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
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)
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)
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.
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
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.
Objective—To investigate postprandial changes in
serum concentrations of unconjugated bile acids in
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
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
Objective—To purify and partially characterize various
isoforms of canine pepsinogen (PG) from gastric
Sample Population—Stomachs obtained from 6
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
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)
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)
Objective—To evaluate the qualitative variation in
bacterial microflora among compartments of the
intestinal tract of dogs by use of a molecular fingerprinting
Animals—14 dogs (similarly housed and fed identical
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
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)
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.