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- Author or Editor: David A. Stein x
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Abstract
Objective—To develop a fecal sample collection strategy and quantification method for measurement of fecal IgA concentrations in dogs.
Sample Population—Fecal samples from 23 healthy pet dogs of various breeds.
Procedures——Immunoglobulin A was extracted from fecal samples. An ELISA for the measurement of fecal IgA concentrations was established and analytically validated. Intraindividual variation of fecal IgA was determined by calculation of coefficients of variation. A sample collection strategy was developed on the basis of results of intraindividual variation of fecal IgA concentrations. A reference range for fecal IgA concentrations was determined.
Results—The method for extraction and quantification of fecal IgA was determined to be sufficiently sensitive, reproducible, accurate, and precise. On the basis of the intraindividual variability of our results, the determined fecal sample collection strategy required analysis of a total of 4 fecal samples/dog, with each fecal sample collected on 2 consecutive days with 28 days between sample collection periods (ie, days 1 and 2 followed by days 28 and 29). Reference range values for fecal IgA concentration were 0.22 to 3.24 mg/g of feces.
Conclusions and Clinical Relevance— Methods of fecal IgA extraction and quantification used in our study allow for identification of dogs with consistently low fecal IgA concentrations. Use of these techniques will enable future investigations into possible associations between low fecal IgA concentrations and signs of gastrointestinal disease in dogs.
Abstract
Objective—To evaluate the qualitative variation in bacterial microflora among compartments of the intestinal tract of dogs by use of a molecular fingerprinting technique.
Animals—14 dogs (similarly housed and fed identical diets).
Procedure—Samples of intestinal contents were collected from the duodenum, jejunum, ileum, colon, and rectum of each dog. Bacterial DNA was extracted from the samples, and the variable V6 to V8 region of 16S ribosomal DNA (gene coding for 16S ribosomal RNA) was amplified by use of universal bacterial primers; polymerase chain reaction amplicons were separated via denaturing gradient gel electrophoresis (DGGE). Similarity indices of DGGE banding patterns were used to assess variation in the bacterial microflora among different compartments of the intestine within and among dogs. Bacterial diversity was assessed by calculating the Simpson diversity index, the Shannon-Weaver diversity index, and evenness.
Results—DGGE profiles indicated marked differences in bacterial composition of intestinal compartments among dogs (range of similarity, 25.6% to 36.6%) and considerable variation among compartments within individual dogs (range of similarity, 36.7% to 57.9%). Similarities between neighboring intestinal compartments were significantly greater than those between non-neighboring compartments. Diversity indices for the colon and rectum were significantly higher than those of the duodenum, jejunum, and ileum.
Conclusions and Clinical Relevance—Results indicated that the different intestinal compartments of individual dogs appear to host different bacterial populations, and these compartmental populations vary among dogs. In dogs, fecal sample analysis may not yield accurate information regarding the composition of bacterial populations in compartments of the gastrointestinal tract. (Am J Vet Res 2005;66:1556–1562)
Abstract
Objective—To compare effects of the locking-loop suture pattern (LLP) and 3-loop pulley (3LP) suture pattern for tenorrhaphy on the intrinsic vasculature of the superficial digital flexor tendon (SDFT) of horses in vitro after surgery.
Sample Population—16 forelimbs obtained from 8 mature horses.
Procedure—Tenotomy and subsequent tenorrhaphy was performed in anesthetized horses. Following systemic administration of heparin, horses were euthanatized and the limbs were removed and placed under tension to load the flexor tendons. The intrinsic vasculature was then perfused with a mixture of barium sulfate and water. Four-millimeter sections of the SDFT were prepared for microangiographic analysis. Mean vessel density was calculated for each section by use of a grid consisting of 1.5-mm2 vascular assessment squares (VAS). Comparisons were made among the control, LLP, and 3LP groups.
Results—Mean ± SD vessel density was 3.11 ± 0.38, 1.47 ± 0.47, and 2.01 ± 0.63 perfused vessels/1.5 mm2 for control, LLP, and 3LP groups, respectively. Significant differences in vascular density were detected between the control and 3LP groups, control and LLP groups, and LLP and 3LP groups.
Conclusions and Clinical Relevance—Use of the LLP and 3LP pattern has deleterious effects in vitro on the intrinsic vasculature of the SDFT. However, the 3LP pattern was less disruptive to the intrinsic vasculature, compared with the effects for the LLP. Use of the 3LP tenorrhaphy suture pattern in clinical situations may result in less damage to the intrinsic vasculature of the SDFT of horses during convalescence. ( Am J Vet Res 2004;65:279–282)
Abstract
Objective—To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species.
Sample Population—Stomachs of 6 cats.
Procedure—A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition.
Results—Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10- fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum.
Conclusions and Clinical Relevance—PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats. (Am J Vet Res 2004;65:1195–1199)
Abstract
Objective—To test the hypothesis that exchange of medium-chain triglycerides (MCTs) for long-chain triglycerides (LCTs) in the diet of dogs with well-managed exocrine pancreatic insufficiency (EPI) changes serum biochemical variables and to subjectively assess the well-being of dogs with EPI in response to experimental diets.
Animals—21 dogs with EPI and 6 healthy control dogs.
Procedure—The effects of 3 diets containing 0%, 16%, or 35% of the total fat content as MCTs were examined in a randomized controlled double-blind crossover trial. The 3 diets were fed for 12 weeks each. Dietary effects were evaluated by both subjective and objective variables.
Results—Analysis of subjective data revealed no significant difference in appetite, attitude, drinking behavior, volume of feces, defecation frequency, color of feces, consistency of feces, flatulence, or borborygmus among dogs fed the 3 experimental diets. A high MCT content in the diet was associated with significantly higher serum vitamin E, cholesterol, triglyceride, retinyl stearate, retinyl palmitate, and total vitamin A concentrations in dogs with EPI and significantly higher serum vitamin E concentrations in control dogs, compared with low MCT content. High MCT content in the diet was also associated with significantly lower concentrations of serum linoleic acid (C18:2[n-6]) in dogs with EPI and in control dogs, compared with low MCT content.
Conclusions and Clinical Relevance—A high MCT content in the diet leads to increases in serum concentrations of cholesterol and certain fat-soluble vitamins. However, no effect was found on the subjective well-being of the dogs as evaluated by their owners. (Am J Vet Res 2004;65:1293–1302)
Abstract
Objective—To determine changes in serum feline trypsin-like immunoreactivity (fTLI) in response to administration of ceruletide to healthy cats.
Animals—11 healthy cats.
Procedures—Serum fTLI was determined, using a radioimmunoassay, before and 10, 20, 30, 40, and 50 minutes after IM administration of ceruletide (0.3 mg/kg [0.14 mg/lb]).
Results—Mean ± SD baseline serum fTLI was 23.1 ± 4.1 mg/L. There was a statistically significant, but clinically unimportant, increase in serum fTLI 10 and 30 minutes after ceruletide administration.
Conclusions and Clinical Relevance—In healthy cats, administration of ceruletide induced a statistically significant, but clinically unimportant, increase in serum fTLI. Whether responses in cats with exocrine pancreatic disorders would be different is unknown, but results suggest that a ceruletide stimulation test would likely not be useful for differentiating between healthy cats and cats with subclinical chronic exocrine pancreatic disorders. (Am J Vet Res 2000;61:925–927)
Abstract
Objective—To develop and validate an ELISA for measurement of serum canine pepsinogen A (cPG A) as a diagnostic marker of gastric disorders in dogs and to measure serum cPG A in healthy dogs after food deprivation and after feeding.
Sample Population—Sera from 72 healthy dogs.
Procedure—A sandwich ELISA was developed and validated. The reference range for serum concentrations of cPG A was determined in 64 healthy dogs. Postprandial changes in serum concentrations of cPG A were evaluated in 8 healthy dogs.
Results—Assay sensitivity was 18 µg/L, and the maximum detectable concentration was 1,080 µg/L. The observed-to-expected ratio (O:E) for 3 serial dilutions of 3 serum samples ranged from 69.3 to 104.1%. The O:E for 3 serum samples spiked with 8 concentrations of cPG A ranged from 58.8 to 120.4%. Coefficients of variation for intra- and interassay variability of 3 serum samples ranged from 7.6 to 11.9% and from 10.1 to 13.1%, respectively. Mean ± SD serum concentration of cPG A in healthy dogs was 63.8 ± 31.0 µg/L and the reference range was 18 to 129 µg/L. Significant increases in serum concentrations of cPG A were observed between 1 and 7 hours after feeding.
Conclusions and Clinical Relevance—The ELISA for measuring cPG A was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. Serum concentrations of cPG A increase substantially after feeding, which should be taken into account when conducting clinical studies. (Am J Vet Res 2003;64:1146–1150)
Abstract
Objective—To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa.
Sample Population—Stomachs obtained from 6 euthanatized dogs.
Procedure—Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, sizeexclusion chromatography, and strong anionexchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing.
Results—Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IEP. Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0. The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species.
Conclusions and Clinical Relevance—Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs. (Am J Vet Res 2002;63:1585–1590)
Abstract
Objective—To determine an optimal dose of carbon 13 (13C)-labeled aminopyrine for use in a 13C-aminopyrine demethylation blood test in healthy dogs.
Animals—9 adult dogs.
Procedures—Food was withheld from each dog for 12 hours. A 2-mL baseline blood sample was obtained from each dog and placed into an evacuated tube containing sodium heparin. Carbon 13-labeled aminopyrine was administered IV at doses of 1, 2, 5, or 10 mg/kg. Additional blood samples (2 mL) were obtained and placed into evacuated tubes containing sodium heparin 30, 45, 60, and 75 minutes after 13C-aminopyrine administration. Hydrochloric acid was used to extract CO2 from blood samples. The extracted gas was analyzed by fractional mass spectrometry to determine the percentage dose of 13C administered as 13C-aminopyrine and recovered in extracted gas (PCD).
Results—Gross evidence of clinical adverse effects was not detected in any dog after administration of 13C-aminopyrine. The mean coefficient of variation (CV) for PCD was significantly lower than the mean CV for the summation of PCD values up to a given sampling time (CUMPCD). Mean PCD values among the 4 doses for each sample time were not significantly different. Administration of 13C-aminopyrine at a dose of 2 mg/kg resulted in the lowest interindividual variability.
Conclusions and Clinical Relevance—The PCD is superior to CUMPCD for the quantification of aminopyrine demethylation. Administration of 13C-13C-aminopyrine at a dose of 2 mg/kg is appropriate for use in the 13C-aminopyrine demethylation blood test in healthy dogs.
Abstract
Objective—To indirectly assess the pancreatic response in healthy dogs that were fed diets of different fat compositions with or without supplemental pancreatic enzymes and medium-chain triglycerides (MCTs).
Animals—10 healthy adult dogs.
Procedures—Dogs were fed 4 diets once in random order at 1-week intervals; food was withheld from the dogs for ≥ 12 hours prior to the feeding of each diet. Diets A and B contained 16% and 5% crude fat, respectively; diet C was composed of diet A with pancreatic enzymes; and diet D was composed of diet B with pancreatic enzymes and MCTs. Serum canine trypsin–like immunoreactivity (cTLI) and canine pancreatic lipase immunoreactivity (cPLI) concentrations were measured before (0 hours) and at 1 to 2 and 6 hours after feeding. Serum gastrin concentration was measured at 0 hours and at 5 to 10 minutes and 1 to 2 hours after feeding. A gastrin assay validation study was performed to confirm accuracy of test results in dogs. Data were analyzed by use of a repeated-measures general ANOVA.
Results—Serum cTLI, cPLI, or gastrin concentrations in the dogs did not differ among the different diets fed, among dogs, or over time. When multiple comparisons were analyzed, diet D caused the least amount of measurable pancreatic response, although this difference was not significant.
Conclusions and Clinical Relevance—Results did not indicate a significant effect of dietary fat content or addition of supplemental MCT oil or pancreatic enzymes in diets on serum cTLI, cPLI, or gastrin concentrations in healthy dogs.
