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.

The effect of phenobarbital treatment on behavioral comorbidities and on the composition and function of the fecal microbiome in dogs with idiopathic epilepsy.

Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Effects of leukoreduction on N-methylhistamine concentration in stored units of canine whole blood.

OBJECTIVE: To determine the effects of leukoreduction on N-methylhistamine (NMH; a stable histamine metabolite) concentration in units of canine whole blood during storage and incubation at room temperature (approx 22 °C) to simulate temperature conditions during transfusion. ANIMALS: 8 healthy adult Walker Hounds. PROCEDURES: A standard unit of blood (450 mL) was obtained from each dog twice, with at least 28 days between donations. Blood units collected from 4 dogs during the first donation underwent leukoreduction, whereas the blood units collected from the other 4 dogs did not undergo leukoreduction, prior to storage at 4 °C. The alternate treatment was applied to blood units collected during the second donation. A sample from each unit was obtained for determination of plasma NMH concentration the day of donation (before and after leukoreduction when applicable) and before and after incubation at room temperature for 5 hours on days 14 and 28 of storage. RESULTS: Units that underwent leukoreduction had substantially lower leukocyte and platelet counts than nonleukoreduced units. Plasma NMH concentration increased immediately after leukoreduction but did not change significantly during the subsequent 28 days of storage, nor did it differ between units that did and did not undergo leukoreduction. CONCLUSIONS AND CLINICAL RELEVANCE: Leukoreduction and simulated transfusion temperature did not affect the histamine load in units of canine whole blood during the first 28 days of storage. Further research is necessary to determine whether histamine contributes to the development and severity of blood transfusion reactions in dogs.

Developmental stages in microbiota, bile acids, and clostridial species in healthy puppies.

BACKGROUND: The fecal microbiota, fecal bile acid concentrations, and abundance of Clostridium perfringens and Clostridium difficile are altered in acute and chronic gastrointestinal disease in adult dogs. However, less is known in young puppies. HYPOTHESIS/OBJECTIVES: To determine composition of the fecal microbiota, assess development of fecal bile acid profiles, and determine the abundance of Clostridial species in puppies, young adult dogs, and adult dogs. ANIMALS: Healthy puppies from a whelping kennel (n = 53) and healthy client-owned dogs <1 year old (n = 20) were separated into 6 age groups, then compared to client-owned dogs over 1 year of age (n = 13). METHODS: Prospective observational study. Naturally voided fecal samples were analyzed by quantitative polymerase chain reaction to measure bacterial abundances. Fecal bile acids were quantified using gas chromatography-mass spectrometry. RESULTS: Puppies up to 5 to 6 weeks of age had increased Dysbiosis Index (median [min-max]: 5.39 [1.32-8.6], P < .001), increased abundance of C. difficile (4.1 [0.01-4.85] log DNA, P < .001), decreased secondary bile acid concentrations (0.61 [0.28-5.06] µg/mg, P = .006), and decreased abundance of C. hiranonis (0.84 [0.01-6.71], P = .005) compared to adult dogs (-4.62 [-8.36 to -0.61], 0.01 [0.01-0.01], 4.12 [0.32-8.94], and 6.02 [5.06-7.00], respectively). Secondary bile acid concentration positively correlated with C. hiranonis abundance (ρ = 0.77; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The increase in secondary bile acids and simultaneous decrease of C. difficile and C. perfringens after 5 to 6 weeks of age warrants further investigation into regulatory impacts that secondary bile acids could have on clostridial species in 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.