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.

Effects of a milk oligosaccharide biosimilar on fecal characteristics, microbiota, and bile acid, calprotectin, and immunoglobulin concentrations of healthy adult dogs treated with metronidazole.

In recent dog and cat experiments, a novel milk oligosaccharide biosimilar (GNU100) positively modulated fecal microbiota and metabolite profiles, suggesting benefits to gastrointestinal health. The objective of this study was to investigate the effects of GNU100 on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Twelve healthy adult female dogs (mean age: 3.74 ± 2.4 yr) were used in an 8-wk crossover design study (dogs underwent both treatments). All dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were orally administered metronidazole (20 mg/kg BW) twice daily. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, and 8 for measurement of pH, dry matter, microbiota populations, and BA, immunoglobulin A, and calprotectin concentrations. On weeks 0, 4, and 8, blood samples were collected for serum chemistry and hematology analysis. All data were analyzed as repeated measures using the Mixed Models procedure of SAS version 9.4, with significance considered P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools) and modified (P < 0.05) fecal microbiota and BA profiles. Using qPCR, metronidazole reduced fecal Blautia, Fusobacterium, Turicibacter, Clostridium hiranonis, and Faecalibacterium abundances, and increased fecal Streptococcus and Escherichia coli abundances. DNA sequencing analysis demonstrated that metronidazole reduced microbial alpha diversity and influenced the relative abundance of 20 bacterial genera and families. Metronidazole also increased primary BA and reduced secondary BA concentrations. Most antibiotic-induced changes returned to baseline by week 8. Fecal scores were more stable (P = 0.01) in GNU100-fed dogs than controls after antibiotic administration. GNU100 also influenced fecal microbiota and BA profiles, reducing (P < 0.05) the influence of metronidazole on microbial alpha diversity and returning some fecal microbiota and secondary BA to baseline levels at a quicker (P < 0.05) rate than controls. In conclusion, our results suggest that GNU100 supplementation provides benefits to dogs treated with antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and secondary BA profiles which play an essential role in gut health.

Our objective was to test the effects of a novel milk oligosaccharide biosimilar (GNU100) on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were given an oral antibiotic. Fecal scores were recorded and fresh fecal samples were collected over time to assess fecal characteristics, microbiota populations, and BA concentrations. The antibiotic was shown to increase fecal scores (looser stools) and modify fecal microbiota populations (altered diversity and ~20 bacterial genera and families) and BA profiles (increased primary and reduced secondary BA). Most antibiotic-induced changes returned to baseline by week 8. In dogs fed GNU100, fecal scores were more stable and changes to microbial diversity were lower than controls after antibiotic administration. Fecal microbiota and secondary BA of GNU100-fed dogs also returned to baseline levels at a quicker rate than controls. These results suggest that GNU100 provides benefits to dogs given antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and BA profiles.

Dietary supplementation with fiber, "biotics," and spray-dried plasma affects apparent total tract macronutrient digestibility and the fecal characteristics, fecal microbiota, and immune function of adult dogs.

A variety of functional ingredients, including fibers, prebiotics, probiotics, and postbiotics may be added to pet foods to support gastrointestinal and immune health. While many of these ingredients have been tested individually, commercial foods often include blends that also require testing. This study was conducted to evaluate the effects of diets containing blends of fibers, "biotics," and/or spray-dried plasma on apparent total tract digestibility (ATTD), stool quality, fecal microbiota and metabolites, and immune health outcomes of adult dogs. A total of 12 healthy adult intact English pointer dogs (6 M, 6 F; age = 6.4 ± 2.0 yr; BW = 25.8 ± 2.6 kg) were used in a replicated 3 × 3 Latin square design to test diets formulated to: 1) contain a low concentration of fermentative substances (control diet, CT); 2) be enriched with a fiber-prebiotic-probiotic blend (FPPB); and 3) be enriched with a fiber-prebiotic-probiotic blend + immune-modulating ingredients (iFFPB). In each 28-d period, 22 d of diet adaptation was followed by a 5-d fecal collection phase and 1 d for blood sample collection. All data were analyzed using SAS 9.4, with significance being P < 0.05 and trends being P < 0.10. FPPB and iFPPB diets led to shifts in numerous outcome measures. Dry matter (DM), organic matter, fat, fiber, and energy ATTD were lower (P < 0.01), fecal scores were lower (P < 0.01; firmer stools), and fecal DM% was higher (P < 0.0001) in dogs fed FPPB or iFPPB than those fed CT. Serum triglycerides and cholesterol were lower (P < 0.01) in dogs fed FPPB or iFPPB than those fed CT. Fecal protein catabolites (isobutyrate, isovalerate, indole, and ammonia) and butyrate were lower (P < 0.05), while fecal immunoglobulin A (IgA) was higher (P < 0.01) in dogs fed FPPB and iFPPB than those fed CT. Fecal microbiota populations were affected by diet, with alpha-diversity being lower (P < 0.05) in dogs fed iFPPB and the relative abundance of 20 bacterial genera being altered in dogs fed FPPB or iFPPB compared with CT. The circulating helper T cell:cytotoxic T cell ratio was higher (P < 0.05) in dogs fed iFPPB than those fed CT. Circulating B cells were lower (P < 0.05) in dogs fed FPPB than those fed iFPPB, and lower (P < 0.05) in dogs fed iFPPB than those fed CT. Our results demonstrate that feeding a fiber-prebiotic-probiotic blend may provide many benefits to canine health, including improved stool quality, beneficial shifts to fecal microbiota and metabolite profiles, reduced blood lipids, and increased fecal IgA.

A variety of functional ingredients­those that provide benefits beyond their nutritional value­may be added to pet foods to support gastrointestinal and immune health. While many of these ingredients have been tested individually, commercial foods often include blends that also require testing. This study was conducted to evaluate the effects of diets containing blends of dietary fibers and other functional ingredients on nutrient digestibility and the stool characteristics and immune health outcomes of adult dogs consuming them. Treatments included a control diet containing low amounts of dietary fiber, a diet containing a fiber­prebiotic­probiotic blend, and a diet containing the fiber­prebiotic­probiotic blend as well as immune-modulating ingredients. The test diets were shown to shift many outcome measures. First, they were shown to reduce nutrient digestibility and decrease fecal scores (more firm stool). Second, test diets reduced blood lipids and beneficially altered fecal metabolite concentrations. Third, test diets increased fecal immunoglobulin A concentrations, suggesting enhanced gut immunity. Lastly, the test diets shifted fecal bacterial populations. Our results demonstrate that feeding a fiber­prebiotic­probiotic blend may provide many benefits to canine health, including improved stool quality, beneficial shifts to fecal bacteria and metabolite profiles, reduced blood lipids, and enhanced gut immunity.

Weight loss and high-protein, high-fiber diet consumption impact blood metabolite profiles, body composition, voluntary physical activity, fecal microbiota, and fecal metabolites of adult dogs.

Canine obesity is associated with reduced lifespan and metabolic dysfunction, but can be managed by dietary intervention. This study aimed to determine the effects of restricted feeding of a high-protein, high-fiber (HPHF) diet and weight loss on body composition, physical activity, blood metabolites, and fecal microbiota and metabolites of overweight dogs. Twelve spayed female dogs (age: 5.5 ± 1.1 yr; body weight [BW]: 14.8 ± 2.0 kg, body condition score [BCS]: 7.9 ± 0.8) were fed a HPHF diet during a 4-wk baseline phase to maintain BW. After baseline (week 0), dogs were first fed 80% of baseline intake and then adjusted to target 1.5% weekly weight loss for 24 wk. Body composition using dual-energy x-ray absorptiometry and blood samples (weeks 0, 6, 12, 18, and 24), voluntary physical activity (weeks 0, 7, 15, and 23), and fresh fecal samples for microbiota and metabolite analysis (weeks 0, 4, 8, 12, 16, 20, and 24) were measured over time. Microbiota data were analyzed using QIIME 2. All data were analyzed statistically over time using SAS 9.4. After 24 wk, dogs lost 31.2% of initial BW and had 1.43 ± 0.73% weight loss per week. BCS decreased (P < 0.0001) by 2.7 units, fat mass decreased (P < 0.0001) by 3.1 kg, and fat percentage decreased (P < 0.0001) by 11.7% with weight loss. Many serum metabolites and hormones were altered, with triglycerides, leptin, insulin, C-reactive protein, and interleukin-6 decreasing (P < 0.05) with weight loss. Relative abundances of fecal Bifidobacterium, Coriobacteriaceae UCG-002, undefined Muribaculaceae, Allobaculum, Eubacterium, Lachnospira, Negativivibacillus, Ruminococcus gauvreauii group, uncultured Erysipelotrichaceae, and Parasutterella increased (P < 0.05), whereas Prevotellaceae Ga6A1 group, Catenibacterium, Erysipelatoclostridium, Fusobacterium, Holdemanella, Lachnoclostridium, Lactobacillus, Megamonas, Peptoclostridium, Ruminococcus gnavus group, and Streptococcus decreased (P < 0.01) with weight loss. Despite the number of significant changes, a state of dysbiosis was not observed in overweight dogs. Fecal ammonia and secondary bile acids decreased, whereas fecal valerate increased with weight loss. Several correlations between gut microbial taxa and biological parameters were observed. Our results suggest that restricted feeding of a HPHF diet and weight loss promotes fat mass loss, minimizes lean mass loss, reduces inflammatory marker and triglyceride concentrations, and modulates fecal microbiota phylogeny and activity in overweight dogs.

Canine obesity is associated with reduced lifespan and metabolic dysfunction, but dietary intervention may aid in its management. This study aimed to determine the effects of restricted feeding of a high-protein, high-fiber (HPHF) diet and weight loss on body composition, physical activity, blood metabolites, and fecal bacteria and metabolites of overweight dogs. Twelve overweight dogs were fed a HPHF diet during a 4-wk baseline to maintain body weight and then fed to lose weight for 24 wk. Body composition, blood samples, voluntary physical activity, and fresh fecal samples were measured over time. After 24 wk, dogs lost over 30% of their initial body weight and had 1.4% weight loss per week. As expected, serum triglycerides, leptin, insulin, C-reactive protein, and interleukin-6 decreased with weight loss. The relative abundances of 4 bacterial phyla and over 30 bacterial genera were altered with weight loss. Fecal ammonia and secondary bile acid concentrations decreased, whereas fecal valerate concentrations increased with weight loss. Several correlations between fecal bacteria and physiological parameters were identified. Our results suggest that a HPHF diet and weight loss promote fat mass loss, reduce inflammatory marker and triglyceride concentrations, and modulate fecal bacterial populations and activity in overweight dogs.