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.

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.

Background: In previous studies, only a subset of dogs with chronic enteropathy (CE) had an increased dysbiosis index (DI) or altered fecal metabolites or both, suggesting differences in underlying intestinal pathophysiology between these subsets. Objectives: To compare microbial functional genes and fecal metabolites between healthy dogs with DI < 0 (HC) and dogs with CE and DI > 0 (increased DI-CE) or DI < 0 (normal DI-CE). Animals: Retrospective cross-sectional study including 78 HC and 138 CE dogs. Methods: Fecal microbiome was assessed by DNA shotgun sequencing. Dysbiosis index was quantified by qPCR. Targeted fecal metabolites, long-chain fatty acids, sterols, bile acids (BAs), and carbohydrates were measured using gas chromatography–mass spectrometry (GC–MS). Results: In permutational analysis of variance (PERMANOVA), functional gene profiles showed larger shifts in increased DI-CE (median R2 [95% confidence interval (CI)] = 0.12 [0.08–0.17]) than normal DI-CE (0.02 [0.01–0.04]) compared with HC (adjusted-p < 0.02), characterized by increased counts of carbohydrate and lipid degradation genes. Similarly, increased DI-CE (PERMANOVA, median R2 [95% CI] = 0.23 [0.14–0.34]) had larger shifts in fecal metabolome than normal DI-CE (0.10 [0.04–0.20]; adjusted-p < 0.02). Increased DI-CE had lower fecal unconjugated secondary BAs percentage (95% CI; HC, 88.4%–96.4%; normal DI-CE, 79.8%–99.0%; increased DI-CE, 28.1%–64.1%) and transporter-independent carbohydrates (combined ribose, xylose, rhamnose, and arabinose) concentrations (1.6–2.6; 0.7–1.8; 0.3–1.3 ng/mg; adjusted-p < 0.01). Conclusions: Results indicate differences in fecal microbial gene profiles and metabolome in increased DI-CE versus normal DI-CE and HC, suggesting dogs with an increased DI have more severe intestinal changes in metabolic functions.

Bile acids (BAs) are important signaling molecules in the gastrointestinal (GI) tract and are associated with health and disease in humans and animals. Intestinal bacteria transform BA through deconjugation, dehydroxylation, and epimerization reactions, producing various isoforms, many of which have not been investigated in companion animal diseases. We aimed to develop and analytically validate a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of 30 BAs in dog feces, with a simple extraction procedure and on-line solid-phase extraction. Validation demonstrated good accuracy, precision, sensitivity, spiking recovery, dilution, and stability for 29 BAs. The method was applied to fecal samples from healthy dogs (H; n = 121) and dogs with chronic enteropathy (CE; n = 58). The immediate and downstream products of bacterial 7α-dehydroxylation reactions with cholic acid were lower in concentration in dogs with CE when compared to healthy dogs (deoxycholic acid, 3-oxo-deoxycholic acid, and 12-oxo-lithocholic acid; q < 0.001). Across all fecal samples, the products of hydroxysteroid dehydrogenase (including oxo- and iso-BA) made up an average of 30% of the total measured fecal BA pool (glycine-BA, 0.1%; taurine-BA, 2.2%; unconjugated BA, 53%).

Protein-losing enteropathy (PLE) is a spectrum of gastrointestinal disorders in which protein loss occurs through the gastrointestinal tract. One of the underlying causes is chronic inflammatory enteropathy (CIE). Conventional therapies for CIE often include diet, immunosuppressives, anti-microbials, probiotics, and, recently, fecal microbial transplantation (FMT). This case report highlights the use of lyophilized material-based FMT through oral capsules and enema in a dog with PLE and concurrent protein-losing nephropathy (PLN). The patient initially had a significantly increased dysbiosis index (DI) and required repeated FMT treatments, resulting in a positive clinical response through improvement in body weight, serum albumin concentrations, fecal scores, and normalization of the DI over time. To maintain clinical responses, FMT had to be performed monthly. Approximately 1 year after starting FMT therapy, the patient then developed an episode of acute hemorrhagic diarrhea syndrome (AHDS) associated with netF-gene-encoding Clostridium perfringens strains, after which the DI became abnormal again. The patient responded clinically well to monthly FMT treatments again, but it took several months for normalization of the DI after the AHDS episode. In summary, this case report highlights the continued use of adjunct lyophilized FMT in a dog with PLE resulting in improved clinical control over time.