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Introduction: Anxiety and cognitive dysfunction are frequent, difficult to treat and burdensome comorbidities in human and canine epilepsy. Fecal microbiota transplantation (FMT) has been shown to modulate behavior in rodent models by altering the gastrointestinal microbiota (GIM). This study aims to investigate the beneficial effects of FMT on behavioral comorbidities in a canine translational model of epilepsy., Methods: Nine dogs with drug-resistant epilepsy (DRE) and behavioral comorbidities were recruited. The fecal donor had epilepsy with unremarkable behavior, which exhibited a complete response to phenobarbital, resulting in it being seizure-free long term. FMTs were performed three times, two weeks apart, and the dogs had follow-up visits at three and six months after FMTs. Comprehensive behavioral analysis, including formerly validated questionnaires and behavioral tests for attention deficit hyperactivity disorder (ADHD)- and fear- and anxiety-like behavior, as well as cognitive dysfunction, were conducted, followed by objective computational analysis. Blood samples were taken for the analysis of antiseizure drug (ASD) concentrations, hematology, and biochemistry. Urine neurotransmitter concentrations were measured. Fecal samples were subjected to analysis using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based Dysbiosis Index (DI) assessment, and short-chain fatty acid (SCFA) quantification., Results: Following FMT, the patients showed improvement in ADHD-like behavior, fear- and anxiety-like behavior, and quality of life. The excitatory neurotransmitters aspartate and glutamate were decreased, while the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and GABA/glutamate ratio were increased compared to baseline. Only minor taxonomic changes were observed, with a decrease in Firmicutes and a Blautia_A species, while a Ruminococcus species increased. Functional gene analysis, SCFA concentration, blood parameters, and ASD concentrations remained unchanged., Discussion: Behavioral comorbidities in canine IE could be alleviated by FMT. This study highlights FMT's potential as a novel approach to improving behavioral comorbidities and enhancing the quality of life in canine patients with epilepsy., Competing Interests: HV has served as a paid consultant in the field of epilepsy for Boehringer Ingelheim, CEVA Animal Health, Nestle Purina and served as contract researcher for: Nestle Purina, Desitin Pharma and Boehringer Ingelheim. JS and RP were employees of the Gastrointestinal Laboratory at Texas A&M University which offers microbiome assessment on a fee-for-service basis. JS was the Purina PetCare Endowed Chair for Microbiome Research and receives support for microbiome research through the Purina PetCare Research Excellence Funds. JS has also received consulting or speaking fees from Nestle Purina, IDEXX Laboratories, Royal Canin and Hill’s Pet Nutrition, Inc. AF participates in academia-industry partnerships with Nestle Purina and Vetoquinol. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AW declared a past collaboration with the authors HV to the handling editor., (Copyright © 2024 Watanangura, Meller, Farhat, Suchodolski, Pilla, Khattab, Lopes, Bathen-Nöthen, Fischer, Busch-Hahn, Flieshardt, Gramer, Richter, Zamansky and Volk.)

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., Competing Interests: AF-P, AS-M, and CT were employed by the pet food company Affinity Petcare, which funded the study. JS and RP were employed by the Gastrointestinal Laboratory at Texas A&M University, which provides microbiome assessment on a fee-for-service basis. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fernández-Pinteño, Pilla, Manteca, Suchodolski, Torre and Salas-Mani.)

Objectives: While feline chronic bronchitis (CB) is known as neutrophilic bronchial inflammation (NI), feline asthma (FA) is defined as an eosinophilic airway inflammation (EI). Feline chronic bronchial disease refers to both syndromes, with similar clinical presentations and applied treatment strategies. Recent studies described alterations of the microbiota composition in cats with FA, but little is known about the comparison of the lung microbiota between different types of feline bronchial disease. The study aimed to describe the bacterial microbiota of the lower respiratory tracts of cats with FA and CB and to identify potential differences., Methods: Twenty-two client-owned cats with FA ( n = 15) or CB ( n = 7) confirmed via bronchoalveolar-lavage (BALF)-cytology were included. Next-generation sequencing analysis of 16S rRNA genes was performed on bacterial DNA derived from BALF samples. QIIME was used to compare microbial composition and diversity between groups., Results: Evenness and alpha-diversity-indices did not significantly differ between cats with FA and CB (Shannon p = 0.084, Chao 1 p = 0.698, observed ASVs p = 0.944). Based on a PERMANOVA analysis, no significant differences were observed in microbial composition between animals of both groups (Bray-Curtis metric, R -value 0.086, p = 0.785; unweighted UniFrac metric, R -value -0.089, p = 0.799; weighted Unifrac metric, R -value -0.072, p = 0.823). Regarding taxonomic composition, significant differences were detected for Actinobacteria on the phylum level ( p = 0.026), Mycoplasma spp. ( p = 0.048), and Acinetobacteria ( p = 0.049) on the genus level between cats with FA and CB, with generally strong interindividual differences seen. There was a significant difference in the duration of clinical signs before diagnosis in animals dominated by Bacteriodetes (median 12 months, range 2-58 months) compared to animals dominated by Proteobacteria (median 1 month, range 1 day to 18 months; p = 0.003)., Conclusions and Relevance: Lung microbiota composition is very similar in cat populations with spontaneous FA and CB besides small differences in some bacterial groups. However, with disease progression, the lung microbiome of cats with both diseases appears to shift away from dominantly Proteobacteria to a pattern more dominated by Bacteriodetes . A substantial proportion of cats tested positive for Mycoplasma spp. via sequencing, while none of them tested positive using classical PCR., Competing Interests: JP was employed by Vet Med Labor GmbH, Division of IDEXX Laboratories. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Werner, Weeger, Hörner-Schmid, Weber, Palić, Shih, Suchodolski, Pilla and Schulz.)

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., Competing Interests: Author HV served as a paid consultant in the field of epilepsy for Boehringer Ingelheim, CEVA Animal Health, Nestle Purina, and as a contract researcher for Nestle Purina, Desitin Pharma, and Boehringer Ingelheim. Author JS is an employee of the Gastrointestinal Laboratory at Texas A&M University that provides microbiome testing on a fee-for-service basis and is on the scientific advisory board for Nestle Purina and received speaker honoraria from Royal Canin, Nutramax Laboratories, ExeGi Pharma, LLC, and Hill's Pet Nutrition, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Watanangura, Meller, Suchodolski, Pilla, Khattab, Loderstedt, Becker, Bathen-Nöthen, Mazzuoli-Weber and Volk.)

Consumption of diets containing medium chain triglycerides have been shown to confer neuroprotective and behavior modulating effects. We aimed to identify metabolic and microbiome perturbations in feces that are associated with consumption of a medium chain triglyceride ketogenic diet (MCT-KD) in dogs with idiopathic epilepsy. We used 16S rRNA gene sequencing to generate microbiome profiles and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to generate lipidomic profiles of canine feces. We made comparisons between the MCT-KD, standardized placebo diet and baseline pre-trial diet phases. Consumption of the MCT-KD resulted in a significant increase in the species richness (α-diversity) of bacterial communities found in the feces when compared to the baseline diet. However, phylogenetical diversity between samples (beta-diversity) was not affected by diet. An unnamed Bacteroidaceae species within genus 5-7N15 was identified by LEfSe as a potential biomarker associated with consumption of the MCT-KD, showing an increased abundance ( p = 0.005, q = 0.230) during consumption of MCT-KD. In addition, unclassified members of families Erysipelotrichaceae ( p = 0.013, q = 0.335) and Fusobacteriaceae ( p = 0.022, q = 0.358) were significantly increased during MCT-KD consumption compared to baseline. Blautia sp. and Megamonas sp. instead were decreased during consumption of either placebo or MCT-KD ( p = 0.045, q = 0.449, and p = 0.039, q = 0.449, respectively). Bacteroidaceae , including genus 5-7N15 , have previously been associated with non-aggressive behavior in dogs. In addition, 5-7N15 is correlated in humans with Akkermansia , a genus known to be involved in the neuroprotective effect of ketogenic diets in mice models of seizures. Five metabolite features, tentatively identified as long chain triglycerides, were significantly higher after consumption of the placebo diet, but no unique features were identified after consumption of the MCT-KD. The data presented in this study highlight significant changes shown in both the fecal microbiome and lipidome as a result of consumption of the MCT-KD. Elucidating the global canine gut response to MCT consumption will improve our understanding of the potential mechanisms which confer anti-seizure and behavior modulating effects. Further studies should aim to characterize the gut microbiome of both dogs with epilepsy and healthy controls., Competing Interests: YP, BZ, and QL were employed by the company Nestlé Purina Research. YP and BZ are inventors of US9789079B2. The remaining authors possesses any rights of the patent. The commercial sponsor has not been involved in case recruitment, data handling, data analysis, and data storage. Furthermore, the commercial sponsor could not prevent the manuscript from being submitted for publication., (Copyright © 2020 Pilla, Law, Pan, Zanghi, Li, Want, Lidbury, Steiner, Suchodolski and Volk.)

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., (Copyright © 2020 Chaitman, Ziese, Pilla, Minamoto, Blake, Guard, Isaiah, Lidbury, Steiner, Unterer and Suchodolski.)

The gut microbiome contributes to host metabolism, protects against pathogens, educates the immune system, and, through these basic functions, affects directly or indirectly most physiologic functions of its host. Molecular techniques have allowed us to expand our knowledge by unveiling a wide range of unculturable bacteria that were previously unknown. Most bacterial sequences identified in the canine gastrointestinal (GI) tract fall into five phyla: Firmicutes, Fusobacteria, Bacteroidetes, Proteobacteria, and Actinobacteria. While there are variations in the microbiome composition along the GI tract, most clinical studies concentrate on fecal microbiota. Age, diet, and many other environmental factors may play a significant role in the maintenance of a healthy microbiome, however, the alterations they cause pale in comparison with the alterations found in diseased animals. GI dysfunctions are the most obvious association with gut dysbiosis. In dogs, intestinal inflammation, whether chronic or acute, is associated with significant differences in the composition of the intestinal microbiota. Gut dysbiosis happens when such alterations result in functional changes in the microbial transcriptome, proteome, or metabolome. Commonly affected metabolites include short-chain fatty acids, and amino acids, including tryptophan and its catabolites. A recently developed PCR-based algorithm termed "Dysbiosis Index" is a tool that allows veterinarians to quantify gut dysbiosis and can be used to monitor disease progression and response to treatment. Alterations or imbalances in the microbiota affect immune function, and strategies to manipulate the gut microbiome may be useful for GI related diseases. Antibiotic usage induces a rapid and significant drop in taxonomic richness, diversity, and evenness. For that reason, a renewed interest has been put on probiotics, prebiotics, and fecal microbiota transplantation (FMT). Although probiotics are typically unable to colonize the gut, the metabolites they produce during their transit through the GI tract can ameliorate clinical signs and modify microbiome composition. Another interesting development is FMT, which may be a promising tool to aid recovery from dysbiosis, but further studies are needed to evaluate its potential and limitations., (Copyright © 2020 Pilla and Suchodolski.)

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.

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 (ANOSIM Unweighted : 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.