The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea.

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD.

Safety profile and effects on the peripheral immune response of fecal microbiota transplantation in clinically healthy dogs.

BACKGROUND: Fecal microbiota transplantation (FMT) is increasingly used for gastrointestinal and extra-gastrointestinal diseases in veterinary medicine. However, its effects on immune responses and possible adverse events have not been systematically investigated. HYPOTHESIS/OBJECTIVES: Determine the short-term safety profile and changes in the peripheral immune system after a single FMT administration in healthy dogs. ANIMALS: Ten client-owned, clinically healthy dogs as FMT recipients, and 2 client-owned clinically healthy dogs as FMT donors. METHODS: Prospective non-randomized clinical trial. A single rectal enema of 5 g/kg was given to clinically healthy canine recipients. During the 28 days after FMT administration, owners self-reported adverse events and fecal scores. On Days 0 (baseline), 1, 4, 10, and 28 after FMT, fecal and blood samples were collected. The canine fecal dysbiosis index (DI) was calculated using qPCR. RESULTS: No significant changes were found in the following variables: CBC, serum biochemistry, C-reactive protein, serum cytokines (interleukins [IL]-2, -6, -8, tumor necrosis factor [TNF]-α), peripheral leukocytes (B cells, T cells, cluster of differentiation [CD]4+ T cells, CD8+ T cells, T regulatory cells), and the canine DI. Mild vomiting (n = 3), diarrhea (n = 4), decreased activity (n = 2), and inappetence (n = 1) were reported, and resolved without intervention. CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal microbiota transplantation did not significantly alter the evaluated variables and recipients experienced minimal adverse events associated with FMT administration. Fecal microbiota transplantation was not associated with serious adverse events, changes in peripheral immunologic variables, or the canine DI in the short-term.

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.

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.

Fecal supernatants from dogs with idiopathic epilepsy activate enteric neurons.

Introduction: Alterations in the composition and function of the gut microbiome have been reported in idiopathic epilepsy (IE), however, interactions of gut microbes with the enteric nervous system (ENS) in this context require further study. This pilot study examined how gastrointestinal microbiota (GIM), their metabolites, and nutrients contained in intestinal contents communicate with the ENS. Methods: Fecal supernatants (FS) from healthy dogs and dogs with IE, including drug-naïve, phenobarbital (PB) responsive, and PB non-responsive dogs, were applied to cultured myenteric neurons to test their activation using voltage-sensitive dye neuroimaging. Additionally, the concentrations of short-chain fatty acids (SCFAs) in the FS were quantified. Results: Our findings indicate that FS from all examined groups elicited neuronal activation. Notably, FS from PB non-responsive dogs with IE induced action potential discharge in a higher proportion of enteric neurons compared to healthy controls, which exhibited the lowest burst frequency overall. Furthermore, the highest burst frequency in enteric neurons was observed upon exposure to FS from drug-naïve dogs with IE. This frequency was significantly higher compared to that observed in PB non-responsive dogs with IE and showed a tendency to surpass that of healthy controls. Discussion: Although observed disparities in SCFA concentrations across the various FS samples might be associated with the induced neuronal activity, a direct correlation remains elusive at this point. The obtained results hint at an involvement of the ENS in canine IE and set the basis for future studies.

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.

Temporal Variability of the Dominant Fecal Microbiota in Healthy Adult Cats.

While shifts in gut microbiota have been studied in diseased states, the temporal variability of the microbiome in cats has not been widely studied. This study investigated the temporal variability of the feline dysbiosis index (DI) and the abundance of core bacterial groups in healthy adult cats. The secondary aim was to evaluate the relationship between the fecal abundance of Clostridium hiranonis and the fecal concentrations of unconjugated bile acids. A total of 142 fecal samples collected from 17 healthy cats were prospectively included: nine cats with weekly collection over 3 weeks (at least four time points), five cats with monthly collection over 2 months (three time points), and three cats with additional collections for up to 10 months. The DI remained stable within the reference intervals over two months for all cats (Friedman test, p > 0.2), and 100% of the DI values (n = 142) collected throughout the study period remained within the RI. While some temporal individual variation was observed for individual taxa, the magnitude was minimal compared to cats with chronic enteropathy and antibiotic exposure. Additionally, the abundance of Clostridium hiranonis was significantly correlated with the percentage of fecal primary bile acids, supporting its role as a bile acid converter in cats.

Giardiasis and diarrhea in dogs: Does the microbiome matter?

BACKGROUND: Giardia duodenalis (Gd) causes intestinal parasitosis. The involvement of the intestinal microbiome in determining the infection's clinical phenotype is unknown. OBJECTIVE: Investigate the fecal microbiome features in dogs with giardiasis. ANIMALS AND METHODS: Cross-sectional study, including fecal samples of kenneled dogs with Gd diagnosed by fecal Giardia antigen dot ELISA. The fecal microbial compositional characteristics and dysbiosis index (DI) were compared between diarrheic and nondiarrheic dogs. RESULTS: Fecal samples of 38 Gd-infected dogs (diarrheic, 21; nondiarrheic, 17) were included. No differences were found in Faith's phylogenic diversity and beta diversity (weighted UniFrac distances) and in specific taxa abundances at the phylum, genus, and species levels, as well as in alpha and beta diversities between diarrheic and nondiarrheic dogs, and also when divided by sex or age. Among diarrheic dogs, alpha diversity was higher in males than in females (pairwise Kruskal-Wallis, q = 0.01). Among males, fecal abundances of the genus Clostridium (W = 19) and Clostridium spiroforme species (W = 33) were higher in diarrheic compared to nondiarrheic dogs. In diarrheic dog fecal samples, Proteobacteria were more prevalent (W = 1), whereas Verrucomicrobia were less prevalent in dogs <1 year of age than in older dogs. The fecal sample DI of 19 diarrheic and 19 nondiarrheic dogs was similar (median, -0.2; range, -4.3 to 4.5 and median, -1.0; range, -4.3 to 5.8, respectively). CONCLUSIONS: The fecal microbial composition of symptomatic and asymptomatic dogs with giardiasis is similar. Based on fecal DI, giardiasis is not characterized by prominent dysbiosis. Other host and parasite characteristics might determine the severity of giardiasis in dogs.

Gut microbiome responds to alteration in female sex hormone status and exacerbates metabolic dysfunction.

Women are at significantly greater risk of metabolic dysfunction after menopause, which subsequently leads to numerous chronic illnesses. The gut microbiome is associated with obesity and metabolic dysfunction, but its interaction with female sex hormone status and the resulting impact on host metabolism remains unclear. Herein, we characterized inflammatory and metabolic phenotypes as well as the gut microbiome associated with ovariectomy and high-fat diet feeding, compared to gonadal intact and low-fat diet controls. We then performed fecal microbiota transplantation (FMT) using gnotobiotic mice to identify the impact of ovariectomy-associated gut microbiome on inflammatory and metabolic outcomes. We demonstrated that ovariectomy led to greater gastrointestinal permeability and inflammation of the gut and metabolic organs, and that a high-fat diet exacerbated these phenotypes. Ovariectomy also led to alteration of the gut microbiome, including greater fecal ß-glucuronidase activity. However, differential changes in the gut microbiome only occurred when fed a low-fat diet, not the high-fat diet. Gnotobiotic mice that received the gut microbiome from ovariectomized mice fed the low-fat diet had greater weight gain and hepatic gene expression related to metabolic dysfunction and inflammation than those that received intact sham control-associated microbiome. These results indicate that the gut microbiome responds to alterations in female sex hormone status and contributes to metabolic dysfunction. Identifying and developing gut microbiome-targeted modulators to regulate sex hormones may be useful therapeutically in remediating menopause-related diseases.

A Comparison of the Oral Microbiota in Healthy Dogs and Dogs with Oral Tumors.

The aim of this study was to further describe the oral microbiota of healthy dogs by DNA shotgun sequencing and compare those to dogs with oral tumors. Oral swabs (representative of all niches of the oral cavity) were collected from healthy dogs (n = 24) and from dogs with different oral tumors (n = 7). DNA was extracted from the swabs and shotgun metagenomic sequencing was performed. Only minor differences in microbiota composition were observed between the two groups. At the phylum level, the Bacteroidota, Proteobacteria, Actinobacteriota, Desulfobacterota and Firmicutes were most abundant in both groups. Observed Operational Taxonomic Units-OTUs (species richness) was significantly higher in the healthy patients, but there was no significant difference in the Shannon diversity index between the groups. No significant difference was found in beta diversity between the groups. The core oral microbiota consisted of 67 bacterial species that were identified in all 24 healthy dogs. Our study provides further insight into the composition of the oral microbiota of healthy dogs and in dogs with oral tumors.

High-Dose Hydrocortisone Treatment Does Not Affect Serum C-Reactive Protein (CRP) Concentrations in Healthy Dogs.

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.

Alterations of the bacterial ocular surface microbiome are found in both eyes of horses with unilateral ulcerative keratitis.

Next generation sequencing (NGS) studies in healthy equine eyes have shown a more diverse ocular surface microbiota compared to culture-based techniques. This study aimed to compare the bacterial ocular surface microbiota in both eyes of horses with unilateral ulcerative keratitis (UK) with controls free of ocular disease. Conjunctival swabs were obtained from both ulcerated eyes and unaffected eyes of 15 client-owned horses with unilateral UK following informed consent, as well as from one eye of 15 healthy horses. Genomic DNA was extracted from the swabs and sequenced on an Illumina platform using primers that target the V4 region of bacterial 16S rRNA. Data were analyzed using Quantitative Insights Into Molecular Ecology (QIIME2). The ocular surface of ulcerated eyes had significantly decreased species richness compared with unaffected fellow eyes (Chao1 q = 0.045, Observed ASVs p = 0.045) with no differences in evenness of species (Shannon q = 0.135). Bacterial community structure was significantly different between either eye of horses with UK and controls (unweighted UniFrac: control vs. unaffected, p = 0.03; control vs. ulcerated, p = 0.003; unaffected vs. ulcerated, p = 0.016). Relative abundance of the gram-positive taxonomic class, Bacilli, was significantly increased in ulcerated eyes compared with controls (q = 0.004). Relative abundance of the taxonomic family Staphylococcaceae was significantly increased in ulcerated and unaffected eyes compared with controls (q = 0.030). The results suggest the occurrence of dysbiosis in infected eyes and reveal alterations in beta diversity and taxa of unaffected fellow eyes. Further investigations are necessary to better understand the role of the microbiome in the pathophysiology of ocular surface disease.

Fecal Concentrations of Long-Chain Fatty Acids, Sterols, and Unconjugated Bile Acids in Cats with Chronic Enteropathy.

Chronic enteropathy (CE) in cats encompasses food-responsive enteropathy, chronic inflammatory enteropathy (or inflammatory bowel disease), and low-grade intestinal T-cell lymphoma. While alterations in the gut metabolome have been extensively studied in humans and dogs with gastrointestinal disorders, little is known about the specific metabolic profile of cats with CE. As lipids take part in energy storage, inflammation, and cellular structure, investigating the lipid profile in cats with CE is crucial. This study aimed to measure fecal concentrations of various fatty acids, sterols, and bile acids. Fecal samples from 56 cats with CE and 77 healthy control cats were analyzed using gas chromatography-mass spectrometry, targeting 12 fatty acids, 10 sterols, and 5 unconjugated bile acids. Fecal concentrations of nine targeted fatty acids and animal-derived sterols were significantly increased in cats with CE. However, fecal concentrations of plant-derived sterols were significantly decreased in cats with CE. Additionally, an increased percentage of primary bile acids was observed in a subset of cats with CE. These findings suggest the presence of lipid maldigestion, malabsorption, and inflammation in the gastrointestinal tract of cats with CE. Understanding the lipid alterations in cats with CE can provide insights into the disease mechanisms and potential future therapeutic strategies.

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.

Effect of esomeprazole with and without a probiotic on fecal dysbiosis, intestinal inflammation, and fecal short-chain fatty acid concentrations in healthy dogs.

BACKGROUND: Proton pump inhibitors can cause diarrhea and a transient increase in fecal dysbiosis index in dogs. It is unknown if concurrent probiotic administration mitigates these effects. OBJECTIVE/HYPOTHESIS: To assess the fecal Canine Microbial Dysbiosis Index (CMDI), fecal short chain fatty acid (SCFA), and fecal calprotectin concentrations in dogs administered esomeprazole with and without a probiotic. ANIMALS: Eleven healthy dogs. METHODS: Prospective, within-subjects before and after study. All dogs received 7-day courses of esomeprazole (1 mg/kg PO q 24h) alone followed by esomeprazole with a probiotic (15 billion CFU/kg), separated by a 4-week washout period. Data were compared between phases using mixed effects ANOVA or generalized estimating equations with post-hoc Holm adjustment for 2-way comparisons. RESULTS: Compared to baseline (mean CMDI -2.66, SD 3.04), fecal CMDI was not different with esomeprazole administration alone (mean CMDI -1.48, SD 3.32, P = .08), but there was a significant increase (Diff 3.05, 95% CI [1.37, 4.74], P < .001, Effect size 2.02) when esomeprazole and a probiotic were administered concurrently (mean CMDI 0.39, SD 2.83). CMDI was significantly higher when esomeprazole was administered with a probiotic than alone (Diff 1.87, 95% CI [0.19, 1.87], P = .02, Effect size 1.24). Fecal calprotectin and SCFA concentrations did not differ between phases. The occurrence of vomiting and diarrhea was not different from baseline when esomeprazole was administered alone (36%/27%) or with a probiotic (46%/9%). CONCLUSIONS AND CLINICAL IMPORTANCE: In healthy dogs, concurrent administration of a probiotic is unlikely to lessen adverse effects associated with esomeprazole administration.

Age-associated changes in intestinal health biomarkers in dogs.

The gut microbiome is critical for maintaining host health. In healthy humans, the aging process is one of the main factors modulating the changes in the intestinal microbiota. However, little is known about the relationship between gut health, microbiota, and the aging process in dogs. The present study aims to explore the differences in the intestinal microbiota and intestinal health based on fecal biomarkers in a population of dogs of different ages. The study involved 106 dogs of different breeds aged between 0.2 and 15 years categorized as senior (>7 years; n = 40), adult (2-7 years; n = 50), and junior (< 2 years; n = 16). Fecal samples were collected during the same period at the same facilities. The analysis included the following gut health indicators: 16S rRNA gene sequencing to investigate the differences in the fecal microbiota; qPCR to determine the dysbiosis index; fecal short-chain fatty acid concentrations; fecal calprotectin; and immunoglobulin A. Beta diversity analysis revealed a significant difference with a small effect size (p = 0.003; R = 0.087) among age categories based on the unweighted UniFrac metric, but no significance was observed based on the weighted UniFrac metric or Bray-Curtis distances. There were no significant differences in the alpha diversity measures or the fecal dysbiosis index among age categories. Senior dogs had significantly higher relative abundance proportions in phyla Bacteroidota and Pseudomonadota and the genus Faecalibacterium, but not on qPCR analysis. At the family level, Ruminococcaceae, Uncl. Clostridiales.1, Veillonellaceae, Prevotellaceae, Succinivibrionaceae, and Bacteroidaceae abundances were higher in the senior category than in the adult and/or junior categories. Relative proportions, but not concentrations of fecal acetate, were higher in the senior category, while butyrate, isovaleric acid, and valeric acid were lower. The valeric acid concentration was significantly lower in the senior category than in the adult category. Calprotectin and immunoglobulin A levels did not differ significantly across groups. In conclusion, this study observed multiple minor changes in the fecal microbiota composition and the relative amount of short-chain fatty acids in dogs among different age groups, but studies in larger populations representative of all ages are warranted to refine the present results.

Effect of the Probiotic Bacillus subtilis DE-CA9TM on Fecal Scores, Serum Oxidative Stress Markers and Fecal and Serum Metabolome in Healthy Dogs.

BACKGROUND: There is increasing interest in the use of Bacillus species as probiotics since their spore-forming ability favors their survival in the acidic gastric environment over other probiotic species. The subsequent germination of B. subtilis to their vegetative form allows for their growth in the small intestine and may increase their beneficial effect on the host. B. subtilis strains have also previously been shown to have beneficial effects in humans and production animals, however, no reports are available so far on their use in companion animals. STUDY DESIGN: The goal of this study was therefore to investigate the daily administration of 1 × 109 cfu DE-CA9TM orally per day versus placebo on health parameters, fecal scores, fecal microbiome, fecal metabolomics, as well as serum metabolomics and oxidative stress markers in ten healthy Beagle dogs in a parallel, randomized, prospective, placebo-controlled design over a period of 45 days. RESULTS: DE-CA9TM decreased the oxidative status compared to controls for advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS) and reactive oxygen metabolites (d-ROMS), suggesting an antioxidant effect of the treatment. Fecal metabolomics revealed a significant reduction in metabolites associated with tryptophan metabolism in the DE-CA9TM-treated group. DE-CA9TM also significantly decreased phenylalanine and homocysteine and increased homoserine and threonine levels. Amino acid metabolism was also affected in the serum metabolome, with increased levels of urea and cadaverine, and reductions in N-acetylornithine in DE-CA9TM compared to controls. Similarly, changes in essential amino acids were observed, with a significant increase in tryptophan and lysine levels and a decrease in homocysteine. An increase in serum guanine and deoxyuridine was also detected, with a decrease in beta-alanine in the animals that ingested DE-CA9TM. CONCLUSIONS: Data generated throughout this study suggest that the daily administration of 1 × 109 cfu of DE-CA9TM in healthy Beagle dogs is safe and does not affect markers of general health and fecal scores. Furthermore, DE-CA9TM administration had a potential positive effect on some serum markers of oxidative stress, and protein and lipid metabolism in serum and feces.

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, 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.