Characterization and Description of the Fecal Microbiomes of Pet Domestic Ferrets (Mustela putorius furo) Living in Homes.

The domestic ferret (Mustela putorius furo) is a popular companion pet in the United States, with an estimated population of 500,000. Despite being obligate carnivores with a fast digestive system, little is known about their gut microbiomes. This study aims to compare the fecal microbiomes of healthy domestic ferrets and cats, which are both obligate carnivores. We collected and analyzed stool samples from 36 healthy ferrets and 36 healthy cats, sequencing the V4 region of the 16S rRNA gene. Using QIIME 2, we assessed the alpha and beta diversities and identified the taxa differences. Compared to cats, ferrets exhibited a higher representation of Firmicutes and Proteobacteria, while Bacteroidota and Actinomycetota were more prevalent in cats. The ferrets' microbiomes displayed lower alpha diversities. The highly present bacterial genera in the gut microbiomes of ferrets included Clostridium sensu stricto, Streptococcus, Romboutsia, Paeniclostridium, Lactobacillus, Enterococcus, and Lactococcus. Notably, the ferrets' microbiomes significantly differed from those of cats. This research highlights the potential differences in gastrointestinal care for ferrets, emphasizing the need for tailored approaches. Future studies should explore microbiome variations in ferrets with gastrointestinal issues and their responses to dietary and medical interventions.

The Oral Microbiome across Oral Sites in Cats with Chronic Gingivostomatitis, Periodontal Disease, and Tooth Resorption Compared with Healthy Cats.

Feline chronic gingivostomatitis (FCGS) is a chronic mucosal and gingival inflammatory disease in which pathogenesis remains unclear. Interactions between the host inflammatory process, the host immune response, and the oral microbiome are implicated in this pathogenesis. To begin to understand this disease and the impact of the microbiome to host inflammatory disease states, we collected sterile noninvasive plaque biofilm samples from ten distinct sites within the oral cavity in cats with stomatitis (n = 12), healthy cats (n = 9), and cats with tooth resorption or periodontitis (n = 11). Analysis of full-length 16S rRNA gene sequences indicated that the microbiomes of cats with FCGS presented marked dysbiosis at multiple oral sites. Additionally, microbiome beta diversity varied with oral condition, indicating that stomatitis, periodontitis, and/or tooth resorption influence the microbiome differently. Lastly, we found that the microbiomes of swabs taken from the oral cavity were comparable to those taken from plaque using endodontic paper points, validating this as another sampling method. Collectively, our work furthers our understanding of the dysbiosis and composition of bacteria in the oral microbiome in FCGS, with hopes of contributing to the prevention, diagnosis, and treatment of this challenging condition in felines.

Characterization of the Blood Microbiome and Comparison with the Fecal Microbiome in Healthy Dogs and Dogs with Gastrointestinal Disease.

Recent studies have found bacterial DNA in the blood of healthy individuals. To date, most studies on the blood microbiome have focused on human health, but this topic is an expanding research area in animal health as well. This study aims to characterize the blood microbiome of both healthy dogs and those with chronic gastro-enteropathies. For this study, blood and fecal samples were collected from 18 healthy and 19 sick subjects, DNA was extracted through commercial kits, and the V3-V4 regions of the 16S rRNA gene were sequenced on the Illumina platform. The sequences were analyzed for taxonomic annotation and statistical analysis. Alpha and beta diversities of fecal microbiome were significantly different between the two groups of dogs. Principal coordinates analysis revealed that healthy and sick subjects were significantly clustered for both blood and fecal microbiome samples. Moreover, bacterial translocation from the gut to the bloodstream has been suggested because of found shared taxa. Further studies are needed to determine the origin of the blood microbiome and the bacteria viability. The characterization of a blood core microbiome in healthy dogs has potential for use as a diagnostic tool to monitor for the development of gastro-intestinal disease.

Regulatory Role of microRNA of Milk Exosomes in Mastitis of Dairy Cows.

The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows.

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.

Characterization of Microbiome on Feces, Blood and Milk in Dairy Cows with Different Milk Leucocyte Pattern.

Mastitis is an inflammatory disease of the mammary gland, caused by the invasion of microorganism on this site, associated with an altered immune response. Recent studies in this field hypothesize that the origin of these pathogens can also be from the gastrointestinal tract, through the entero-mammary pathway in relation to an increase in gut permeability. In this study, we wanted to investigate if inflammatory status of the mammary gland is related to an alteration of gut permeability. The microbiome of feces, blood and milk of lactating cows, recruited on the basis of the total somatic cell count and of the percentage of polymorphonuclear neutrophils and lymphocytes, was studied. Cows were divided into healthy (G), at risk of mastitis (Y) and with mastitis (R) classifications. The bacterial DNA was extracted and the V3 and V4 regions of 16S rRNA sequenced. Moreover, the quantification of total bacteria was performed with quantitative real-time PCR. A non-parametric Kruskal-Wallis test was applied at the phylum, family and genera levels and beta biodiversity was evaluated with the unweighted UniFrac distance metric. Significant differences between groups were found for the microbial composition of feces (Clostridiaceae, Turicibacteriaceae for family level and Clostridium, Dorea, SMB53 and Turicibacter for genus level), blood (Tenericutes for phylum level and Mycoplasma for genus level) and milk (OD1 and Proteobacteria for phylum level, Enterobacteriaceae and Moraxallaceae for family level and Olsenella and Rhodococcus for genus level). The beta biodiversity of feces and blood did not change between groups. Significant differences (p < 0.05) were observed between the beta diversity in milk of G group and Y group and between Y group and R group. The number of taxa in common between feces, blood and milk were 8 at a phylum, 19 at a family and 15 at a genus level. From these results, the bacterial crossing from gut to milk in cows was not confirmed but remained hypothetical and deserves further investigation.

Blood Microbiome: A New Marker of Gut Microbial Population in Dogs?

The characterization of the microbial population in different compartments of the organism, such as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field have already investigated the fecal microbiome in healthy or sick subjects; however, the methodologies used in the different laboratories and the limited number of animals recruited in each experiment do not allow a straight comparison among published results. Previously, our research focused on the characterization of the microbial taxa variability in 340 fecal samples from 132 healthy dogs, collected serially from several in-house experiments. The results supported the responsiveness of microbiota to dietary and sex factors and allowed us to cluster dogs with high accuracy. For the present study, intestinal and blood microbiota of healthy dogs from different breeds, genders, ages and food habits were collected, with three principal aims: firstly, to confirm the results of our previous study regarding the fecal microbiome affected by the different type of diet; secondly, to investigate the existence of a blood microbial population, even in heathy subjects; and thirdly, to seek for a possible connection between the fecal and the blood microbiota. Limited researches have been published on blood microbiota in humans, and this is the first evidence of the presence of a bacterial population in the blood of dogs. Moreover, gut and blood microbiota can discriminate the animals by factors such as diet, suggesting some relationship between them. These preliminary results make us believe in the use of the blood microbiome for diagnostic purposes, such as researching and preventing gut inflammatory diseases.

Effect of different starch sources in a raw meat-based diet on fecal microbiome in dogs housed in a shelter.

A dietary intervention study was assessed to determine if different sources of starch in homemade diets could significantly modify fecal microbiome of dogs. Twenty-seven adult dogs were enrolled and fed a diet based on a mixture of rice and pasta with fresh raw meat (CD). After 90 d, 8 dogs continued to receive CD diet, 10 dogs received a diet made of a raw meat and a complementary food with rice as the main source of starch (B1), and 9 dogs were fed a diet with the same raw meat and a complementary food with potato as the main source of starch (B2). Samples of feces were collected from each dog in the mornings at the beginning of the study and after 15 d and analyzed for pH, ammonia N (N-NH3) and total N, short chain fatty acids (SCFA) and lactic acid. Relative abundance of fecal microbiota was assessed by sequencing and annotating the V3-V4 regions of the 16S rRNA. Total starch intake was similar between diets but differed in the in vitro rate digestion and in the resistant starch, which was higher in B2 than in B1 and CD diets. Dogs fed B2 diet showed lower (P < 0.05) N-NH3 and pH but higher (P < 0.05) molar proportion of lactic acid. Linear discriminant analysis of the genera relative abundances indicated a significant (P < 0.01) increase of SMB53 genus at the end of the study in B1 diet and of Megamonas genus in B1 and B2 diets in comparison to CD diet. These results suggest that changes of starch source in a raw meat-based diet have limited effects on fecal microbiome in healthy dogs, but underline a high variability of microbiota among dogs.

Learning machine approach reveals microbial signatures of diet and sex in dog.

The characterization of the microbial population of many niches of the organism, as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field already investigated faecal microbiome in healthy or affected subjects, although the methodologies used in the different laboratories and the limited number of animals recruited in each experiment does not allow a straight comparison among published results. In the present study, we report data collected from several in house researches carried out in healthy dogs, with the aim to seek for a variability of microbial taxa in the faeces, caused by factors such as diet and sex. The database contains 340 samples from 132 dogs, collected serially during dietary intervention studies. The procedure of samples collection, storage, DNA extraction and sequencing, bioinformatic and statistical analysis followed a standardized pipeline. Microbial profiles of faecal samples have been analyzed applying dimensional reduction discriminant analysis followed by random forest analysis to the relative abundances of genera in the feces as variables. The results supported the responsiveness of microbiota at a genera taxonomic level to dietary factor and allowed to cluster dogs according this factor with high accuracy. Also sex factor clustered dogs, with castrated males and spayed females forming a separated group in comparison to intact dogs, strengthening the hypothesis of a bidirectional interaction between microbiota and endocrine status of the host. The findings of the present analysis are promising for a better comprehension of the mechanisms that regulate the connection of the microorganisms living the gastrointestinal tract with the diet and the host. This preliminary study deserves further investigation for the identification of the factors affecting faecal microbiome in dogs.

Gut Microbiome of Healthy and Arthritic Dogs.

Several studies have underlined the interplay among host-microbiome and pathophysiological conditions of animals. Research has also focused specifically on whether and how changes in the gut microbiome have provoked the occurrence of pathological phenomena affecting cartilage and joints in humans and in laboratory animals. Here, we tried to evaluate the relationship between the gut microbiome and the hip and elbow arthritis in owned dogs. The study included 14 dogs suffering from chronic arthritis (AD) and 13 healthy dogs (HD). After the first visit and during the period of the study, the dogs, under the supervision of the owner, were fed a semi-moist complete diet supplemented with omega 3 fatty acids. Feces and blood samples were collected in the clinic at the first visit (T0) and after days (T45). The plasma C-reactive protein (CRP) was higher, and the serum vitamin B12 and folate concentrations were lower (p < 0.05) in the AD group in comparison to the HD group. Data of the fecal microbiome showed that the relative abundances of the genus Megamonas were higher in AD (p < 0.001), while the relative abundance of the families Paraprevotellaceae, Porphyromonadaceae, and Mogibacteriaceae was significantly lower in comparison to HD. The results of the study identified several bacterial groups that differed significantly in the fecal microbiome between healthy and diseased dogs. If the observed differences in fecal bacterial composition predispose dogs to hip and elbow arthritis or if these differences reflect a correlation with these conditions deserves further investigation.

MicroRNA Milk Exosomes: From Cellular Regulator to Genomic Marker.

Recent advances in ruminants' milk-derived exosomes (EXO) have indicated a role of microRNAs (miRNAs) in cell-to-cell communication in dairy ruminants. The miRNAs EXO retain peculiar mechanisms of uptake from recipient cells, which enables the selective delivery of cargos, with a specific regulation of target genes. Although many studies have been published on the miRNAs contained in milk, less information is available on the role of miRNAs EXO, which are considered stable over time and resistant to digestion and milk processing. Several miRNAs EXO have been implicated in the cellular signaling pathway, as in the regulation of immune response. Moreover, they exert epigenetic control, as extenuating the expression of DNA methyltransferase 1. However, the study of miRNAs EXO is still challenging due to the difficulty of isolating EXO. In fact, there are not agreed protocols, and different methods, often time-consuming, are used, making it difficult to routinely process a large number of samples. The regulation of cell functions in mammary glands by miRNAs EXO, and their applications as genomic markers in livestock, is presented.

Interplay between Neuroendocrine Biomarkers and Gut Microbiota in Dogs Supplemented with Grape Proanthocyanidins: Results of Dietary Intervention Study.

Several studies on the interaction between gut microbiota and diets, including prebiotics, have been reported in dogs, but no data are available about the effects of dietary administration of grape proanthocyanidins. In the study, 24 healthy adult dogs of different breeds were recruited and divided in 3 groups of 8 subjects each. A group was fed with a control diet (D0), whilst the others were supplemented with 1 (D1) or 3 (D3) mg/kg live weight of grape proanthocyanidins. Samples of feces were collected at the beginning and after 14 and 28 days for microbiota, short chain fatty acid, and lactic acid analysis. Serotonin and cortisol were measured in saliva, collected at the beginning of the study and after 28 days. A significantly higher abundance (p < 0.01) of Enterococcus and Adlercreutzia were observed in D0, whilst Escherichia and Eubacterium were higher in D1. Fusobacterium and Phascolarctobacterium were higher (p < 0.01) in D3. Salivary serotonin increased (p < 0.01) at T28 for D1 and D3 groups but cortisol did not vary. Proanthocyanidins administration influenced the fecal microbiota and neuroendocrine response of dogs, but a high variability of taxa was observed, suggesting a uniqueness and stability of fecal microbiota related to the individual.