Distinct Fermentation and Antibiotic Sensitivity Profiles Exist in Salmonellae of Canine and Human Origin

Background Salmonella enterica is a recognised cause of diarrhoea in dogs and humans, yet the potential for transfer of salmonellosis between dogs and their owners is unclear, with reported evidence both for and against Salmonella as a zoonotic pathogen. A collection of 174 S. enterica isolates from clinical infections in humans and dogs were analysed for serotype distribution, carbon source utilisation, chemical and antimicrobial sensitivity profiles. The aim of the study was to understand the degree of conservation in phenotypic characteristics of isolates across host species. Results Serovar distribution across human and canine isolates demonstrated nine serovars common to both host species, 24 serovars present in only the canine collection and 39 solely represented within the human collection. Significant differences in carbon source utilisation profiles and ampicillin, amoxicillin and chloramphenicol sensitivity profiles were detected in isolates of human and canine origin. Differences between the human and canine Salmonella collections were suggestive of evolutionary separation, with canine isolates better able to utilise several simple sugars than their human counterparts. Generally higher minimum inhibitory concentrations of three broad-spectrum antimicrobials, commonly used in veterinary medicine, were also observed in canine S. enterica isolates. Conclusions Differential carbon source utilisation and antimicrobial sensitivity profiles in pathogenic Salmonella isolated from humans and dogs are suggestive of distinct reservoirs of infection for these hosts. Although these findings do not preclude zoonotic or anthroponotic potential in salmonellae, the separation of carbon utilisation and antibiotic profiles with isolate source is indicative that infectious isolates are not part of a common reservoir shared frequently between these host species

Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by 'universal' primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology

Quantification of Canine Dental Plaque Using Quantitative Light-Induced Fluorescence

The aim of this work was to evaluate Quantitative Light-induced Fluorescence (QLFTM) as an alternative to the established Logan and Boyce method for determining plaque coverage of dogs’ teeth. In a series of studies in conscious and anesthetized dogs, QLF showed good intra-photographer repeatability (coefficient of variation [CV] of 7.5% for undisclosed teeth) and inter-photographer reproducibility (CV of 3.2% for undisclosed teeth and 8.5% for disclosed teeth). The QLF software accurately identifies areas of plaque as demonstrated by comparison to the variability of 5 human scorers, manually marking plaque on QLF-acquired images (P = 0.1). There was good agreement with the modified Logan and Boyce method in the percentage reduction in plaque accumulation measured when dogs were fed an oral care chew versus no chew. To see a 15% difference in plaque accumulation, which is considered sufficient by the Veterinary Oral Health Council to differentiate between 2 treatments, a retrospective power analysis (90%) of the data established that only 7 dogs would be required, compared to 19 dogs for the modified Logan and Boyce method. QLF is a reliable method for measuring dental plaque in dogs with the added advantage that it is not subjective and requires fewer animals

Investigating the prevalence of Salmonella in dogs within the Midlands region of the United Kingdom

Background - The intimate relationship between dogs and their owners has the potential to increase the risk of human exposure to bacterial pathogens. Over the past 40 years, there have been several reports on transmission of salmonellae from dogs to humans. This study therefore aimed to determine the prevalence of Salmonella in the faeces of dogs from the Midlands region of the United Kingdom to assess exposure risk and potential for zoonotic transmission. Results - A total of 436 apparently healthy dogs without diarrhoea from households (n = 126), rescue centres (n = 96), boarding kennels (n = 43), retired greyhound kennels (n = 39) and a pet nutrition facility (n = 132) were investigated for Salmonella shedding. Faecal samples were processed by an enrichment culture based method. The faeces from one dog (0.23 %; 95 % confidence limit 0.006 %, 1.27 %) was positive for Salmonella. The species was S. enterica subspecies arizonae. Conclusion - This study showed that the prevalence of Salmonella from faeces from apparently healthy dogs from a variety of housing conditions is low; however, Salmonella shedding was still identified

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

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

Abstract 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 (r s =0.805), and two periodontal disease associated taxa, Peptostreptococcaceae XI [G-4] sp. COT-019 (r s =0.902) and Clostridiales sp. COT-028 (r s =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

Observed OTUs calculated using the RDP Infernal Aligner and Complete Linkage Clustering tools, and Chao1 richness estimates calculated using Mothur, based on the 10,000 reads analysed from each animal.

<p>Red triangles: observed OTUs; black squares: Chao1 richness estimates; bars represent upper and lower 95% confidence intervals.</p

Details of miniature Schnauzer dogs used in the study, showing genetic relatedness.

<p>Details of miniature Schnauzer dogs used in the study, showing genetic relatedness.</p

Percentages of sequence reads, which were unassigned at the genus level using an RDP bootstrap score of 30%, that could be assigned by phylum.

<p>Percentages of sequence reads, which were unassigned at the genus level using an RDP bootstrap score of 30%, that could be assigned by phylum.</p