Music of metagenomics-a review of its applications, analysis pipeline, and associated tools.

This humble effort highlights the intricate details of metagenomics in a simple, poetic, and rhythmic way. The paper enforces the significance of the research area, provides details about major analytical methods, examines the taxonomy and assembly of genomes, emphasizes some tools, and concludes by celebrating the richness of the ecosystem populated by the "metagenome."

Untargeted fecal metabolome analysis in obese dogs after weight loss achieved by feeding a high-fiber-high-protein diet.

INTRODUCTION: In humans and companion animals, obesity is accompanied by metabolic derangements. Studies have revealed differences in the composition of the fecal microbiome between obese dogs and those with an ideal body weight. OBJECTIVES: We have previously reported that the fecal microbiome in obese dogs changes after controlled weight reduction, induced by feeding a diet high in fiber and protein. Despite these findings, it is unclear if taxonomic differences infer differences at the functional level between obese dogs and those with an ideal body weight. METHODOLOGY: Untargeted fecal metabolome analysis was performed on dogs with obesity before and after weight loss achieved by feeding a high-fiber-high-protein diet. RESULTS: Fecal metabolome analysis revealed a total of 13 compounds that changed in concentration in obese dogs after weight loss. Of these compounds, metabolites associated with bacterial metabolism decreased after weight loss including purine, L-(-)-methionine, coumestrol, and the alkaloids 1-methylxanthine and trigonelline. Conversely, the polyphenols (-)-epicatechin and matairesinol and the quinoline derivatives 1,5-isoquinolinediol and 2-hydroxiquinoline increased after weight loss. CONCLUSION: These results suggest differences in intestinal microbiome at the functional level after weight loss, but further studies are needed to determine the role of these compounds in the etiology of obesity and weight loss.

Effects of a perioperative antibiotic and veterinary probiotic on fecal dysbiosis index in dogs.

Although widely used, the effects of perioperative antibiotics on the gastrointestinal microbiome are still being researched. The role of probiotics to ameliorate adverse effects of perioperative antibiotics is unclear. The dysbiosis index (DI), based on a quantitative polymerase chain reaction (qPCR) technique, is used to assess gastrointestinal health. The DI in dogs receiving perioperative antibiotics and the effects of concurrent probiotics were evaluated in this study. This was a prospective study of 20 dogs undergoing hemilaminectomy. Baseline and 48-hour postoperative fecal DI were evaluated. Eleven dogs received a probiotic and 9 received placebo. Preanesthetic DI was not different between treatment groups (P = 0.378). One bacterial group, Blautia, decreased in the placebo group (P = 0.002); however, there was no change in the probiotic group (P = 0.336). The DI increased numerically after probiotic administration, but the time × treatment interaction was not significant (P = 0.996). Administration of a probiotic failed to improve DI. Further investigation is needed to evaluate long-term effects of perioperative antibiotics on the gut microbiome.

Effets d'un antibiotique périopératoire et d'un probiotique vétérinaire sur l'indice de dysbiose fécale chez le chien. Les antibiotiques périopératoires sont largement utilisés, mais leurs effets sur le microbiome gastro-intestinal sont toujours à l'étude. Le rôle des probiotiques dans l'amélioration des effets indésirables liés aux antibiotiques périopératoires n'est pas clair. L'indice de dysbiose (ID), une technique de PCR quantitative, est utilisé pour évaluer la santé gastro-intestinale. Cette étude a évalué l'ID chez les chiens recevant des antibiotiques périopératoires ainsi que tout effet lié à l'administration d'un probiotique en simultané. Il s'agissait d'une étude prospective portant sur 20 chiens subissant une hémilaminectomie. Les valeurs d'ID de référence ainsi que 48 heures postopératoires ont été évaluées. Onze chiens ont reçu un probiotique; 9 ont reçu un placebo. L'ID pré-anesthésique n'était pas différent entre les deux groupes (P = 0,378). Un groupe bactérien, Blautia, a diminué dans le groupe placebo (P = 0,002); il n'y a eu aucun changement dans le groupe probiotique (P = 0,336). L'ID a augmenté quantitativement après l'administration de probiotiques, mais l'interaction « temps × traitement ¼ n'était pas significative (P = 0,996). L'administration d'un probiotique n'a pas amélioré l'ID. Des recherches supplémentaires sont nécessaires pour évaluer les effets à long terme des antibiotiques périopératoires sur le microbiome intestinal.(Traduit par les auteurs).

BIOMARKERS OF GASTROINTESTINAL DISEASE IN CHEETAHS (ACINONYX JUBATUS).

Gastrointestinal disease is a common clinical problem in captive cheetahs (Acinonyx jubatus). It is reported that gastritis affects the vast majority of the captive population of cheetahs. Pancreatitis and acute and chronic enteritis have also been reported. These issues pose significant long-term health and welfare implications for cheetahs. Cobalamin, folate, methylmalonic acid (MMA), gastrin, feline pancreatic-specific lipase immunoreactivity (fPLI), and feline trypsin-like immunoreactivity (fTLI) immunoassays are important biomarkers of gastrointestinal disease in domestic cats. The goal of this study was to determine if these immunoassays validated in domestic cats could be used clinically in cheetahs, by establishing reference intervals (RI) for these biomarkers in cheetahs. A cohort of 40 clinically healthy cheetahs was selected from three zoological institutions on the basis of being free of clinical gastrointestinal disease and extra-gastrointestinal disease that could affect biomarkers, as well as having banked frozen serum. Cheetah biomarker RI, with domestic cat RI for comparison in parentheses, are as follows: cobalamin 470-618 pg/ml (290-1500 pg/ml), folate 2.2-15.7 ng/ml (9.7-21.6 ng/ml), MMA 365-450 nM/L (139-897 nM/L), fPLI 0.5-1.2 µg/L (0-4 µg/L), and gastrin 30-50 pg/ml (<10-39.5 pg/ml). This study shows that RI for gastrointestinal biomarkers can be notably different, even between species that are as closely related as the domestic cat and the cheetah. Additionally, it was found that the fTLI assay does not cross-immunoreact with cheetahs. In conclusion, this study emphasizes the importance of developing species-specific RI for biomarker assays and using caution when extrapolating RI from other species.

EXOCRINE PANCREATIC INSUFFICIENCY-LIKE SYNDROME IN FOUR CAPTIVE TIGERS (PANTHERA TIGRIS).

Exocrine pancreatic insufficiency (EPI) is a condition characterized by a decreased synthesis and secretion of pancreatic enzymes, which results in weight loss, poor hair coat, and diarrhea. The diagnostic test of choice for EPI in domestic cats is feline serum trypsin-like immunoreactivity (fTLI). This paper details four tigers (Panthera tigris) with clinical signs compatible with EPI. On the basis of domestic cat reference ranges, fTLI assays for all four clinically affected tigers were diagnostic for EPI (median 1.0 µg/L; range 0.5-1.2 µg/L). All four tigers had a rapid clinical response to pancreatic enzyme supplementation. Serum from 10 clinically healthy tigers was submitted for the fTLI assay, for comparative purposes. The healthy tigers' fTLI assays were also within range for a diagnosis of EPI in domestic cats (median 3.1 µg/L; range 1.9-4.5 µg/L); however, clinically affected tigers had significantly lower serum fTLI concentrations than healthy tigers (P = 0.0058). Serum cobalamin was below the detection limit in both the affected and healthy tigers (<150 ng/L). Measuring fTLI appears to be a useful tool in the diagnosis of EPI-like syndrome in tigers. As in other species, EPI-like syndrome in tigers may also be associated with cobalamin deficiency.

Bacterial Biogeography of the Colon in Dogs With Chronic Inflammatory Enteropathy.

The intestinal microbiota is believed to play a role in the pathogenesis of inflammatory bowel disease in humans and chronic inflammatory enteropathy (CIE) in dogs. While most previous studies have described the gut microbiota using sequencing methods, it is fundamental to assess the spatial distribution of the bacteria for a better understanding of their relationship with the host. The microbiota in the colonic mucosa of 22 dogs with CIE and 11 control dogs was investigated using fluorescence in situ hybridization (FISH) with a universal eubacterial probe (EUB338) and specific probes for select bacterial groups. The number of total bacteria labeled with EUB338 probe was lower within the colonic crypts of dogs with CIE compared to controls. Helicobacter spp. and Akkermansia spp. were decreased on the colonic surface and in the crypts of dogs with CIE. Dogs with CIE had increased number of Escherichia coli/Shigella spp. on the colonic surface and within the crypts compared to control dogs. In conclusion, the bacterial microbiota in the colonic mucosa differed between dogs with and without CIE, with depletion of the crypt bacteria in dogs with CIE. The crypt bacterial species that was intimately associated with the host mucosa in control dogs was composed mainly of Helicobacter spp.

Correlating Gastrointestinal Histopathologic Changes to Clinical Disease Activity in Dogs With Idiopathic Inflammatory Bowel Disease.

Prior studies have failed to detect a convincing association between histologic lesions of inflammation and clinical activity in dogs with inflammatory bowel disease (IBD). We hypothesized that use of a simplified histopathologic scoring system would improve the consistency of interpretation among pathologists when describing histologic lesions of gastrointestinal inflammation. Our aim was to evaluate the correlation of histopathologic changes to clinical activity in dogs with IBD using this new system. Forty-two dogs with IBD and 19 healthy control dogs were enrolled in this retrospective study. Endoscopic biopsies from the stomach, duodenum, ileum, and colon were independently scored by 8 pathologists. Clinical disease activity was scored using the Canine Inflammatory Bowel Disease Activity Index (CIBDAI) or the Canine Chronic Enteropathy Clinical Activity Index (CCECAI), depending on the individual study center. Summative histopathological scores and clinical activity were calculated for each tissue (stomach, duodenum, ileum, and colon) and each tissue histologic score (inflammatory/morphologic feature). The correlation between CCECAI/CIBDAI and summative histopathologic score was significant ( P < .05) for duodenum ( r = 0.42) and colon ( r = 0.33). In evaluating the relationship between histopathologic scores and clinical activity, significant ( P < .05) correlations were observed for crypt dilation ( r = 0.42), lamina propria (LP) lymphocytes ( r = 0.40), LP neutrophils ( r = 0.45), mucosal fibrosis ( r = 0.47), lacteal dilation ( r = 0.39), and villus stunting ( r = 0.43). Compared to earlier grading schemes, the simplified scoring system shows improved utility in correlating histopathologic features (both summative histology scores and select histologic scores) to IBD clinical activity.

Effects of prebiotic inulin-type fructans on blood metabolite and hormone concentrations and faecal microbiota and metabolites in overweight dogs.

Because obesity is associated with many co-morbidities, including diabetes mellitus, this study evaluated the second-meal effect of a commercial prebiotic, inulin-type fructans, and the effects of the prebiotic on faecal microbiota, metabolites and bile acids (BA). Nine overweight beagles were used in a replicated 3×3 Latin square design to test a non-prebiotic control (cellulose) against a low (equivalent to 0·5 % diet) and high dose (equivalent to 1·0 % diet) of prebiotic over 14-d treatments. All dogs were fed the same diet twice daily, with treatments provided orally via gelatin capsules before meals. On days 13 or 14 of each period, fresh faecal samples were collected, dogs were fed at 08.00 hours and then challenged with 1 g/kg body weight of maltodextrin in place of the 16.00 hours meal. Repeated blood samples were analysed for glucose and hormone concentrations to determine postprandial incremental AUC (IAUC) data. Baseline glucose, insulin and active glucagon-like peptide-1 levels were similar between all groups (P>0·10). Glucose and insulin IAUC after glucose challenge appeared lower following the high dose, but did not reach statistical relevance. Prebiotic intervention resulted in an increase in relative abundance of some Firmicutes and a decrease in the relative abundance of some Proteobacteria. Individual and total faecal SCFA were significantly increased (P<0·05) following prebiotic supplementation. Total concentration of excreted faecal BA tended to increase in dogs fed the prebiotic (P=0·06). Our results indicate that higher doses of inulin-type prebiotics may serve as modulators of gut microbiota, metabolites and BA pool in overweight dogs.

Development and analytical validation of a radioimmunoassay for the quantification of alpha1 -proteinase inhibitor in serum and feces from the common marmoset (Callithrix jacchus).

BACKGROUND: The objective of this study was to develop and analytically validate a radioimmunoassay (RIA) for the measurement of alpha1 -proteinase inhibitor (α1 -PI) concentrations in serum and feces from the common marmoset. METHODS: Serum samples (n = 30) and 3-day fecal samples (n = 30) were obtained from healthy marmosets. An RIA was established and validated by determination of sensitivity, working range, dilutional parallelism, spiking recovery, and intra- and interassay variability. A reference interval for mα1 -PI in serum and feces was established. RESULTS: Sensitivity and upper limit of the working range were 0.75 and 100.62 µg/L, respectively. Observed-to-expected (O/E) ratios for serial dilutions ranged from 89.9% to 123.0% (mean ±SD: 106.0 ± 11.5%) for 8 serum samples, and from 90.6% to 132.7% (mean ±SD: 107.6 ± 19.2%) for 4 fecal samples. O/E ratios for spiking recovery ranged from 97.6% to 104.4% (mean ±SD: 101.3 ± 3%) for 4 serum samples, and from 97.5% to 101.4% (mean ±SD: 99.2 ± 1.8%) for 4 fecal samples and 3 different spiking concentrations. Coefficients of variation (CV) for intra-assay variability for 8 serum samples ranged from 1.7% to 10.6% and 2.2% to 5.1% in the 8 fecal samples. The interassay CV for eight serum samples ranged from 1.3% to 9.9%, and from 1.0% to 6.7% in the 8 fecal samples. The reference interval in serum was determined to be 1047-1484 µg/L. The reference interval in serum was determined to be 1047-1484 µg/L. The reference interval for the 3-day mean fecal concentration, and 3-day maximum fecal concentration were determined to be 32.4-124.4 µg/g and 39.1-158.7 µg/g of feces, respectively. CONCLUSION: The developed assay is sensitive, linear, accurate, precise, and reproducible. Further studies are needed to determine the clinical utility of this assay.

S100A12 concentrations and myeloperoxidase activities are increased in the intestinal mucosa of dogs with chronic enteropathies.

BACKGROUND: Intestinal mucosal S100A12 and myeloperoxidase (MPO) are inflammatory biomarkers in humans with inflammatory bowel disease (IBD). However, these biomarkers have not been studied in the intestinal mucosa of dogs with chronic enteropathies (CE), even though dogs with CE have increased S100A12 concentrations in feces and serum. This study investigated mucosal S100A12 concentrations and MPO activities in both dogs with CE and healthy Beagles. ELISA (S100A12 concentrations) and spectrophotometric methods (MPO activity) were used. The associations of both biomarkers with canine IBD activity index (CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations were also investigated. We studied intestinal mucosal samples originating from different intestinal regions of 40 dogs with CE and 18 healthy Beagle dogs (duodenum, ileum, colon, and cecum). RESULTS: Compared with healthy Beagles, mucosal S100A12 concentrations in dogs with CE were significantly higher in the duodenum (p < 0.0001) and colon (p = 0.0011), but not in the ileum (p = 0.2725) and cecum (p = 0.2194). Mucosal MPO activity of dogs with CE was significantly higher in the duodenum (p < 0.0001), ileum (p = 0.0083), colon (p < 0.0001), and cecum (p = 0.0474). Mucosal S100A12 concentrations in the duodenum were significantly higher if the inflammatory infiltrate consisted mainly of neutrophils (p = 0.0439) or macrophages (p = 0.037). Mucosal S100A12 concentrations also showed a significant association with the severity of total histopathological injury and epithelial injury in the colon (p < 0.05). Mucosal MPO activity showed a significant association (p < 0.05) with the severity of total histopathological injury, epithelial injury, and eosinophil infiltration in the duodenum. There was no significant association of both biomarkers with CIBDAI or clinical outcome. CONCLUSIONS: This study showed that both mucosal S100A12 concentrations and MPO activities are significantly increased in the duodenum and colon of dogs with CE; mucosal MPO was also increased in the ileum and cecum. Future research should focus on assessing the clinical utility of S100A12 and MPO as diagnostic markers in dogs with CE.

Analysis of Bacterial and Fungal Nucleic Acid in Canine Sterile Granulomatous and Pyogranulomatous Dermatitis and Panniculitis.

Next generation sequencing (NGS) studies are revealing a diverse microbiota on the skin of dogs. The skin microbiota of canine sterile granulomatous and pyogranulomatous dermatitis (SGPD) has yet to be investigated using NGS techniques. NGS targeting the 16S rRNA and ITS-1 region of bacterial and fungal DNA, respectively, were used to investigate if bacterial and fungal DNA were associated with skin lesions in cases of canine SGPD. The study included 20 formalin-fixed paraffin-embedded (FFPE) skin samples and 12 fresh samples from SGPD-affected dogs, and 10 FFPE and 10 fresh samples from healthy dogs. DNA was extracted from deep dermis and panniculus, and microbial DNA was amplified using primers targeting the bacterial 16S rRNA V1-V3 and fungal ITS-1 regions. The amplified DNA was utilized for NGS on an Illumina MiSeq instrument. The sequences were processed using QIIME. No differences in fungal or bacterial alpha diversity were observed between the SGPD and control samples. Beta diversity analysis demonstrated differences in the bacterial communities between SGPD and control, but not in the fungal communities. Compared to controls, the family Erysipelotrichaceae and genus Staphylococcus were significantly more abundant in the SGPD FFPE samples, and genus Corynebacterium were more abundant in fresh samples. The bacteria found to be more abundant in SGPD are common inhabitants of skin surfaces, and likely secondary contaminants in SGPD cases. This study provides additional evidence that SGPD lesions are likely sterile.

Reliable Biomarker discovery from Metagenomic data via RegLRSD algorithm.

BACKGROUND: Biomarker detection presents itself as a major means of translating biological data into clinical applications. Due to the recent advances in high throughput sequencing technologies, an increased number of metagenomics studies have suggested the dysbiosis in microbial communities as potential biomarker for certain diseases. The reproducibility of the results drawn from metagenomic data is crucial for clinical applications and to prevent incorrect biological conclusions. The variability in the sample size and the subjects participating in the experiments induce diversity, which may drastically change the outcome of biomarker detection algorithms. Therefore, a robust biomarker detection algorithm that ensures the consistency of the results irrespective of the natural diversity present in the samples is needed. RESULTS: Toward this end, this paper proposes a novel Regularized Low Rank-Sparse Decomposition (RegLRSD) algorithm. RegLRSD models the bacterial abundance data as a superposition between a sparse matrix and a low-rank matrix, which account for the differentially and non-differentially abundant microbes, respectively. Hence, the biomarker detection problem is cast as a matrix decomposition problem. In order to yield more consistent and solid biological conclusions, RegLRSD incorporates the prior knowledge that the irrelevant microbes do not exhibit significant variation between samples belonging to different phenotypes. Moreover, an efficient algorithm to extract the sparse matrix is proposed. Comprehensive comparisons of RegLRSD with the state-of-the-art algorithms on three realistic datasets are presented. The obtained results demonstrate that RegLRSD consistently outperforms the other algorithms in terms of reproducibility performance and provides a marker list with high classification accuracy. CONCLUSIONS: The proposed RegLRSD algorithm for biomarker detection provides high reproducibility and classification accuracy performance regardless of the dataset complexity and the number of selected biomarkers. This renders RegLRSD as a reliable and powerful tool for identifying potential metagenomic biomarkers.

Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma.

BACKGROUND: Onset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs. RESULTS: S100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%. CONCLUSIONS: S100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

Effect of gastric acid-suppressive therapy and biological variation of serum gastrin concentrations in dogs with chronic enteropathies.

BACKGROUND: Serum gastrin concentration can help diagnose gastrinomas in dogs if >3-10× the upper reference limit (URL), but antisecretory therapy and other conditions can also cause hypergastrinemia. Effects of antisecretory therapy (famotidine or ranitidine, omeprazole) on serum gastrin concentration in dogs with chronic enteropathy (CE) and its biological variation (BV) are unknown. Aim of the study was to evaluate serum gastrin in acid-suppressant-treated or -naïve CE dogs; test the association between serum gastrin and histopathologic findings in acid-suppressant-naïve CE dogs; and evaluate the BV of serum gastrin in dogs not receiving any gastric acid suppressive therapy. Samples from 231 dogs were used and serum gastrin was measured by chemiluminescence assay. Gastric and duodenal histologic lesions were evaluated and graded. BV of serum gastrin was evaluated in serial samples. RESULTS: Serum gastrin concentrations were significantly higher in acid-suppressant-treated than acid-suppressant-naïve dogs (P = 0.0245), with significantly higher concentrations in proton pump inhibitor (PPI)- than H2-antihistamine-treated patients (P = 0.0053). More PPI- than H2-antihistamine-treated dogs had gastrin concentrations above URL (P = 0.0205), but not >3× nor >10× the URL. Serum gastrin concentrations correlated with the severity of gastric antral epithelial injury (P = 0.0069) but not with any other lesions or the presence/numbers of spiral bacteria in gastric biopsies. Intra- and inter-individual BV were 43.4 and 21.6%, respectively, in acid-suppressant-naïve dogs, with a reciprocal individuality index of 0.49 and a critical difference of ≥29.5 ng/L. CONCLUSIONS: Antisecretory (particularly PPI) treatment leads to hypergastrinemia in CE dogs, but the concentrations seen in this study are unlikely to compromise a diagnosis of gastrinoma. Use of a population-based URL for canine serum gastrin and a URL of ≤27.8 ng/L are appropriate.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues.

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

The fecal microbiome of dogs with exocrine pancreatic insufficiency.

Exocrine pancreatic insufficiency (EPI) in dogs is a syndrome of inadequate synthesis and secretion of pancreatic enzymes. Small intestinal bacterial dysbiosis occurs in dogs with EPI, and is reversed with pancreatic enzyme therapy. However, there are no studies evaluating the fecal microbiome of dogs with EPI. The objective of this study was to evaluate the fecal microbiome of dogs with EPI. Three day pooled fecal samples were collected from healthy dogs (n = 18), untreated (n = 7) dogs with EPI, and dogs with EPI treated with enzyme replacement therapy (n = 19). Extracted DNA from fecal samples was used for Illumina sequencing of the bacterial 16S rRNA gene and analyzed using Quantitative Insights Into Microbial Ecology (QIIME) and PICRUSt was used to predict the functional gene content of the microbiome. Linear discriminant analysis effect size (LEfSe) revealed significant differences in bacterial groups and functional genes between the healthy dogs and dogs with EPI. There was a significant difference in fecal microbial communities when healthy dogs were compared to treated and untreated dogs with EPI (unweighted UniFrac distance, ANOSIM P = 0.001, and 0.001 respectively). Alpha diversity was significantly decreased in untreated and treated EPI dogs when compared to the healthy dogs with respect to Chao1, Observed OTU, and Shannon diversity (P = 0.008, 0.003, and 0.002 respectively). The families Bifidobacteriaceae (P = 0.005), Enterococcaceae (P = 0.018), and Lactobacillaceae (P = 0.001) were significantly increased in the untreated and treated dogs with EPI when compared to healthy dogs. In contrast, Lachnospiraceae (P < 0.001), and Ruminococcaceae (P < 0.01) were significantly decreased in dogs with EPI. Dogs with EPI (before treatment) had significant increases in functional genes associated with secretion system, fatty acid metabolism, and phosphotransferase system. In contrast, healthy dogs had a significant increase in genes related to phenylalanine, tyrosine and tryptophan biosynthesis, transcription machinery and sporulation. In conclusion, this study shows that the fecal microbiome of dogs with EPI (both treated and untreated) is different to that of healthy dogs.

Characterization of the cutaneous mycobiota in healthy and allergic cats using next generation sequencing.

BACKGROUND: Next generation sequencing (NGS) studies have demonstrated a diverse skin-associated microbiota and microbial dysbiosis associated with atopic dermatitis in people and in dogs. The skin of cats has yet to be investigated using NGS techniques. HYPOTHESIS/OBJECTIVES: We hypothesized that the fungal microbiota of healthy feline skin would be similar to that of dogs, with a predominance of environmental fungi, and that fungal dysbiosis would be present on the skin of allergic cats. ANIMALS: Eleven healthy cats and nine cats diagnosed with one or more cutaneous hypersensitivity disorders, including flea bite, food-induced and nonflea nonfood-induced hypersensitivity. METHODS: Healthy cats were sampled at twelve body sites and allergic cats at six sites. DNA was isolated and Illumina sequencing was performed targeting the internal transcribed spacer region of fungi. Sequences were processed using the bioinformatics software QIIME. RESULTS: The most abundant fungal sequences from the skin of all cats were classified as Cladosporium and Alternaria. The mucosal sites, including nostril, conjunctiva and reproductive tracts, had the fewest number of fungi, whereas the pre-aural space had the most. Allergic feline skin had significantly greater amounts of Agaricomycetes and Sordariomycetes, and significantly less Epicoccum compared to healthy feline skin. CONCLUSIONS: The skin of healthy cats appears to have a more diverse fungal microbiota compared to previous studies, and a fungal dysbiosis is noted in the skin of allergic cats. Future studies assessing the temporal stability of the skin microbiota in cats will be useful in determining whether the microbiota sequenced using NGS are colonizers or transient microbes.

The skin microbiome in allergen-induced canine atopic dermatitis.

BACKGROUND: Studies focusing on next-generation sequencing of the bacterial 16S rRNA gene have allowed detailed surveys of skin bacterial populations (microbiota) of the skin. HYPOTHESIS/OBJECTIVES: This study evaluated temporal changes in the skin microbiota in a canine model of atopic dermatitis. ANIMALS: Eight atopic dogs previously sensitized with house dust mites (HDM). METHODS: The dogs were topically challenged on the right groin with HDM allergens. Swabs were collected from the challenged and the contralateral nonchallenged sites prior to provocation (pre-challenge; baseline sample) and on days 1, 7, 14, 21 and 28 after allergen challenge. The 16S rRNA gene was amplified, sequenced and analysed. Staphylococcus spp. and Staphylococcus pseudintermedius were quantified with quantitative PCR (RT-qPCR). RESULTS: Skin lesions developed in all dogs on the challenged sites. Differences in bacterial groups were observed on the challenged site over time. Relatively lower abundances of Fusobacteriaceae on Day 7, and, based on LEfSe, increased abundances of Corynebacteriaceae on Day 1, and Staphylococcaceae on days 7, 14 and 21, were observed on the challenged site, compared to the contralateral site. Results of RT-qPCR correlated with those of next-generation sequencing, with significantly increased numbers of Staphylococcus spp. and S. pseudintermedius on Day 21, and days 7 and 21 on the challenged site compared to the contralateral site, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrates that an allergen challenge in sensitized dogs leads to bacterial dysbiosis with increased abundance of S. pseudintermedius at the site of lesion induction.

Modulation of the faecal microbiome of healthy adult dogs by inclusion of potato fibre in the diet.

Inclusion of fermentable fibres in the diet can have an impact on the hindgut microbiome and provide numerous health benefits to the host. Potato fibre (PF), a co-product of potato starch isolation, has a favourable chemical composition of pectins, resistant and digestible starch, cellulose, and hemicelluloses. The objective of the present study was to evaluate the effect of increasing dietary PF concentrations on the faecal microbiome of healthy adult dogs. Fresh faecal samples were collected from ten female dogs with hound bloodlines (6·13 (SEM 0·17) years; 22·0 (SEM 2·1) kg) fed five test diets containing graded concentrations of PF (0, 1·5, 3, 4·5 or 6% as-fed; Roquette Frères) in a replicated 5 × 5 Latin square design. Extraction of DNA was followed by amplification of the V4-V6 variable region of the 16S rRNA gene using barcoded primers. Sequences were classified into taxonomic levels using Basic Local Alignment Search Tool (BLASTn) against a curated GreenGenes database. Inclusion of PF increased (P< 0·05) the faecal proportions of Firmicutes, while those of Fusobacteria decreased (P< 0·05). Similar shifts were observed at the genus level and were confirmed by quantitative PCR (qPCR) analysis. With increasing concentrations of PF, faecal proportions of Faecalibacterium increased (P< 0·05). Post hoc Pearson's correlation analysis showed positive (P< 0·05) correlations with Bifidobacterium spp. and butyrate production and Lactobacillus spp. concentrations. Overall, increases in the proportion of Faecalibacterium (not Lactobacillus/Bifidobacterium, as confirmed by qPCR analysis) and faecal SCFA concentrations with increasing dietary PF concentrations suggest that PF is a possible prebiotic fibre.