Marco A. F. Lopes

Thrombelastography in 26 healthy horses with and without activation by recombinant human tissue factor

OBJECTIVES To develop a standardized technique for thrombelastography (TEG) analysis in healthy adult horses, with and without the ex vivo addition of tissue factor (TF) as an activator. To determine reference intervals for TEG parameters in the horse, and to determine if traditional coagulation tests correlate with TEG. DESIGN Prospective, observational. SETTING Veterinary teaching hospital. ANIMALS Twenty-six healthy adult horses. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Thrombelastography with (TF-TEG) and without (TEG) the addition of TF performed by 4 operators. Coagulation profiles (prothrombin time, activated partial thromboplastin time, platelet count, fibrinogen, antithrombin, and fibrinogen degradation products) were assessed in a subset of horses. Mean values (SD) for TEG parameters in healthy horses were: reaction time (R)=17.0 minutes (3.0 min), K time (K)=5.8 minutes (2.3 min), clotting rate (Ang)=42 degrees (14 degrees ), maximum clot strength (maximum amplitude [MA])=60.3 mm (5.7 mm), CL30=97.0% (2.0%), LY30=0.8% (0.6%), CL60=92% (5.9%), LY60=3.2% (2.5%). Mean values (SD) for TF-TEG parameters were: R-TF=6.6 minutes (1.4 min), K-TF=3.1 minutes (1.0 min), Ang-TF=50.9 degrees (9 degrees ), MA-TF=62.3 mm (5.1 mm), CL30-TF=97.8% (1.6%), LY30-TF=0.6% (0.5%), CL60-TF=90.8% (4.2%), and LY60-TF=3.6% (1.9%). The addition of TF decreased R and K and increased Ang. TF-TEG had a narrower SD for R, K, Ang, CL60 and LY60 compared with TEG. Interoperator differences were reduced by the addition of TF. Regression analysis indicated a positive relationship between MA and fibrinogen concentrations (P=0.02) and R-TF time and prothrombin time (P=0.03). CONCLUSION TF-TEG using the described protocol may minimize variability in data obtained across institutions or users. However, due to the variability associated with different operators, it is recommended that each laboratory set up individual reference intervals with the personnel who will perform the assay, and that the assay protocols and data obtained are compared on a regular basis.

Thrombelastography in Horses with Acute Gastrointestinal Disease

BACKGROUND Coagulopathies in horses with gastrointestinal disease are frequently identified and associated with morbidity and fatality. OBJECTIVE Determine if thrombelastography (TEG) identifies abnormalities associated with lesion type, presence of systemic inflammatory response syndrome (SIRS), morbidity, and fatality more consistently than traditional coagulation testing. ANIMALS One-hundred and one horses examined for gastrointestinal disease and 20 healthy horses. METHODS TEG, tissue factor (TF)-TEG, and traditional coagulation panels parameters and percentages of horses with coagulopathies were compared for lesion type, presence of SIRS, complications, and survival. RESULTS Changes in individual parameters and increased incidence of coagulopathies were associated with fatality (R, P= .007; k-value [K], P= .004; clot lysis [CL]30, P= .037; CL60, P= .050; angle [Ang], P= .0003; maximum amplitude [MA], P= .006; lysis [Ly]30, P= .042; Ly60, P= .027; CI, P= .0004; ≥ 2 TEG coagulopathies, P= .013; ≥ 3 TEG coagulopathies, P= .038; TF-R, P= .037; TF-K, P= .004; TF-CL30, P < .0001; TF-CL60, P < .0001; TF-Ang, P= .005; TF-Ly30, P= .0002; TF-Ly60, P < .0001; TF-CI, P= .043; ≥ 1 TF-TEG coagulopathies, P= .003; ≥ 2 TF-TEG coagulopathies, P= .0004; prothrombin tme [PT], P < .0001; activated partial throboplastin time [aPTT], P= .021), inflammatory lesions (MA, P= .013; TF-CL30, P= .033; TF-CL60, P= .010; TF-Ly60, P= .011; ≥ 1 TF-TEG coagulopathy, P= .036; ≥ 2 TF-TEG coagulopathy, P= .0007; PT, P= .0005; fibrinogen, P= .019), SIRS (MA, P= .004; TF-CL30, P= .019; TF-CL60, P= .013; TF-Ly30, P= .020; TF-Ly60, P= .010; PT, P < .0001; aPTT, P= .032; disseminated intravascular coagulation, P= .005), and complications (ileus: aPTT, P= .020; diarrhea: TF-CL30, P= .040; TF-Ly30, P= .041; thrombophlebitis: ≥ 1 TF-TEG coagulopathy, P= .018; laminitis: MA, P= .004; CL60, P= .045; CI, P= .036; TF-MA, P= .019; TF-TEG CI, P= .019). Abnormalities in TEG and TF-TEG parameters were indicative of hypocoagulation and hypofibrinolysis. CONCLUSIONS AND CLINICAL IMPORTANCE TEG identifies changes in coagulation and fibrinolysis associated with lesion type, SIRS, morbidity, and fatality in horses with gastrointestinal disease.

Expression of inflammation-associated genes in circulating leukocytes collected from horses with gastrointestinal tract disease.

OBJECTIVE To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome. ANIMALS 10 healthy horses and 50 horses with GIT disease. PROCEDURES A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. Results were grouped by GIT disease type and disease outcome for comparison. RESULTS Horses with GIT disease had colic of unknown etiology (n = 8 horses), GIT inflammation or strangulation (19), or nonstrangulating GIT obstruction (23). Among the 45 horses receiving treatment, 38 were discharged from the hospital, and 7 died or were euthanized. Compared with healthy horses, horses with colic of unknown etiology had similar gene expression. Significant differences in expression of the interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected between healthy horses and horses with GIT disease. Significant differences in expression of the interleukin-1 receptor antagonist, interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected among healthy horses and horses grouped by disease outcome. CONCLUSIONS AND CLINICAL RELEVANCE Inflammatory gene expression in leukocytes of horses with GIT disease appeared to be related to disease pathogenesis and prognosis.

Expression of genes associated with inflammation induced by ex vivo exposure to lipopolysaccharide in peripheral blood leukocytes from horses with gastrointestinal disease

OBJECTIVE To investigate the effect of ex vivo exposure to lipopolysaccharide (LPS) on the expression of inflammatory genes in leukocytes from horses with gastrointestinal (Gl) disease and determine whether the pattern or magnitude of the response to LPS correlated with the type of disease and outcome. ANIMALS 49 horses with Gl disease and 10 healthy horses PROCEDURES Leukocytes were isolated from blood samples and submitted to 3 protocols: immediate freezing, freezing after 4-hour incubation in medium, and freezing after 4-hour incubation in medium containing LPS. Expression of 14 genes associated with inflammation was assessed via PCR assay. Results were compared by disease type and outcome RESULTS Horses with Gl disease had colic of unknown etiology (n=8), Gl inflammation or strangulation (18), or nonstrangulating Gl obstruction (23). Among the 44 horses receiving treatment, 38 were discharged from the hospital and 6 died or were euthanized. Incubation of leukocytes in medium alone changed the expression of several genes. Incubation with LPS resulted in increased expression of interleukin-10 and monocyte chemotactic protein-3 in leukocytes from healthy and sick horses. Leukocytes from horses with nonstrangulating obstruction and horses that survived had less pronounced LPS-induced increases in interleukin-10 expression than did cells from healthy horses. The opposite was evident for monocyte chemotactic protein-3. CONCLUSIONS AND CLINICAL RELEVANCE No evidence existed for a reduced response of leukocytes from horses with gastrointestinal disease to ex vivo exposure to LPS. Leukocyte expression of inflammatory genes after ex vivo incubation with LPS appeared to be related to pathogenesis and prognosis.

An attempt to detect lameness in galloping horses by use of body-mounted inertial sensors

OBJECTIVE To evaluate head, pelvic, and limb movement to detect lameness in galloping horses. ANIMALS 12 Thoroughbreds. PROCEDURES Movement data were collected with inertial sensors mounted on the head, pelvis, and limbs of horses trotting and galloping in a straight line before and after induction of forelimb and hind limb lameness by use of sole pressure. Successful induction of lameness was determined by measurement of asymmetric vertical head and pelvic movement during trotting. Differences in gallop strides before and after induction of lameness were evaluated with paired-sample statistical analysis and neural network training and testing. Variables included maximum, minimum, range, and time indices of vertical head and pelvic acceleration, head rotation in the sagittal plane, pelvic rotation in the frontal plane, limb contact intervals, stride durations, and limb lead preference. Difference between median standardized gallop strides for each limb lead before and after induction of lameness was calculated as the sum of squared differences at each time index and assessed with a 2-way ANOVA. RESULTS Head and pelvic acceleration and rotation, limb timing, stride duration measurements, and limb lead preference during galloping were not significantly different before and after induction of lameness in the forelimb or hind limb. Differences between limb leads before induction of lameness were similar to or greater than differences within limb leads before and after lameness induction. CONCLUSIONS AND CLINICAL RELEVANCE Galloping horses maintained asymmetry of head, pelvic, and limb motion between limb leads that was unrelated to lameness.

Objective Detection and Quantification of Irregular Gait With a Portable Inertial Sensor-Based System in Horses During an Endurance Race—a Preliminary Assessment

Abstract Detection of irregular gait (IG) during endurance competitions is based on a brief subjective veterinary examination. Horse elimination due to IG is common and may elicit competitors’ complaints. We hypothesized that detection of IG by objectively assessing motion asymmetry can be performed during endurance competitions. The aim of this preliminary study was to start investigating the feasibility of detecting and quantifying IG with a portable inertial sensor‐based system (PISBS) during endurance races. Horses participating in two qualifying endurance rides were simultaneously and independently evaluated by Fédération Equestre Internationale veterinarians and the PISBS, and the results were compared after the rides. Asymmetric vertical displacement of the head and/or pelvis measured with the PISBS indicated severity of IG and identified the affected limb(s). Veterinarians and competitors answered questionnaires about the use of the PISBS. The PISBS detected IG in 21/22 horses (48/70 evaluations). Significant disagreement between the PISBS and veterinarians was detected. Disagreement between the PISBS and veterinarians was no longer detected after reducing the sensitivity of the PISBS by reclassifying horses with mild IG as sound. Competitors and veterinarians had favorable impressions about the use of the PISBS but recommended reducing instrumentation time and trotting distance for expediency. Inherent human limitations can explain the lower sensitivity of veterinary evaluation relative to PISBS evaluation. Simple methodological changes are likely to address the issues raised in this study by competitors and Fédération Equestre Internationale veterinarians. Additional studies are needed to complete the feasibility assessment of objective detection and quantification of IG during endurance competitions with a PISBS. HighlightsA portable inertial sensor‐based system (PISBS) can be used for objective gait evaluation in endurance races.There was significant disagreement between the PISBS and veterinarians.Inherent human limitations explain the lower sensitivity of veterinary evaluation.Riders and veterinarians had positive impression about the use of the PISBS.More studies are needed to fully test the PISBS for IG detection during endurance races.