logarithmic dilutions of virulent and avirulent strains of R equi were added to equine buffy coat and tracheal wash fluid samples. The DNA was extracted and amplified by polymerase chain reaction (PCR), using primers specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi.

Results

PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasmid primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacteria. Virulent strains of R equi could be identified by PCR and differentiated from avirulent strains within 12 to 24 hours after sample collection, with as few as 10 to 100 organisms present.

Conclusions

PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and can differentiate between virulent and avirulent strains of the organism.

Clinical Relevance

Because PCR can confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination. (Am J Vet Res 1997;58:1232–1237)

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in American Journal of Veterinary Research

Abstract

Objective

To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry.

Animals

16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, ElAV-positive horses and ponies; and 2 thrombocytopenic, ElAV-negative horses.

Procedure

Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms.

Results

Healthy ponies (n = 9) had 1.28 to 2.83% (mean ± SD, 2.03 ± 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 ± 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41 % (26.51 ± 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 ± 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 ± 20,500 and 39,300 ± 13,500 platelets/μl, respectively (reference range, 94,000 to 232,000 platelets/μl).

Conclusion

Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses.

Clinical Relevance

Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses. (Am J Vet Res 1997;58:1092–1096)

Free access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To determine the plasma pharmacokinetics and safety of 1% diclofenac sodium cream applied topically to neonatal foals every 12 hours for 7 days.

ANIMALS Twelve 2- to 14-day old healthy Arabian and Arabian-pony cross neonatal foals.

PROCEDURES A 1.27-cm strip of cream containing 7.3 mg of diclofenac sodium (n = 6 foals) or an equivalent amount of placebo cream (6 foals) was applied topically to a 5-cm square of shaved skin over the anterolateral aspect of the left tarsometatarsal region every 12 hours for 7 days. Physical examination, CBC, serum biochemistry, urinalysis, gastric endoscopy, and ultrasonographic examination of the kidneys and right dorsal colon were performed before and after cream application. Venous blood samples were collected at predefined intervals following application of the diclofenac cream, and plasma diclofenac concentrations were determined by liquid chromatography–mass spectrometry.

RESULTS No foal developed any adverse effects attributed to diclofenac application, and no significant differences in values of evaluated variables were identified between treatment groups. Plasma diclofenac concentrations peaked rapidly following application of the diclofenac cream, reaching a maximum of < 1 ng/mL within 2 hours, and declined rapidly after application ceased.

CONCLUSIONS AND CLINICAL RELEVANCE Topical application of the 1% diclofenac sodium cream to foals as described appeared safe, and low plasma concentrations of diclofenac suggested minimal systemic absorption. Practitioners may consider use of this medication to treat focal areas of pain and inflammation in neonatal foals.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Sample Population—Blood samples obtained from 4 Thoroughbreds.

Sample Population—Blood samples obtained from 4 Thoroughbreds.

Procedure—Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein- conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode.

Results—Number of platelets (mean ± SEM) in samples containing LMWH (109.5 ± 11.3 × 103 cells/µl) was significantly less than the number in samples containing sodium citrate (187.3 ± 30.3 × 103 cells/µl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding.

Conclusion and Clinical Relevance—A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation. (Am J Vet Res 2001; 62:547–554)

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in American Journal of Veterinary Research

Abstract

Objectives—To assess safety and determine effects of IV administration of formaldehyde on hemostatic variables in healthy horses.

Animals—7 healthy adult horses.

Procedure—Clinical signs and results of CBC, serum biochemical analyses, and coagulation testing including template bleeding time (TBT) and activated clotting time (ACT) were compared in horses given a dose of 0.37% formaldehyde or lactated Ringer’s solution (LRS), IV, in a 2-way crossover design. In a subsequent experiment, horses received an infusion of 0.74% formaldehyde or LRS. In another experiment, horses were treated with aspirin to impair platelet responses prior to infusion of formaldehyde or LRS.

Results—Significant differences were not detected in any variable measured between horses when given formaldehyde or any other treatment. Infusion of higher doses of formaldehyde resulted in adverse effects including muscle fasciculations, tachycardia, tachypnea, serous ocular and nasal discharge, agitation, and restlessness.

Conclusions and Clinical Relevance—Intravenous infusion of formaldehyde at doses that do not induce adverse reactions did not have a detectable effect on measured hemostatic variables in healthy horses. (Am J Vet Res 2000;61:1191–1196)

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in American Journal of Veterinary Research

Abstract

Objective—To investigate the potential use of fluorescent- labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry.

Sample Population—Platelets obtained from 6 Thoroughbreds.

Procedure—Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion.

Results—Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817- activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation.

Conclusions and Clinical Relevance—Activation of equine platelets can be detected by use of fluorescent- labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders. (Am J Vet Res 2002;63:513–519)

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in American Journal of Veterinary Research