Stratification of Companion Animal Life Stages from Electronic Medical Record Diagnosis Data.

Aging is a complex, multifactorial process, where different life stages reflect changes in metabolic processes, immune capacities, and genetic/epigenetic repertoires. With accumulating exposure to environmental stresses and deterioration of physiological functions, body systems become more prone to low-grade chronic inflammation and an increasing range of pathologies. We hypothesized that differential susceptibility to diseases across life span reflects phased changes in an organism's physiological capacity that may highlight when interventions may be appropriately used. Furthermore, the number of life stages may vary between species and be impacted by signalment such as breed. We tested this hypothesis using disease diagnoses data from veterinary electronic medical records containing almost 2 million cats and over 4 million dogs. Bi-clustering (on rates of disease diagnoses) and adaptive branch pruning were used to identify age clusters that could be used to define adult life stages. Clustering among diagnoses were then interpreted within the context of each defined life stage. The analyses identified 5 age clusters in cats and 4 age clusters within each of the 4 canine breed size categories used. This study, using population scale data for two species, one with differential size and life expectancies, is the first to our knowledge to use disease diagnosis data to define adult life stages. The life stages presented here are a result of a data-driven approach to age and disease stratification and are intended to support conversations between clinicians and clients about appropriate health care recommendations.

An early prediction model for canine chronic kidney disease based on routine clinical laboratory tests.

The aim of this study was to derive a model to predict the risk of dogs developing chronic kidney disease (CKD) using data from electronic health records (EHR) collected during routine veterinary practice. Data from 57,402 dogs were included in the study. Two thirds of the EHRs were used to build the model, which included feature selection and identification of the optimal neural network type and architecture. The remaining unseen EHRs were used to evaluate model performance. The final model was a recurrent neural network with 6 features (creatinine, blood urea nitrogen, urine specific gravity, urine protein, weight, age). Identifying CKD at the time of diagnosis, the model displayed a sensitivity of 91.4% and a specificity of 97.2%. When predicting future risk of CKD, model sensitivity was 68.8% at 1 year, and 44.8% 2 years before diagnosis. Positive predictive value (PPV) varied between 15 and 23% and was influenced by the age of the patient, while the negative predictive value remained above 99% under all tested conditions. While the modest PPV limits its use as a stand-alone diagnostic screening tool, high specificity and NPV make the model particularly effective at identifying patients that will not go on to develop CKD.

Responses in randomised groups of healthy, adult Labrador retrievers fed grain-free diets with high legume inclusion for 30 days display commonalities with dogs with suspected dilated cardiomyopathy.

Early responses in healthy adult dogs fed grain-free diets with high inclusion of split peas (20%) and lentils (40%) that may lead to canine diet-induced dilated cardiomyopathy (DCM) were investigated. To help understand the clinical relevance of the findings, a survey of electronic health records (EHR) was conducted of dogs with and without suspected DCM for comparison. Control and Test diets were fed to Labrador retriever dogs for 30 days (n = 5 and 6, respectively). Blood and urine samples collected at baseline and days 3, 14 and 28/30 were analyzed for hematology, clinical biochemistry and taurine concentrations. The EHRs of dogs at Banfield® Pet Hospitals in the 2-year period 2018-2019 were surveyed, revealing 420 dogs diagnosed with DCM, which were compared with 420 breed, gender and age-matched healthy control dogs. Compared to baseline values, feeding the Test diet for 28 days caused progressive, significant (p < 0.001) decreases in red blood cell counts (RBC), hematocrit and total hemoglobin by 7.7, 8.3 and 6.3%, respectively, and a 41.8% increase in plasma inorganic phosphate. Commonalities in these parameters were observed in clinical DCM cases. Regarding taurine status, Test dogs transiently increased whole-blood (23.4%) and plasma (47.7%) concentrations on day 14, while taurine:creatinine ratio in fresh urine and taurine in pooled urine were reduced by 77 and 78%, respectively, on day 28/30. Thus grain-free, legume-rich Test diets caused reduced RBC and hyperphosphatemia, findings also indicated in dogs with suspected DCM. Changes in taurine metabolism were indicated. The data will aid in generating hypotheses for future studies.

Effect of feeding a daily oral care chew on the composition of plaque microbiota in dogs.

The objective of this study was to investigate the influence of daily feeding of an oral care chew on the composition of canine supragingival plaque microbiota. Twelve beagle dogs were recruited to the randomized cross-over study. The dogs were fed one of two dietary regimes, both consisting of a commercially available wet and dry diet mix, either with or without daily supplementation with an oral care chew. After each 28-day test phase, supragingival plaque samples were collected and processed via Illumina sequencing to determine the microbiota composition. A comparative analysis of bacterial species associated with health and periodontal disease, identified from prior clinical studies, revealed differences between the dietary regimes. Consumption of the daily oral care chew, resulted in a significant increase in proportion of 6 health associated taxa but only 3 disease associated taxa compared to no chew. In contrast, 8 disease and 1 health associated taxa showed increased proportions for no chew versus the oral care chew. Daily feeding of the oral care chew tested in this study has therefore been shown to increase the proportion of health associated bacteria, over bacteria associated with periodontal disease, in supragingival plaque compared to no chew. By influencing plaque microbiota towards a bias for health associated bacteria, feeding of the oral care chew provides a means to reduce the prevalence of bacterial species shown to be associated with periodontal disease in dogs.

Rapid Reconstitution of the Fecal Microbiome after Extended Diet-Induced Changes Indicates a Stable Gut Microbiome in Healthy Adult Dogs.

The gut microbiome has an important role in health, and diet represents a key lever for shaping the gut microbiome across all stages of life. Maternal milk consumption in neonates leads to long-term health effects, indicating that pliability in the infant gut microbiome in response to diet can drive enduring change. The ability of diet to drive lasting changes in the adult gut microbiome is less understood. We studied the effect of an extreme dietary shift on the fecal microbiome of 46 Labrador retriever dogs (mean age, 4.6 years) over 11 months. Dogs were fed a nutritionally complete, commercially available complex diet (CD) for a minimum of 5 weeks, followed by highly purified diets (PDs) for 36 weeks, and the initial CD for at least a further 4 weeks. Fecal samples were collected at regular intervals for DNA extraction. By analyzing 16S rRNA genes and the metagenomes, we observed minor effects on microbial diversity but significant changes in bacterial taxa and genetic potential when a PD was fed. Specifically, metagenomics identified an enrichment of quinone- and GABA-related pathways on PD, providing insights into dietary effects on cross-feeding strategies impacting community structure. When dogs returned to the CD, no significant differences were found with the initial time point. These findings are consistent with the gut microbiome being rapidly adaptable but capable of being reconstituted when provided with similar diets. These data highlight that long-term changes in the adult dog gut microbiome may only be achieved through long-term maintenance on a specified diet, rather than through feeding a transitionary diet.IMPORTANCE Diet can influence the adult gut microbiome (the community of bacteria) and health outcomes, but the ability to make changes persisting beyond feeding of a particular diet is poorly understood. We investigated whether feeding highly purified diets to adult dogs for 36 weeks would alter bacterial populations sufficiently to result in a persistent change following the dogs' return to a commercial diet. As expected, the microbiome changed when the purified diet was fed, but the original microbiome was reconstituted within weeks of the dogs returning to the commercial diet. The significance of these findings is in identifying an intrinsic stability of the host microbiome in healthy dogs, suggesting that dietary changes to support adult dog health through modifying the gut microbiome may be achieved only through maintenance on a specified diet, rather than through feeding transitionary diets.

A transcriptomic atlas of mammalian olfactory mucosae reveals an evolutionary influence on food odor detection in humans.

The mammalian olfactory system displays species-specific adaptations to different ecological niches. To investigate the evolutionary dynamics of olfactory sensory neuron (OSN) subtypes across mammalian evolution, we applied RNA sequencing of whole olfactory mucosa samples from mouse, rat, dog, marmoset, macaque, and human. We find that OSN subtypes, representative of all known mouse chemosensory receptor gene families, are present in all analyzed species. Further, we show that OSN subtypes expressing canonical olfactory receptors are distributed across a large dynamic range and that homologous subtypes can be either highly abundant across all species or species/order specific. Highly abundant mouse and human OSN subtypes detect odorants with similar sensory profiles and sense ecologically relevant odorants, such as mouse semiochemicals or human key food odorants. Together, our results allow for a better understanding of the evolution of mammalian olfaction in mammals and provide insights into the possible functions of highly abundant OSN subtypes.

Genomes from bacteria associated with the canine oral cavity: A test case for automated genome-based taxonomic assignment.

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach required fewer subjective decisions, and required far less labor. The primary differences in the non-identical taxonomic assignments involved cases where GTDB has proposed taxonomic revisions.

A Longitudinal Study of the Feline Faecal Microbiome Identifies Changes into Early Adulthood Irrespective of Sexual Development.

Companion animals provide an excellent model for studies of the gut microbiome because potential confounders such as diet and environment can be more readily controlled for than in humans. Additionally, domestic cats and dogs are typically neutered early in life, enabling an investigation into the potential effect of sex hormones on the microbiome. In a longitudinal study to investigate the potential effects of neutering, neutering age and gender on the gut microbiome during growth, the faeces of kittens (16 male, 14 female) were sampled at 18, 30 and 42 weeks of age. DNA was shotgun sequenced on the Illumina platform and sequence reads were annotated for taxonomy and function by comparison to a database of protein coding genes. In a statistical analysis of diversity, taxonomy and functional potential of the microbiomes, age was identified as the only factor with significant associations. No significant effects were detected for gender, neutering, or age when neutered (19 or 31 weeks). At 18 weeks of age the microbiome was dominated by the genera Lactobacillus and Bifidobacterium (35% and 20% average abundance). Structural and functional diversity was significantly increased by week 30 but there was no further significant increase. At 42 weeks of age the most abundant genera were Bacteroides (16%), Prevotella (14%) and Megasphaera (8%). Significant differences in functional potential included an enrichment for genes in energy metabolism (carbon metabolism and oxidative phosphorylation) and depletion in cell motility (flagella and chemotaxis). We conclude that the feline faecal microbiome is predominantly determined by age when diet and environment are controlled for. We suggest this finding may also be informative for studies of the human microbiome, where control over such factors is usually limited.

Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis.

Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity.

A Pyrosequencing Investigation of Differences in the Feline Subgingival Microbiota in Health, Gingivitis and Mild Periodontitis.

Periodontitis is the most frequently diagnosed health problem in cats yet little is known about the bacterial species important for the disease. The objective of this study was to identify bacterial species associated with health, gingivitis or mild periodontitis (<25% attachment loss) in feline plaque. Knowledge of these species is a first step in understanding the potential for improving oral health of cats via dietary interventions that alter the proportions of influential species. Subgingival plaque samples were collected from 92 cats with healthy gingiva, gingivitis or mild periodontitis. Pyrosequencing of the V1-V3 region of the 16S rDNA from these plaque samples generated more than one million reads and identified a total of 267 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all gingival health categories, particularly in health along with Moraxella and Fusobacteria. The Peptostreptococcaceae were the most abundant family in gingivitis and mild periodontitis. Logistic regression analysis identified species from various genera that were significantly associated with health, gingivitis or mild periodontitis. The species identified were very similar to those observed in canine plaque in the corresponding health and disease states. Such similarities were not observed between cat and human at the bacterial species level but with disease progression similarities did emerge at the phylum level. This suggests that interventions targeted at human pathogenic species will not be effective for use in cats but there is more potential for commonalities in interventions for cats and dogs.

A longitudinal assessment of changes in bacterial community composition associated with the development of periodontal disease in dogs.

Periodontal disease is the most widespread oral disease in dogs. Whilst the involvement of bacteria in the aetiology of periodontitis is well established the role of individual species and their complex interactions with the host is not well understood. The objective of this research was therefore to perform a longitudinal study in dogs to identify the changes that occur in subgingival bacterial communities during the transition from mild gingivitis to the early stages of periodontitis (<25% attachment loss). Subgingival plaque samples were collected from individual teeth of 52 miniature schnauzer dogs every six weeks for up to 60 weeks. The microbial composition of plaque samples was determined using 454-pyrosequencing of the 16S rDNA. A group of aerobic Gram negative species, including Bergeyella zoohelcum COT-186, Moraxella sp. COT-017, Pasteurellaceae sp. COT-080, and Neisseria shayeganii COT-090 decreased in proportion as teeth progressed to mild periodontitis. In contrast, there was less evidence that increases in the proportion of individual species were associated with the onset of periodontitis, although a number of species (particularly members of the Firmicutes) became more abundant as gingivitis severity increased. There were small increases in Shannon diversity, suggesting that plaque community membership remains relatively stable but that bacterial proportions change during progression into periodontitis. This is the first study to demonstrate the temporal dynamics of the canine oral microbiota; it showed that periodontitis results from a microbial succession predominantly characterised by a reduction of previously abundant, health associated taxa.

Draft genome sequences of 26 porphyromonas strains isolated from the canine oral microbiome.

We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease.

Early canine plaque biofilms: characterization of key bacterial interactions involved in initial colonization of enamel.

Periodontal disease (PD) is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops.

Deep Illumina-based shotgun sequencing reveals dietary effects on the structure and function of the fecal microbiome of growing kittens.

BACKGROUND: Previously, we demonstrated that dietary protein:carbohydrate ratio dramatically affects the fecal microbial taxonomic structure of kittens using targeted 16S gene sequencing. The present study, using the same fecal samples, applied deep Illumina shotgun sequencing to identify the diet-associated functional potential and analyze taxonomic changes of the feline fecal microbiome. METHODOLOGY & PRINCIPAL FINDINGS: Fecal samples from kittens fed one of two diets differing in protein and carbohydrate content (high-protein, low-carbohydrate, HPLC; and moderate-protein, moderate-carbohydrate, MPMC) were collected at 8, 12 and 16 weeks of age (n = 6 per group). A total of 345.3 gigabases of sequence were generated from 36 samples, with 99.75% of annotated sequences identified as bacterial. At the genus level, 26% and 39% of reads were annotated for HPLC- and MPMC-fed kittens, with HPLC-fed cats showing greater species richness and microbial diversity. Two phyla, ten families and fifteen genera were responsible for more than 80% of the sequences at each taxonomic level for both diet groups, consistent with the previous taxonomic study. Significantly different abundances between diet groups were observed for 324 genera (56% of all genera identified) demonstrating widespread diet-induced changes in microbial taxonomic structure. Diversity was not affected over time. Functional analysis identified 2,013 putative enzyme function groups were different (p<0.000007) between the two dietary groups and were associated to 194 pathways, which formed five discrete clusters based on average relative abundance. Of those, ten contained more (p<0.022) enzyme functions with significant diet effects than expected by chance. Six pathways were related to amino acid biosynthesis and metabolism linking changes in dietary protein with functional differences of the gut microbiome. CONCLUSIONS: These data indicate that feline feces-derived microbiomes have large structural and functional differences relating to the dietary protein:carbohydrate ratio and highlight the impact of diet early in life.