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  • Author or Editor: Craig G. Ruaux x
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Abstract

Objective—To validate an automated chemiluminescent immunoassay for measuring serum cobalamin concentration in cats, to establish and validate gas chromatography-mass spectrometry techniques for use in quantification of methylmalonic acid, homocysteine, cysteine, cystathionine, and methionine in sera from cats, and to investigate serum concentrations of methylmalonic acid, methionine, homocysteine, cystathionine, and cysteine as indicators of biochemical abnormalities accompanying severe cobalamin (vitamin B12) deficiency in cats.

Sample Population—Serum samples of 40 cats with severe cobalamin deficiency (serum cobalamin concentration < 100 ng/L) and 24 control cats with serum cobalamin concentration within the reference range.

Procedure—Serum concentrations of cobalamin were measured, using a commercial automated chemiluminescent immunoassay. Serum concentrations of methylmalonic acid, methionine, homocysteine, cystathionine, and cysteine were measured, using gas chromatography-mass spectrometry, selected ion monitoring, stable-isotope dilution assays.

Results—Cats with cobalamin deficiency had significant increases in mean serum concentrations of methylmalonic acid (9,607 nmol/L), compared with healthy cats (448 nmol/L). Affected cats also had substantial disturbances in amino acid metabolism, compared with healthy cats, with significantly increased serum concentrations of methionine (133.8 vs 101.1 µmol/L) and significantly decreased serum concentrations of cystathionine (449.6 vs 573.2 nmol/L) and cysteine (142.3 vs 163.9 µmol/L). There was not a significant difference in serum concentrations of homocysteine between the 2 groups.

Conclusions and Clinical Relevance—Cats with gastrointestinal tract disease may have abnormalities in amino acid metabolism consistent with cobalamin deficiency. Parenteral administration of cobalamin may be necessary to correct these biochemical abnormalities. (Am J Vet Res 2001;62:1852–1858)

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in American Journal of Veterinary Research

Abstract

Objective—To characterize historical, clinicopathologic, ultrasonographic, microbiological, surgical, and histopathologic features of bacterial cholecystitis and bactibilia in dogs and evaluate response to treatment and outcomes in these patients.

Design—Retrospective case-control study.

Animals—40 client-owned dogs (10 with bacterial cholecystitis on histologic analysis or bactibilia on cytologic examination [case dogs] and 30 without bactibilia [controls]) evaluated at a veterinary teaching hospital between 2010 and 2014.

Procedures—Signalment, history, clinicopathologic findings, ultrasonographic features, microbiological results, surgical findings, histopathologic changes, treatments, and outcomes of case dogs were derived from medical records and summarized. Demographic and clinicopathologic data and ultrasonographic findings were compared between case and control dogs. Relationships among prior antimicrobial treatment, sediment formation in the gallbladder, presence of immobile biliary sludge, and presence of bactibilia or bacterial cholecystitis were assessed.

Results—No finding was pathognomonic for bactibilia or bacterial cholecystitis in dogs. Case dogs were significantly more likely to have immobile biliary sludge and had a greater degree of biliary sediment formation than did control dogs. All case dogs for which gallbladders were examined histologically (6/6) had bacterial cholecystitis. Five of 10 case dogs were Dachshunds. Medical or surgical treatment resulted in good outcomes.

Conclusions and Clinical Relevance—Bactibilia and bacterial cholecystitis were important differential diagnoses in dogs with signs referable to biliary tract disease. Dachshunds were overrepresented, which may suggest a breed predisposition. Cytologic evaluation of bile should be considered in the routine assessment of dogs with hepatobiliary disease if immobile biliary sludge is present. (J Am Vet Med Assoc 2015;246:982–989)

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To develop and validate a gas chromatography–mass spectrometry (GC-MS) method for determination of Nτ-methylhistamine (NMH) concentration in canine urine and fecal extracts and to assess urinary NMH concentrations in dogs with mast cell neoplasia and fecal NMH concentrations in dogs with protein-losing enteropathy.

Sample Population—Urine specimens were collected from 6 healthy dogs and 7 dogs with mast cell neoplasia. Fecal extracts were obtained from fecal specimens of 28 dogs with various severities of protein-losing enteropathy, as indicated by fecal concentration of α1-proteinase inhibitor.

Procedures—NMH was extracted directly from urine, and fecal specimens were first extracted into 5 volumes of PBSS containing 1% newborn calf serum. Nτ-methylhistamine in specimens was quantified via stable isotope dilution GC-MS. The assay was validated via determination of percentage recovery of known amounts of NMH and interassay coefficients of variation. Urinary excretion of NMH was evaluated by means of NMH-to-creatinine concentration ratios.

Results—Recovery of NMH in urine and fecal extracts averaged 104.6% and 104.5%, respectively. Interassay coefficients of variation ranged from 5.4% to 11.7% in urine and 12.6% to 18.1% in fecal extracts. Urinary NMH excretion was significantly increased in dogs with mast cell neoplasia, compared with that in healthy dogs. No correlation was detected between severity of protein-losing enteropathy and fecal NMH concentration.

Conclusions and Clinical Relevance—This method provided a sensitive, reproducible means of measuring NMH in canine urine and fecal extracts. High urinary NMH-to-creatinine concentration ratios in dogs with mast cell neoplasia are consistent with increased histamine release in this disease.

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in American Journal of Veterinary Research
History

A 4-year-old 7.3-kg (16-lb) castrated male Pug was evaluated because of a sudden onset of rapidly progressive neurologic abnormalities. Four days prior to referral evaluation, the owner perceived the dog to have right forelimb lameness. The following day, a neurologic examination by the primary care veterinarian revealed ambulatory tetraparesis with decreased conscious proprioception in all limbs. Clinical signs were refractory to empirical fluid therapy administered SC and an NSAID administered orally.

Clinical and Gross Findings

At the referral evaluation, the dog had markedly diminished mentation. Physical examination revealed nonambulatory tetraparesis, vertical nystagmus of the right eye, rotary nystagmus of

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in Journal of the American Veterinary Medical Association

Abstract

OBJECTIVE To assess clinical effects of an omega-3 fatty acid and protein-enriched diet, physical rehabilitation, or both in dogs following tibial plateau leveling osteotomy (TPLO) and arthroscopic surgery for cranial cruciate ligament (CCL) disease.

DESIGN Randomized, prospective clinical trial.

ANIMALS 48 dogs with unilateral CCL disease.

PROCEDURES Dogs were randomly assigned to receive a dry omega-3 fatty acid and protein-enriched dog food formulated to support joint health (test food [TF]), a dry food formulated for maintenance of adult dogs (control food [CF]), TF plus rehabilitation (TF-R), or CF plus rehabilitation (CF-R). Data collected over 6 months included body weight, body condition score, ground reaction force data, tibial plateau angle, limb circumference measurements, subjective pain and lameness scores assigned by surgeons and dog owners, and daily activity measured by accelerometry.

RESULTS Peak vertical force and vertical impulse were greater after surgery for dogs in the TF groups than in the CF groups; peak vertical force was greater after surgery in dogs that underwent rehabilitation than in those that did not. Owner scores indicated lower frequencies of lameness and signs of pain during some activities for the TF group, compared with other groups, and for the TF-R and CF-R groups, compared with the CF group. Sedentary time decreased and time spent in light-to-moderate or vigorous activity increased in all groups over time. Rehabilitation was significantly associated with greater time spent in light-to-moderate activity, regardless of diet.

CONCLUSIONS AND CLINICAL RELEVANCE Feeding the TF and providing physical rehabilitation during the first 6 months after TPLO were associated with improvements in some indices of clinical outcome and function in dogs. Significant interactions between time and some outcome variables were observed, indicating further research is warranted.

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To investigate postprandial changes in serum concentrations of unconjugated bile acids in healthy Beagles.

Animals—7 healthy Beagles.

Procedure—Blood samples were obtained from dogs at regular intervals up to 8 hours after consumption of a meal. Serum concentrations of 5 unconjugated bile acids were determined at each time point, using gas chromatography-mass spectrometry with selected ion monitoring.

Results—Total serum unconjugated bile acid concentration was significantly increased, relative to baseline values, at 360, 420, and 480 minutes after feeding. Unconjugated cholic acid was significantly increased at 360, 420, and 480 minutes. The proportion of total unconjugated bile acids represented by cholic acid was significantly increased at 240 to 480 minutes. Deoxycholic acid was significantly increased at 360 and 420 minutes. Chenodeoxycholic acid was significantly increased at 360 to 480 minutes. Lithocholic acid was significantly increased at 180 minutes, whereas no significant changes in ursodeoxycholic acid were detected at any time point.

Conclusion and Clinical Relevance—Healthy Beagles had significant increases in serum concentrations and changes in the profile of unconjugated bile acids after a meal. These increases persisted > 8 hours, indicating that prolonged withholding of food is necessary when to avoid the risk of a false-positive diagnosis when assessing serum unconjugated bile acid concentrations in dogs. (Am J Vet Res 2002;63:789–793

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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)

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in American Journal of Veterinary Research

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)

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in American Journal of Veterinary Research

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)

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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