Objective—To evaluate the use of a commercially available 5-carboxyfluorescein–based, intramolecularly quenched, fluorescence resonance energy transfer (FRET) peptide substrate of renin for measurement of plasma renin concentration in cats.
Sample Population—Plasma samples obtained during a previous study of renal autograft ischemia-reperfusion injury in 10 cats and samples of fetal bovine serum containing recombinant human renin (rh-renin).
Procedures—Experiments involving samples of fetal bovine serum containing rh-renin were conducted to identify a suitable control vehicle, optimal substrate concentration, and appropriate duration of incubation. With the use of the identified assay conditions, a standard curve was constructed to allow conversion of relative fluorescent units into values of renin concentration (ng/mL). Subsequently, plasma samples obtained from cats before and after renal autograft ischemia-reperfusion injury were assayed to determine endogenous renin concentration.
Results—Under conditions of a 1:50 substrate dilution and 4-hour incubation period, the assay detected small amounts of rh-renin in fetal bovine serum. A linear relationship (R2 = 0.996) between the relative fluorescent units generated and exogenous rh-renin concentration was evident. The assay detected renin in plasma samples obtained from cats after renal autograft ischemia-reperfusion, and renin concentrations on days 1 and 2 after transplant differed significantly.
Conclusions and Clinical Relevance—The study data indicated that the assay involving the FRET peptide substrate of renin is potentially a rapid and specific method for measurement of plasma renin concentration in cats.
Objective—To evaluate the performance of a chemiluminescent endotoxin activity assay in horses with colic and healthy horses.
Animals—20 horses with colic and systemic inflammatory response syndrome (SIRS group), 8 horses with colic with no SIRS (NSIRS group), and 20 healthy horses.
Procedures—Venous blood was collected into EDTA blood collection tubes after completion of a physical examination, and a chemiluminescent endotoxin activity assay was performed within 60 minutes of collection. Medical or surgical interventions and outcome were recorded for each horse.
Results—Mean ± SE endotoxin activity was 0.16 ± 0.05 for healthy horses, 0.18 ± 0.07 for the NSIRS group, and 0.53 ± 0.05 for the SIRS group and was significantly different among the groups. Mean endotoxin activity was significantly higher in the SIRS group than in the NSIRS group and the healthy group. No significant difference between the healthy and NSIRS groups was present. The higher the measured endotoxin activity, the more likely it was for horses to be euthanized.
Conclusions and Clinical Relevance—The chemiluminescent endotoxin assay was easy to use, required a short time to perform, could be completed at the patient's side, and with some modifications, may be a useful component in the clinical assessment and prognostication of horses with colic.
Objective—To assess the acetaminophen absorption test (APAT) for use in determining function of the reticular groove reflex in lambs.
Animals—12 Baluchi lambs.
Procedures—2 consecutive APATs were performed at each of 3 developmental stages (stage 1, before weaning; stage 2, at weaning; and stage 3, after weaning). Lambs suckled a test solution consisting of acetaminophen and barium sulfate and 1 week later were tube fed the same test solution. Abdominal radiographs were obtained immediately after administration of the test solution. Plasma acetaminophen concentrations were determined before and 30, 60, 90, 120, 150, and 180 minutes after intake of the test solution.
Results—Closure of the reticular groove after suckling the test solution was confirmed in all 12 lambs at stage 1, in 8 lambs at stage 2, and in 0 lambs at stage 3. Maximum plasma acetaminophen concentrations and area under the plasma acetaminophen concentration-time curves from 0 to 180 minutes were significantly higher in lambs suckling the test solution, compared with values for tube-fed lambs. Receiver operating characteristics analysis revealed that the plasma acetaminophen concentration at 60 minutes after administration was best suited to determine closure of the reticular groove in lambs.
Conclusions and Clinical Relevance—Results suggested that the APAT can be a useful diagnostic instrument to assess function of the reticular groove reflex in lambs. We propose a cutoff value for the plasma acetaminophen concentration of 25 μg/mL at 60 minutes after administration to determine function of the reticular groove mechanism in lambs.
Objective—To determine whether epidurally derived evoked potentials (EPs) can be used to reliably assess nociception and antinociception in ponies.
Procedures—EPs and electromyograms (EMGs) from the quadriceps femoris muscles were recorded simultaneously, following electrical stimulation applied to the distal portion of the hind limb. The effect of increasing stimulus intensity, conduction velocities of the stimulated nerves, effect of epidurally applied methadone, and effect of systemically administered propofol were evaluated.
Results—In the EP and EMG waveforms, 2 distinct complexes, the EP N25 and P50 and the EMG P27 and N62, respectively, were identified. On the basis of their latency and calculated conduction velocities, the EP P50 and EMG N62 were considered to be related to nociception (AD-mediated). All complexes increased significantly in amplitude with increasing stimulus intensity and decreased significantly following epidural administration of methadone or systemic administration of propofol.
Conclusions and Clinical Relevance—Although the experimental setup allowed successful discrimination between tactile- and nociceptive-associated responses, the identified EPs, considered to reflect activity in the spinal cord, could not be definitively differentiated from activity in the lumbosacral epaxial musculature. Further research is required to refine measurement techniques to allow for discrimination between these 2 signals. Similar to other species, neurophysiologic variables such as EPs could potentially become a useful additional tool in quantifying nociception in equidae.
Objective—To develop and evaluate an immunoassay based on time-resolved immunofluorometry (TR-IFM) for measurement of haptoglobin concentrations in samples of various body fluids of swine.
Animals—20 pigs without clinical signs of disease and seronegative for antibodies against major viruses that affect pigs and 30 pigs with clinical signs of disease.
Procedures—Haptoglobin concentrations were measured in samples of serum, saliva, and meat juice obtained from both groups of pigs to evaluate the ability of TR-IFM to differentiate between healthy and diseased pigs. Performance of TR-IFM was evaluated by means of its calibration curve and detection limit, analytic precision during routine operation, and linearity of results for serial dilutions for the 3 types of samples. In addition, performance of TR-IFM was compared with that of a commercial spectrophotometric assay.
Results—The TR-IFM assay involved only 1 step, and the results were obtained in 20 minutes, with good analytic sensitivity and reproducibility. The analytic limit of detection was 0.52 ng/mL. Intra-assay and interassay coefficients of variation ranged from 1.13% to 4.81% and 5.97% to 13.57%, respectively. The method yielded linear results for all sample types. Serum haptoglobin concentrations determined by use of TR-IFM and spectrophotometric assays were highly correlated (r = 0.96). Differences between healthy and diseased pigs with respect to median haptoglobin concentrations were significant for all types of samples.
Conclusions and Clinical Relevance—The 1-step TR-IFM assay accurately quantified haptoglobin concentrations in serum, saliva, and meat juice samples from swine and may be useful in laboratory and meat inspection settings.
Objective—To determine the feasibility of performing serial laminar and skin biopsies on sedated horses and whether sampling affected adjacent tissues.
Procedures—Laminar tissues were harvested via biopsy through the hoof wall from healthy conscious horses via sedation and regional anesthesia. Eight specimens were collected at 4 time points during 24 hours from a single foot. Laminar biopsy specimens were harvested with a 6-mm-diameter biopsy punch after burring through the horny corium to the stratum medium. Skin biopsy specimens were collected from an area proximal to the coronary band. All tissues were examined via light microscopy. Total RNA was extracted and quantified, and gene expression analysis was completed for 2 housekeeping genes and the inflammatory mediator cyclooxygenase-2.
Results—Laminar and skin biopsies yielded adequate specimens for histologic and gene expression evaluation. There was no extension of inflammation or detectable damage to adjacent tissues during the 24-hour period in either laminar or skin specimens as judged via histologic findings and cyclooxygenase-2 expression. Lameness and discomfort induced by the procedure were minimal.
Conclusions and Clinical Relevance—Laminar biopsy provided a satisfactory method of collecting laminar specimens and allowed serial sampling of individual horses.
Objective—To compare the McMaster and centrifugal flotation techniques and larval culture for recovery of cyathostomin (small strongyle) eggs from the feces of horses.
Sample Population—Fecal samples from 101 horses.
Procedures—In experiment I, homogenized fresh feces from a single horse were randomly subsampled by use of each technique for 10 replicates. In experiment II, samples from 43 horses that had no anthelmintic treatment were analyzed by use of McMaster, centrifugal flotation, and larval culture techniques. In experiment III, 57 horses were treated with an anthelmintic by owners, and fecal samples were analyzed as for experiment II.
Results—In experiment I, use of the McMaster technique recovered 72% of the eggs obtained by use of centrifugal flotation from paired subsamples. In experiment II, use of the McMaster technique recovered 81% of the eggs obtained by use of centrifugal flotation. Only cyathostomins resulted from individual larval cultures. In experiment III, 24 samples had negative results for all 3 tests, 18 samples had positive results only with larval cultures, and 15 samples had positive results of centrifugal flotation (only 5 of which had positive results via the McMaster technique).
Conclusions and Clinical Relevance—Centrifugal flotation consistently was superior to the McMaster technique, especially at low fecal egg numbers. The combination of centrifugal flotation and larval culture may provide the best accuracy for evaluation of anthelmintic efficacy.
Objective—To detect changes in serum lipoprotein and apolipoprotein profiles via precipitation and electrophoresis in ketotic cows and in those cows treated with different methods.
Animals—21 cows with clinical and subclinical ketosis, 7 healthy cows in the early lactation period, and 7 healthy cows in the nonlactation period.
Procedures—Ketotic cows were allocated into 3 groups; the first group was treated with dextrose and dexamethasone, the second group with dextrose and prednisolone, and the third group with dextrose and insulin. The β and α lipoproteins were precipitated with dextran sulfate-magnesium chloride in ketotic cows after treatment and healthy cows in the nonlactation and lactation periods. The serum samples, precipitates, and supernatants were examined via agarose gel electrophoresis for detection of alterations in serum lipoproteins. Subsequently, alterations in serum apolipoproteins were detected via SDS-PAGE of precipitates.
Results—Compared with serum β and α lipoprotein concentrations in healthy cows during nonlactation, those in cows during lactation were higher; however, those in cows with ketosis were lower. The SDS-PAGE analysis of serum β lipoproteins revealed that apolipoprotein E (approx 36 and 40 kDa) decreased in ketotic cows, in comparison with healthy cows in the nonlactation and lactation periods, but increased after treatment. Decreases in apolipo-protein B (approx 222 kDa), apolipoprotein A-I (19 and 24 kDa), apolipoprotein A-IV (55 kDa), apolipoprotein C-III (8.8 and 10.2 kDa), and albumin (66 kDa) concentrations were detected in ketotic cows, in comparison with the healthy cows in the lactation period.
Conclusions and Clinical Relevance—Serum lipoprotein and apolipoproteins may routinely be determined via precipitation and electrophoresis in the diagnosis and treatment of ketosis.
Objective—To determine by use of an accelerometer the sampling interval that has the least variable total activity counts from one week to the next in companion (ie, nonlaboratory) dogs.
Procedures—Dogs wore an accelerometer continuously for 2 weeks. Between-dog and within-dog day-to-day variability in total activity counts were evaluated. The changes in counts between week 1 and week 2 were compared for weekdays, weekends, and full weeks.
Results—Significant between-dog variability in total activity counts was detected. Within dogs, there was significant day-to-day variability, with highest counts recorded on weekends. In comparison of data from the first week with data from the second week, the greatest differences were in weekend counts (median difference, 21%; range, 0% to 154%) and the smallest differences were in full 7-day counts (median difference, 10%; range, 0% to 74%). Comparison of weekday counts revealed a median change of 12% (range, 0% to 104%).
Conclusions and Clinical Relevance—Significant between-dog variability in total daily activity counts was detected. Within dogs, a full 7-day comparison of total activity counts from one week to the next provided the least variable estimate of the dogs' activity. For dogs in their home environment, the activity monitor may be most useful in following changes in activity over time. For dogs that have no change in routine according to the owner's report, the least variable estimates of activity can be collected by comparing activity in 7-day intervals.
Objective—To develop and analytically validate a gas chromatography–mass spectrometry (GC-MS) method for the quantification of lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose in canine serum.
Sample Population—Pooled serum samples from 200 dogs.
Procedures—Serum samples spiked with various sugars were analyzed by use of GC-MS. The method was analytically validated by determination of dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability.
Results—Standard curves ranging from 0.5 to 500 mg/L for each sugar revealed a mean r2 of 0.997. The lower detection limit was 0.03 mg/L for lactulose, rhamnose, xylose, and methylglucose and 0.12 mg/L for sucrose. The observed-to-expected ratios for dilutional parallelism had a mean ± SD of 105.6 ± 25.4% at dilutions of 1:2, 1:4, and 1:8. Analytic recoveries for the GC-MS assays of sugars ranged from 92.1% to 124.7% (mean ± SD, 106.2 ± 13.0%). Intra-assay coefficients of variation ranged from 6.8% to 12.9% for lactulose, 7.1% to 12.8% for rhamnose, 7.2% to 11.2% for xylose, 8.9% to 11.5% for methylglucose, and 8.9% to 12.0% for sucrose. Interassay coefficients of variation ranged from 7.0% to 11.5% for lactulose, 6.4% to 9.4% for rhamnose, 6.8% to 13.2% for xylose, 7.0% to 15.9% for methylglucose, and 5.5% to 9.4% for sucrose.
Conclusions and Clinical Relevance—The GC-MS method described here was accurate, precise, and reproducible for the simultaneous measurement of sugar probes in canine serum.