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Abstract Cardiovascular-kidney-metabolic health reflects the interactions between metabolic risk factors, chronic kidney disease, and the cardiovascular system. A growing body of literature suggests that metabolic syndrome (MetS) in individuals of normal weight is associated with a high prevalence of cardiovascular diseases and an increased mortality. The aim of this study was to establish a non-invasive preclinical model of MetS in support of future research focusing on the effects of novel antidiabetic therapies beyond glucose reduction, independent of obesity. Eighteen healthy adult Beagle dogs were fed an isocaloric Western diet (WD) for ten weeks. Biospecimens were collected at baseline (BAS1) and after ten weeks of WD feeding (BAS2) for measurement of blood pressure (BP), serum chemistry, lipoprotein profiling, blood glucose, glucagon, insulin secretion, NT-proBNP, angiotensins, oxidative stress biomarkers, serum, urine, and fecal metabolomics. Differences between BAS1 and BAS2 were analyzed using non-parametric Wilcoxon signed-rank testing. The isocaloric WD model induced significant variations in several markers of MetS, including elevated BP, increased glucose concentrations, and reduced HDL-cholesterol. It also caused an increase in circulating NT-proBNP levels, a decrease in serum bicarbonate, and significant changes in general metabolism, lipids, and biogenic amines. Short-term, isocaloric feeding with a WD in dogs replicated key biological features of MetS while also causing low-grade metabolic acidosis and elevating natriuretic peptides. These findings support the use of the WD canine model for studying the metabolic effects of new antidiabetic therapies independent of obesity.

ABSTRACTWomen 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 β-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.

Abstract 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

Abstract 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,

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.

Abstract 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

Abstract Background Straight‐ and branched‐chain (BCFA) short‐chain fatty acids (SCFAs) are produced by colonic microbiota and have both beneficial and deleterious effects in humans with chronic kidney disease (CKD). Fecal SCFAs in cats with CKD have not been described. Objective To characterize fecal SCFA concentrations in cats with CKD as compared to healthy geriatric cats and correlate SCFA to serum indoxyl sulfate (IS) and p‐cresol sulfate (pCS) concentrations. Animals Twenty‐eight cats with CKD (International Renal Interest Society [IRIS] stages 2, 3, and 4) and 11 older (≥ 8 years) healthy geriatric cats. Methods Prospective, cross‐sectional study. Voided feces were analyzed using stable isotope dilution gas chromatography‐mass spectrometry to determine fecal concentrations of SCFAs. Serum concentrations of IS and pCS were measured using liquid chromatography tandem mass spectrometry. Results Fecal isovaleric acid concentrations were significantly higher in CKD cats(P = .02) Cats with IRIS CKD stage 3 and 4 had significantly higher fecal isovaleric acid concentrations compared to healthy geriatric cats (P = .03), but not compared to IRIS CKD stage 2 cats. Total fecal concentrations of BCFAs were found to correlate weakly with serum creatinine concentration (rho, 0.33; P = .05), blood urea nitrogen concentration (rho, 0.40; P = .01), and pCS concentration (rho, 0.35; P = .04). Conclusions and Clinical Importance Fecal isovaleric acid concentrations were higher in CKD cats, particularly in late stage disease, compared to healthy geriatric cats. Fecal BCFA concentrations correlated with pCS and were higher in cats with muscle wasting, providing evidence for malassimilation of protein in CKD cats.

The purpose of the study was to quantify serum and fecal amino acids (AA) in cats with chronic kidney disease (CKD) and compare to healthy cats. Thirty-five cats with International Renal Interest Society Stage 1–4 CKD and 16 healthy mature adult and senior client-owned cats were included in this prospective cross-sectional study. Sera were analyzed for 25 AA concentrations using an ion exchange chromatography AA analyzer with post column ninhydrin derivatization. Voided fecal samples were analyzed for 22 AA concentrations using liquid chromatography with tandem mass spectrometry. CKD cats had lower serum concentrations of phenylalanine (mean difference ± standard error of the mean: 12.7 ± 4.3 µM; p = 0.03), threonine (29.6 ± 9.2 µM; p = 0.03), tryptophan (18.4 ± 5.4 µM; p = 0.005), serine (29.8 ± 12.6 µM; p = 0.03), and tyrosine (11.6 ± 3.8 µM; p = 0.01) and higher serum concentrations of aspartic acid (4.7 ± 2.0 µM; p = 0.01), β-alanine (3.4 ± 1.2 µM; p = 0.01), citrulline (5.7 ± 1.6 µM; p = 0.01), and taurine (109.9 ± 29.6 µM; p = 0.01) when compared to healthy cats. Fecal AA concentrations did not differ between healthy cats and CKD cats. 3-Methylhistidine-to-creatinine did not differ between healthy cats with and without muscle loss. Cats with CKD IRIS Stages 1–4 have a deranged serum amino acid profile compared to healthy cats.

Diarrhea is an adverse effect of antimicrobial therapy in horses. This matched, case-controlled study compared the fecal microbiome and metabolome of horses on antibiotics that developed diarrhea (AAD, n = 17) to those that did not develop diarrhea (ABX, n = 15) and to a control population not exposed to antibiotics (CON, n = 31). Fecal samples were collected from horses that were matched for diet and antimicrobial agent (including dose, route, and duration of therapy). Illumina sequencing of 16S rRNA genes was performed, and QIIME 2.0 was used to generate alpha and beta diversity metrics. Untargeted metabolomics using GC-MS platforms was performed and analyzed using Metaboanalyst 5.0. Microbiome composition was significantly different in AAD compared to CON (ANOSIM, R = 0.568, p = 0.001) but not to ABX (ANOSIM, R = 0.121, p = 0.0012). AAD and ABX horses had significantly decreased richness and evenness compared to CON horses (p < 0.05). Horses on antimicrobials (AAD and ABX) had significant changes in 14 phyla compared to CON horses. Only Verrucomicrobia distinguished AAD from ABX and CON horses (q = 0.0005). Metabolite profiles of horses with AAD clustered separately from ABX and CON horses. Seven metabolites were found to be significantly different between groups (p < 0.05): L-tyrosine, kynurenic acid, xanthurenic acid, 5-hydroxyindole-3-acetic acid, docosahexaenoic acid ethyl ester, daidzein, and N-acetyltyramine. Metabolite profiles of horses on antimicrobials, especially those with AAD, are altered compared to CON horses.

Abstract Background Biomarker detection presents itself as a major means of translating biological data into clinical applications. Due to the recent advances in high throughput sequencing technologies, an increased number of metagenomics studies have suggested the dysbiosis in microbial communities as potential biomarker for certain diseases. The reproducibility of the results drawn from metagenomic data is crucial for clinical applications and to prevent incorrect biological conclusions. The variability in the sample size and the subjects participating in the experiments induce diversity, which may drastically change the outcome of biomarker detection algorithms. Therefore, a robust biomarker detection algorithm that ensures the consistency of the results irrespective of the natural diversity present in the samples is needed. Results Toward this end, this paper proposes a novel Regularized Low Rank-Sparse Decomposition (RegLRSD) algorithm. RegLRSD models the bacterial abundance data as a superposition between a sparse matrix and a low-rank matrix, which account for the differentially and non-differentially abundant microbes, respectively. Hence, the biomarker detection problem is cast as a matrix decomposition problem. In order to yield more consistent and solid biological conclusions, RegLRSD incorporates the prior knowledge that the irrelevant microbes do not exhibit significant variation between samples belonging to different phenotypes. Moreover, an efficient algorithm to extract the sparse matrix is proposed. Comprehensive comparisons of RegLRSD with the state-of-the-art algorithms on three realistic datasets are presented. The obtained results demonstrate that RegLRSD consistently outperforms the other algorithms in terms of reproducibility performance and provides a marker list with high classification accuracy. Conclusions The proposed RegLRSD algorithm for biomarker detection provides high reproducibility and classification accuracy performance regardless of the dataset complexity and the number of selected biomarkers. This renders RegLRSD as a reliable and powerful tool for identifying potential metagenomic biomarkers.

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.

In equids, susceptibility to disease caused by Rhodococcus equi occurs almost exclusively in foals. This distribution might be attributable to the age-dependent maturation of immunity following birth undergone by mammalian neonates that renders them especially susceptible to infectious diseases. Expansion and diversification of the neonatal microbiome contribute to development of immunity in the gut. Moreover, diminished diversity of the gastrointestinal microbiome has been associated with risk of infections and immune dysregulation. We thus hypothesized that varying composition or reduced diversity of the intestinal microbiome of neonatal foals would contribute to increased susceptibility of their developing R. equi pneumonia. The composition and diversity indices of the fecal microbiota at 3 and 5 weeks of age were compared among 3 groups of foals: 1) foals that subsequently developed R. equi pneumonia after sampling; 2) foals that subsequently developed ultrasonographic evidence of pulmonary abscess formation or consolidation but not clinical signs (subclinical group); and, 3) foals that developed neither clinical signs nor ultrasonographic evidence of pulmonary abscess formation or consolidation. No significant differences were found among groups at either sampling time, indicating absence of evidence of an influence of composition or diversity of the fecal microbiome, or predicted fecal metagenome, on susceptibility to subsequent R. equi pneumonia. A marked and significant difference identified between a relatively short interval of time appeared to reflect ongoing adaptation to transition from a milk diet to a diet including available forage (including hay) and access to concentrate fed to the mare.