OBJECTIVE To analytically validate a gas concentration of chromatography–mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at −80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at −80°C for 1 week had no effect on GC-MS results.

OBJECTIVE To assess the urinary protein-to-creatinine ratio (UPCR) of healthy sexually intact male dogs and to compare the UPCR of these dogs before and after castration. ANIMALS 19 client- or shelter-owned healthy adult sexually intact male dogs. PROCEDURES Physical, hematologic, and biochemical examinations and urinalysis (including calculation of the UPCR) were performed on each dog. Dogs were then castrated, and physical examination and urinalysis (including calculation of the UPCR) were performed again at least 15 days after castration. RESULTS A dipstick test yielded positive results for protein in the urine of 10 sexually intact male dogs, but the UPCR was < 0.5 for all sexually intact male dogs. Mean UPCR for sexually intact male dogs was 0.12 (range, 0.10 to 0.32). The UPCR was < 0.2 for all castrated dogs, except for 1. Mean UPCR for all castrated dogs was 0.08 (range, 0.05 to 0.69). There was a significant difference between mean UPCR before and after castration. CONCLUSIONS AND CLINICAL RELEVANCE In this study, pathological proteinuria was not detected in sexually intact male dogs. Positive results for a urine dipstick test should be interpreted with caution in sexually intact male dogs and should be confirmed by assessment of the UPCR. An increased UPCR in sexually intact male dogs may be considered abnormal.

OBJECTIVE To determine whether thioredoxin (TRX)-1 can be used as a valid biomarker for oxidative stress in dogs. ANIMALS AND SAMPLES 10 Beagles and Madin-Darby canine kidney cells. PROCEDURES Madin-Darby canine kidney cells were used to verify antigen cross-reactivity between human and canine anti–TRX-antibodies. Dogs were assigned to receive 21% or 100% O2 (5 dogs/group) via an artificial respirator during a 3-hour period of isoflurane anesthesia (starting at 0 hours). Blood and urine samples were collected before (baseline) and at 6, 12, 24, and 48 hours after commencement of inhalation anesthesia. Concentrations of TRX-1 and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in plasma and urine samples were analyzed; urine concentrations were reported as ratios against urine creatinine concentration. RESULTS Canine TRX-1 was recognized by monoclonal human anti-TRX-1 antibodies (clones of adult T-cell leukemia-derived factor [ADF]-11 and ADF21) by western blot analysis. Results of an ELISA indicated that plasma TRX-1 concentration and urine TRX-1-to-creatinine concentration ratio increased rapidly after the 3-hour period of hyperoxia with maximal peaks at 12 and 6 hours, respectively. Urine 8-OHdG-to-creatinine concentration ratio also increased significantly after hyperoxia induction. However, unlike the rapid increase in urine TRX-1-to-creatinine concentration ratio, maximal urine 8-OHdG-to-creatinine concentration ratio was attained at 48 hours after hyperoxia induction. These variables remained unchanged from baseline in the control group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that human anti-TRX monoclonal antibodies cross-reacted with canine TRX, and plasma TRX-1 concentrations were rapidly increased in dogs following an oxidative stress challenge. Thus, TRX may be a valuable clinical biomarker for detecting oxidative stress more rapidly than 8-OHdG in dogs.

Brucella melitensis is the Brucella species most frequently associated with brucellosis in humans. It is also the causative agent of the disease in goats and other ruminants. Although significant aspects of the pathogenesis of infection by this intracellular pathogen have been clarified, several events during invasion of host cells remain to be elucidated. In this study, infections of human macrophages from the THP-1 monocyte cell line were conducted with B. melitensis Bm133 wild-type strain and a strain of Salmonella serovar Enteritidis as a control. A multiplicity of infection of 100 was used in trials focused on defining the relative expression of syntaxin 4 (STX4), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, in the early events of phagocytosis (at 15, 30, 45, and 60 min). Immunoblot assays were also done to visualize expression of the protein in cells infected with either bacterial strain. The expression of STX4 was not significantly different in cells infected with B. melitensis strain Bm133 compared to that observed in cells infected with S. Enteritidis. When the expression of STX4 mRNA was inhibited with short or small interfering, or silencing, RNA in the THP-1 cells, the survival of B. melitensis was significantly reduced at time 0, when gentamicin treatment of cultures was begun (after 1 h of phagocytosis), and also at 2 h and 12 h after infection.

OBJECTIVE To compare gait mechanics and limb loading in Icelandic horses tölting and trotting at equal speeds and estimate their impact on orthopedic health. ANIMALS 12 orthopedically normal Icelandic horses. PROCEDURES Kinetic and kinematic gait variables were simultaneously recorded as each horse was ridden at a tölt and trot on an instrumented treadmill at 3.4 m/s and 3.9 m/s. Differences between gaits were tested via 1-factor repeated-measures ANOVA. RESULTS Horses had a higher stride rate and lower stride impulses at a tölt than at a trot. For forelimbs at a tölt, shorter relative stance duration resulted in higher peak vertical force (Fz(peak)). Conversely, for hind limbs, longer relative stance duration resulted in lower Fz(peak). The higher head-neck position at a tölt versus trot caused no weight shift to the hind limbs, but a higher forehoof flight arc and lower proretraction movement were identified. Stance durations for forelimbs were briefer than for hind limbs at a tölt, and the inverse was observed at a trot. Minimal height of the horse's trunk at the point of Fz(peak) of the respective limb suggested a spring-like mechanism for all limbs at a tölt. Hind limb measurements revealed no evidence of increased collection. Stride-to-stride limb timing varied more at a tölt than at a trot. At a trot, horses had brief or no suspension phases and a slightly 4-beated footfall rhythm was common. Post hoc energetic estimations revealed that tölting at the measured speeds was less advantageous than trotting. CONCLUSIONS AND CLINICAL RELEVANCE High forelimb action in Icelandic horses and higher head-neck position at a tölt were associated with more restricted limb proretraction, higher Fzpeak, and faster force onset than at a trot. The impact of these differences on orthopedic health needs to be investigated more in detail.

OBJECTIVE To determine whether high doses of enalapril and benazepril would be more effective than standard doses of these drugs in suppressing the furosemide-activated renin-angiotensin-aldosterone system (RAAS). ANIMALS 6 healthy Beagles. PROCEDURES 2 experiments were conducted; each lasted 10 days, separated by a 2-week washout period. In experiment 1, all dogs received furosemide (2 mg/kg, PO, q 12 h) and enalapril (1 mg/kg, PO, q 12 h) for 8 days (days 0 through 7). In experiment 2, dogs received furosemide (2 mg/kg, PO, q 12 h) and benazepril (1 mg/kg, PO, q 12 h) for 8 days. Effects on the RAAS were determined by assessing serum angiotensin-converting enzyme (ACE) activity on days −1, 3, and 7; serum aldosterone concentration on days −2, −1, 1, 3, and 7; and the urinary aldosterone-creatinine ratio (UAldo:C) in urine collected in the morning and evening of days −2, −1, 1, 3, and 7. RESULTS High doses of enalapril and benazepril caused significant reductions in serum ACE activity on all days but were not more effective than standard doses used in other studies. Mean UAldo:C remained significantly higher on days 2 through 7, compared with baseline values. Serum aldosterone concentration also increased after drug administration, which mirrored changes in the UAldo:C. CONCLUSIONS AND CLINICAL RELEVANCE In this study, administration of high doses of enalapril and benazepril significantly inhibited ACE activity, yet did not prevent increases in mean urine and serum aldosterone concentrations resulting from furosemide activation of RAAS. This suggested that aldosterone breakthrough from ACE inhibition was a dose-independent effect of ACE inhibitors.

OBJECTIVE To determine the scan delay for use in performing cardiac CT angiography in dogs. ANIMALS 4 clinically normal adult Beagles. PROCEDURES In a crossover study, 12 formulations of iohexol solutions differing in iodine dose (300, 400, and 800 mg/kg) and concentration (undiluted and diluted 1:1, 1:2, and 1:3 with saline [0.9% NaCl] solution) were administered IV to each dog. Dynamic CT angiography was performed to evaluate enhancement characteristics of each formulation, with the region of interest set over the aorta. Time-attenuation curves (TACs) were obtained and analyzed. RESULTS Peak arc–type TACs were obtained after administration of all undiluted formulations. Curve shape changed from peak arc type to plateau type as the total volume of the contrast solution (ie, dilution) increased. Prolonged peaks characteristic of plateau-type TACs suggested that a sufficient period of homogeneous attenuation could be achieved for CT scanning with administration of higher iohexol dilutions (1:2 or 1:3) containing higher iodine doses (400 or 800 mg/kg). In particular, attenuation values for plateau-type TACs remained between 200 and 300 Hounsfield units for > 16 seconds after the plateau endpoint was reached for 1:2 and 1:3 dilutions containing an iodine dose of 800 mg/kg. Scan delays of 13 to 17 seconds were computed for those 2 formulations. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that for clinically normal dogs, a scan delay of 13 to 17 seconds could be used to perform cardiac CT angiography with iohexol solutions containing an iodine dose of 800 mg/kg at dilutions of 1:2 or 1:3.

OBJECTIVE To compare ultrasound biomicroscopy (UBM) with standard ocular ultrasonography for detection of canine uveal cysts and to determine the sensitivity, specificity, and interobserver agreement for detection of uveal cysts with UBM. SAMPLE 202 enucleated eyes from 101 dogs. PROCEDURES 2 examiners examined 202 eyes by means of UBM (50 MHz) to identify uveal cysts. A board-certified radiologist then examined 98 of the 202 eyes by means of standard ocular ultrasonography (7- to 12-MHz linear transducer). Subsequently, 1 examiner dissected all 202 eyes under magnification from an operating microscope to definitively identify uveal cysts. Each examiner was masked to other examiners’ findings. Sensitivity, specificity, and interobserver agreement were calculated for detection of cysts by UBM. RESULTS Cysts were detected by use of UBM in 55 of 202 (27%) eyes by one examiner and 29 of 202 (14%) eyes by the other. No cysts were detected in the 98 eyes examined with standard ocular ultrasonography. Dissection results revealed that cysts were present in 64 of 202 (32%) eyes, including 29 of 98 (30%) eyes examined by standard ocular ultrasonography. Mean sensitivity of UBM for cyst detection was 47%; mean specificity was 92%. Uveal cysts not identified with UBM were often small (mean diameter, 490 üm). Interobserver agreement was high (κP = 0.81). CONCLUSIONS AND CLINICAL RELEVANCE UBM was more effective than standard ocular ultrasonography for detection of uveal cysts in enucleated eyes. Small-diameter cysts were difficult to visualize even with UBM.

OBJECTIVE To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. SAMPLE Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PROCEDURES PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. RESULTS The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. CONCLUSIONS AND CLINICAL RELEVANCE The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

OBJECTIVE To compare the attenuation of the angiotensin I–induced blood pressure response by once-daily oral administration of various doses of angiotensin receptor blockers (irbesartan, telmisartan, and losartan), benazepril hydrochloride, or lactose monohydrate (placebo) for 8 days in clinically normal cats. ANIMALS 6 healthy cats (approx 17 months old) with surgically implanted arterial telemetric blood pressure–measuring catheters. PROCEDURES Cats were administered orally the placebo or each of the drug treatments (benazepril [2.5 mg/cat], irbesartan [6 and 10 mg/kg], telmisartan [0.5, 1, and 3 mg/kg], and losartan [2.5 mg/kg]) once daily for 8 days in a crossover study. Approximately 90 minutes after capsule administration on day 8, each cat was anesthetized and arterial blood pressure measurements were recorded before and after IV administration of each of 4 boluses of angiotensin I (20, 100, 500, and 1,000 ng/kg). This protocol was repeated 24 hours after benazepril treatment and telmisartan (3 mg/kg) treatment. Differences in the angiotensin I–induced change in systolic arterial blood pressure (ΔSBP) among treatments were determined. RESULTS At 90 minutes after capsule administration, only losartan did not significantly reduce ΔSBP in response to the 3 higher angiotensin doses, compared with placebo. Among drug treatments, telmisartan (3 mg/kg dosage) attenuated ΔSBP to a significantly greater degree than benazepril and all other treatments. At 24 hours, telmisartan was more effective than benazepril (mean ± SEM ΔSBP, 15.7 ± 1.9 mm Hg vs 55.9 ± 12.42 mm Hg, respectively). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that telmisartan administration may have advantages over benazepril administration for cats with renal or cardiovascular disease.