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.

Correlation between Peptacetobacter hiranonis, the baiCD Gene, and Secondary Bile Acids in Dogs.

Bile acid metabolism is a key pathway modulated by intestinal microbiota. Peptacetobacter (Clostridium) hiranonis has been described as the main species responsible for the conversion of primary into secondary fecal unconjugated bile acids (fUBA) in dogs. This multi-step biochemical pathway is encoded by the bile acid-inducible (bai) operon. We aimed to assess the correlation between P. hiranonis abundance, the abundance of one specific gene of the bai operon (baiCD), and secondary fUBA concentrations. In this retrospective study, 133 fecal samples were analyzed from 24 dogs. The abundances of P. hiranonis and baiCD were determined using qPCR. The concentration of fUBA was measured by gas chromatography-mass spectrometry. The baiCD abundance exhibited a strong positive correlation with secondary fUBA (ρ = 0.7377, 95% CI (0.6461, 0.8084), p < 0.0001). Similarly, there was a strong correlation between P. hiranonis and secondary fUBA (ρ = 0.6658, 95% CI (0.5555, 0.7532), p < 0.0001). Animals displaying conversion of fUBA and lacking P. hiranonis were not observed. These results suggest P. hiranonis is the main converter of primary to secondary bile acids in dogs.

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.

Correlation between Targeted qPCR Assays and Untargeted DNA Shotgun Metagenomic Sequencing for Assessing the Fecal Microbiota in Dogs.

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.

Analytical Validation of an Assay for Concurrent Measurement of Amino Acids in Dog Serum and Comparison of Amino Acid Concentrations between Whole Blood, Plasma, and Serum from Dogs.

Amino acids play an important role in metabolism. Comprehensive analytical validation of an assay for the concurrent measurement of a large number of amino acids in dogs is lacking, which precludes its usefulness in a clinical setting. Amino acids are often measured in plasma or whole blood. However, serum is commonly used for gastrointestinal diagnostic testing in dogs and is therefore convenient to use. This study aimed to analytically validate an assay for the concurrent measurement of amino acids in dog serum and to evaluate differences in amino acid concentrations in whole blood, plasma, and serum in dogs. Analytical validation of the assay (Biochrom 30+ Amino Acid Analyzer) was performed on fresh or banked serum samples from dogs. Whole blood, plasma, and serum from 36 healthy dogs were analyzed, and concentrations of the three sample types were compared. The assay was demonstrated to be precise, reproducible, accurate, linear, and stable for the measurement of the majority of compounds detected in dog serum. Cystine, glutamic acid, and ethanolamine were shown to be unstable at conditions commonly encountered in clinical settings. Significant differences in concentrations were identified between whole blood, plasma, and serum for 33 of 42 compounds. Amino acid profiles in serum and plasma were more similar to each other than to those in whole blood. While some amino acids are present in similar concentrations in whole blood, plasma, and serum, others are highly dependent on the type of biofluid, and measurements warrant strict adherence to sample type-based reference intervals.

Detection of invasive Escherichia coli in dogs with granulomatous colitis using immunohistochemistry.

Granulomatous colitis in dogs can be associated with infection of the colonic mucosa by invasive strains of Escherichia coli. To date, fluorescence in situ hybridization (FISH) is the gold-standard method to assess intramucosal and intracellular bacterial invasion. However, FISH requires expensive fluorescence microscopy equipment and is therefore not widely available. We investigated the use of immunohistochemistry (IHC) as an alternative method to detect invasive E. coli in dogs with granulomatous colitis. Archived paraffin-embedded blocks were selected from 26 dogs with colitis, in which FISH had been performed by an outside laboratory. Using a polyclonal antibody, IHC for E. coli was performed on sections cut from the same blocks, and the presence of invasive E. coli was recorded. All 11 specimens in which FISH had detected E. coli were also positive on IHC, with strong immunolabeling in the cytoplasm of macrophages and extracellularly in the lamina propria; all 15 specimens that were negative for invasive bacteria on FISH were also negative on IHC. We found that IHC is a sensitive technique for the detection of invasive E. coli in dogs with granulomatous colitis.

The effect of combined carprofen and omeprazole administration on gastrointestinal permeability and inflammation in dogs.

BACKGROUND: Proton pump inhibitors (eg, omeprazole) commonly are administered concurrently with nonsteroidal anti-inflammatory drugs (NSAIDs; eg, carprofen) as prophylaxis to decrease the risk of gastrointestinal (GI) injury. However, evidence to support this practice is weak, and it might exacerbate dysbiosis and inflammation. HYPOTHESIS/OBJECTIVES: To evaluate the effect of carprofen alone or combined with omeprazole in dogs. We hypothesized that coadministration of omeprazole and carprofen would significantly increase GI permeability and dysbiosis index (DI) compared to no treatment or carprofen alone. ANIMALS: Six healthy adult colony beagle dogs. METHODS: Gastrointestinal permeability and inflammation were assessed by serum lipopolysaccharide (LPS) concentration, plasma iohexol concentration, fecal DI, and fecal calprotectin concentration in a prospective, 3-period design. In the first 7-day period, dogs received no intervention (baseline). During the 2nd period, dogs received 4 mg/kg of carprofen q24h PO for 7 days. In the 3rd period, dogs received 4 mg/kg of carprofen q24h and 1 mg/kg of omeprazole q12h PO for 7 days. Gastrointestinal permeability testing was performed at the end of each period. Data were analyzed using repeated measures mixed model analysis of variance with Tukey-Kramer post hoc tests (P < .05). RESULTS: Serum LPS and plasma iohexol concentrations did not differ between treatments. Fecal calprotectin concentrations differed between treatments (P = .03). The DI varied over time based on the treatment received (P = .03). Coadministration of omeprazole and carprofen significantly increased fecal calprotectin concentration and DI compared to baseline and carprofen alone. CONCLUSIONS AND CLINICAL IMPORTANCE: Omeprazole prophylaxis induces fecal dysbiosis and increases intestinal inflammatory markers when coadministered with carprofen to otherwise healthy dogs with no other risk factors for GI bleeding.

Monitoring sheep and Culicoides midges in Montana for evidence of Bunyamwera serogroup virus infection.

INTRODUCTION: A serological and entomological investigation was performed to monitor for potential Bunyamwera (BUN) serogroup virus activity in Montana. RESULTS: To facilitate the serological investigation, sera were collected from 104 sheep in 2013 and 2014 and assayed by plaque reduction neutralization test using all six BUN serogroup viruses known to occur in the United States: Cache Valley virus (CVV), Lokern virus (LOKV), Main Drain virus (MDV), Northway virus, Potosi virus and Tensaw virus. BUN serogroup virus-specific antibodies were detected in 41 (39%) sheep. Of these, three were seropositive for MDV, one was seropositive for CVV, one was seropositive for LOKV and 36 had antibodies to an undetermined BUN serogroup virus. Additionally, 30,606 Culicoides sonorensis were collected in 2013 using Centers for Disease Control and Prevention (CDC) light traps and assayed for cytopathic virus by virus isolation in African Green Monkey kidney (Vero) cells. All midges were negative. Almost one-third of the midges were further tested by reverse transcription-polymerase chain reaction using BUN serogroup virus-reactive primers and all were negative. CONCLUSIONS: We provide evidence of BUN serogroup virus infection in sheep but not C. sonorensis in Montana in 2013-2014. This study also provides the first evidence of CVV, MDV and LOKV activity in Montana.