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Background Folate and cobalamin are essential cofactors for homocysteine ( HCY) metabolism. Hyperhomocysteinemia, a multifactorial condition, may reflect B vitamin deficiency and is associated with increased risk of cardiovascular disease, thrombosis, and neurodegenerative and chronic gastrointestinal diseases in humans. Hyperhomocysteinemia has been reported in Greyhounds with suspected chronic enteropathy. Objectives To evaluate the frequencies of and the association between hypofolatemia and hyperhomocysteinemia in Greyhounds. Animals Data and serum samples from 559 Greyhounds. Methods Nested case-control study. The frequency of hypofolatemia in Greyhounds was determined by a laboratory database search. The relationship between hyperhomocysteinemia (measured by gas chromatography-mass spectrometry) and hypocobalaminemia and hypofolatemia was evaluated, and its frequency compared between healthy Greyhounds and Greyhounds with thrombosis or chronic diarrhea. Results Hypofolatemia was identified in 172 of 423 (41%) Greyhounds and was more common in hypo- than in normocobalaminemic dogs (49% vs. 35%; P = .0064). Hyperhomocysteinemia was detected in 53 of 78 (68%) of Greyhounds, being more common in hypo- than in normofolatemic dogs (88% vs. 59%; P = .0175). All healthy Greyhounds, 21 of 30 (70%) of dogs with chronic diarrhea and 6 of 8 (75%) of those with thrombosis, were hyperhomocysteinemic. Serum HCY concentrations were inversely correlated with serum folate concentration (ρ = −0.28; P = .0386) and were positively associated with serum albumin concentration (ρ = 0.66; P = .0022). Conclusions and Clinical Relevance Hyperhomocysteinemia occurs frequently in the Greyhound population. Its association with hypofolatemia suggests decreased intracellular availability of B vitamins, but the functional implications warrant further investigation. Hyperhomocysteinemia in Greyhounds potentially may serve as a spontaneous canine model to further investigate hyperhomocysteinemia in humans. [ABSTRACT FROM AUTHOR]

Measuring C-reactive protein (CRP) in serum is a useful surrogate marker for assessing disease progression and treatment response in dogs with autoinflammatory diseases. Affected dogs often receive high-dose glucocorticoid treatment, but the effect of such treatment alone on serum CRP concentrations is unknown. We evaluated serum CRP concentrations via immunoassay (sandwich enzyme-linked immunosorbent assay and particle-enhanced turbidimetric immunoassay) in 12 healthy beagle dogs administered high-dose hydrocortisone (8 mg/kg q12 h) per os vs. placebo over 28 days (days 0, 1, 5, and 28) in a randomized parallel study design. Serum CRP concentrations slightly decreased during treatment or placebo but without a significant association with hydrocortisone administration ( p = 0.761). Compared to baseline, serum CRP concentrations were decreased by >2.7-fold (minimum critical difference) in three hydrocortisone-treated dogs and two dogs in the placebo group on day 28, whereas an increase to >2.7-fold was seen in one dog receiving placebo. These results suggest a lack of confounding effects of high-dose hydrocortisone administration on serum CRP concentrations in healthy dogs. This might also hold in dogs with autoinflammatory conditions and/or administration of other high-dose corticosteroids, suggesting that CRP presents a suitable biomarker to monitor inflammatory disease processes. However, this needs confirmation by further studies evaluating corticosteroid-induced cellular (e.g., hepatic) transcriptome and proteome changes.

Miniature Schnauzers are predisposed to develop pancreatitis, with familial hypertriglyceridemia (HTG) described as a potential risk factor. Diagnosing pancreatitis in dogs is based on the integration of serum canine-specific pancreatic lipase (cPLI) concentration, clinical presentation, and diagnostic imaging findings. However, markers of systemic inflammation and antiprotease activity have not been extensively investigated in the characterization and prognostication of pancreatitis in dogs. Serum concentrations of alpha 1 -proteinase inhibitor (α 1 PI; as a marker of systemic antiprotease response) and calprotectin and S100A12 (as markers of systemic inflammation) were measured in serum samples from 35 Miniature Schnauzers diagnosed with pancreatitis (serum cPLI concentration >400 μg/L, clinical signs, abdominal imaging findings). These markers were evaluated for possible associations with patient characteristics, clinical presentation, risk factors for pancreatitis, and outcome. The study showed that biomarkers of systemic inflammation and antiprotease activity are commonly increased in Miniature Schnauzers with pancreatitis. Whereas serum calprotectin and S100A12 concentrations were found to have limited utility in differentiating pancreatitis presentations, serum α 1 PI concentrations and potentially also the serum calprotectin-to-S100A12 ratio might be non-invasive surrogate markers of disease severity in dogs with pancreatitis.

Background: Immunoglobulin A (IgA) deficiency, chronic enteropathies and exocrine pancreatic insufficiency (EPI) have a high prevalence in German Shepherd dogs (GSD). This prospective study determined the prevalence of faecal IgA deficiency (IgAD) in GSD and investigated several candidate genes and the canine genome for a region or locus co-segregating with IgAD in GSD. Faecal IgA concentrations were quantified and genomic DNA was extracted from 8 GSD with an undetectable faecal IgA (classified as IgAD) and 80 non-IgAD GSD. The canine minimal screening set II microsatellite markers were genotyped, with evidence of an association at p < 1.0 × 10 -3 . Faecal IgA concentrations were also tested for an association with patient clinical and biochemical variables., Results: Allele frequencies observed using the candidate gene approach were not associated with faecal IgAD in GSD. In the genome-wide association study (GWAS), the microsatellite marker FH2361 on canine chromosome 33 approached statistical significance for a link with IgAD in GSD (p = 1.2 × 10 -3 ). A subsequent GWAS in 11 GSD with EPI and 80 control GSD revealed a significant association between EPI and FH2361 (p = 8.2 × 10 -4 )., Conclusions: The lack of an association with the phenotype of faecal IgAD in GSD using the candidate gene approach and GWAS might suggests that faecal IgAD in GSD is a relative or transient state of deficiency. However, the prevalence of faecal IgAD in GSD appears to be low (<3%). The relationship between faecal IgAD, EPI and loci close to FH2361 on canine chromosome 33 in GSD warrants further investigation., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Background: Fecal S100/calgranulin (S100A12 and calprotectin) concentrations are useful markers of gastrointestinal inflammation in dogs. In people, fecal S100/calgranulin concentrations are affected by age, obesity, diet and other lifestyle factors. Knowledge about the effects of such factors on fecal S100/calgranulin concentrations in dogs is currently scarce., Objective: To evaluate the association between several factors and fecal S100/calgranulin concentrations in a large cohort of healthy adult dogs., Methods: Single-spot fecal samples from 181 healthy pet dogs and data derived from a standard questionnaire served to evaluate the effect of age, sex, reproductive status, body weight and body condition, breed type and size, vaccination, endoparasite treatment, diet, environment and travel history on fecal S100/calgranulin concentrations and the fecal calgranulin ratio (fCalR)., Results: Univariate analysis showed a significant association of reproductive status (in female dogs) and breed size with fecal S100A12, fecal calprotectin and fCalR. Breed type was linked to fecal S100A12 concentrations and fCalR; recent vaccination (particularly with a vaccine against canine parvovirus) to fCalR. In multivariate models, breed size was linked to fecal S100A12 and calprotectin concentrations, and recent vaccination affected S100A12 concentrations., Conclusions: Breed size, recent vaccination and reproductive status in female dogs can affect fecal S100/calgranulin concentrations, and these biomarkers should be interpreted in light of those confounding factors. The utility of reference intervals for fecal canine S100/calgranulin concentrations might be improved through stratification by sex/reproductive status and breed size. Fecal canine S100/calgranulin concentrations are not confounded by age, body condition, deworming, diet, environment or travel history., (© 2021 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Folate (vitamin B 9 ) and cobalamin (vitamin B 12 ) play an important role in amino acid metabolism, nucleic acid synthesis, and methyl group transfer. Two intracellular enzymes, methionine synthase and methylmalonyl-CoA mutase, are folate and/or cobalamin-dependent, respectively. At the cellular level, a lack of folate and cobalamin leads to accumulation of serum homocysteine (HCY) and a lack of cobalamin leads to increased methylmalonic acid (MMA) concentrations. Altered serum HCY and MMA concentrations can influence amino acid metabolism and nucleic acid synthesis in pigs. Therefore, we aimed to evaluate serum folate, cobalamin, HCY, and MMA concentrations in postweaning pigs between 6 and 26 weeks of age. Serum samples from 12 pigs collected at week 6, 7, 8, 9, 10, 14, 18, 22, and 26 as part of an unrelated study were analyzed. Serum folate (p < .0001), cobalamin (p = .0001), HCY (p < .0001), and MMA (p < .0001) concentrations differed significantly during the postweaning period between 6 and 26 weeks of age; with significantly higher serum HCY (at weeks 6 and 7 compared to weeks 9, 14, 18, 22, and 26) and MMA concentrations (at weeks 6, 7, and 8 compared to weeks 14, 18, 22, and 26) and an overall decrease of serum MMA concentrations from week 6 to week 14 in the pigs studied. This study suggests age-dependent changes in intracellular folate- and cobalamin-dependent metabolites (i.e., HCY and MMA) in pigs between 6 and 26 weeks of age, possibly reflecting decreased availability of intracellular folate and/or cobalamin for amino acid metabolism, nucleic acid synthesis, and methyl group transfer., Competing Interests: Declaration of Competing Interest None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Disorders of cobalamin (vitamin B 12 ) metabolism are increasingly recognized in small animal medicine and have a variety of causes ranging from chronic gastrointestinal disease to hereditary defects in cobalamin metabolism. Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in clinical practice. While the detection of hypocobalaminemia has therapeutic implications, interpretation of cobalamin status in dogs can be challenging. The aim of this review is to define hypocobalaminemia and cobalamin deficiency, normocobalaminemia, and hypercobalaminemia in dogs, describe known cobalamin deficiency states, breed predispositions in dogs, discuss the different biomarkers of importance for evaluating cobalamin status in dogs, and discuss the management of dogs with hypocobalaminemia., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Measuring S100A12 concentrations in serum and feces is a sensitive and specific marker of inflammation, such as seen with chronic gastrointestinal inflammation in people and dogs. Biomarkers of inflammation in cats are currently lacking., Objectives: We aimed to analytically cross-validate the canine S100A12-ELISA for the measurement of S100A12 in feline specimens., Methods: The ELISA was analytically validated by assessing dilutional linearity, spiking/recovery, intra- and inter-assay variability. Reference intervals for serum and fecal feline S100A12 concentrations were calculated using samples from healthy cats, and the short-term biological variation of fecal S100A12 was assessed., Results: Observed-to-expected ratios (O/E) for serial dilutions of serum and fecal extracts ranged from 91%-159% (mean, 120%) and 100%-128% (mean, 114%), and for the spiking/recovery method ranged from 106%-263% (mean, 154%) and 52%-171% (mean, 112%). Intra- and inter-assay CV% for serum were ≤5.6% and ≤14.0%, and for fecal extracts were ≤3.8% and ≤19.1%, repsectively. RIs for feline serum and fecal S100A12 concentrations were <43 µg/L and < 20 ng/g, respectively. A mild short-term biologic variation, but large individuality were detected when measuring fecal S100A12 concentrations in healthy cats., Conclusions: The canine S100A12-ELISA is accurate, reproducible, and sufficiently linear and precise for the measurement of S100A12 in feline serum and fecal samples. The use of this assay is a reasonable option for the measurement of S100A12 concentrations in feline specimens and provides a basis for the further evaluation of  S100A12 in cats with gastrointestinal disease. Using a population-based RI for fecal feline S100A12 appears to be of limited value., (© 2019 American Society for Veterinary Clinical Pathology.)

S100A12 and S100A8/A9 (calprotectin) are released from activated mononuclear cells and belong to the group of damage associated molecular patterns. Fecal S100A12 and S100A8/A9 concentrations have been suggested as biomarkers of intestinal inflammation in dogs with chronic inflammatory enteropathies (CIE). However, the mucosal cellular infiltrate in dogs with CIE is primarily lymphocytic-plasmacytic. Whether fecal S100A12 and S100A8/A9 levels reflect the number and/or activity of intestinal mucosal mononuclear cells, or whether these proteins are also produced by other cells has not been investigated. Thus, the aim of this study was to evaluate intestinal mucosal S100A12 and S100A8/A9 positivity and a potential relationship with the respective protein concentrations in serum and fecal samples in dogs with CIE. Serum (single sample), fecal samples (from 3 consecutive days), and gastrointestinal tissue biopsies (i.e., stomach, duodenum, ileum, and colon) were evaluated from 21 dogs with CIE. Serum and fecal S100A12 and S100A8/A9 concentrations were measured by analytically validated in-house ELISAs. Tissue biopsies underwent routine histopathology and immunohistochemical evaluation for S100A12 and S100A8/A9 positivity (S100A12 + and S100A8/A9 + , each recorded as positive cells/mm 2 ). S100A12 + and S100A8/A9 + cells were identified in all segments of the gastrointestinal tract, but were predominantly localized in the lamina propria (LP). Duodenal LP S100A12 positivity correlated statistically significantly with that in the stomach and ileum (ρ = 0.66 and 0.69, both p < 0.01), but was inversely correlated with the severity of macrophage infiltration in the duodenum (ρ=-0.47, p = 0.042). Ileal LP S100A8/A9 positivity correlated positively with the extent of ileal neutrophil and macrophage infiltration (ρ=0.61, p = 0.047). Fecal S100A12 concentrations strongly correlated with the number of S100A12 + cells along the entire gastrointestinal tract (ρ = 0.76, p = 0.028), whereas serum S100A12 concentrations were inversely correlated to colonic S100A12 + cell counts (ρ=-0.50, p = 0.043). Mucosal S100A8/A9 + cell counts were not associated with the corresponding fecal or serum S100A8/A9 concentrations. These results suggest that the intestinal mucosa in dogs with CIE contains an increased number of activated (pro-inflammatory) phagocytes expressing and secreting the S100A12 protein, but the macrophage population seen on routine histopathology is predominantly mature (anti-inflammatory) with a reduced or absent expression of S100A12 and a normal or increased expression of S100A8/A9. However, the distribution of intestinal S100A8/A9 expression requires further study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Background: Calprotectin is a marker of inflammatory disorders in people, and serum and fecal calprotectin were shown to be increased in dogs with gastrointestinal inflammation. Biomarkers of gastrointestinal inflammation are currently lacking in cats., Objectives: The purpose of the study was to analytically validate the canine calprotectin radioimmunoassay for quantification of calprotectin in feline specimens., Methods: The immunoassay was analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for fecal calprotectin were established from healthy cats, and the influence of age, sex, and housing condition on fecal calprotectin was determined., Results: The working range of the assay was 1.5-346.2 μg/g of feces and 11.2-8654.4 μg/L of serum. Observed-to-expected ratios (O/E) for serial dilutions of fecal extracts ranged from 77.3% to 112.0% (mean: 99.2%) and from 95.7% to 161.4% (mean: 118.5%) for spiking recovery. Intra- and inter-assay coefficients of variation for fecal samples were ≤ 11.0% and ≤ 12.8%, respectively. Fecal calprotectin concentrations ranged 1.5-66.5 μg/g (3-day sample mean) and 1.5-126.1 μg/g (3-day sample maximum). Housing conditions, sex, or age did not affect fecal calprotectin (all P > .05). For serial dilutions of serum samples, O/E ranged from 96.0% to 152.0% (mean: 115.7%). Serum calprotectin concentrations in healthy cats ranged from 108.8 to 255.3 μg/L (median: 158.2 μg/L)., Conclusions: The canine radioimmunoassay for the measurement of calprotectin is analytically sensitive, linear, reproducible, accurate, and sufficiently precise (CV A  ≤ 43.2%) for use with feline feces (with a loss of accuracy at high calprotectin concentrations). The RIs for feline fecal calprotectin are comparable to those established for dogs. Independence of fecal calprotectin from age and sex agrees with findings in dogs., (© 2018 American Society for Veterinary Clinical Pathology.)

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., (Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Detection of subclinical Brachyspira hyodysenteriae infection in pig herds using feces is challenging. However, the ability to detect the pathogen in intestinal samples of slaughtered pigs has not been investigated, to our knowledge. Therefore, we determined the detection of B. hyodysenteriae in the colon, cecum, and rectum from slaughtered pigs. We analyzed the correlation between detection rates and intestinal lesions, ingesta or fecal consistency, and time from sample collection until processing. A total of 400 ingesta-mucosal (colon, cecum) and 200 fecal (rectum) samples from 200 pigs originating from 20 different herds were bacteriologically examined using selective culture followed by Brachyspira spp. identification by PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ingesta or fecal consistency and intestinal lesions were scored. Brachyspira hyodysenteriae was detected in 23 samples from 16 intestines originating from 7 herds. Brachyspira spp. were detected in 96 samples. More intestinal (16) than fecal (7) samples tested positive for B. hyodysenteriae. For Brachyspira spp., this difference was significant (69 vs. 27; p < 0.01). In particular, colon samples tested positive ( n = 42, p = 0.06). Most (91%) of the intestines showed no lesions typical for clinical B. hyodysenteriae infection, and median ingesta or fecal consistency was "soft and formed," indicating subclinical infection, colonization, or absence of infection. Ingesta from slaughtered pigs, in particular from the colon and sites with lesions, is useful material for detection of B. hyodysenteriae.

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 H 2 -antihistamine-treated patients (P = 0.0053). More PPI- than H 2 -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.

Objective: To determine whether the concentration of serum canine alpha 1 -proteinase inhibitor (cα 1 -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 (APPLE full ) and 5-parameter (APPLE fast ) score. Serum cα 1 -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 cα 1 -PI concentrations were significantly lower in dogs with SIRS/sepsis (P < 0.001) than in healthy controls. While day 1 serum cα 1 -PI concentrations did not differ between dogs with SIRS and those with sepsis (P = 0.592), septic dogs had significantly lower serum cα 1 -PI concentrations on days 2 (P = 0.017) and 3 (P = 0.036) than dogs with SIRS. Serum cα 1 -PI concentrations did not differ between survivors and nonsurvivors (P = 1.000), but were inversely correlated with the APPLE full 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 cα 1 -PI as a negative acute phase protein in dogs. The concentration of serum cα 1 -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., (© Veterinary Emergency and Critical Care Society 2017.)

Respiratory disorders in fattening pigs are of major concern worldwide. Particularly Enzootic Pneumonia remains a problem for the pig industry. This chronic respiratory disease is primarily caused by Mycoplasma hyopneumoniae (M. hyopneumoniae). However, more recently it was hypothesised that M. hyorhinis can also cause similar lung lesions. To investigate the relevance of M. hyorhinis as a cause of pneumonia in fattening pigs 10 farms in Switzerland (considered free of Enzootic Pneumonia) and 20 farms in Germany (regarded as endemic for Enzootic Pneumonia) with a history of chronic and/or recurrent respiratory diseases were included in the study. During a one-time farm visit the coughing index was determined in the batch of oldest fattening pigs in each farm before submission to slaughter. In total, 1375 lungs from these pigs were collected at the abattoir and individually scored for lesions. Furthermore, 600 lungs with, if present, indicative lesions for Enzootic Pneumonia (purple to grey areas of tissue consolidation in the cranio-ventral lung lobes) were tested for mycoplasma species by culture and by real-time PCR for the presence of M. hyorhinis and M. hyopneumoniae. In total, 15.7% of the selected lungs were tested positive for M. hyorhinis by real-time PCR. The prevalence of M. hyorhinis was 10% in Switzerland and 18.5% in Germany and differed significantly between these two countries (p=0.007). M. hyorhinis was detected significantly more often in pneumonic lungs (p=0.004) but no significant association was found between M. hyorhinis and the coughing index or the M. hyopneumoniae status of the pig. M. hyopneumoniae was detected in 0% and 78.5% of the selected lungs in Switzerland and Germany, respectively. We found no evidence that M. hyorhinis alone can lead to similar lung lesions as seen by an infection with M. hyopneumoniae in fattening pigs. In addition, a simultaneous infection with both M. hyorhinis and M. hyopneumoniae did not aggravate the observed lung lesions. Moreover, the presence of M. hyorhinis showed no clinical effect in terms of coughing at least at the end of the fattening phase. However, different levels of virulence of M. hyorhinis isolates as well as interactions with viral pathogens like porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were reported in the literature and need to be further investigated., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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/A9 USG ; S100A12 USG ], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs., Results: S100A8/A9 USG 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/A9 USG  ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%., Conclusions: S100A8/A9 USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9 USG may be a good screening test for canine TCC/PCA.

S100A12 is a potential biomarker of gastrointestinal inflammation in dogs and fecal S100A12 concentrations are correlated with disease severity and outcome. The aim of the present study was to investigate whether there was any association between pre-treatment fecal S100A12 concentrations in dogs affected with chronic enteropathy (CE) and the response to treatment. Dogs affected with CE were recruited into the study and were classified as antibiotic-responsive diarrhea (ARD; n = 9), food-responsive diarrhea (FRD; n = 30) or idiopathic inflammatory bowel disease (IBD; n = 25). They were also grouped based on their response to treatment as complete remission (n = 35), partial response (n = 25) or no response (n = 4). Fecal S100A12 concentrations, measured by ELISA, were elevated in dogs affected with IBD compared with those from dogs affected with FRD (P = 0.010) or ARD (P = 0.025). Dogs with IBD that did not respond to treatment (n = 4) had significantly greater fecal S100A12 concentrations than dogs in complete remission (P = 0.009). Measurement of fecal S100A12 at the time of diagnosis discriminated between dogs with IBD that were refractory to therapy (≥2700 ng/g fecal S100A12) from those with at least a partial response (<2700 ng/g fecal S100A12), with a sensitivity of 100% and a specificity of 76%. These preliminary results suggest that testing of fecal S100A12 may be useful for predicting the lack of response to treatment in dogs affected with CE. The utility of serial fecal S100A12 measurements for monitoring dogs undergoing treatment for CE warrants further investigation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2016 American Society for Veterinary Clinical Pathology.)

Background: Cobalamin (vitamin B12 ) and folate (vitamin B9 ) are important for the amino acid metabolism and nucleic acid synthesis. Immunoassays for the measurement of both vitamins in serum are routinely used in people, cats, and dogs, serving as indicators for clinical disorders including cobalamin and/or folate deficiency, small intestinal dysbiosis, or inadequate dietary supply of these vitamins. The analysis of these analytes may also be clinically useful in pigs., Objective: The purpose of this study was to analytically validate immunoassays for the measurement of cobalamin and folate concentrations in porcine serum, and to determine serum cobalamin and folate concentrations in healthy newborn pigs pre- and postweaning., Methods: Assay validation for both vitamins included the determination of linearity, accuracy, and intra- and inter-assay variability using serum samples from 10 pigs. Also, serum cobalamin and folate concentrations were compared in piglets between pre- and postweaning., Results: For both vitamins, observed-to-expected ratios for linearity and accuracy were 93.2 ± 14.3% and 100.3 ± 8.1% (mean ± standard deviation), respectively. Intra- and inter-assay coefficients of variation for serum were ≤ 8.7% and ≤12.5%, respectively. Significantly higher serum cobalamin and lower folate concentrations were observed in piglets at the time of weaning than at postweaning (P < .0061; P < .0001, respectively)., Conclusions: Both immunoassays are linear, accurate, precise, and reproducible for measurement of porcine serum cobalamin and folate concentrations. Piglets differing in age by only 12 days had significantly different serum cobalamin and folate concentrations. The implications of differing serum cobalamin and folate concentrations in pigs at different stages of life should be further investigated., (© 2016 American Society for Veterinary Clinical Pathology.)

Background: Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers., Objective: The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces., Methods: Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated., Results: Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations., Conclusions: The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs., (© 2016 American Society for Veterinary Clinical Pathology.)

Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs. Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P <0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56-92%) and specificity (67-81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: 3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27)., Results: Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, -20°C, and -80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] μmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] μmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] μmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] μmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] μmol/L) were not different compared to healthy control dogs., Conclusions: The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio is highly beneficial in many research areas. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, Western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 24-45 min.

Chinese Shar-Pei dogs have a high prevalence of hypocobalaminemia and are commonly presented with clinical signs suggestive of severe and long-standing gastrointestinal disease such as diarrhea, vomiting, and/or weight loss. The aim of the current study was to evaluate serum concentrations of inflammatory markers, markers for intestinal disease, and immunological markers in Shar-Peis with hypocobalaminemia or normocobalaminemia (serum cobalamin concentrations within the reference interval). Serum samples from Shar-Peis were collected from various parts of the United States. Serum concentrations of inflammatory markers (i.e., C-reactive protein [CRP], calprotectin [CP], and S100A12), hyaluronic acid (HA, a marker for cutaneous mucinosis), and analytes commonly altered in chronic intestinal diseases (i.e., albumin, zinc, alpha1-proteinease inhibitor [α1PI], immunoglobulin [Ig]A, and IgM) were compared between Shar-Peis with hypocobalaminemia and Shar-Peis with normocobalaminemia. Serum concentrations of CRP, CP, S100A12, HA, zinc, and cα1-PI concentrations did not differ between hypocobalaminemic and normocobalaminemic Shar-Peis (P > 0.05). Serum concentrations of albumin were significantly lower in hypocobalaminemic Shar-Peis (median: 2.5 g/dl) than in normocobalaminemic Shar-Peis (median: 2.9 g/dl; P < 0.0001). Higher serum IgA concentrations and lower serum IgM concentrations were observed in hypocobalaminemic Shar-Peis (median: 1.7 g/l and 0.8 g/l, respectively) than in normocobalaminemic Shar-Peis (median: 0.7 g/l and 1.9 g/l, respectively; both P < 0.0001). In conclusion, no difference was found in serum concentrations of CRP, CP, S100A12, and HA between hypocobalaminemic and normocobalaminemic Shar-Peis whereas some differences were observed in analytes (e.g., albumin, IgA, and IgM) that may be altered in patients with chronic enteropathies., (© 2014 The Author(s).)

Background: Increased serum concentrations of homocysteine (HCY) and methylmalonic acid (MMA), the 2 main cobalamin-dependent metabolites, as well as decreased serum albumin and canine alpha1 -proteinase inhibitor (cα1 -PI) concentrations have previously been described in hypocobalaminemic dogs with gastrointestinal disease. However, no studies have been conducted to evaluate potential relationships between these serum biomarkers., Objective: The aim of this study was to evaluate the relationship between HCY and MMA, 2 cobalamin-dependent metabolites, and both serum albumin and cα1 -PI concentrations in hypocobalaminemic dogs., Methods: Serum samples from 285 dogs including 7 different breeds (Beagle, Boxer, Cocker Spaniel, German Shepherd, Labrador Retriever, Chinese Shar-Pei, and Yorkshire Terrier) with hypocobalaminemia were used. Serum HCY, MMA, albumin, and cα1 -PI concentrations were determined., Results: There was a significant correlation between serum HCY and albumin concentrations, as well as serum HCY and cα1 -PI concentrations (ρ = 0.62 and ρ = 0.37, respectively; P < .0001). No correlations were observed between serum MMA and albumin concentrations, or cα1 -PI concentrations (ρ = 0.01 and ρ = 0.08, respectively; P > .05). In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, and serum HCY and MMA concentrations in Chinese Shar-Peis with hypocobalaminemia., Conclusions: This study shows a correlation between serum albumin and cα1 -PI and HCY concentrations, but not with serum MMA concentration in dogs with hypocobalaminemia. In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, as well as serum HCY and MMA concentrations in Chinese Shar-Peis, emphasizing the unique metabolic interactions in those dog breeds affected by hypocobalaminemia., (© 2014 American Society for Veterinary Clinical Pathology.)

Inflammatory bowel disease (IBD) is a common condition in dogs, and a dysregulated innate immunity is believed to play a major role in its pathogenesis. S100A12 is an endogenous damage-associated molecular pattern molecule, which is involved in phagocyte activation and is increased in serum/fecal samples from dogs with IBD. S100A12 binds to the receptor of advanced glycation end products (RAGE), a pattern-recognition receptor, and results of studies in human patients with IBD and other conditions suggest a role of RAGE in chronic inflammation. Soluble RAGE (sRAGE), a decoy receptor for inflammatory proteins (e.g., S100A12) that appears to function as an anti-inflammatory molecule, was shown to be decreased in human IBD patients. This study aimed to evaluate serum sRAGE and serum/fecal S100A12 concentrations in dogs with IBD. Serum and fecal samples were collected from 20 dogs with IBD before and after initiation of medical treatment and from 15 healthy control dogs. Serum sRAGE and serum and fecal S100A12 concentrations were measured by ELISA, and were compared between dogs with IBD and healthy controls, and between dogs with a positive outcome (i.e., clinical remission, n=13) and those that were euthanized (n=6). The relationship of serum sRAGE concentrations with clinical disease activity (using the CIBDAI scoring system), serum and fecal S100A12 concentrations, and histologic disease severity (using a 4-point semi-quantitative grading system) was tested. Serum sRAGE concentrations were significantly lower in dogs with IBD than in healthy controls (p=0.0003), but were not correlated with the severity of histologic lesions (p=0.4241), the CIBDAI score before (p=0.0967) or after treatment (p=0.1067), the serum S100A12 concentration before (p=0.9214) and after treatment (p=0.4411), or with the individual outcome (p=0.4066). Clinical remission and the change in serum sRAGE concentration after treatment were not significantly associated (p=0.5727); however, serum sRAGE concentrations increased only in IBD dogs with complete clinical remission. Also, dogs that were euthanized had significantly higher fecal S100A12 concentrations than dogs that were alive at the end of the study (p=0.0124). This study showed that serum sRAGE concentrations are decreased in dogs diagnosed with IBD compared to healthy dogs, suggesting that sRAGE/RAGE may be involved in the pathogenesis of canine IBD. Lack of correlation between sRAGE and S100A12 concentrations is consistent with sRAGE functioning as a non-specific decoy receptor. Further studies need to evaluate the gastrointestinal mucosal expression of RAGE in healthy and diseased dogs, and also the formation of S100A12-RAGE complexes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The enzyme-linked immunosorbent assay (ELISA) constitutes an important clinical diagnostic approach. However, the prolonged incubation times involved lead to turnaround times of typically ⩾1 day, potentially delaying a definitive diagnosis or an adequate treatment plan for individual patients. Here cold-microwave technology (CMT) was employed to significantly reduce the times required for diagnostic ELISAs. The new approach was validated and compared to a conventional ELISA setup measuring canine calprotectin (cCP). Canine serum and fecal specimens were used for the analytical validation of cCP ELISA by conventional and CMT-ELISA. Cross-validation of both ELISA methods consisted of the determination of analytic sensitivity, linearity, accuracy, precision, and reproducibility. The long-term stability of antibody-coated ELISA plates was also evaluated up to 33 days. The ELISA approaches were comparable to each other. The observed-to-expected ratios for linearity and accuracy were 100.2±11.8 and 98.1±10.8% (mean±standard deviation), respectively. Precision and reproducibility were ⩽17.2%. For samples run on precoated ELISA plates over 33 days %CVs were ⩽12.5%. While both ELISA approaches were analytically sensitive, linear, accurate, precise, and reproducible with measurements of cCP concentrations, CMT-ELISA offered a reduction in incubation times by 90-95%, facilitating a very fast turnaround time and suggesting CMT-ELISA for improved human and veterinary clinical diagnostics., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Idiopathic inflammatory bowel disease (IBD) in dogs can be challenging to diagnose and fecal markers of disease that correlate with its severity could potentially be clinically useful. Surrogate inflammatory markers, such as the concentration of fecal S100A12, are used to detect active IBD in humans. The aim of this study was to determine the relationship between fecal canine S100A12 concentrations and clinical, endoscopic, and histologic disease severity. Twenty-six dogs with IBD and 90 healthy control dogs were enrolled. Spot fecal samples were collected and fecal canine S100A12 concentrations measured by an in-house ELISA. The correlation of fecal canine S100A12 concentrations with clinical disease activity (using the canine chronic enteropathy clinical activity index scoring system) and with endoscopic and histologic disease severity (using semi-quantitative grading systems) was assessed in dogs with IBD. Concentrations of fecal canine S100A12 were significantly higher in dogs with IBD (median [interquartile range]: 223 [21-3477]ng/g) than in healthy controls (median [interquartile range]: 9 [5-31]ng/g; P<0.0001). Fecal canine S100A12 concentrations correlated with the CCECAI score (ρ=0.4778; P=0.0408) and the severity of endoscopic lesions in the duodenum (ρ=0.4703; P=0.0354) and colon (ρ=0.9747; P=0.0144), but not with the severity of histopathologic changes except for inflammatory lesions in the colon (ρ=0.8669; P=0.0230). A concentration of 273ng fecal canine S100A12/g feces or greater distinguished (a) dogs with moderate to severe endoscopic disease in any GI section from dogs with at most mild endoscopic disease, and (b) dogs with very severe clinical disease (i.e., a CCECAI score of ≥12) from dogs with a CCECAI score of <12, with a sensitivity of 71% and 90%, respectively, and a specificity of 89% and 75%, respectively. This study showed that fecal canine S100A12 concentrations are increased in dogs with IBD. Further, this study showed that fecal canine S100A12 is associated with the clinical disease activity, the severity of endoscopic lesions, and the severity of colonic inflammation in dogs with IBD. Fecal S100A12 concentrations are potentially useful as a biomarker of inflammation in dogs with IBD., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Research suggests that dietary composition influences gastrointestinal function and bacteria-derived metabolic products in the dog colon. We previously reported that dietary composition impacts upon the faecal microbiota of healthy dogs. This study aims at evaluating the dietary influences on bacteria-derived metabolic products associated with the changes in faecal microbiota that we had previously reported. We fed high-carbohydrate starch based (HCS), [crude protein: 194 g/kg, starch: 438 g/kg], high-protein greaves-meal (HPGM), [crude protein: 609 g/kg, starch: 54 g/kg] and dry commercial (DC), [crude protein: 264 g/kg, starch: 277 g/kg] diets, and studied their effects on the metabolism of the colonic microbiota and faecal calprotectin concentrations in five Beagle dogs, allocated according to the Graeco-Latin square design. Each dietary period lasted for three weeks and was crossed-over with washout periods. Food intake, body weight, and faecal consistency scores, dry matter, pH, ammonia, volatile fatty acids (VFAs), and faecal canine calprotectin concentrations were determined., Results: Faecal ammonia concentrations decreased with the HCS diet. All dogs fed the HPGM diet developed diarrhoea, which led to differences in faecal consistency scores between the diets. Faecal pH was higher with the HPGM diet. Moreover, decreases in propionic and acetic acids coupled with increases in branched-chain fatty acids and valeric acid caused changes in faecal total VFAs in dogs on the HPGM diet. Faecal canine calprotectin concentration was higher with the HPGM diet and correlated positively with valeric acid concentration., Conclusions: The HPGM diet led to diarrhoea in all dogs, and there were differences in faecal VFA profiles and faecal canine calprotectin concentrations.

Pancreatic enzymes, such as trypsin and lipase, are essential for the digestion of dietary components in the small intestine. Measurement of both enzymes in jejunal fluid and fecal specimens from dogs has not been reported and will be a prelude for further investigations. Therefore, the aim of the study was to validate radioimmunoassays (RIAs) for the measurement of canine trypsin-like immunoreactivity (cTLI) and pancreatic lipase immunoreactivity (cPLI) in jejunal fluid and fecal specimens from dogs. Jejunal fluid and fecal specimens were collected from five healthy Beagles. A commercial (125)I-RIA was used for measuring cTLI concentrations and an in-house (125)I-RIA was modified for the quantification of cPLI in jejunal fluid and fecal specimens. Both RIAs were analytically validated for canine jejunal fluid and fecal specimens by determining dilutional parallelism, spiking recovery, and intra- and inter-assay variability. For both cTLI and cPLI in jejunal fluid, observed-to-expected ratios for dilutional parallelism and spiking recovery ranged from ≥77.0% to ⩽115.3% and ≥79.0% to ≤ 120.0%, respectively, and from ≥87.2% to ≤ 118.5% and ≥74.6% to ≤ 116.1%, respectively, for fecal specimens. Intra- and inter-assay coefficients of variation (%CV) for both cTLI and cPLI in jejunal fluid were ≤ 7.6% and ≤ 10.0%, respectively, and were ≤ 10.8% and ≤ 9.0%, respectively, for fecal specimens. Both RIAs were demonstrated to be linear, accurate, precise, and reproducible for use with jejunal fluid and fecal specimens from dogs. These results are important for the investigation of pancreatic enzyme concentrations in the gastrointestinal lumen in response to changes in dietary components., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cobalamin deficiency is suspected to be hereditary in Chinese Shar-Pei dogs (Shar-Peis), and inherited causes of cobalamin deficiency may affect the cellular processing of cobalamin. In humans, a defect of the two main cobalamin-dependent intracellular enzymes (i.e., methionine synthase and methylmalonyl-CoA mutase) may lead to hyperhomocysteinemia and hypermethylmalonic acidemia. The aim of this retrospective study was to evaluate serum homocysteine (HCY) and methylmalonic acid (MMA) concentrations in cobalamin-deficient Shar-Peis and dogs of six other breeds. Serum samples (n=297) from cobalamin-deficient dogs (Shar-Peis, German Shepherd dogs, Labrador Retrievers, Yorkshire Terriers, Boxers, Cocker Spaniels, and Beagles) were analyzed for serum HCY and MMA concentrations. A Fisher's exact test was used to evaluate if cobalamin deficiency in Shar-Peis is associated with hyperhomocysteinemia. Serum HCY and MMA concentrations were higher in cobalamin-deficient Shar-Peis compared to cobalamin-deficient dogs of the six other breeds (P<0.0001). Hyperhomocysteinemia was associated with cobalamin deficiency in Shar-Peis (P=0.009). In addition, serum HCY and MMA concentrations did not differ between cobalamin-deficient German Shepherd dogs with and without exocrine pancreatic insufficiency (EPI), a potential cause of secondary cobalamin deficiency. These findings suggest that the function of the two intracellular cobalamin-dependent enzymes is impaired in Shar-Peis with cobalamin deficiency., (Published by Elsevier Ltd.)

Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.

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&#95;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&#95;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&#95;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&#95;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., (© 2012 American Society for Veterinary Clinical Pathology.)

Altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders such as exocrine pancreatic insufficiency (EPI) or gastrointestinal inflammation. The aims of the current study were 1) to identify breeds with a higher proportion of dogs with a decreased serum cobalamin concentration, 2) to determine whether dogs with such decreased concentrations tend to have serum canine trypsin-like immunoreactivity (cTLI) concentrations diagnostic for EPI, and 3) to compare the number of submissions for serum cobalamin analysis by breed to the American Kennel Club (AKC) breed ranking list of 2009. In this retrospective study, results of 28,675 cobalamin tests were reviewed. Akitas, Chinese Shar-Peis, German Shepherd Dogs, Greyhounds, and Labrador Retrievers had increased proportions of serum cobalamin concentrations below the lower limit of the reference interval (<251 ng/l; all P < 0.0001). Akitas, Chinese Shar-Peis, German Shepherd Dogs, and Border Collies had increased proportions of serum cobalamin concentrations below the detection limit of the assay (<150 ng/l; all P < 0.0001). Akitas, Border Collies, and German Shepherd Dogs with serum cobalamin concentrations <150 ng/l were more likely to have a serum cTLI concentration considered diagnostic for EPI (≤2.5 µg/l; all P ≤ 0.001). The breed with the highest proportion of samples submitted for serum cobalamin analysis in comparison with the AKC ranking list was the Greyhound (odds ratio: 84.6; P < 0.0001). In Akitas and Border Collies, further investigations are warranted to clarify if a potentially breed-specific gastrointestinal disorder is responsible for the increased frequency of decreased serum cobalamin and cTLI concentrations.

A total of 22,462 serum sample results from dogs being evaluated for gastrointestinal disease at the Gastrointestinal Laboratory, College of Veterinary Medicine, Texas A&M University were evaluated retrospectively. The proportion of dogs with serum cobalamin concentrations below the reference interval and median serum concentrations were compared between Shar Peis and other dog breeds. Serum samples were also obtained prospectively from 22 healthy and 32 Shar Peis with chronic gastrointestinal disease and 59 healthy dogs of other breeds, and serum concentrations of cobalamin, folate, and methylmalonic acid were determined and compared. Overall, 64.0% (89/139) of serum samples from Shar Peis showed serum cobalamin concentrations below the limit of the reference interval and 38.1% (53/139) of these were below the detectable limit for the assay. The median serum cobalamin concentration in Shar Peis was significantly lower than in other breeds. Shar Peis with gastrointestinal disease had significantly lower serum cobalamin and higher serum methylmalonic acid concentrations compared to healthy Shar Peis. Healthy Shar Peis had significantly increased serum methylmalonic acid concentrations compared to healthy dogs of other breeds. There were no meaningful differences in folate concentrations between groups. In conclusion, Shar Peis have a high prevalence of cobalamin deficiency compared to other breeds and healthy Shar Peis may have subclinical cobalamin deficiency., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

S100A12 (calgranulin C) is a Ca(2+)-binding protein that has been proposed to play a central role in both innate and acquired immune responses. In humans, S100A12 has been reported to be increased in serum and/or plasma in patients with various inflammatory disorders, and this protein has been suggested to be a sensitive and specific marker for inflammatory bowel disease (IBD). An immunoassay for S100A12 is currently available for use in humans, but antibodies against the human protein do not cross-react with canine S100A12 (cS100A12). Both sensitive and specific markers for canine patients with systemic or localized inflammatory diseases are currently lacking, thus the aim of this study was to develop and analytically validate a radioimmunoassay (RIA) for the quantification of cS100A12 in serum and fecal specimens and to determine the biological variation of cS100A12 in serum from healthy dogs. A competitive liquid-phase RIA was developed and analytically validated by determining assay working range, dilutional parallelism, spiking recovery, and intra- and inter-assay variability. Reference intervals for serum and fecal concentrations of cS100A12 were established from 124 and 65 healthy dogs, respectively, and components of variation for serum cS100A12 were determined from 11 dogs over 2.6 months. The working range of the assay was 0.6-432.7 μg/L. No cross-reactivity was observed with the cS100A8/A9 protein complex, the closest structural analogues available. Observed-to-expected ratios (O/E) for the serial dilution of serum and fecal extracts ranged from 97.2 to 146.8% and from 75.3 to 129.8%, respectively. O/E for spiking recovery for serum and fecal extracts ranged from 87.8 to 130.4% and from 84.8 to 143.8%, respectively. Coefficients of variation (CV) for intra- and inter-assay variability for sera were ≤ 8.1% and ≤ 7.8%, respectively, and were ≤ 7.8% and ≤ 8.7%, respectively, for fecal extracts. Reference intervals for serum and fecal cS100A12 were 33.2-225.1 μg/L and <24-745 ng/g, respectively. For biological variability testing, analytical, intra-individual, inter-individual, and total CV were 5.7, 29.2, 31.2, and 66.0%, respectively, yielding an index of individuality of 0.95 and a minimum critical difference (p<0.05) for sequential values of 84.9%. The RIA for cS100A12 measurement described here is analytically sensitive and specific, linear, accurate, precise, and reproducible, and will facilitate further research into the clinical utility of quantifying serum and fecal cS100A12 in canine patients with inflammatory diseases. Moderate changes in serum cS100A12 concentrations may be clinically relevant; however, the use of a population-based reference interval may require caution., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of a multi-species synbiotic on the fecal microbiota of healthy cats (n = 12) and dogs (n = 12) was evaluated. The synbiotic (containing 5 × 10(9)  CFU of a mixture of seven probiotic strains, and a blend of fructooligosaccharides and arabinogalactans) was administered daily for 21 days. Fecal and serum samples were collected before, during, and up to 3 weeks after administration. Changes in the fecal microbiota were analyzed using denaturing gradient gel electrophoresis, 16S rRNA gene libraries, quantitative real-time PCR, and 16S rRNA gene 454-pyrosequencing. Probiotic species were detectable in 10/12 dogs and 11/12 cats during product administration. Abundances of Enterococcus and Streptococcus spp. were significantly increased in at least one time point during administration, and returned to baseline abundance after treatment was discontinued. No changes in the major bacterial phyla were identified on 454-pyrosequencing. No adverse gastrointestinal effects were recorded and no significant changes in gastrointestinal function or immune markers were observed during the study period. This study shows that while the ingestion of probiotics and prebiotics does not appear to alter the predominant bacterial phyla present in feces, supplementation with the investigated synbiotic leads to an increased abundance of probiotic bacteria in the feces of healthy cats and dogs., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Cobalamin deficiency is a common disorder in Chinese Shar Peis (Shar Peis) and is thus suspected to be hereditary. The objective of this study was to identify a genomic region or locus that cosegregates with the phenotype of cobalamin deficiency in Shar Peis. Serum cobalamin concentrations were measured, and blood for genomic DNA extraction was collected from 14 cobalamin-deficient Shar Peis and 28 Shar Peis with a serum cobalamin concentration in the reference range. The 327 microsatellite markers from the canine minimal screening set 2 and 4 additional markers were amplified by polymerase chain reaction and genotyped by automated capillary electrophoresis. Two microsatellite markers, DTR13.6 (P = 1.4 x 10(-6)) and REN13N11 (P = 1.5 x 10(-5)), both on canine chromosome 13, showed evidence of linkage disequilibrium. These findings indicate that the region of chromosome 13 near these markers should be mapped and closely examined for potential mutations associated with this disease in Shar Peis.