A Randomized Controlled Trial to Evaluate the Impact of a Novel Probiotic and Nutraceutical Supplement on Pruritic Dermatitis and the Gut Microbiota in Privately Owned Dogs.

Pruritic dermatitis (PD) is a common presentation of canine allergic skin diseases, with diversity in severity and treatment response due to complex etiopathogenesis. Evidence suggests the gut microbiota (GM) may contribute to the development of canine allergies. A 10-week double-blind randomized controlled trial evaluated a novel probiotic and nutraceutical blend (PNB) on clinical signs of skin allergy, health measures, and the GM of privately owned self-reported pruritic dogs. A total of 105 dogs were enrolled, with 62 included in pruritus and health analysis and 50 in microbiome analysis. The PNB supported greater improvement of owner-assessed clinical signs of PD at week 2 than the placebo (PBO). More dogs that received the PNB shifted to normal pruritus (digital PVAS10-N: <2) by week 4, compared to week 7 for the PBO. While a placebo effect was identified, clinical differences were supported by changes in the GM. The PNB enriched three probiotic bacteria and reduced abundances of species associated with negative effects. The PBO group demonstrated increased abundances of pathogenic species and reduced abundances of several beneficial species. This trial supports the potential of the PNB as a supplemental intervention in the treatment of PD; however, further investigation is warranted, with stricter diagnostic criteria, disease biomarkers and direct veterinary examination.

Dietary enrichment of resistant starches or fibers differentially alter the feline fecal microbiome and metabolite profile.

BACKGROUND: Cats are strict carnivores but possess a complex gastrointestinal (GI) microbial community that actively ferments dietary substrates that are not digested and reach the colon. The GI microbiota responses to dietary inclusion of resistant starches versus fibers have not been tested in cats. Thus, our objective was to evaluate the effects of diets enriched in resistant starch or fibers on the fecal characteristics, microbiome, and metabolite profiles of cats. Twelve healthy adult domestic shorthair cats (age = 9.6 ± 4.0 year; body weight = 3.9 ± 1.0 kg) were used in a replicated 3 × 3 Latin square design to test diets that were enriched with: (1) resistant starch (ERS), (2) a fiber-prebiotic-probiotic blend (FPPB), or (3) a fiber-prebiotic-probiotic blend + immune-modulating ingredients (iFPPB). In each 28-day period, 22 days of diet adaptation was followed by fecal and blood sample collection. Fecal samples were used for shotgun metagenomic sequencing. In addition, fecal and blood metabolite measurements and white blood cell stimulation was performed to assess immune function. RESULTS: A total of 1690 bacterial species were identified, with 259 species differing between fiber-rich and ERS treatments. In comparison with fiber-rich treatments that increased diversity and promoted Firmicutes and Bacteroidetes populations, resistant starch reduced microbial diversity and fecal pH, led to a bloom in Actinobacteria, and modified Kyoto Encyclopedia of Genes and Genomes orthology (KO) terms pertaining to starch and sucrose metabolism, fatty acid biosynthesis and metabolism, epithelial cell signaling, among others. Resistant starch also differentially modified fecal metabolite concentrations with relevance to GI and overall host health (increased butyrate; decreased propionate and protein catabolites - branched-chain fatty acids; phenols and indoles; ammonia) and reduced blood cholesterol, which correlated strongly with microbial taxa and KO terms, and allowed for a high predictive efficiency of diet groups by random forest analysis. CONCLUSION: Even though domestic cats and other carnivores evolved by eating low-carbohydrate diets rich in protein and fat, our results demonstrate that the feline microbiome and metabolite profiles are highly responsive to dietary change and in directions that are predictable.

Longitudinal fecal microbiome and metabolite data demonstrate rapid shifts and subsequent stabilization after an abrupt dietary change in healthy adult dogs.

BACKGROUND: Diet has a large influence on gut microbiota diversity and function. Although previous studies have investigated the effect of dietary interventions on the gut microbiome, longitudinal changes in the gut microbiome, microbial functions, and metabolite profiles post dietary interventions have been underexplored. How long these outcomes require to reach a steady-state, how they relate to one another, and their impact on host physiological changes are largely unknown. To address these unknowns, we collected longitudinal fecal samples following an abrupt dietary change in healthy adult beagles (n = 12, age: 5.16 ± 0.87 year, BW: 13.37 ± 0.68 kg) using a crossover design. All dogs were fed a kibble diet (control) from d1-14, and then fed that same diet supplemented with fiber (HFD) or a protein-rich canned diet (CD) from d15-27. Fresh fecal samples were collected on d13, 16, 20, 24, and 27 for metabolite and microbiome assessment. Fecal microbial diversity and composition, metabolite profiles, and microbial functions dramatically diverged and stabilized within a few days (2 d for metabolites; 6 d for microbiota) after dietary interventions. Fecal acetate, propionate, and total short-chain fatty acids increased after change to HFD, while fecal isobutyrate, isovalerate, total branched-chain fatty acids, phenol, and indole increased after dogs consumed CD. Relative abundance of ~ 100 bacterial species mainly belonging to the Firmicutes, Proteobacteria, and Actinobacteria phyla increased in HFD. These shifts in gut microbiome diversity and composition were accompanied by functional changes. Transition to HFD led to increases in the relative abundance of KEGG orthology (KO) terms related to starch and sucrose metabolism, fatty acid biosynthesis, and amino sugar and nucleotide sugar metabolism, while transition to CD resulted in increased relative abundance of KO terms pertaining to inositol phosphate metabolism and sulfur metabolism. Significant associations among fecal microbial taxa, KO terms, and metabolites were observed, allowing for high-accuracy prediction of diet group by random forest analysis. CONCLUSIONS: Longitudinal sampling and a multi-modal approach to characterizing the gastrointestinal environment allowed us to demonstrate how drastically and quickly dietary changes impact the fecal microbiome and metabolite profiles of dogs following an abrupt dietary change and identify key microbe-metabolite relationships that allowed for treatment prediction.

Heterogeneity of gut microbial responses in healthy household dogs transitioning from an extruded to a mildly cooked diet.

BACKGROUND: The gut microbiota (GM) is associated with canine health and can be impacted by diet. Dog owners in the U.S. have increasingly shown an interest in feeding their dogs a mildly cooked (MC) diet. However, its impact on canine GM and health remains largely unknown. METHODS: Healthy household dogs were tracked upon switching from various brands of extruded to MC diets for four weeks. A health assessment was completed and stool samples were collected by each owner before (day 0) and after the diet transition (day 28). Shotgun metagenomic sequencing was performed at both time points to characterize the GM. RESULTS: Dogs completed the study by either completing the health assessments (n = 31) or providing stool samples at both time points (n = 28). All owners reported either better or no change in overall health at the end of the study (61% and 39%, respectively), and none reported worse overall health. Defecation frequency was also reported to be lower (58%) or about the same (35%). Principal coordinate (PCo) analysis showed a significant shift (p = 0.004) in the ß-diversity of the GM upon diet transition (34.2% and 10.3% explained by the first two axes). The abundances of 70 species increased after the diet change (adjusted p < 0.05), 67% and 24% of which belonged to the Lactobacillales and the Enterobacterales orders respectively. The abundances of 28 species decreased (adjusted p < 0.05), 46%, 18%, and 11% of which belonged to the Clostridiales, Bacillales, and Bacteroidales orders, respectively. Lower Lactobacillales and Enterobacterales, and higher Bacteroidales at baseline were associated with a greater shift along the PCo1 axis. Protein content of the baseline diet was correlated with the shift along the PCo1 axis (ρ = 0.67, p = 0.006). CONCLUSION: Owners reported either improvement or no change in health in dogs transitioning from extruded kibble to MC diets for 4 weeks, but this report of health perception requires further exploration in a controlled trial. Diet change also led to a significant shift in the GM profile of healthy dogs. The magnitude of shift was associated with baseline GM and dietary protein, and warrants further examination of individualized responses and personalized nutrition in companion dogs. These results also support future investigation of the impact of a MC diet on health maintenance given its increasing popularity.

Apparent total tract nutrient digestibility and metabolizable energy estimation in commercial fresh and extruded dry kibble dog foods.

Commercial fresh cooked foods have started gaining popularity among American dog owners in recent years. However, nutrient digestibility and the estimation of metabolizable energy (ME) of commercial fresh dog foods remain inadequately understood, even though both measures are critical to provide the intended calories for the target animal. In this preliminary study, different cohorts of normal-weight dogs were fed one of five test diets of comparable macronutrient composition: a chicken-based extruded dry kibble diet (n = 12), and chicken- (n = 12), beef- (n = 6), pork- (n = 6), or turkey-based fresh food (n = 6) for 10 d. Daily food intake and fecal characteristics were recorded, and fecal samples were collected for nutrient analysis. Despite comparable dry matter (DM) and caloric intakes between the two chicken-based diets, the fresh diet led to lower defecation frequency (1.2 ± 0.2 vs. 1.7 ± 0.5 times/d, adjusted P < 0.001), lower fecal DM (24 ± 8 vs. 47 ± 10 g/d, adjusted P < 0.001), and lower fecal calories (92 ± 31 vs. 189 ± 43 kcal/d, adjusted P < 0.001) than the kibble diet. The apparent total tract digestibility of DM, protein, fat, nitrogen-free extract, and calories of the kibble diet were all significantly lower than any of the fresh diets (adjusted P < 0.001 for all). Measured ME per food DM in all of the fresh diets, except the pork-based recipe, was significantly higher than that of the kibble diet (adjusted P < 0.001 for all). For the kibble diet, the modified Atwater calculation underestimated the ME and the NRC 2006 calculation was the most accurate predictor of ME. The standard Atwater calculation performed best for the two fresh diets that had the highest fat content (chicken, beef) and the NRC 2006 calculation performed best for the fresh diet that had the highest protein content (pork). ME of the turkey-based diet was equally overestimated and underestimated with the standard Atwater and NRC 2006 methods, respectively. We propose that commercial and home-prepared fresh diets should be assessed using standard Atwater factors as commonly done in human nutrition, or preferably for commercial products, by direct measurement in conforming feeding trials.

The microbiota of healthy dogs demonstrates individualized responses to synbiotic supplementation in a randomized controlled trial.

BACKGROUND: Probiotics have been demonstrated to ameliorate clinical signs of gastrointestinal diseases in dogs in various studies. However, the effect of probiotics in a healthy population, as well as factors contributing individualized responses, remain largely unknown. This trial examined gut microbiota (GM) and health outcomes in household dogs after synbiotic (SN) supplementation containing probiotics and inulin (a prebiotic). Healthy dogs were randomized to receive SN (50 mg/d inulin and 20 billion total CFU/d of L. reuteri, P. acidilactici, E. faecium, L. acidophilus, B. animalis, L. fermentum, L. rhamnosus) or placebo (PL) for 4 weeks. Owners completed a health survey and collected stool samples for GM profiling (shotgun metagenomic sequencing) at baseline and week 4 in both groups, and at week 6 in the SN group. RESULTS: A significant shift (p < 0.001) in ß-diversity was observed in the SN (n = 24), but not PL group (n = 19), at week 4 relative to baseline. Forty-five bacterial species, 43 (96%) of which were Lactobacillales, showed an increase in the relative abundances (≥2 fold change, adjusted p < 0.05) in the SN group at week 4. E. coli also decreased at week 4 in the SN group (2.8-fold, adjusted p < 0.01). The altered taxa largely returned to baseline at week 6. The degree of changes in ß-diversity was associated with GM at baseline. Specifically, dogs with higher Proteobacteria and lower Lactobacillales responded more robustly to supplementation in terms of the change in ß-diversity. Dogs fed SN tended to have lower diarrhea incidence (0% vs 16%, p = 0.08). CONCLUSIONS: SN supplement had a short-term impact on the gut microbiota in healthy household dogs as characterized by shotgun metagenomic sequencing. Findings warrant further investigation with longer duration and populations at risk of gastrointestinal diseases. The magnitude of response to the supplement was associated with microbial profile at baseline. To our knowledge, this is the first study documenting such association and may provide a basis for personalized nutrition in companion dogs.

Characterization of gut microbiomes of household pets in the United States using a direct-to-consumer approach.

The role of gut microbiomes as important regulators of mammalian health is increasingly recognized, although feline and canine gut microbiomes remain poorly characterized. In this proof-of-concept study, we assessed the utility of a direct-to-consumer approach to executing pet microbiome studies. We characterized the gut microbiomes of 238 pets (46 cats and 192 dogs) by generating ~11 million merged reads that were mapped to the V4 region of 16S ribosomal RNA gene at a sequencing depth of 45,806 (±22,325) reads per sample. Analyses of these reads revealed that both feline and canine gut microbiomes are dominated by three major phyla, namely Firmicutes, Proteobacteria, and Bacteroides and that alpha diversity is higher in the feline gut. In addition to interspecies differences between the feline and canine gut, we also detected appreciable intraspecies bacterial variation within the canine population. While the dogs in this dataset could be assigned to three distinct clusters based on their gut microbiome, no clustering was observed within the feline population. Integration of additional data obtained from survey questionnaires revealed that geography and body weight may be associated with canine gut microbiome composition. Furthermore, we found that both the inter and intraspecies differences are more pronounced at finer taxonomic levels, indicating that strain-level investigations may be necessary in the future. This study demonstrates that the direct-to-consumer approach overcomes existing limitations in pet microbiome research, for example, it allows collection of large numbers of pet samples. The direct-to-consumer approach has proven successful in human genomics as well as human microbiomics and this study demonstrates that by building partnerships with an engaged general public this approach can also propel the field of pet microbiomics forward.

Whole-Genome Sequences of Phages p000v and p000y, Which Infect the Bacterial Pathogen Shiga-Toxigenic Escherichia coli.

We report here the genome sequences and morphological characterizations of phages p000v and p000y, which infect the bacterial pathogen Shiga-toxigenic Escherichia coli O157:H7 and which are potential candidates for phage therapy against such pathogens.

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands.

BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

DosS responds to a reduced electron transport system to induce the Mycobacterium tuberculosis DosR regulon.

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.

Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy.

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.

Mycobacterium bovis BCG vaccine strains lack narK2 and narX induction and exhibit altered phenotypes during dormancy.

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world's population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T cells of M. tuberculosis-infected individuals, especially those harboring latent infections. Differences in the expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for the induction of two dormancy genes: narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.