Effect of selected gastrointestinal parasites and viral agents on fecal S100A12 concentrations in puppies as a potential comparative model.

BACKGROUND: Previous data suggest that fecal S100A12 has clinical utility as a biomarker of chronic gastrointestinal inflammation (idiopathic inflammatory bowel disease) in both people and dogs, but the effect of gastrointestinal pathogens on fecal S100A12 concentrations is largely unknown. The role of S100A12 in parasite and viral infections is also difficult to study in traditional animal models due to the lack of S100A12 expression in rodents. Thus, the aim of this study was to evaluate fecal S100A12 concentrations in a cohort of puppies with intestinal parasites (Cystoisospora spp., Toxocara canis, Giardia sp.) and viral agents that are frequently encountered and known to cause gastrointestinal signs in dogs (coronavirus, parvovirus) as a comparative model. METHODS: Spot fecal samples were collected from 307 puppies [median age (range): 7 (4-13) weeks; 29 different breeds] in French breeding kennels, and fecal scores (semiquantitative system; scores 1-13) were assigned. Fecal samples were tested for Cystoisospora spp. (C. canis and C. ohioensis), Toxocara canis, Giardia sp., as well as canine coronavirus (CCV) and parvovirus (CPV). S100A12 concentrations were measured in all fecal samples using an in-house radioimmunoassay. Statistical analyses were performed using non-parametric 2-group or multiple-group comparisons, non-parametric correlation analysis, association testing between nominal variables, and construction of a multivariate mixed model. RESULTS: Fecal S100A12 concentrations ranged from < 24-14,363 ng/g. Univariate analysis only showed increased fecal S100A12 concentrations in dogs shedding Cystoisospora spp. (P = 0.0384) and in dogs infected with parvovirus (P = 0.0277), whereas dogs infected with coronavirus had decreased fecal S100A12 concentrations (P = 0.0345). However, shedding of any single enteropathogen did not affect fecal S100A12 concentrations in multivariate analysis (all P > 0.05) in this study. Only fecal score and breed size had an effect on fecal S100A12 concentrations in multivariate analysis (P < 0.0001). CONCLUSIONS: An infection with any single enteropathogen tested in this study is unlikely to alter fecal S100A12 concentrations, and these preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation.

Association of fecal calprotectin concentrations with disease severity, response to treatment, and other biomarkers in dogs with chronic inflammatory enteropathies.

BACKGROUND: Calprotectin is a marker of inflammation, but its clinical utility in dogs with chronic inflammatory enteropathies (CIE) is unknown. OBJECTIVE: Evaluation of fecal calprotectin in dogs with biopsy-confirmed CIE. ANIMALS: 127 dogs. METHODS: Prospective case-control study. Dogs were assigned a canine chronic enteropathy clinical activity index (CCECAI) score, and histologic lesions severity was assessed. Fecal calprotectin, fecal S100A12, and serum C-reactive protein (CRP) were measured. Food- or antibiotic-responsive cases (FRE/ARE, n = 13) were distinguished from steroid-/immunosuppressant-responsive or -refractory cases (SRE/IRE, n = 20). Clinical response to treatment in SRE/IRE dogs was classified as complete remission (CR), partial response (PR), or no response (NR). RESULTS: Fecal calprotectin correlated with CCECAI (ρ = 0.27, P = .0065) and fecal S100A12 (ρ = 0.90, P < .0001), some inflammatory criteria, and cumulative inflammation scores, but not serum CRP (ρ = 0.16, P = .12). Dogs with SRE/IRE had higher fecal calprotectin concentrations (median: 2.0 µg/g) than FRE/ARE dogs (median: 1.4 µg/g), and within the SRE/IRE group, dogs with PR/NR had higher fecal calprotectin (median: 37.0 µg/g) than dogs with CR (median: 1.6 µg/g). However, both differences did not reach statistical significance (both P = .10). A fecal calprotectin ≥15.2 µg/g separated both groups with 80% sensitivity (95% confidence interval [95%CI]: 28%-100%) and 75% specificity (95%CI: 43%-95%). CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal calprotectin could be a useful surrogate marker of disease severity in dogs with CIE, but larger longitudinal studies are needed to evaluate its utility in predicting the response to treatment.

Effect of gastric acid-suppressive therapy and biological variation of serum gastrin concentrations in dogs with chronic enteropathies.

BACKGROUND: Serum gastrin concentration can help diagnose gastrinomas in dogs if >3-10× the upper reference limit (URL), but antisecretory therapy and other conditions can also cause hypergastrinemia. Effects of antisecretory therapy (famotidine or ranitidine, omeprazole) on serum gastrin concentration in dogs with chronic enteropathy (CE) and its biological variation (BV) are unknown. Aim of the study was to evaluate serum gastrin in acid-suppressant-treated or -naïve CE dogs; test the association between serum gastrin and histopathologic findings in acid-suppressant-naïve CE dogs; and evaluate the BV of serum gastrin in dogs not receiving any gastric acid suppressive therapy. Samples from 231 dogs were used and serum gastrin was measured by chemiluminescence assay. Gastric and duodenal histologic lesions were evaluated and graded. BV of serum gastrin was evaluated in serial samples. RESULTS: Serum gastrin concentrations were significantly higher in acid-suppressant-treated than acid-suppressant-naïve dogs (P = 0.0245), with significantly higher concentrations in proton pump inhibitor (PPI)- than H2-antihistamine-treated patients (P = 0.0053). More PPI- than H2-antihistamine-treated dogs had gastrin concentrations above URL (P = 0.0205), but not >3× nor >10× the URL. Serum gastrin concentrations correlated with the severity of gastric antral epithelial injury (P = 0.0069) but not with any other lesions or the presence/numbers of spiral bacteria in gastric biopsies. Intra- and inter-individual BV were 43.4 and 21.6%, respectively, in acid-suppressant-naïve dogs, with a reciprocal individuality index of 0.49 and a critical difference of ≥29.5 ng/L. CONCLUSIONS: Antisecretory (particularly PPI) treatment leads to hypergastrinemia in CE dogs, but the concentrations seen in this study are unlikely to compromise a diagnosis of gastrinoma. Use of a population-based URL for canine serum gastrin and a URL of ≤27.8 ng/L are appropriate.

Serum alpha1 -proteinase inhibitor concentrations in dogs with systemic inflammatory response syndrome or sepsis.

OBJECTIVE: To determine whether the concentration of serum canine alpha1 -proteinase inhibitor (cα1 -PI) has diagnostic or prognostic utility in dogs with sepsis or noninfectious systemic inflammatory response syndrome (SIRS). DESIGN: Prospective, observational study from May to December 2010. SETTING: University teaching hospital ICU. ANIMALS: Sixty-nine client-owned dogs: 19 dogs with SIRS or sepsis and 50 healthy control dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum and plasma samples were collected from dogs with SIRS or sepsis on the day of hospital admission and once on the following 2 days, and on a single day in healthy controls. Patients were assessed using the 10-parameter Acute Patient Physiologic and Laboratory Evaluation (APPLEfull ) and 5-parameter (APPLEfast ) score. Serum cα1 -PI concentrations were measured, compared among groups of dogs, and evaluated for a correlation with the concentration of serum C-reactive protein, plasma interleukin-6, tumor necrosis factor-α, the APPLE scores, and survival to discharge. Serum cα1 -PI concentrations were significantly lower in dogs with SIRS/sepsis (P < 0.001) than in healthy controls. While day 1 serum cα1 -PI concentrations did not differ between dogs with SIRS and those with sepsis (P = 0.592), septic dogs had significantly lower serum cα1 -PI concentrations on days 2 (P = 0.017) and 3 (P = 0.036) than dogs with SIRS. Serum cα1 -PI concentrations did not differ between survivors and nonsurvivors (P = 1.000), but were inversely correlated with the APPLEfull score (ρ = -0.48; P = 0.040) and plasma interleukin-6 concentrations (ρ = -0.50; P = 0.037). CONCLUSIONS: These results suggest a role of cα1 -PI as a negative acute phase protein in dogs. The concentration of serum cα1 -PI at the time of hospital admission does not have utility to identify dogs with sepsis from those with noninfectious SIRS, but may be a useful surrogate marker for early stratification of illness severity.

Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma.

BACKGROUND: Onset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs. RESULTS: S100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%. CONCLUSIONS: S100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

Development and analytic validation of an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-bromotyrosine in canine serum.

BACKGROUND: The activation of eosinophils causes the release of eosinophil peroxidase and subsequent production of 3-bromotyrosine (3-BrY), a stable byproduct. In people, 3-BrY is used as a biomarker for eosinophil activation. The method for measuring 3-BrY concentrations in biologic samples from dogs has not previously been described. OBJECTIVES: The objective of this study was to develop and analytically validate an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-BrY in canine serum samples. METHODS: Pooled canine serum samples were utilized to validate the assay. Serum samples from healthy control dogs (n = 41) were used to evaluate 3-BrY concentrations and establish a reference interval. RESULTS: The analytic validation revealed that the limit of blank and limit of detection were 0.33 and 0.63 µmol/L, respectively. The coefficients of variation for precision and reproducibility for 3-BrY were < 13.9% and < 11.0%, respectively. The means ± SD of observed-to-expected ratios for linearity and accuracy were 109.6 ± 17.2% and 98.7 ± 11.3%, respectively. The reference interval was determined as ≤ 1.12 µmol/L (median [range]: ≤ 0.63 µmol/L [≤ 0.63-1.13]). CONCLUSIONS: The EI-GC/MS assay described here for the measurement of 3-BrY in canine serum samples was precise, reproducible, linear, and accurate. Further studies are underway to determine the diagnostic utilities in canine patients with eosinophilic diseases.

Analytic validation of a gas chromatography-mass spectrometry method for quantification of six amino acids in canine serum samples.

OBJECTIVE: To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES: Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES: GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS: Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE: GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS results.

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection.

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B(9)) and cobalamin (vitamin B(12)) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

Evaluation of the MYC_CANFA gene in Chinese Shar Peis with cobalamin deficiency.

BACKGROUND: A recent genome-wide scan using the canine minimal screening set 2 (MSS-2) showed that cobalamin deficiency appears to be hereditary in Chinese Shar Peis and is linked to the microsatellite markers DTR13.6 and REN13N11 on canine chromosome 13. OBJECTIVE: The goal of this study was to evaluate the MYC_CANFA gene, which is the closest known gene with a distance of approximately 0.06 megabases (Mb) to the microsatellite marker DTR13.6, for any mutations in this breed. METHODS: Microsatellite markers (Myc and G15987) for genotyping and primers for sequencing were used to evaluate the MYC_CANFA gene. The genotype and gene sequence were compared between cobalamin-deficient Shar Peis, Shar Peis with normal serum cobalamin concentrations, and the DNA sequences published as part of the Ensemble Genomic map. RESULTS: Neither the microsatellite markers (Myc and G15987) nor the sequences of the MYC_CANFA gene showed a significant difference among both groups of Shar Peis and the published canine DNA sequence. CONCLUSIONS: The data presented here suggest that cobalamin deficiency in Shar Peis is not related to any mutations of the MYC_CANFA gene according to the genotyping and sequencing results in this study. Further investigations are warranted to find a potential genomic locus in proximity to DTR13.6 and REN13N11 that shows mutations in cobalamin-deficient Shar Peis.