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  • Author or Editor: Jan S. Suchodolski x
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Abstract

Objective—To develop and analytically validate a radioimmunoassay (RIA) for the quantification of canine calprotectin (cCP) in serum and fecal extracts of dogs.

Sample Population—Serum samples (n = 50) and fecal samples (30) were obtained from healthy dogs of various breeds and ages.

Procedures—A competitive, liquid-phase, double-antibody RIA was developed and analytically validated by assessing analytic sensitivity, working range, linearity, accuracy, precision, and reproducibility. Reference intervals for serum and fecal cCP concentrations were determined.

Results—Sensitivity and upper limit of the working range were 29 and 12,774 μg/L for serum and 2.9 and 1,277.4 μg/g for fecal extracts, respectively. Observed-to-expected ratios for serial dilutions of 6 serum samples and 6 fecal extracts ranged from 95.3% to 138.2% and from 80.9% to 118.1%, respectively. Observed-to-expected ratios for spiking recovery for 6 serum samples and 6 fecal extracts ranged from 84.6% to 121.5% and from 80.3% to 132.1%, respectively. Coefficients of variation for intra-assay and interassay variability were < 3.9% and < 8.7% for 6 serum samples and < 8.5% and < 12.6% for 6 fecal extracts, respectively. Reference intervals were 92 to 1,121 μg of cCP/L for serum and < 2.9 to 137.5 μg of cCP/g for fecal extracts.

Conclusions and Clinical Relevance—The RIA described here was analytically sensitive, linear, accurate, precise, and reproducible for the quantification of cCP in serum and fecal extracts. This assay should facilitate research into the clinical use of serum and fecal cCP measurements in dogs with inflammatory bowel disease.

Full access
in American Journal of Veterinary Research

Abstract

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 r 2 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.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To elucidate the relationship between plasma ammonia concentration and severity of hepatic encephalopathy and determine whether factors that precipitate hepatic encephalopathy in humans are associated with the presence of clinical signs of hepatic encephalopathy in dogs previously treated for the disease.

Design—Retrospective case series.

Animals—118 dogs with hepatic encephalopathy.

Procedures—The medical records database of a veterinary teaching hospital was searched for records of dogs in which hepatic encephalopathy was diagnosed between October 1, 1991, and September 1, 2014. Hepatic encephalopathy severity was graded on a 5-point scale, and the correlation between disease severity and plasma ammonia concentration was determined. Respective associations between hepatic encephalopathy and systemic inflammatory response syndrome, gastrointestinal hemorrhage, dietary indiscretion, constipation, furosemide treatment, azotemia, hypokalemia, hyponatremia, alkalosis, and hyperammonemia were assessed by Fisher exact tests followed by multivariable logistic regression.

Results—Severity of hepatic encephalopathy at hospital admission was not significantly correlated with plasma ammonia concentration. Dogs treated for hepatic encephalopathy prior to hospital admission were significantly less likely to have clinical signs of the disease at hospital admission, compared with dogs that were not treated for the disease (OR, 0.36; 95% confidence interval, 0.17 to 0.78). None of the putative precipitating factors for hepatic encephalopathy were significantly associated with the presence of clinical signs of the disease at hospital admission.

Conclusions and Clinical Relevance—Results indicated that hepatic encephalopathy treatment alleviated clinical signs of the disease. Further investigation is necessary to identify precipitating factors for hepatic encephalopathy in dogs. (J Am Vet Med Assoc 2015;247:176–183)

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To test the hypothesis that intestinal pathologic changes are often concurrent with gastric pathologic changes in dogs and to characterize the historical, physical, clinicopathologic, imaging, and endoscopic findings in dogs with gastric histopathologic abnormalities.

Design—Retrospective case series.

Animals—67 dogs with gastric histopathologic abnormalities.

Procedures—Medical records from dogs that had undergone gastrotomy, gastroduodenoscopy, or gastroscopy between September 2002 and September 2007 were identified. Dogs were included in the study when histopathologic abnormalities were detected during evaluation of gastric tissue sections. History, clinical examination findings, results of diagnostic tests, diagnoses, treatments, and outcome were recorded for each dog.

Results—67 dogs with gastric histopathologic abnormalities were included in the study. The most frequent clinical sign recorded was vomiting (36/67 [53.7%] dogs). The most common biochemical abnormality recorded was panhypoproteinemia (27/64 [42.2%] dogs). Lymphoplasmacytic gastritis was the most frequent histopathologic finding recorded (34/67 [50.7%] dogs). For dogs in which both intestinal biopsy specimens and gastric biopsy specimens were collected, concurrent pathologic changes were recorded in 43 of 60 (71.7%) dogs.

Conclusions and Clinical Relevance—Results of this study suggested that intestinal pathologic changes are commonly concurrent in dogs with gastric pathologic changes. This supports the practice of collecting both gastric and duodenal biopsy specimens every time gastroduodenoscopy is performed. Lymphoplasmacytic gastritis was the most commonly recorded gastric histopathologic finding and was often of minimal or mild severity.

Full access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine the prevalence of hypocobalaminemia in dogs with multicentric lymphoma and to investigate any relationship between serum cobalamin concentration and disease outcome.

Design—Cohort study.

Animals—58 dogs with multicentric lymphoma.

Procedures—Serum cobalamin concentrations were measured in 58 dogs with multicentric lymphoma. Clinical signs, stage, and immunophenotype for dogs with hypocobalaminemia were compared with those for dogs with serum cobalamin concentrations above the lower end of the reference range. Survival times for dogs undergoing a cyclic multidrug chemotherapy protocol (n = 53) were similarly compared. Serum cobalamin concentrations for treated dogs that died or were euthanized before day 60 were compared with those of dogs still alive at day 60.

Results—Serum cobalamin concentrations ranged from < 150 to 1,813 ng/L, with a median concentration of 401 ng/L. Nine of the 58 (16%) dogs had hypocobalaminemia (serum cobalamin concentration < 252 ng/L). Three of 9 dogs with hypocobalaminemia survived to at least day 60, compared with 40 of 44 (91%) dogs without hypocobalaminemia (serum cobalamin concentration ≥ 252 ng/L). Ten (10/53 [19%]) dogs undergoing a cyclic multidrug chemotherapy protocol died before day 60, and the median serum cobalamin concentration for these dogs (232 ng/L) was significantly lower than for those still alive at the end point of the study (556 ng/L).

Conclusions and Clinical Relevance—Hypocobalaminemia was relatively uncommon in this population of dogs with multicentric lymphoma, but was associated with a poor outcome. Serum cobalamin concentrations may provide prognostic information in dogs with multicentric lymphoma.

Full access
in Journal of the American Veterinary Medical Association

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.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation.

Sample Population—Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs.

Procedures—cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile.

Results—cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 μg/L. The reference range was established as < 2,239 μg/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability.

Conclusions and Clinical Relevance—The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To evaluate the effects of withholding food on the results for measurements of serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin-like immunoreactivity (cTLI) in healthy dogs.

ANIMALS

11 healthy employee- or student-owned dogs.

PROCEDURES

Food was withheld from the dogs for 12 hours, baseline blood samples were collected, then dogs were fed. Postprandial blood samples collected 1, 2, 4, and 8 hours later were assessed. A mixed-effects ANOVA model with fasting duration (time) as a fixed factor and dog as a random effect was fit for each analyte variable. Additionally, a mixed-effects ANOVA model controlling for the variable of time was fit to assess whether lipemia affected serum concentrations of the analytes.

RESULTS

The median serum cobalamin concentration was lower at 4 hours (428 ng/L) and 8 hours (429 ng/L) postprandially, compared with baseline (479 ng/L), but this difference was not clinically meaningful. Although there were no substantial differences in serum concentrations of folate, cPLI, or cTLI, postprandial changes in serum concentrations of cTLI or folate could potentially affect diagnoses in some dogs.

CONCLUSIONS AND CLINICAL RELEVANCE

Although results indicated that feedings rarely resulted in clinically important differences in the median serum concentrations of cobalamin, folate, cPLI, or cTLI in healthy dogs, given the further processing required for lipemic samples, withholding food for at least 8 hours is an appropriate recommendation when measuring these analytes. Similar research is needed in dogs with gastrointestinal disease to determine whether the withholding of food is necessary when measuring these analytes in affected dogs.

Full access
in American Journal of Veterinary Research

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)

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To develop and validate a sandwich ELISA for the measurement of α1-proteinase inhibitor (α1-PI) concentrations in serum and fecal samples obtained from common marmosets (Callithrix jacchus).

SAMPLE Leftover serum (n = 42) and fecal (23) samples submitted for diagnostic testing; paired serum and fecal samples obtained from 30 common marmosets at 2 research colonies.

PROCEDURES A sandwich ELISA was developed and analytically validated by determining the lower limit of detection, linearity, accuracy, precision, and reproducibility. Reference intervals for α1-PI concentrations in serum and feces of common marmosets were calculated.

RESULTS The standard curve was generated for concentrations between 1 and 100 ng/mL. Mean ± SD observed-to-expected ratio for serial dilutions of serum and fecal samples was 117.1 ± 5.6% (range, 112.2% to 123.0%) and 106.1 ± 19.7% (range, 82.6% to 130.2%), respectively. Mean observed-to-expected ratio for spiking recovery of serum and fecal samples was 102.9 ± 12.1% (range, 86.8% to 115.8%) and 97.9 ± 19.0% (range, 83.0% to 125.1%), respectively. Reference interval for serum concentrations of α1-PI was 1,254 to 1,813 μg/mL, for 3-day mean fecal concentrations was 11.5 to 42.2 μg/g of feces, and for 3-day maximum fecal concentrations was 13.2 to 51.2 μg/g of feces.

CONCLUSIONS AND CLINICAL RELEVANCE The ELISA was linear, accurate, precise, and reproducible for quantification of α1-PI concentrations in serum and feces of common marmosets. However, the ELISA had limited linearity and accuracy for spiking recovery of fecal samples.

Full access
in American Journal of Veterinary Research