Stability of 3-bromotyrosine in serum and serum 3-bromotyrosine concentrations in dogs with gastrointestinal diseases.

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.

Long-Term Recovery of the Fecal Microbiome and Metabolome of Dogs with Steroid-Responsive Enteropathy.

The long-term impact of treatment of dogs with steroid-responsive enteropathy (SRE) on the fecal microbiome and metabolome has not been investigated. Therefore, this study aimed to evaluate the fecal microbiome and metabolome of dogs with SRE before, during, and following treatment with standard immunosuppressive therapy and an elimination diet. We retrospectively selected samples from 9 dogs with SRE enrolled in a previous clinical trial, which received treatment for 8 weeks, and had achieved remission as indicated by the post-treatment clinical scores. Long-term (1 year) samples were obtained from a subset (5/9) of dogs. Samples from 13 healthy dogs were included as controls (HC). We evaluated the microbiome using 16S rRNA sequencing and qPCR. To evaluate the recovery of gut function, we measured fecal metabolites using an untargeted approach. While improvement was observed for some bacterial taxa after 8 weeks of treatment, several bacterial taxa remained significantly different from HC. Seventy-five metabolites were altered in dogs with SRE, including increased fecal amino acids and vitamins, suggesting malabsorption as a component of SRE. One year after treatment, however, all bacterial species were evaluated by qPCR and 16S rRNA gene sequencing, and all but thirteen metabolites were no longer different from healthy controls.

Fecal Microbial and Metabolic Profiles in Dogs With Acute Diarrhea Receiving Either Fecal Microbiota Transplantation or Oral Metronidazole.

The aim was to characterize differences in fecal consistency, and fecal microbiota and metabolome profiles in dogs with acute diarrhea (AD) treated with either fecal microbiota transplantation as enema (FMT; n = 11) or oral metronidazole (MET; n = 7) for 7 days. On days 0, 7, and 28 fecal samples were obtained. Fecal samples from healthy dogs (HC; n = 14) were used for comparison. Samples were analyzed by the previously validated qPCR based canine Dysbiosis Index (DI; increased values indicate microbiota dysbiosis) and 16S rRNA gene sequencing. The fecal metabolome was analyzed using a previously validated targeted canine assay for fecal unconjugated bile acids, and untargeted metabolomics. Fecal consistency improved significantly in dogs treated with FMT and MET by day 7 and day 28 (p < 0.01) compared to day 0. However, on day 28 fecal consistency was significantly better in FMT compared to MET (p = 0.040). At day 0, dogs with AD had an altered microbiota indicated by significantly increased DI, decreased alpha-diversity, and altered beta-diversity. In the FMT group, the DI decreased over time, while MET led to a significant increase in the dysbiosis index at day 7 and 28 compared to FMT. Sequencing data revealed that in FMT microbial diversity and beta-diversity was similar to HC at day 28, while in MET these parameters were still significantly different from HC. In dogs treated with FMT, a decrease in cholic acid and the percentage of primary bile acids was observed, whereas treatment with metronidazole led to an increase in cholic acid at day 7 and an increase in percentage of primary bile acids over time. Based on untargeted metabolomics, dogs with AD had an altered fecal metabolome compared to HC. Dogs treated with FMT clustered closer to HC at day 28, while dogs treated with MET did not. In this pilot study, dogs with AD had significant differences in fecal microbiota and metabolome profiles. Dogs treated with MET still had altered microbial and metabolic profiles at day 28 compared to dogs treated with FMT or healthy dogs.

Effects of metronidazole on the fecal microbiome and metabolome in healthy dogs.

BACKGROUND: Metronidazole has a substantial impact on the gut microbiome. However, the recovery of the microbiome after discontinuation of administration, and the metabolic consequences of such alterations have not been investigated to date. OBJECTIVES: To describe the impact of 14-day metronidazole administration, alone or in combination with a hydrolyzed protein diet, on fecal microbiome, metabolome, bile acids (BAs), and lactate production, and on serum metabolome in healthy dogs. ANIMALS: Twenty-four healthy pet dogs. METHODS: Prospective, nonrandomized controlled study. Dogs fed various commercial diets were divided in 3 groups: control group (no intervention, G1); group receiving hydrolyzed protein diet, followed by metronidazole administration (G2); and group receiving metronidazole only (G3). Microbiome composition was evaluated with sequencing of 16S rRNA genes and quantitative polymerase chain reaction (qPCR)-based dysbiosis index. Untargeted metabolomics analysis of fecal and serum samples was performed, followed by targeted assays for fecal BAs and lactate. RESULTS: No changes were observed in G1, or G2 during diet change. Metronidazole significantly changed microbiome composition in G2 and G3, including decreases in richness (P < .001) and in key bacteria such as Fusobacteria (q < 0.001) that did not fully resolve 4 weeks after metronidazole discontinuation. Fecal dysbiosis index was significantly increased (P < .001). Those changes were accompanied by increased fecal total lactate (P < .001), and decreased secondary BAs deoxycholic acid and lithocholic acid (P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Our results indicate a minimum 4-week effect of metronidazole on fecal microbiome and metabolome, supporting a cautious approach to prescription of metronidazole in dogs.

Effects of the Probiotic Mixture Slab51® (SivoMixx®) as Food Supplement in Healthy Dogs: Evaluation of Fecal Microbiota, Clinical Parameters and Immune Function.

The gut microbiota plays a crucial role in several physiologic functions of the host. In humans and animals, manipulation of the intestinal microbiota by oral administration of probiotic lactic acid bacteria plays a significant role in modulating the immune system. The aim of this study was to evaluate the safety of the probiotic mixture Slab51® and the capacity of this mixture to stimulate immune function in healthy dogs. Twenty dogs were divided in two groups and received a control diet or the same diet supplemented with a dose of 400 billion cfu of lyophilized bacteria for a period of 60 days. Body weight, food intake, body condition score (BCS), fecal score (FSS), fecal immunoglobulin IgA concentration, plasma IgG concentration, and fecal microbiota composition were monitored. Weight, food intake, BCS, FSS, and biochemical parameters remained unchanged during the treatment in both groups of animals. The fecal microbiota showed a significant decrease in the abundance of Clostridium perfringens and a significant increase in the abundance of beneficial Bifidobacterium and Lactobacillus organisms (p < 0.05). Fecal IgA and plasma IgG levels were significantly higher in the group receiving the probiotic compared to healthy controls. These data show that dietary supplementation with the probiotic mixture Slab51® is safe and well-tolerated, modulating the composition of the intestinal microbiota, and enhancing specific immune functions in healthy dogs.

Cholestyramine decreases apparent total tract macronutrient digestibility and alters fecal characteristics and metabolites of healthy adult dogs.

Absorption of dietary lipids in the small intestine is dependent on the emulsification by bile acids (BA) and the formation of chylomicrons. Cholestyramine is a common drug used in humans-and potentially dogs-to treat BA malabsorption associated with chronic diarrhea. It is known to bind BA to form insoluble complexes, preventing their reabsorption and possibly proper emulsification and absorption of dietary fats. The objective of this study was to evaluate the effects of cholestyramine on 1) macronutrient apparent total tract digestibility (ATTD), and 2) fecal characteristics and metabolites of healthy adult dogs. We hypothesized that cholestyramine would decrease ATTD of fat and organic matter (OM), increase fecal dry matter (DM) content, and increase fecal output. Twelve healthy beagles (3.2 ± 0.8 yr; 10.4 ± 0.9 kg) were used in a randomized crossover design. All procedures were approved by the University of Illinois Institutional Animal Care and Use Committee before the study. The study included a baseline period and two 14-d experimental periods separated by a 14-d washout. All dogs were fed the same experimental diet, formulated to meet all nutrient needs recommended by AAFCO, throughout the study. Dogs were randomized into 2 groups [diet only (control) or diet + 11.4 g/d cholestyramine (8 g/d active ingredient)] in Period 1 and received the other treatment in Period 2. During the washout, all dogs were fed the diet only. Dogs were fed once daily (0800 h) to maintain BW. Total fecal output was collected during the last 4 d of each period for ATTD analysis. On day 14 of each of period, fresh fecal and blood samples were collected for metabolite analysis. Dogs fed cholestyramine had lower (P < 0.001) ATTD of DM, OM, energy, crude protein, and fat and lower (P < 0.01) fecal scores (firmer stools) than controls. Dogs fed cholestyramine had greater (P < 0.01) as-is and dry fecal output than controls. Dogs fed cholestyramine had lower (P < 0.05) fecal ammonia and phenol concentrations, but greater (P < 0.05) fecal indole, acetate, butyrate, and total short-chain fatty acid concentrations than controls. Fecal DM% and pH were greater (P < 0.01) in dogs fed cholestyramine. Our results indicate that cholestyramine, when given with a meal, is safe and well tolerated but significantly decreases nutrient digestibility and alters fecal characteristics. Future studies are required to explore the effects of cholestyramine on dogs with gastrointestinal disease.

Altered microbiota, fecal lactate, and fecal bile acids in dogs with gastrointestinal disease.

The intestinal microbiota plays an important role in health and disease and produces, through fermentative reactions, several metabolic products, such as lactate, that can affect the host. The microbiota also interacts with and metabolizes compounds produced by the host, such as primary bile acids. Lactate and bile acids (BA) are of particular interest in gastrointestinal diseases because they have been associated with metabolic acidosis and bile acid diarrhea, respectively. The objectives of this study were to validate an enzymatic assay to quantify D-, L-, and total lactate in canine feces, and to characterize fecal lactate and BA concentrations as well as bacterial abundances in healthy dogs and dogs with gastrointestinal diseases. Fecal samples were collected from 34 healthy dogs, 15 dogs with chronic enteropathy (CE), and 36 dogs with exocrine pancreatic insufficiency (EPI). Lactate was quantified with an enzymatic assay, BA with gas chromatography-mass spectrometry, and 11 bacterial groups with qPCR. A fecal lactate reference interval was established from 34 healthy dogs and was 0.7-1.4 mM, 0.3-6.0 mM, and 1.0-7.0 mM for D-, L-, and total lactate, respectively. The assay to measure D-, L-, and total lactate in canine fecal samples was linear, accurate, precise, and reproducible. Significant increases in fecal lactate and decreases in secondary BA concentrations were observed in dogs with CE and dogs with EPI. Dogs with EPI had an increased abundance of Escherichia coli, Lactobacillus, and Bifidobacterium; a decreased abundance of Fusobacterium and Clostridium hiranonis; and a higher Dysbiosis Index when compared to healthy dogs. Further studies are necessary to determine the clinical utility of lactate and BA quantification in canine feces. These metabolites suggest functional alterations of intestinal dysbiosis and may become promising targets for further elucidating the role of the microbiota in health and disease.

Untargeted metabolomic profiling of urine from healthy dogs and dogs with chronic hepatic disease.

Chronic hepatic disease can present a diagnostic challenge with different etiologies being associated with similar clinical and laboratory findings. The histopathological assessment of a liver biopsy specimen is usually required in order to make a definitive diagnosis and the availability of non-invasive prognostic biomarkers is limited. The emerging science of metabolomics is used to detect changes in endogenous low molecular weight metabolites in biological samples and offers the possibility of identifying noninvasive markers of disease. The objective of this study was to investigate differences in the urine metabolome between healthy dogs, dogs with chronic hepatitis, dogs with hepatocellular carcinoma, and dogs with a congenital portosystemic shunt. Stored urine samples from 10 healthy dogs, 10 dogs with chronic hepatitis, 6 dogs with hepatocellular carcinoma, and 5 dogs with a congenital portosystemic shunt were analyzed. The urine metabolome was analyzed by gas chromatography-quadrupole time of flight mass spectrometry and 220 known metabolites were identified. Principal component analysis and heat dendrogram plots of the metabolomics data showed clustering between groups. Random forest analysis showed differences in the abundance of various metabolites including putrescine, gluconic acid, sorbitol, and valine. Based on univariate statistics, 37 metabolites were significantly different between groups. In, conclusion, the urine metabolome varies between healthy dogs, dogs with chronic hepatitis, dogs with hepatocellular carcinoma, and dogs with a congenital portosystemic shunt. Further targeted assessment of these metabolites is needed to assess their diagnostic utility.

Administration of a Synbiotic Containing Enterococcus faecium Does Not Significantly Alter Fecal Microbiota Richness or Diversity in Dogs With and Without Food-Responsive Chronic Enteropathy.

Background: Canine chronic enteropathies (CE) are a group of intestinal diseases that can be categorized based on treatment response into diet- or food- responsive enteropathy (FRD), antibiotic-responsive enteropathy, steroid-responsive enteropathy, and non-responsive enteropathy. CE can often be associated with intestinal dysbiosis and thus administration of probiotic or synbiotic products may provide a useful tool for the management of some of these patients. Enterococcus faecium (EF) is a probiotic strain included in a commercially available synbiotic for small animals, however its impact on the microbial communities in dogs with FRD has not yet been evaluated. Hypothesis/Objectives: The administration of a synbiotic will lead to a significant difference of the fecal microbial composition and/or diversity in dogs with FRD, and these changes are not attributable to diet change alone. Animals/Samples: Twelve dogs with FRD fed a hydrolyzed protein diet received either a synbiotic (EF, fructooligosaccharides, gum Arabic) or placebo. Fecal samples were taken before and 6 weeks into treatment. Fecal samples were also acquired from 8 healthy dogs before and 6 weeks after being switched to the same hydrolyzed protein diet as their sole food. Methods: Bacterial DNA was extracted from fecal samples and next generation sequencing based on the 16S rRNA genes was performed. Microbial composition and diversity between groups were compared using QIIME. Results: There was a small increase in species diversity in the feces of dogs with FRD treated with synbiotics. However, there were no significant differences in microbial community composition before and after 6 weeks in either the synbiotic or placebo treated dogs with FRD or the healthy dogs. In all groups, large individual variations were observed. Conclusions: No changes in microbial composition were observed in diseased or healthy dogs with diet change alone. However, administration of a synbiotic increased bacterial richness in both groups.

Microbiota-Related Changes in Unconjugated Fecal Bile Acids Are Associated With Naturally Occurring, Insulin-Dependent Diabetes Mellitus in Dogs.

Diabetes mellitus (DM) in humans has recently been associated with altered intestinal microbiota. The consequences of intestinal dysbiosis, such as increased intestinal permeability and altered microbial metabolites, are suspected to contribute to the host inflammatory state and peripheral insulin resistance. Human diabetics have been shown to have changes in bile acid (BA) metabolism which may be detrimental to glycemic control. The purpose of this study was to examine BA metabolism in dogs with naturally-occurring, insulin-dependent DM and to relate these findings to changes in the intestinal microbiota. A prospective observational study of adult dogs with a clinical diagnosis of DM (n = 10) and healthy controls (HC, n = 10) was performed. The fecal microbiota were analyzed by 16S rRNA gene next-generation (Illumina) sequencing. Concentrations of fecal unconjugated BA (fUBA) were measured using gas chromatography and mass spectrometry. Analysis of bacterial communities showed no significant difference for any of the alpha-diversity measures between DM vs. HC dogs. Principal coordinate analysis based on unweighted Unifrac distance metric failed to show significant clustering between dog groups (ANOSIMUnweighted: R = 0.084; p = 0.114). However, linear discriminate analysis effects size (LEfSe) detected differentially abundant bacterial taxa (α = 0.01, LDA score >2.0) on various phylogenetic levels. While Enterobacteriaceae was overrepresented in dogs with DM, the proportions of Erysipelotrichia, Mogibacteriaceae, and Anaeroplasmataceae were increased in HC dogs. Dogs with DM had increased concentration of total primary fUBA compared to HC dogs (p = 0.028). The concentrations of cholic acid and the cholic acid percentage of the total fUBA were increased (p = 0.028 and p = 0.035, respectively) in the feces of DM dogs relative to HC dogs. The levels of lithocholic acid (both absolute value and percentage of the total fUBA) were decreased (p = 0.043 and p < 0.01, respectively) in DM dogs vs. HC dogs. Results indicate that dogs with DM have both intestinal dysbiosis and associated fUBA alterations. The pattern of dysbiosis and altered BA composition is similar to that seen in humans with Type 2 DM. The dog represents a novel large animal model for advancing translational medicine research efforts (e.g., investigating pathogenesis and therapeutics) in DM affecting humans.

Longitudinal assessment of microbial dysbiosis, fecal unconjugated bile acid concentrations, and disease activity in dogs with steroid-responsive chronic inflammatory enteropathy.

BACKGROUND: Mounting evidence from human studies suggests that bile acid dysmetabolism might play a role in various human chronic gastrointestinal diseases. It is unknown whether fecal bile acid dysmetabolism occurs in dogs with chronic inflammatory enteropathy (CE). OBJECTIVE: To assess microbial dysbiosis, fecal unconjugated bile acids (fUBA), and disease activity in dogs with steroid-responsive CE. ANIMALS: Twenty-four healthy control dogs and 23 dogs with steroid-responsive CE. METHODS: In this retrospective study, fUBA were measured and analyzed. Fecal microbiota were assessed using a dysbiosis index. The canine inflammatory bowel disease activity index was used to evaluate remission of clinical signs. This was a multi-institutional study where dogs with steroid-responsive CE were evaluated over time. RESULTS: The dysbiosis index was increased in dogs with CE (median, 2.5; range, -6.2 to 6.5) at baseline compared with healthy dogs (median, -4.5; range, -6.5 to -2.6; P = .002) but did not change in dogs with CE over time. Secondary fUBA were decreased in dogs with CE (median, 29%; range, 1%-99%) compared with healthy dogs (median, 88%; 4%-96%; P = .049). The percent of secondary fUBA in dogs with CE increased from baseline values (median, 28%; range, 1%-99%) after 2-3 months of treatment (median, 94%; range, 1%-99%; P = 0.0183). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings suggest that corticosteroids regulate fecal bile acids in dogs with CE. Additionally, resolution of clinical activity index in dogs with therapeutically managed CE and bile acid dysmetabolism are likely correlated. However, subclinical disease (i.e., microbial dysbiosis) can persist in dogs with steroid-responsive CE.

The fecal microbiome and metabolome differs between dogs fed Bones and Raw Food (BARF) diets and dogs fed commercial diets.

INTRODUCTION: Feeding a Bones and Raw Food (BARF) diet has become an increasing trend in canine nutrition. Bones and Raw Food diets contain a high amount of animal components like meat, offal, and raw meaty bones, combined with comparatively small amounts of plant ingredients like vegetables and fruits as well as different sorts of oil and supplements. While many studies have focused on transmission of pathogens via contaminated meat and on nutritional imbalances, only few studies have evaluated the effect of BARF diets on the fecal microbiome and metabolome. The aim of the study was to investigate differences in the fecal microbiome and the metabolome between dogs on a BARF diet and dogs on a commercial diet (canned and dry dog food). METHODS: Naturally passed fecal samples were obtained from 27 BARF and 19 commercially fed dogs. Differences in crude protein, fat, fiber, and NFE (Nitrogen-Free Extract) between diets were calculated with a scientific nutrient database. The fecal microbiota was analyzed by 16S rRNA gene sequencing and quantitative PCR assays. The fecal metabolome was analyzed in 10 BARF and 9 commercially fed dogs via untargeted metabolomics approach. RESULTS: Dogs in the BARF group were fed a significantly higher amount of protein and fat and significantly lower amount of NFE and fiber. There was no significant difference in alpha-diversity measures between diet groups. Analysis of similarity (ANOSIM) revealed a significant difference in beta-diversity (p < 0.01) between both groups. Linear discriminant analysis effect size (LefSe) showed a higher abundance of Lactobacillales, Enterobacteriaceae, Fusobacterium and, Clostridium in the BARF group while conventionally fed dogs had a higher abundance of Clostridiaceae, Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae. The qPCR assays revealed significantly higher abundance of Escherichia coli (E. coli) and Clostridium (C.). perfringens and an increased Dysbiosis Index in the BARF group. Principal component analysis (PCA) plots of metabolomics data showed clustering between diet groups. Random forest analysis showed differences in the abundance of various components, including increased 4-hydroxybutryric acid (GBH) and 4-aminobutyric acid (GABA) in the BARF group. Based on univariate statistics, several metabolites were significantly different between diet groups, but lost significance after adjusting for multiple comparison. No differences were found in fecal bile acid concentrations, but the BARF group had a higher fecal concentration of cholesterol in their feces compared to conventionally fed dogs. CONCLUSION: Microbial communities and metabolome vary significantly between BARF and commercially fed dogs.

Comparison of intestinal expression of the apical sodium-dependent bile acid transporter between dogs with and without chronic inflammatory enteropathy.

BACKGROUND: Intestinal absorption of bile acids is mediated by the apical sodium-dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). OBJECTIVE: Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. ANIMALS: Twenty-four dogs with CIE and 11 control dogs. METHODS: The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography-mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. RESULTS: In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = -0.40; Pcorr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

Characterization of the fecal microbiome during neonatal and early pediatric development in puppies.

Limited information is available describing the development of the neonatal fecal microbiome in dogs. Feces from puppies were collected at 2, 21, 42, and 56 days after birth. Feces were also collected from the puppies' mothers at a single time point within 24 hours after parturition. DNA was extracted from fecal samples and 454-pyrosequencing was used to profile 16S rRNA genes. Species richness continued to increase significantly from 2 days of age until 42 days of age in puppies. Furthermore, microbial communities clustered separately from each other at 2, 21, and 42 days of age. The microbial communities belonging to dams clustered separately from that of puppies at any given time point. Major phylogenetic changes were noted at all taxonomic levels with the most profound changes being a shift from primarily Firmicutes in puppies at 2 days of age to a co-dominance of Bacteroidetes, Fusobacteria, and Firmicutes by 21 days of age. Further studies are needed to elucidate the relationship between puppy microbiota development, physiological growth, neonatal survival, and morbidity.

Characterization of microbial dysbiosis and metabolomic changes in dogs with acute diarrhea.

Limited information is available regarding the metabolic consequences of intestinal dysbiosis in dogs with acute onset of diarrhea. The aim of this study was to evaluate the fecal microbiome, fecal concentrations of short-chain fatty acids (SCFAs), as well as serum and urine metabolites in healthy dogs (n=13) and dogs with acute diarrhea (n=13). The fecal microbiome, SCFAs, and serum/urine metabolite profiles were characterized by 454-pyrosequencing of the 16S rRNA genes, GC/MS, and untargeted and targeted metabolomics approach using UPLC/MS and HPLC/MS, respectively. Significantly lower bacterial diversity was observed in dogs with acute diarrhea in regards to species richness, chao1, and Shannon index (p=0.0218, 0.0176, and 0.0033; respectively). Dogs with acute diarrhea had significantly different microbial communities compared to healthy dogs (unweighted Unifrac distances, ANOSIM p=0.0040). While Bacteroidetes, Faecalibacterium, and an unclassified genus within Ruminococcaceae were underrepresented, the genus Clostridium was overrepresented in dogs with acute diarrhea. Concentrations of fecal propionic acid were significantly decreased in acute diarrhea (p=0.0033), and were correlated to a decrease in Faecalibacterium (ρ=0.6725, p=0.0332). The predicted functional gene content of the microbiome (PICRUSt) revealed overrepresentations of genes for transposase enzymes as well as methyl accepting chemotaxis proteins in acute diarrhea. Serum concentrations of kynurenic acid and urine concentrations of 2-methyl-1H-indole and 5-Methoxy-1H-indole-3-carbaldehyde were significantly decreased in acute diarrhea (p=0.0048, 0.0185, and 0.0330, respectively). These results demonstrate that the fecal dysbiosis present in acute diarrhea is associated with altered systemic metabolic states.