Alison Colyer

Geometric analysis of macronutrient selection in breeds of the domestic dog, Canis lupus familiaris.

Although many herbivores and omnivores have been shown to balance their intake of macronutrients when faced with nutritionally variable foods, study of this ability has been relatively neglected in carnivores, largely on the assumption that prey are less variable in nutrient composition than the foods of herbivores and omnivores and such mechanisms therefore unnecessary. We performed diet selection studies in 5 breeds of adult dog (Canis lupus familiaris) to determine whether these domesticated carnivores regulate macronutrient intake. Using nutritional geometry, we show that the macronutrient content of the diet was regulated to a protein:fat:carbohydrate ratio of approximately 30%:63%:7% by energy, a value that was remarkably similar across breeds. These values, which the analysis suggests are dietary target values, are based on intakes of dogs with prior experience of the respective experimental food combinations. On initial exposure to the diets (i.e., when naive), the same dogs self-selected a diet that was marginally but significantly lower in fat, suggesting that learning played a role in macronutrient regulation. In contrast with the tight regulation of macronutrient ratios, the total amount of food and energy eaten was far higher than expected based on calculated maintenance energy requirements. We interpret these results in relation to the evolutionary history of domestic dogs and compare them to equivalent studies on domestic cats.

A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva, Gingivitis or Mild Periodontitis

Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.

Pyrosequencing the Canine Faecal Microbiota: Breadth and Depth of Biodiversity

Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5′ region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a “core microbiota”. Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs) at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs.

Effect of dietary water intake on urinary output, specific gravity and relative supersaturation for calcium oxalate and struvite in the cat

It has been reported that daily fluid intake influences urinary dilution, and consequently the risk of urolithiasis in human subjects and dogs. The aim of the present study was to investigate the role of dietary moisture on urinary parameters in healthy adult cats by comparing nutritionally standardised diets, varying only in moisture content. A total of six cats were fed a complete dry food (6.3 % moisture) hydrated to 25.4, 53.2 and 73.3 % moisture for 3 weeks in a randomised block cross-over design. Urinary specific gravity (SG), urine volume, water drunk and total fluid intake were measured daily; relative supersaturation (RSS) for calcium oxalate (CaOx) and struvite was calculated using the SUPERSAT computer program. Cats fed the 73.3 % moisture diet produced urine with a significantly lower SG (P < 0.001) compared with diets containing 53.2 % moisture or lower. Mean RSS for CaOx was approaching the undersaturated zone (1.14 (sem 0.21); P = 0.001) for cats fed the diet with 73.3 % moisture and significantly lower than the 6.3 % moisture diet (CaOx RSS 2.29 (sem 0.21)). The effect of diet on struvite RSS was less clear, with no significant difference between treatment groups. Total fluid intake was significantly increased (P < 0.001) in the 73.3 % moisture diet (144.7 (SEM 5.2) ml, or 30 ml/kg body weight per d) compared with the 6.3 % (103.4 (SEM 5.3) ml), 25.4 % (98.6 (SEM 5.3) ml) and 53.3 % (104.7 (SEM 5.3) ml) moisture diets, despite voluntary water intake decreasing as dietary moisture intake increased. Cats fed the 73.3 % moisture diet had a higher total daily fluid intake resulting in a more dilute urine with a lower risk of CaOx when compared with the lower-moisture diets.

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.

The effect of dietary starch level on postprandial glucose and insulin concentrations in cats and dogs

A charge made against feeding dry foods to cats is that the high carbohydrate (i.e. starch) content results in high blood glucose levels which over time may have detrimental health effects. The present study determined the post-meal concentrations of plasma glucose and insulin in adult cats (seven males and four females) and dogs (Labrador retrievers; four males and five females) fed dry diets with low-starch (LS), moderate-starch (MS) or high-starch (HS) levels. In a cross-over design with at least 7 d between the test meals, plasma glucose and insulin concentrations were measured following a single meal of a LS, MS and HS diet (209 kJ/kg bodyweight). Only the HS diet resulted in significant post-meal increases in plasma glucose concentration in cats and dogs although the time-course profiles were different between the species. In cats, plasma glucose concentration was significantly increased above the pre-meal concentration from 11 h until 19 h after the meal, while in dogs, a significant increase above baseline was seen only at the 7 h time point. Plasma insulin was significantly elevated in dogs 4-8 h following the MS diet and 2-8 h after the HS diet. In cats, plasma insulin was significantly greater than baseline from 3-7 and 11-17 h after the HS diet. The time lag (approximately 11 h) between eating the HS diet and the subsequent prolonged elevation of plasma glucose concentration seen in cats may reflect metabolic adaptations that result in a slower digestive and absorptive capacity for complex carbohydrate.

Postprandial glucose and insulin profiles following a glucose-loaded meal in cats and dogs

Data from intravenous (i.v.) glucose tolerance tests suggest that glucose clearance from the blood is slower in cats than in dogs. Since different physiological pathways are activated following oral administration compared with i.v. administration, we investigated the profiles of plasma glucose and insulin in cats and dogs following ingestion of a test meal with or without glucose. Adult male and female cats and dogs were fed either a high-protein (HP) test meal (15 g/kg body weight; ten cats and eleven dogs) or a HP + glucose test meal (13 g/kg body-weight HP diet + 2 g/kg body-weight D-glucose; seven cats and thirteen dogs) following a 24 h fast. Marked differences in plasma glucose and insulin profiles were observed in cats and dogs following ingestion of the glucose-loaded meal. In cats, mean plasma glucose concentration reached a peak at 120 min (10.2, 95 % CI 9.7, 10.8 mmol/l) and returned to baseline by 240 min, but no statistically significant change in plasma insulin concentration was observed. In dogs, mean plasma glucose concentration reached a peak at 60 min (6.3, 95 % CI 5.9, 6.7 mmol/l) and returned to baseline by 90 min, while plasma insulin concentration was significantly higher than pre-meal values from 30 to 120 min following the glucose-loaded meal. These results indicate that cats are not as efficient as dogs at rapidly decreasing high blood glucose levels and are consistent with a known metabolic adaptation of cats, namely a lack of glucokinase, which is important for both insulin secretion and glucose uptake from the blood.

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.

Development of real-time PCR assays for the detection of Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar) and European plaice (Pleuronectes platessa) in complex food samples

We have developed species-specific real-time PCR assays for the identification of Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar) and European plaice (Pleuronectes platessa) in food products. The species-specific assays, comprising a set of primers and probe for each species, were designed using genomic genes (pantophysin for Atlantic cod, growth hormone for Atlantic salmon and parvalbumin for European plaice) which were then optimised for specificity and selectivity. The sensitivity and the effect of heat and pressure on amplification efficiency were then determined for each assay. These assays were then used to analyse DNA extracted from commercial fish products and model food samples spiked with each of the fish species. The target species was successfully identified in all samples analysed, demonstrating the applicability of these assays to the analysis of food products.