Untargeted fecal metabolome analysis in obese dogs after weight loss achieved by feeding a high-fiber-high-protein diet.

INTRODUCTION: In humans and companion animals, obesity is accompanied by metabolic derangements. Studies have revealed differences in the composition of the fecal microbiome between obese dogs and those with an ideal body weight. OBJECTIVES: We have previously reported that the fecal microbiome in obese dogs changes after controlled weight reduction, induced by feeding a diet high in fiber and protein. Despite these findings, it is unclear if taxonomic differences infer differences at the functional level between obese dogs and those with an ideal body weight. METHODOLOGY: Untargeted fecal metabolome analysis was performed on dogs with obesity before and after weight loss achieved by feeding a high-fiber-high-protein diet. RESULTS: Fecal metabolome analysis revealed a total of 13 compounds that changed in concentration in obese dogs after weight loss. Of these compounds, metabolites associated with bacterial metabolism decreased after weight loss including purine, L-(-)-methionine, coumestrol, and the alkaloids 1-methylxanthine and trigonelline. Conversely, the polyphenols (-)-epicatechin and matairesinol and the quinoline derivatives 1,5-isoquinolinediol and 2-hydroxiquinoline increased after weight loss. CONCLUSION: These results suggest differences in intestinal microbiome at the functional level after weight loss, but further studies are needed to determine the role of these compounds in the etiology of obesity and weight loss.

EXOCRINE PANCREATIC INSUFFICIENCY-LIKE SYNDROME IN FOUR CAPTIVE TIGERS (PANTHERA TIGRIS).

Exocrine pancreatic insufficiency (EPI) is a condition characterized by a decreased synthesis and secretion of pancreatic enzymes, which results in weight loss, poor hair coat, and diarrhea. The diagnostic test of choice for EPI in domestic cats is feline serum trypsin-like immunoreactivity (fTLI). This paper details four tigers (Panthera tigris) with clinical signs compatible with EPI. On the basis of domestic cat reference ranges, fTLI assays for all four clinically affected tigers were diagnostic for EPI (median 1.0 µg/L; range 0.5-1.2 µg/L). All four tigers had a rapid clinical response to pancreatic enzyme supplementation. Serum from 10 clinically healthy tigers was submitted for the fTLI assay, for comparative purposes. The healthy tigers' fTLI assays were also within range for a diagnosis of EPI in domestic cats (median 3.1 µg/L; range 1.9-4.5 µg/L); however, clinically affected tigers had significantly lower serum fTLI concentrations than healthy tigers (P = 0.0058). Serum cobalamin was below the detection limit in both the affected and healthy tigers (<150 ng/L). Measuring fTLI appears to be a useful tool in the diagnosis of EPI-like syndrome in tigers. As in other species, EPI-like syndrome in tigers may also be associated with cobalamin deficiency.

Bacterial Biogeography of the Colon in Dogs With Chronic Inflammatory Enteropathy.

The intestinal microbiota is believed to play a role in the pathogenesis of inflammatory bowel disease in humans and chronic inflammatory enteropathy (CIE) in dogs. While most previous studies have described the gut microbiota using sequencing methods, it is fundamental to assess the spatial distribution of the bacteria for a better understanding of their relationship with the host. The microbiota in the colonic mucosa of 22 dogs with CIE and 11 control dogs was investigated using fluorescence in situ hybridization (FISH) with a universal eubacterial probe (EUB338) and specific probes for select bacterial groups. The number of total bacteria labeled with EUB338 probe was lower within the colonic crypts of dogs with CIE compared to controls. Helicobacter spp. and Akkermansia spp. were decreased on the colonic surface and in the crypts of dogs with CIE. Dogs with CIE had increased number of Escherichia coli/Shigella spp. on the colonic surface and within the crypts compared to control dogs. In conclusion, the bacterial microbiota in the colonic mucosa differed between dogs with and without CIE, with depletion of the crypt bacteria in dogs with CIE. The crypt bacterial species that was intimately associated with the host mucosa in control dogs was composed mainly of Helicobacter spp.

Multiple-Choice Questions in Small Animal Medicine: An Analysis of Cognitive Level and Structural Reliability, and the Impact of these Characteristics on Student Performance.

Students entering the final year of the veterinary curriculum need to integrate information and problem solve. Assessments used to document competency prior to entry to the clinical environment should ideally provide a reliable measurement of these essential skills. In this study, five internal medicine specialists evaluated the cognitive grade (CG) and structural integrity of 100 multiple-choice questions (MCQs) used to assess learning by third-year students at a United States (US) veterinary school. Questions in CG 1 tested factual recall and simple understanding; those in CG 2 required interpretation and analysis; CG 3 MCQs tested problem solving. The majority (53%) of questions could be answered correctly using only recall or simple understanding (CG 1); 12% of MCQs required problem solving (CG 3). Less than half of the questions (43%) were structurally sound. Overall student performance for the 3 CGs differed significantly (92% for CG 1 vs. 84% for CG 3; p = .03. Structural integrity did not appear to impact overall performance, with a median pass rate of 90% for flawless questions versus 86% for those with poor structural integrity (p = .314). There was a moderate positive correlation between individual student outcomes for flawless CG 1 versus CG 3 questions (rs = 0.471; p = < .001), although 13% of students failed to achieve an aggregate passing score (65%) on the CG 3 questions. These findings suggest that MCQ-based assessments may not adequately evaluate intended learning outcomes and that instructors may benefit from guidance and training for this issue.

Development and analytical validation of a radioimmunoassay for the quantification of alpha1 -proteinase inhibitor in serum and feces from the common marmoset (Callithrix jacchus).

BACKGROUND: The objective of this study was to develop and analytically validate a radioimmunoassay (RIA) for the measurement of alpha1 -proteinase inhibitor (α1 -PI) concentrations in serum and feces from the common marmoset. METHODS: Serum samples (n = 30) and 3-day fecal samples (n = 30) were obtained from healthy marmosets. An RIA was established and validated by determination of sensitivity, working range, dilutional parallelism, spiking recovery, and intra- and interassay variability. A reference interval for mα1 -PI in serum and feces was established. RESULTS: Sensitivity and upper limit of the working range were 0.75 and 100.62 µg/L, respectively. Observed-to-expected (O/E) ratios for serial dilutions ranged from 89.9% to 123.0% (mean ±SD: 106.0 ± 11.5%) for 8 serum samples, and from 90.6% to 132.7% (mean ±SD: 107.6 ± 19.2%) for 4 fecal samples. O/E ratios for spiking recovery ranged from 97.6% to 104.4% (mean ±SD: 101.3 ± 3%) for 4 serum samples, and from 97.5% to 101.4% (mean ±SD: 99.2 ± 1.8%) for 4 fecal samples and 3 different spiking concentrations. Coefficients of variation (CV) for intra-assay variability for 8 serum samples ranged from 1.7% to 10.6% and 2.2% to 5.1% in the 8 fecal samples. The interassay CV for eight serum samples ranged from 1.3% to 9.9%, and from 1.0% to 6.7% in the 8 fecal samples. The reference interval in serum was determined to be 1047-1484 µg/L. The reference interval in serum was determined to be 1047-1484 µg/L. The reference interval for the 3-day mean fecal concentration, and 3-day maximum fecal concentration were determined to be 32.4-124.4 µg/g and 39.1-158.7 µg/g of feces, respectively. CONCLUSION: The developed assay is sensitive, linear, accurate, precise, and reproducible. Further studies are needed to determine the clinical utility of this assay.

S100A12 concentrations and myeloperoxidase activities are increased in the intestinal mucosa of dogs with chronic enteropathies.

BACKGROUND: Intestinal mucosal S100A12 and myeloperoxidase (MPO) are inflammatory biomarkers in humans with inflammatory bowel disease (IBD). However, these biomarkers have not been studied in the intestinal mucosa of dogs with chronic enteropathies (CE), even though dogs with CE have increased S100A12 concentrations in feces and serum. This study investigated mucosal S100A12 concentrations and MPO activities in both dogs with CE and healthy Beagles. ELISA (S100A12 concentrations) and spectrophotometric methods (MPO activity) were used. The associations of both biomarkers with canine IBD activity index (CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations were also investigated. We studied intestinal mucosal samples originating from different intestinal regions of 40 dogs with CE and 18 healthy Beagle dogs (duodenum, ileum, colon, and cecum). RESULTS: Compared with healthy Beagles, mucosal S100A12 concentrations in dogs with CE were significantly higher in the duodenum (p < 0.0001) and colon (p = 0.0011), but not in the ileum (p = 0.2725) and cecum (p = 0.2194). Mucosal MPO activity of dogs with CE was significantly higher in the duodenum (p < 0.0001), ileum (p = 0.0083), colon (p < 0.0001), and cecum (p = 0.0474). Mucosal S100A12 concentrations in the duodenum were significantly higher if the inflammatory infiltrate consisted mainly of neutrophils (p = 0.0439) or macrophages (p = 0.037). Mucosal S100A12 concentrations also showed a significant association with the severity of total histopathological injury and epithelial injury in the colon (p < 0.05). Mucosal MPO activity showed a significant association (p < 0.05) with the severity of total histopathological injury, epithelial injury, and eosinophil infiltration in the duodenum. There was no significant association of both biomarkers with CIBDAI or clinical outcome. CONCLUSIONS: This study showed that both mucosal S100A12 concentrations and MPO activities are significantly increased in the duodenum and colon of dogs with CE; mucosal MPO was also increased in the ileum and cecum. Future research should focus on assessing the clinical utility of S100A12 and MPO as diagnostic markers in dogs with CE.

The fecal microbiome of dogs with exocrine pancreatic insufficiency.

Exocrine pancreatic insufficiency (EPI) in dogs is a syndrome of inadequate synthesis and secretion of pancreatic enzymes. Small intestinal bacterial dysbiosis occurs in dogs with EPI, and is reversed with pancreatic enzyme therapy. However, there are no studies evaluating the fecal microbiome of dogs with EPI. The objective of this study was to evaluate the fecal microbiome of dogs with EPI. Three day pooled fecal samples were collected from healthy dogs (n = 18), untreated (n = 7) dogs with EPI, and dogs with EPI treated with enzyme replacement therapy (n = 19). Extracted DNA from fecal samples was used for Illumina sequencing of the bacterial 16S rRNA gene and analyzed using Quantitative Insights Into Microbial Ecology (QIIME) and PICRUSt was used to predict the functional gene content of the microbiome. Linear discriminant analysis effect size (LEfSe) revealed significant differences in bacterial groups and functional genes between the healthy dogs and dogs with EPI. There was a significant difference in fecal microbial communities when healthy dogs were compared to treated and untreated dogs with EPI (unweighted UniFrac distance, ANOSIM P = 0.001, and 0.001 respectively). Alpha diversity was significantly decreased in untreated and treated EPI dogs when compared to the healthy dogs with respect to Chao1, Observed OTU, and Shannon diversity (P = 0.008, 0.003, and 0.002 respectively). The families Bifidobacteriaceae (P = 0.005), Enterococcaceae (P = 0.018), and Lactobacillaceae (P = 0.001) were significantly increased in the untreated and treated dogs with EPI when compared to healthy dogs. In contrast, Lachnospiraceae (P < 0.001), and Ruminococcaceae (P < 0.01) were significantly decreased in dogs with EPI. Dogs with EPI (before treatment) had significant increases in functional genes associated with secretion system, fatty acid metabolism, and phosphotransferase system. In contrast, healthy dogs had a significant increase in genes related to phenylalanine, tyrosine and tryptophan biosynthesis, transcription machinery and sporulation. In conclusion, this study shows that the fecal microbiome of dogs with EPI (both treated and untreated) is different to that of healthy dogs.

Evaluation of Intestinal Barrier Dysfunction with Serum Iohexol Concentration in Dogs with Acute Hemorrhagic Diarrhea Syndrome.

Histopathologic examination of intestinal biopsies from dogs with acute hemorrhagic diarrhea syndrome (AHDS) reveals necrotizing enteritis and epithelial integrity loss. Serum iohexol measurement has been utilized to assess intestinal permeability. Our hypothesis is that dogs with AHDS have increased intestinal permeability, which is associated with the severity of clinical signs. In this prospective case-control study, 53 client-owned dogs (28 AHDS, 25 healthy controls) were evaluated. Clinical severity was assessed using the AHDS index and systemic inflammatory response syndrome (SIRS) criteria. Simultaneously, dogs received oral iohexol, and serum iohexol concentrations (SICs) were measured two hours later. Results indicated significantly higher (p = 0.002) SIC in AHDS dogs (median: 51 µg/mL; min-max: 9-246) than in healthy controls (30 µg/mL; 11-57). There was a significant positive correlation between AHDS index and SIC (rS = 0.4; p = 0.03) and a significant negative between SIC and serum albumin concentrations (Pearson r = -0.55; p = 0.01). Dogs with severe AHDS (mean 106 µg/mL; range: 17-246) demonstrated significantly higher (p = 0.002) SIC than those with mild to moderate disease (29 µg/mL; 9-54). These findings underscore the association between intestinal permeability and clinical severity in dogs with AHDS assessed by iohexol.

Reply to Piewbang et al. Domestic Cat Hepadnavirus Antigens in Lymphoma Tissues. Comment on "Beatty et al. Domestic Cat Hepadnavirus and Lymphoma. Viruses 2023, 15, 2294".

We are grateful to the authors for providing additional data to demonstrate the presence of domestic cat hepadnavirus in lymphoma tissues [...].

Unbiased serum metabolomic analysis in cats with naturally occurring chronic enteropathies before and after medical intervention.

Chronic enteropathies (CE) are common disorders in cats and the differentiation between the two main underlying diseases, inflammatory bowel disease (IBD) and low-grade intestinal T-cell lymphoma (LGITL), can be challenging. Characterization of the serum metabolome could provide further information on alterations of disease-associated metabolic pathways and may identify diagnostic or therapeutic targets. Unbiased metabolomics analysis of serum from 28 cats with CE (14 cats with IBD, 14 cats with LGITL) and 14 healthy controls identified 1,007 named metabolites, of which 129 were significantly different in cats with CE compared to healthy controls at baseline. Random Forest analysis revealed a predictive accuracy of 90% for differentiating controls from cats with chronic enteropathy. Metabolic pathways found to be significantly altered included phospholipids, amino acids, thiamine, and tryptophan metabolism. Several metabolites were found to be significantly different between cats with IBD versus LGITL, including several sphingolipids, phosphatidylcholine 40:7, uridine, pinitol, 3,4-dihydroxybenzoic acid, and glucuronic acid. However, random forest analysis revealed a poor group predictive accuracy of 60% for the differentiation of IBD from LGITL. Of 129 compounds found to be significantly different between healthy cats and cats with CE at baseline, 58 remained different following treatment.

Urinary 15-F2t-Isoprostane Concentrations in Dogs with Liver Disease.

Isoprostanes are stable end products of lipid peroxidation that can be used as markers of oxidative stress. It was previously reported that a cohort of dogs with various liver diseases had increased urinary isoprostane concentrations compared to healthy control (HC) dogs. The aim of this study was to measure and report urinary isoprostane concentrations in dogs with different types of liver diseases. Urine was collected from 21 HC dogs and from 40 dogs with liver disease, including 25 with chronic hepatitis (CH), 7 with steroid hepatopathy (SH), and 8 with a congenital portosystemic shunt (CPSS). In this prospective, observational study, urinary 15-F2t-isoprostane (F2-IsoP) concentrations were measured by liquid chromatography/mass spectrometry and normalized to urinary creatinine concentrations. Concentrations were compared between groups using a Kruskal-Wallis test followed by Dunn's multiple comparisons tests. Significance was set at p < 0.05. The median (range) urinary F2-IsoP to creatinine ratios (ng/mg UCr) were 3.6 (2.2-12.4) for HC dogs, 5.7 (2.4-11.3) for dogs with CH, 4.8 (2.4-8.6) for dogs with SH, and 12.5 (2.9-22.9) for dogs with CPSS. CPSS dogs had significantly higher urinary F2-IsoP concentrations than HC dogs (p = 0.004), suggesting increased oxidative stress among this cohort.

Serum cobalamin and methylmalonic acid concentrations in juvenile dogs with parvoviral enteritis or other acute enteropathies.

BACKGROUND: Low serum cobalamin concentrations have been associated with ileal malabsorption in dogs with chronic enteropathy. Increased serum methylmalonic acid (MMA) concentrations indicate cobalamin deficiency on a cellular level. Few studies have evaluated serum cobalamin concentrations or methylmalonic acid concentrations in juvenile dogs with parvoviral enteritis or nonparvoviral acute enteropathies. OBJECTIVES: Evaluate serum cobalamin and methylmalonic acid concentrations in juvenile dogs (6 weeks to 10 months old) with parvoviral enteritis or nonparvoviral acute enteropathy. ANIMALS: Thirty-one juvenile dogs with parvoviral enteritis, 29 dogs with nonparvoviral acute diarrhea (NPVAD), and 40 healthy juvenile control dogs. METHODS: Single-center, prospective, observational, cross-sectional study. Serum cobalamin and, when sufficient serum was available, MMA concentrations were measured. RESULTS: Most serum cobalamin concentrations were within the adult reference interval. Serum cobalamin concentrations in healthy dogs (median, 848 ng/L; range, 293-1912 ng/L) were significantly higher than in dogs with parvoviral enteritis (P = .0002; median, 463 ng/L; range, <150-10 000 ng/L) or dogs with NPVAD (P = .02; median, 528 ng/L; range, 160-8998 ng/L). Serum MMA concentrations were not significantly different between groups (healthy dogs: median, 796 nmol/L; range, 427-1933 nmol/L; parvoviral enteritis: median, 858 nmol/L; range, 554-3424 nmol/L; NPVAD: median, 764 nmol/L; range, 392-1222 nmol/L; P = .1). CONCLUSIONS AND CLINICAL IMPORTANCE: Juvenile dogs with parvoviral enteritis or NPVAD had lower serum cobalamin concentrations than healthy juvenile dogs. However, based on serum MMA concentrations cellular cobalamin deficiency was not apparent.

Fecal S100A12 concentrations in cats with chronic enteropathies.

OBJECTIVES: The aim of this study was to compare fecal S100A12 concentrations in cats diagnosed with chronic enteropathy (CE) with healthy control cats. METHODS: This was a prospective, cross-sectional study. Forty-nine cats that had gastrointestinal signs for >3 weeks and a complete diagnostic work-up, including bloodwork, abdominal ultrasound and upper and/or lower gastrointestinal endoscopic biopsies, were enrolled into the CE group. Nineteen cats from the CE group were diagnosed with inflammatory bowel disease (IBD) or chronic inflammatory enteropathy (CIE) and 30 with alimentary lymphoma (LSA), based on histopathology results and additional testing with immunohistochemistry or molecular clonality testing with PCR if indicated. Nineteen apparently healthy control cats were included in the study. One fecal sample was collected from each cat and S100A12 concentrations were quantified by an analytically validated in-house ELISA. RESULTS: Fecal S100A12 concentrations differed between cats with LSA (median 110 ng/g; interquartile range [IQR] 18-548) and control cats (median 4 ng/g; IQR 2-25 [P <0.001]) and between cats with IBD (median 34 ng/g; IQR 15-973) and control cats (P <0.003). S100A12 concentrations in CE cats (median 94 ng/g; IQR 16-548) were statistically significantly higher compared with control cats (P <0.001). The area under the receiver operating characteristic curve (AUROC) to separate healthy cats from CE cats was 0.81 (95% confidence interval [CI] 0.70-0.92) and was statistically significant (P <0.001). The AUROC to separate cats with IBD from cats with LSA was 0.51 (95% CI 0.34-0.68) and was not statistically significant (P = 0.9). CONCLUSIONS AND RELEVANCE: Fecal S100A12 concentrations at the time of diagnostic investigation were higher in cats with CIE and LSA than in healthy controls but did not differ between cats with LSA and those with CIE/IBD. This study is an initial step toward evaluating a novel non-invasive marker of feline CIE. Further studies are needed to determine the diagnostic utility of fecal S100A12 concentrations in cats with CE, including comparing cats with IBD/CIE and LSA, and to compare them with cats with extra-gastrointestinal disease.

Hip sensor location for flash interstitial glucose monitoring system provides adequate clinical accuracy in non-diabetic hypoglycemic dogs.

OBJECTIVE: Flash glucose monitoring systems (FGMS) are frequently used for interstitial glucose monitoring in dogs with diabetes mellitus and are typically placed between the scapulae. We aimed to evaluate the variability between glucose measurements from FGMS placed in 2 locations (between the scapulae and over the hip) in non-diabetic dogs during rapidly induced hypoglycemia. ANIMALS: 24 apparently healthy colony dogs that were subjects in a teaching laboratory. PROCEDURES: Prospective interventional study. FGMS sensors (FreeStyle Libre 14-day system) were placed between the scapulae and over the hip of all dogs. Regular insulin was administered (0.3 u/kg IV) and subsequent hypoglycemia was corrected. Before insulin administration and every 10 minutes over 90 minutes, interstitial glucose was recorded from both locations, and blood glucose was measured with a point-of-care blood glucose monitor (AlphaTRAK 2). RESULTS: There was a constant bias of 5.6 mg/dL (95% limits of agreement: -26.3 to 37.5 mg/dL) between locations, but the proportional bias was not apparent. There was a correlation between FGMS locations (r = 0.731, P = < .001). Sensor site B was clinically accurate with 100% of paired samples within Parkes error grid zones A (83%) and B (17%) but did not meet the criteria for analytical accuracy. CLINICAL RELEVANCE: In this model of induced hypoglycemia in healthy dogs, variation between measurements from FGMS locations was unlikely to have affected the clinical outcome. Placement of FGMS over the hip may be an acceptable alternative to placement between the scapulae, but the utility in hyperglycemic dogs is unknown.

Analytical validation, sample stability, and clinical evaluation of a new high-sensitivity cardiac troponin I immunoassay for use in dogs, with comparison to a previous ultrasensitive assay.

Cardiac troponin I (cTnI) is considered the gold standard biomarker for myocardial injury and shows a high degree of homology between humans and dogs. The ADVIA Centaur XP High-Sensitivity Troponin I (AC-cTnI-HS) assay has been validated for use in humans but not dogs. The study objectives were to analytically validate the AC-cTnI-HS assay in dogs, to assess correlation between the AC-cTnI-HS and a previous ADVIA Centaur TnI-Ultra (AC-cTnI-U) assay, to assess cTnI sample storage stability, and to clinically evaluate the AC-cTnI-HS assay in healthy dogs and dogs with cardiac disease. Canine serum samples were used for analytical validation. Intra- and inter-assay variability, dilutional parallelism, and spiking recovery were assessed. Samples from 196 client-owned dogs were evaluated (healthy dogs (n = 39) or dogs with congenital heart disease (n = 54), myxomatous mitral valve disease (n = 68), dilated cardiomyopathy (n = 15), or myocarditis (n = 20)). Inter- and intra-assay coefficient of variation (%CV) was between 2.8-41.4% and 3.8-30.2%, respectively, with pools with concentrations >20 pg/mL all having %CVs <10%. The observed to expected ratios for dilutional parallelism and spiking recovery experiments ranged between 92.3 and 266.7.0% and 84.3 and 108%, respectively. A strong correlation between the AC-cTnI-HS and AC-cTnI-U assays was observed (Spearman's ρ = 0.927), though a proportional bias existed, with AC-cTnI-HS assay concentrations being proportionally lower than AC-cTnI-U assay concentrations. Serum samples stored at -80°C had stable cTnI measurements for up to 2.7 years and after a single freeze-thaw cycle. Healthy dogs and dogs with congenital heart disease had significantly lower cTnI concentrations than dogs in the other three groups. The AC-cTnI-HS assay precisely, reproducibly, and accurately measures cTnI concentrations in dog serum with cTnI concentrations >20 pg/mL.

Analytical validation of an ELISA for the measurement of feline pancreas-specific lipase and re-evaluation of the reference interval and decision threshold for diagnosing pancreatitis.

BACKGROUND: The diagnosis of feline pancreatitis can be challenging. The clinical presentation often includes mild, nonspecific clinical signs, such as vomiting, anorexia, and weight loss. Measurement of feline pancreatic lipase immunoreactivity (fPLI) concentration in serum has been reported to be sensitive and specific for a diagnosis of pancreatitis in cats. However, analytical validation for a widely available commercial assay for the measurement of fPLI concentration has not been published. OBJECTIVE: We aimed to analytically validate the Spec fPL assay (IDEXX Laboratories, Westbrook, ME), a commercial ELISA for the measurement of fPLI concentration, and re-evaluate its reference interval and decision threshold for diagnosing pancreatitis in cats. METHODS: Dilutional linearity, accuracy, precision, and the effect of interfering substances were assessed. The upper limit of the reference interval was calculated based on the 95th percentile of results from clinically healthy cats (n = 107), and a decision threshold for diagnosing pancreatitis was calculated with an expected specificity of 99%. RESULTS: Analytical validation demonstrated good linearity, accuracy, and precision, as well as the absence of interference from lipemia, hemolysis, or icterus. The upper limit of the reference interval for Spec fPL was determined to be 4.4 µg/L, and the decision threshold (a theoretical cut-off) for diagnosing pancreatitis was determined to be 8.8 µg/L based on a desired specificity of 99%. CONCLUSIONS: The Spec fPL assay is analytically valid, and results suggest that a decision threshold of 8.8 µg/L would have high diagnostic specificity for excluding clinically healthy cats.

Quantitative bioimaging of copper in frozen liver specimens from cats using laser ablation-inductively coupled plasma-mass spectrometry.

OBJECTIVES: The aim of this study was to assess laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as a tool for measuring concentrations and determining accumulation of copper in frozen liver specimens from cats. METHODS: Six frozen liver specimens were evaluated by qualitative copper staining and quantitative flame atomic absorption spectroscopy. Tissue specimens were cryo-sectioned and quantitative bioimaging of copper was performed using LA-ICP-MS. Results were compared with those obtained using conventional methods. RESULTS: Of the six specimens, only one showed positive staining for copper with rhodanine. Using flame atomic absorption spectroscopy (FAAS), one specimen showed a deficient copper level (<100 µg/g dry weight), two specimens had copper within the reference interval (RI; 150-180 µg/g) and three specimens had copper concentrations above the RI. Bioimaging from LA-ICP-MS showed inhomogeneous distribution of hepatic copper. The areas with dense copper accumulation were represented as hotspots in the liver specimens. Hepatic copper quantification by LA-ICP-MS correlated well with copper quantified by FAAS (r = 0.96, P = 0.002). CONCLUSIONS AND RELEVANCE: Our findings suggest that quantitative bioimaging by LA-ICP-MS could be used to demonstrate the distribution and concentration of copper in frozen liver specimens from cats. The distribution of copper in these specimens was inhomogeneous with dense accumulation represented as hotspots on tissue sections. A positive correlation of hepatic copper concentrations determined by LA-ICP-MS and FAAS was found. Further studies to establish an RI for hepatic copper using this technique and to further determine its clinical utility are warranted.

Prevalence of Clostridioides difficile in Canine Feces and Its Association with Intestinal Dysbiosis.

The role of Clostridioides (C.) difficile as an enteropathogen in dogs is controversial. In humans, intestinal bile acid-dysmetabolism is associated with C. difficile prevalence. The relationship between fecal qPCR-based dysbiosis index (DI) and especially the abundance of bile acid-converting Clostridium hiranonis with the presence of C. difficile in dogs was explored across the following 4 cohorts: 358 fecal samples submitted for routine diagnostic work-up, 33 dogs with chronic enteropathy, 14 dogs with acute diarrhea, and 116 healthy dogs. Dogs that tested positive for C. difficile had significantly higher DI (median, 4.4 (range from 0.4 to 8.6)) and lower C. hiranonis (median, 0.1 (range from 0.0 to 7.5) logDNA/g) than dogs that tested negative for C. difficile (median DI, -1 (range from -7.2 to 8.9); median C. hiranonis abundance, 6.2 (range from 0.1 to 7.5) logDNA/g; p < 0.0001, respectively). In 33 dogs with CE and 14 dogs with acute diarrhea, the treatment response did not differ between C. difficile-positive and -negative dogs. In the group of clinically healthy dogs, 9/116 tested positive for C. difficile, and 6/9 of these had also an abnormal DI. In conclusion, C. difficile is strongly linked to intestinal dysbiosis and lower C. hiranonis levels in dogs, but its presence does not necessitate targeted treatment.

Serum and Fecal 3-Bromotyrosine Concentrations in Dogs with Chronic Inflammatory Enteropathy: Clinical Parameters and Histopathological Changes.

Chronic inflammatory enteropathies (CIEs) in dogs involve the infiltration of gastrointestinal tissue with inflammatory cells. This study aimed to assess the sensitivity of serum and fecal 3-bromotyrosine (3-BrY) concentrations in dogs with CIE. The difference in 3-BrY concentrations in dogs with different gastrointestinal (GI) pathological changes was also assessed. In total, 68 dogs with CIE were enrolled in the study. The median serum 3-BrY concentration was 3.3 µmol/L, while the median 3-day mean and maximum fecal 3-BrY concentrations were 38.9 and 63.2 mmol/g of feces, respectively. The median serum C-reactive protein concentration was 45.0 mg/L. The median 3-day mean and maximum fecal α1-proteinase inhibitor concentrations were 6.1 and 9 µg/g of feces, respectively. Increased 3-BrY concentrations were observed in 90.9% of CIE dogs based on serum concentrations, 75.8% based on mean fecal concentrations, and 69.4% based on maximum fecal concentrations. A weak correlation (ρ = 0.31, p < 0.0118) was found between serum CRP and serum 3-BrY concentrations. There was no correlation between the canine chronic enteropathy clinical activity index and serum or fecal 3-BrY concentrations (p > 0.05). Additionally, no significant difference in serum or fecal 3-BrY concentrations was found among CIE dogs with different GI pathological changes (p > 0.05). In conclusion, dogs with CIE have increased 3-BrY concentrations in serum and fecal samples. However, 3-BrY concentrations may not accurately indicate the severity of gastrointestinal inflammation.