Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

Dietary Supplementation with Nucleotides, Short-Chain Fructooligosaccharides, Xylooligosaccharides, Beta-Carotene and Vitamin E Influences Immune Function in Kittens.

Nucleotides, short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS), ß-carotene and vitamin E are reported to enhance immune function; however, the evidence of this in cats is limited. The aim of this study was to determine the immunomodulatory effects of these ingredients in kittens. Forty domestic short hair kittens were designated in litters to control or test diet for 28 weeks. Test diet was fortified with 0.33 g nucleotides, 0.45 g scFOS, 0.3 g XOS, 0.7 mg ß-carotene and 66.5 mg vitamin E per 100 g diet. Kittens were vaccinated against feline parvovirus (FPV) and herpesvirus (FHV) at 10, 14 and 18 weeks. Kittens remained healthy, with no measured evidence of adverse health. Serum FPV and FHV antibody titres were significantly (p < 0.05) higher in the test diet group at week 23 and 27, respectively. A significantly (p < 0.05) higher proportion of test diet group kittens demonstrated an adequate response (four-fold titre increase) to FHV vaccination and a significantly (p < 0.05) higher proportion reached a protective antibody titre for FHV. Serum IgM was significantly (p < 0.05) higher in the test diet group. The test diet group demonstrated a stronger humoral immune response to vaccination, suggesting the diet supports immune defence, enabling a greater response to immune challenges.

Developing diagnostic tools for canine periodontitis: combining molecular techniques and machine learning models.

BACKGROUND: Dental plaque microbes play a key role in the development of periodontal disease. Numerous high-throughput sequencing studies have generated understanding of the bacterial species associated with both canine periodontal health and disease. Opportunities therefore exist to utilise these bacterial biomarkers to improve disease diagnosis in conscious-based veterinary oral health checks. Here, we demonstrate that molecular techniques, specifically quantitative polymerase chain reaction (qPCR) can be utilised for the detection of microbial biomarkers associated with canine periodontal health and disease. RESULTS: Over 40 qPCR assays targeting single microbial species associated with canine periodontal health, gingivitis and early periodontitis were developed and validated. These were used to quantify levels of the respective taxa in canine subgingival plaque samples collected across periodontal health (PD0), gingivitis (PD1) and early periodontitis (PD2). When qPCR outputs were compared to the corresponding high-throughput sequencing data there were strong correlations, including a periodontal health associated taxa, Capnocytophaga sp. COT-339 (rs =0.805), and two periodontal disease associated taxa, Peptostreptococcaceae XI [G-4] sp. COT-019 (rs=0.902) and Clostridiales sp. COT-028 (rs=0.802). The best performing models, from five machine learning approaches applied to the qPCR data for these taxa, estimated 85.7% sensitivity and 27.5% specificity for Capnocytophaga sp. COT-339, 74.3% sensitivity and 67.5% specificity for Peptostreptococcaceae XI [G-4] sp. COT-019, and 60.0% sensitivity and 80.0% specificity for Clostridiales sp. COT-028. CONCLUSIONS: A qPCR-based approach is an accurate, sensitive, and cost-effective method for detection of microbial biomarkers associated with periodontal health and disease. Taken together, the correlation between qPCR and high-throughput sequencing outputs, and early accuracy insights, indicate the strategy offers a prospective route to the development of diagnostic tools for canine periodontal disease.

Development of Yorkshire Terrier Dentition.

The development of dentition in dogs has been associated with several problems including tooth over-crowding, missing permanent dentition, and persistent deciduous teeth (PDT). Information on dentition development in different breeds is lacking. This study of 61 Yorkshire terriers aimed to determine the (i) average age at deciduous tooth exfoliation, (ii) average age at permanent tooth eruption, (iii) PDT incidence, and influencing factors such as body weight. The ages of exfoliation of deciduous teeth and eruption of permanent dentition were influenced by body weight and tooth type. These dentition changes tended to occur later in dogs ≤ 3 kg versus dogs > 5 kg. Generally, incisors were exfoliated first, followed by premolars and then canines. At a body weight of 4.5 kg, the middle of the data range, the estimated age at loss of deciduous teeth (with 95% confidence intervals) was 21.9 (21.1, 22.9) weeks for incisors, 26.1 (24.9, 27.4) weeks for canines, and 23.9 (22.9, 24.9) weeks for premolar. The estimated age at eruption of permanent dentition was 22.3 (21.6, 23.0) weeks for incisors, 23.8 (23.0, 24.6) weeks for canines, 24.7 (24.0, 25.5) weeks for premolars, and 26.4 (25.5, 27.3) for molar teeth. However, this sequence was disrupted in dogs ≤ 3 kg. Yorkshire terriers had a high incidence of PDT. At a body weight of 4.5 kg, the estimated proportion of PDT was: incisors 0.86% (0.32, 2.31), canines 15.62% (7.62, 29.37) and premolars 3.57% (1.62, 7.66). Canines constituted the most frequently retained tooth type, with 89.1% retained in dogs ≤ 3 kg compared to 12.0% in dogs > 5 kg. This information will enable veterinarians to provide personalised advice regarding the oral care requirements for Yorkshire terriers and highlights the need to regularly monitor this breed between the ages of two and seven months, during the active phases of tooth development.

Healthy cats tolerate long-term daily feeding of Cannabidiol.

Cannabidiol (CBD)-containing products are widely commercially available for companion animals, mirroring popularity in human use. Although data on the safety and efficacy of long-term oral supplementation are increasing in dogs, evidence remains lacking in cats. The purpose of these studies was to address gaps in the knowledge around the long-term suitability and tolerance of a tetrahydrocannabinol (THC)-free CBD distillate in clinically healthy cats. The studies were randomized, blinded, and placebo-controlled. The first study supplemented cats with either a placebo oil (n = 10) or with 4 mg/kg body weight (BW) CBD in placebo oil (n = 9) daily, with a meal, for 4 weeks. The concentration of CBD in plasma was measured over 4 h at d0 (first dose) and again at d14 (after 2 weeks of daily dosing). The second study supplemented cats daily with either placebo oil (n = 10) or 4 mg/kg BW CBD in placebo oil (n = 10) for a period of 26 weeks. A comprehensive suite of physiological health measures was performed throughout the study at baseline (week 0) and after 4, 10, 18, and 26 weeks of feeding, followed by a 4-week washout sample (week 30). Postprandial plasma CBD time course data, at both d0 and d14, showed a peak plasma CBD concentration at 2 h after the dose. This peak was 251 (95% CI: 108.7, 393.4) and 431 (95% CI, 288.7, 573.4) ng/mL CBD at d0 and d14, respectively, and the area under the curve concentration was higher by 91.5 (95% CI, 33.1, 149.9) ng-h/mL after 2 weeks of supplementation (p = 0.002). While in the first study the CBD group displayed increased alanine aminotransferase (ALT; 68.7 (95% CI, 43.23, 109.2) U/L) at week 4 compared to the placebo control group [1.44-fold increase (95% CI, 0.813, 2.54)], statistical equivalence (at 2-fold limits) was found for ALT across the duration of the second, long-term study. All other biochemistry and hematology data showed no clinically significant differences between supplement groups. Data presented here suggest that a THC-free, CBD distillate fed at a dose of 4 mg/kg BW was absorbed into plasma and well tolerated by healthy cats when supplemented over a period of 26 weeks.

Long-term daily feeding of cannabidiol is well-tolerated by healthy dogs.

Cannabidiol (CBD) containing dog food and treats are widely commercially available, mirroring the growing popularity of CBD as a supplement for humans. Despite this, experimental evidence of the safety and efficacy of long-term oral exposure in dogs is lacking. The purpose of this study was to address the gap in knowledge around the longer-term suitability and tolerance of a broad-spectrum CBD (THC-free) distillate in clinically healthy dogs. The study was a randomized, placebo-controlled, and blinded study where one group of twenty dogs received daily CBD capsules at a dose of 4 mg/kg of body weight (BW) for a period of 6 months. The control group of twenty dogs received placebo capsules. A comprehensive suite of physiological health measures was performed throughout the study at baseline, and after 2, 4, 10, 18, and 26 weeks of exposure, followed by 4 weeks of washout. CBD concentrations were measured at the same cadence in plasma, feces and urine. Health measures included biochemistry, hematology, urinalysis, in addition to fortnightly veterinary examinations, twice daily well-being observations, and a daily quality-of-life survey. Biochemistry and hematology showed no clinically significant alterations apart from a transient elevation in alkaline phosphatase (ALP) in just over half of the dogs receiving CBD. This elevation was observed in the absence of concurrent elevations of other liver parameters, and without any adverse effects on health and wellbeing. Furthermore, bone alkaline phosphatase (BALP) was simultaneously elevated with a significant, strong (r > 0.9) positive correlation between the two measures, suggesting that the elevation of total ALP was at least partly due to the bone-derived isoform. This study provides evidence that a once-daily oral dose of 4 mg CBD/kg BW is well tolerated in clinically healthy dogs for a duration of 6-months.

Effectiveness of oral care interventions on malodour in dogs.

BACKGROUND: Oral malodour is identified by pet owners as an unpleasant inconvenience, but they may not recognise this likely indicates underlying disease. The primary cause of oral malodour relates to the presence of bacteria in the oral cavity often associated with gingivitis and periodontitis. The purpose of this study was to determine the effect of feeding two oral care chews with different textural properties on oral malodour and the proportion of bacterial species involved in the production of volatile sulphur compounds (VSCs). METHODS: Fourteen dogs (9 Petit Basset Griffon Vendéen (PBGV) and 5 Beagle dogs) participated in the randomised cross-over study for a total of 14 weeks. The cohort was divided into four groups with each exposed to a different intervention per week: chew A, chew B, tooth brushing control or a no intervention control. An induced malodour method was used to assess VSCs in breath samples using a portable gas chromatograph (OralChroma™). Microbiological samples (supragingival plaque and tongue coating scrapes) were analysed for VSC-producing bacteria using Oral Hydrogen Sulfide agar with lead acetate. RESULTS: VSCs were detected in the dogs' breath samples and levels of hydrogen sulphide and methyl mercaptan were found to be reduced following an intervention. Chew B significantly reduced the levels of both hydrogen sulphide (p < 0.001) and methyl mercaptan (p < 0.05) compared to no intervention. Reductions in methyl mercaptan were also observed for chew A and tooth brushing but these were not statistically significant. When compared to no intervention, all interventions significantly reduced the total bacterial load and VSC producing bacterial load in plaque (p < 0.001). For tongue samples, only chew B significantly reduced the total bacterial load and VSC-producing bacterial load (p < 0.001) compared to no intervention. CONCLUSIONS: By inducing oral malodour and subsequently applying the one-time interventions, significant reductions in the levels of VSCs were observed. The use of oral care chews texturally designed to deliver a deep, all-round cleaning action can be particularly effective at managing oral malodour in dogs, likely through an enhanced ability to remove bacteria.

A Mass Spectrometric Approach to the Proteomic Profiling of the Canis lupus familiaris Acquired Enamel Pellicle on Hydroxyapatite Discs.

The acquired enamel pellicle (AEP) is a multi-protein film attached to the surface of teeth, which functions to lubricate the dental surface, form an anti-erosive barrier and exhibits antimicrobial properties. The initiation of AEP formation occurs within seconds of exposure to saliva, a biofluid rich in protein species. While there have been many publications on the formation of human AEP there is little research on the composition of canine AEP during its acquisition. The aim of these studies was to explore the composition of canine AEP formation, utilising hydroxyapatite (HA) discs as a tooth substitute matrix, over time. Qualitative and quantitative proteomics techniques using tandem mass tag labelled peptides and LC-MS/MS were used to follow the formation of canine AEP on hydroxyapatite discs over the course of an hour. Proteins adsorbed to the HA surface included highly abundant proteins in canine saliva, antimicrobial proteins, protease inhibitors and the buffering agent carbonic anhydrase. Greater understanding of the canine AEP deepens fundamental knowledge of the early processes driving bacterial colonisation of the tooth surface and subsequent plaque accumulation.

Comparison of subgingival and gingival margin plaque microbiota from dogs with healthy gingiva and early periodontal disease.

The purpose of the investigation was to determine whether canine gingival margin (GM) plaque is a reliable surrogate for subgingival (SG) plaque from a microbial community (microbiota) perspective. SG and GM plaque samples were collected from 381 dogs visiting pet hospitals in the USA, China and Thailand. Dogs with clinically healthy gingivae through to early periodontitis were included in the study. The samples were subject to next generation Illumina sequence analysis to allow microbiota comparisons to be made between the two plaque sources. Overall, the SG and GM samples indicated commonality via the majority community that were shared between them; health associations led to the identification of some significant taxa-specific differences. GM microbiota exhibited lower variability and diversity and were shown to reflect a sub-population of those associated with SG plaque. Both plaque niches, however, demonstrated similar changes in microbial signatures with health and early periodontal disease and did not indicate divergent trends. The key, most abundant microbiota of GM plaque strongly reflect those observed with SG plaque across health and early periodontitis. Microbiota in plaque from above the gum line may therefore be employed as a biomarker of oral health. This opens up the potential to use plaque, sampled from conscious dogs, to define oral health status and improve the diagnosis, treatments and interventions for periodontal disease.

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.

The canine oral microbiome: variation in bacterial populations across different niches.

BACKGROUND: Microbiota from different niches within the canine oral cavity were profiled and compared. Supragingival plaque and stimulated saliva, were collected alongside samples from the buccal and tongue dorsum mucosa, from 14 Labrador retrievers at three timepoints within a 1 month timeframe. The V3-V4 region of the 16S rRNA gene was sequenced via Illumina MiSeq. RESULTS: Supragingival plaque microbiota had the highest bacterial diversity and the largest number of significant differences in individual taxa when compared to the other oral niches. Stimulated saliva exhibited the highest variability in microbial composition between dogs, yet the lowest bacterial diversity amongst all the niches. Overall, the bacteria of the buccal and tongue dorsum mucosa were most similar. CONCLUSIONS: The bacterial community profiles indicated three discrete oral niches: soft tissue surfaces (buccal and tongue dorsum mucosa), hard tissue surface (supragingival plaque) and saliva. The ability to distinguish the niches by their microbiota signature offers the potential for microbial biomarkers to be identified in each unique niche for diagnostic use.

A longitudinal assessment of periodontal disease in Yorkshire terriers.

BACKGROUND: Periodontal disease is the most common oral disease of dogs and has been associated with systemic disease. The purpose of the present study was to determine the extent of periodontal disease in a population of Yorkshire terrier dogs with and without a tooth brushing regimen. Each dog was assessed under general anaesthesia two to five times between 37 and 78 weeks of age. The extent of gingivitis and periodontitis was ascertained for every tooth in the mouth. Gingivitis was measured using time to bleeding on probing, and periodontitis was based on extent of clinical attachment loss (probing depth, gingival recession and furcation exposure). RESULTS: Of the 49 dogs assessed at 37 weeks of age, 98% had at least one tooth or aspect with early periodontitis (PD2, < 25% attachment loss). The average percentage of teeth with periodontitis in the mouth was 29.6% with 95% confidence interval (23.6, 36.4). The odds of early periodontitis was 2.74 (2.23, 3.37) times higher at 78 weeks of age compared to 37 weeks of age. The canine teeth had a significantly higher probability of periodontitis compared to all other tooth types at both 37 and 78 weeks of age (p < 0.001). In addition, at the same time points, the incisors had a significantly higher probability of periodontitis compared to the molars and premolars (p < 0.001). CONCLUSIONS: Breeds of dog that are susceptible to developing periodontitis, such as Yorkshire terriers, require effective treatments for the prevention of periodontal disease from a young age. Although tooth brushing is one of the most effective methods when it comes to preventative homecare, this is not always realistic, as was found in this study. Therefore alternative ways to retard or prevent plaque accumulation that are practical for both dogs and their owners are required.

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.

Validation of Quantitative Light-Induced Fluorescence for Quantifying Calculus on Dogs' Teeth.

Periodontal disease is a common disease of dogs and is initiated by the buildup of plaque on the tooth surface. As plaque matures, it becomes mineralized to form calculus, which although not directly involved in the etiology of periodontal disease, provides an irregular surface to which plaque can adhere. Evaluation of the quantity of plaque and calculus on dogs' teeth is therefore essential to enable the efficacy of products, designed to prevent or retard plaque and calculus accumulation, to be evaluated. The objective of this study was to determine whether quantitative light-induced fluorescence (QLFTM) is a suitable tool to quantify the amount of calculus on the buccal surface of dogs' teeth following the removal of disclosed plaque by tooth brushing. The amount of calculus on the teeth of 26 miniature schnauzers was measured, using QLF and a calculus index method (Warrick-Gorrel), during a 28-day phase crossover study comparing feeding a daily dental chew versus providing no daily chew. Quantification of calculus using the Warrick-Gorrel method showed a 43.8% reduction in calculus buildup, with 95% confidence interval of 27.3 to 60.3 ( P < .001). With QLF, the percentage reduction in calculus accumulation was 65.8% (58.1-73.4, P < .001). A retrospective sample size analysis showed that fewer dogs were required for QLF analysis compared to the Warrick-Gorrel method. This study demonstrated that QLF is a sensitive and precise method for quantification of calculus on dogs' teeth. It removes the subjective element of human examiners and has greater accuracy and reduced variability through the continuous nature of the data.

The Saliva Proteome of Dogs: Variations Within and Between Breeds and Between Species.

Saliva is a complex multifunctional fluid that bathes the oral cavity to assist in soft and hard tissue maintenance, lubrication, buffering, defense against microbes, and initiating digestion of foods. It has been extensively characterized in humans but its protein composition in dogs remains poorly characterized, yet saliva composition could explain (patho) physiological differences between individuals, breeds and with humans. This pilot discovery study aimed to characterize canine saliva from two breeds, Labrador retrievers and Beagles, and to compare this with human saliva using quantitative mass spectrometry. The analysis demonstrated considerable inter-individual variation and difference between breeds; however these were small in comparison to the differences between species. Functional mapping suggested roles of detected proteins similar to those found in human saliva with the exception of the initiation of digestion as salivary amylase was lacking or at very low abundance in canine saliva samples. Many potential anti-microbial proteins were detected agreeing with the notion that the oral cavity is under continuous microbial challenge.

Assessment of dental plaque coverage by Quantitative Light-induced Fluorescence (QLF) in domestic short-haired cats.

Dietary means of reducing plaque and calculus deposits are frequently sought for the maintenance of oral health in cats and dogs. In the development of such products sensitive, reliable, reproducible methods of measuring plaque and calculus are key. The aim of this study was to assess Quantitative Light-induced Fluorescence (QLF™) for the detection of dental plaque coverage in cats compared to the modified Logan and Boyce technique. The techniques were utilised in a crossover study, which compared two diets for their effect on plaque deposition in a cohort of 24 adult cats. Analysis of the effect of diet on plaque coverage by both the modified Logan and Boyce technique and QLF showed a significant effect of feeding regime (p=0.024 and p≤0.0001, respectively) with good agreement between the techniques in the percentage reduction of plaque accumulation. A within study assessment of QLF demonstrated excellent intra-operator repeatability (coefficient of variation 2.2%). Similarly, inter-operator reproducibility was also good (coefficient of variation 2.3%). A retrospective analysis, using the data to estimate the sample size required for at least 90% power to detect a 15% difference between treatments in a two-way crossover study, established that 10 cats would be sufficient for plaque measurement by QLF, while assessment by the modified Logan and Boyce method required over 30 cats. QLF was determined to be a reliable, reproducible method for the assessment of plaque deposition in cats and requires fewer subjects for the detection of differences between treatment effects compared to the modified Logan and Boyce method.

The Prevalence of Canine Oral Protozoa and Their Association with Periodontal Disease.

Periodontal disease is one of the most important health concerns for companion animals. Research into canine forms of periodontitis has focused on the identification and characterization of the bacterial communities present. However, other microorganisms are known to inhabit the oral cavity and could also influence the disease process. A novel, broad spectrum 18S PCR was developed and used, in conjunction with next-generation sequencing analyses to target the identification of protists. Trichomonas sp. and Entamoeba sp. were identified from 92 samples of canine plaque. The overall prevalence of trichomonads was 56.52% (52/92) and entamoebae was 4.34% (4/92). Next-generation sequencing of pooled healthy, gingivitis, early-stage periodontitis, and severe periodontitis samples revealed the proportion of trichomonad sequences to be 3.51% (health), 2.84% (gingivitis), 6.07% (early periodontitis), and 35.04% (severe periodontitis), respectively, and entamoebae to be 0.01% (health), 0.01% (gingivitis), 0.80% (early-stage periodontitis), and 7.91% (severe periodontitis) respectively. Both genera of protists were statistically associated with plaque from dogs with periodontal disease. These findings provide the first conclusive evidence for the presence of oral protozoa in dog plaque and suggest a possible role for protozoa in the periodontal disease process.

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.

Glycogen metabolic genes are involved in trehalose-6-phosphate synthase-mediated regulation of pathogenicity by the rice blast fungus Magnaporthe oryzae.

The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a Δagl1Δgph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae.

The bacterial secondary metabolite 2,4-diacetylphloroglucinol impairs mitochondrial function and affects calcium homeostasis in Neurospora crassa.

The bacterial secondary metabolite 2,4-diacetylphloroglucinol (DAPG) is of interest as an active ingredient of biological control strains of Pseudomonas fluorescens and as a potential lead pharmaceutical molecule because of its capacity to inhibit growth of diverse microbial and non-microbial cells. The mechanism by which this occurs is unknown and in this study the filamentous fungus Neurospora crassa was used as a model to investigate the effects of DAPG on a eukaryotic cell. Colony growth, conidial germination and cell fusion assays confirmed the inhibitory nature of DAPG towards N. crassa. A number of different fluorescent dyes and fluorescent protein reporters were used to assess the effects of DAPG treatment on mitochondrial and other cellular functions. DAPG treatment led to changes in mitochondrial morphology, and rapid loss of mitochondrial membrane potential. These effects are likely to be responsible for the toxicity of DAPG. It was also found that DAPG treatment caused extracellular calcium to be taken up by conidial germlings leading to a transient increase in cytosolic free Ca(2+) with a distinct concentration dependent Ca(2+) signature.