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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."
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Metagenómica , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenoma , Metagenómica/métodos , Programas InformáticosRESUMEN
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.
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Dieta Rica en Proteínas , Microbioma Gastrointestinal , Animales , Fibras de la Dieta , Perros , Metaboloma , Obesidad/dietoterapia , Pérdida de PesoRESUMEN
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).
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Enfermedades de los Perros , Probióticos , Animales , Antibacterianos/uso terapéutico , Perros , Disbiosis/veterinaria , Heces , Probióticos/uso terapéutico , Estudios ProspectivosRESUMEN
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.
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Acinonyx , Enfermedades de los Gatos , Gastritis , Animales , Animales de Zoológico , Biomarcadores , Enfermedades de los Gatos/diagnóstico , Gatos , Gastritis/veterinaria , Valores de Referencia , Especificidad de la EspecieRESUMEN
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.
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Enfermedades de los Gatos , Insuficiencia Pancreática Exocrina , Tigres , Animales , Gatos , Diarrea/veterinaria , Insuficiencia Pancreática Exocrina/diagnóstico , Insuficiencia Pancreática Exocrina/veterinaria , Valores de Referencia , TripsinaRESUMEN
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.
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Bacterias/patogenicidad , Enfermedades de los Perros/microbiología , Microbioma Gastrointestinal , Helicobacter/patogenicidad , Enfermedades Inflamatorias del Intestino/veterinaria , Animales , Bacterias/genética , Enfermedad Crónica/veterinaria , Colon/microbiología , Colon/patología , Enfermedades de los Perros/patología , Perros , Femenino , Helicobacter/genética , Hibridación Fluorescente in Situ/veterinaria , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , MasculinoRESUMEN
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.
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Colon , Heces , Fructanos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Inulina/farmacología , Obesidad , Prebióticos , Animales , Área Bajo la Curva , Ácidos y Sales Biliares/metabolismo , Glucemia/metabolismo , Colon/metabolismo , Colon/microbiología , Perros , Ácidos Grasos Volátiles/metabolismo , Heces/química , Heces/microbiología , Femenino , Firmicutes/crecimiento & desarrollo , Fructanos/uso terapéutico , Péptido 1 Similar al Glucagón/sangre , Insulina/sangre , Inulina/uso terapéutico , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Obesidad/microbiología , Obesidad/veterinaria , Periodo Posprandial , Proteobacteria/crecimiento & desarrolloRESUMEN
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.
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Callithrix/metabolismo , Heces/química , Radioinmunoensayo/métodos , alfa 1-Antitripsina/metabolismo , Animales , Valores de Referencia , Reproducibilidad de los Resultados , alfa 1-Antitripsina/sangreRESUMEN
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.
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Enfermedades de los Perros/metabolismo , Enfermedades Intestinales/veterinaria , Mucosa Intestinal/metabolismo , Peroxidasa/metabolismo , Proteína S100A12/metabolismo , Animales , Biomarcadores/análisis , Enfermedad Crónica , Perros , Femenino , Enfermedades Intestinales/metabolismo , Mucosa Intestinal/química , Masculino , Proteína S100A12/análisisRESUMEN
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.
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Dermatitis/veterinaria , Enfermedades de los Perros/patología , Paniculitis/veterinaria , Animales , ADN Bacteriano/genética , ADN de Hongos/genética , Dermatitis/microbiología , Dermatitis/patología , Enfermedades de los Perros/microbiología , Perros , Femenino , Granuloma/microbiología , Granuloma/patología , Granuloma/veterinaria , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Paniculitis/microbiología , Paniculitis/patología , ARN Ribosómico 16S/genética , Piel/microbiología , Piel/patologíaRESUMEN
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.
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Enfermedades de los Perros/diagnóstico , Complejo de Antígeno L1 de Leucocito/orina , Neoplasias Urogenitales/veterinaria , Neoplasias Urológicas/veterinaria , Animales , Biomarcadores/orina , Calgranulina A/análisis , Calgranulina B/orina , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/orina , Carcinoma de Células Transicionales/veterinaria , Estudios de Casos y Controles , Creatinina/orina , Enfermedades de los Perros/orina , Perros , Femenino , Masculino , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/orina , Neoplasias de la Próstata/veterinaria , Proteinuria/orina , Proteinuria/veterinaria , Radioinmunoensayo/veterinaria , Neoplasias Urogenitales/diagnóstico , Neoplasias Urogenitales/orina , Enfermedades Urológicas/diagnóstico , Enfermedades Urológicas/orina , Enfermedades Urológicas/veterinaria , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/orinaRESUMEN
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.
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Enfermedades de los Perros/tratamiento farmacológico , Gastrinas/sangre , Antagonistas de los Receptores H2 de la Histamina/farmacología , Enfermedades Intestinales/veterinaria , Inhibidores de la Bomba de Protones/farmacología , Gastropatías/veterinaria , Animales , Variación Biológica Poblacional/efectos de los fármacos , Enfermedades de los Perros/sangre , Perros , Femenino , Gastrinas/efectos de los fármacos , Helicobacter/aislamiento & purificación , Infecciones por Helicobacter/veterinaria , Enfermedades Intestinales/sangre , Enfermedades Intestinales/tratamiento farmacológico , Enfermedades Intestinales/patología , Masculino , Gastropatías/sangre , Gastropatías/tratamiento farmacológico , Gastropatías/patologíaRESUMEN
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.
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Micosis/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/microbiología , Enfermedades de los Gatos/patología , Gatos , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/patología , ADN de Hongos/genética , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/patología , Perros , Enfermedades de los Caballos/diagnóstico , Enfermedades de los Caballos/microbiología , Enfermedades de los Caballos/patología , Caballos , Micosis/diagnóstico , Micosis/microbiología , Micosis/patología , Adhesión en Parafina/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Alineación de Secuencia/veterinariaRESUMEN
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.
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Bacterias/clasificación , Bacterias/genética , Enfermedades de los Perros/microbiología , Disbiosis , Terapia de Reemplazo Enzimático , Insuficiencia Pancreática Exocrina/veterinaria , Microbioma Gastrointestinal , Animales , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/patología , Perros , Insuficiencia Pancreática Exocrina/complicaciones , Insuficiencia Pancreática Exocrina/tratamiento farmacológico , Insuficiencia Pancreática Exocrina/patología , Heces/microbiología , Femenino , Masculino , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
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.
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Enfermedades de los Gatos/microbiología , Gatos/microbiología , Dermatitis Atópica/veterinaria , Microbiota/genética , Piel/microbiología , Animales , Enfermedades de los Gatos/inmunología , ADN de Hongos/genética , Dermatitis Atópica/inmunología , Dermatitis Atópica/microbiología , Femenino , Infestaciones por Pulgas/inmunología , Infestaciones por Pulgas/microbiología , Infestaciones por Pulgas/veterinaria , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/microbiología , Hipersensibilidad a los Alimentos/veterinaria , Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/veterinaria , MasculinoRESUMEN
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.
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Antígenos Dermatofagoides/inmunología , Bacterias/clasificación , Dermatitis Atópica/veterinaria , Enfermedades de los Perros/microbiología , Piel/microbiología , Animales , Bacterias/genética , Dermatitis Atópica/microbiología , Enfermedades de los Perros/patología , Perros , Femenino , Masculino , Pyroglyphidae/microbiología , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
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.
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Dieta/veterinaria , Fibras de la Dieta/administración & dosificación , Perros/microbiología , Heces/microbiología , Microbioma Gastrointestinal , Solanum tuberosum/química , Animales , Bifidobacterium/aislamiento & purificación , Clostridiales/aislamiento & purificación , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Heces/química , Femenino , Fermentación , Firmicutes/aislamiento & purificación , Fusobacterias/aislamiento & purificación , Tracto Gastrointestinal/microbiología , Prebióticos/administración & dosificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificaciónRESUMEN
BACKGROUND/AIMS: Five million people currently live with Crohn's disease (CD) or ulcerative colitis, the two major forms of inflammatory bowel disease. Available treatments frequently result in side effects that compromise the immune health of the patient. Consequently, alternative therapies that cause fewer systemic effects are needed. Dioctahedral smectite clays have been utilized to treat medical conditions, including diarrheal and enteric disease. Herein, we report the ability of a refined dioctahedral smectite (NovaSil, NS) to sorb inflammatory proteins and reduce inflammation in a TNBS (2,4,6-trinitrobenzenesulfonic acid) mouse model of CD. We also investigated whether NS could rescue gut microbial diversity in TNBS-induced mice. METHODS: ELISA, X-ray diffraction, and transmission electron microscopy were employed to characterize the NS-cytokine interaction in vitro. A TNBS mouse colitis model was utilized to study the efficacy of NS supplementation for 4 weeks. The three treatment groups included control, TNBS, and TNBS + NS. DNA was extracted from feces and sorted for bacterial phylogenetic analysis. RESULTS: Results suggest that NS binds TNFα in vitro. In TNBS-treated mice, supplementation with NS significantly reduced weight loss, and serum proinflammatory cytokine levels (IL-2, IL-6, and IL-12, TNFα, IFNγ) compared with the TNBS group. TNBS-treated mice demonstrated a significant reduction in gut microbiota species richness when compared with the TNBS + NS group and control group. CONCLUSIONS: NovaSil mitigated the effects of TNBS-induced colitis based on reduction in systemic markers of inflammation, significant improvement in weight gain, and intestinal microbial profile.
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Silicatos de Aluminio/farmacología , Antiinflamatorios/farmacología , Colitis/prevención & control , Colon/efectos de los fármacos , Fármacos Gastrointestinales/farmacología , Silicatos/farmacología , Silicatos de Aluminio/química , Animales , Antiinflamatorios/química , Bacterias/clasificación , Bacterias/aislamiento & purificación , Arcilla , Colitis/sangre , Colitis/inducido químicamente , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Cristalografía por Rayos X , Citocinas/sangre , Modelos Animales de Enfermedad , Heces/microbiología , Femenino , Fármacos Gastrointestinales/química , Mediadores de Inflamación/sangre , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Difracción de Polvo , Ribotipificación , Silicatos/química , Factores de Tiempo , Ácido Trinitrobencenosulfónico , Aumento de Peso/efectos de los fármacosRESUMEN
BACKGROUND: 3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27). RESULTS: Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, -20°C, and -80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] µmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] µmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] µmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] µmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] µmol/L) were not different compared to healthy control dogs. CONCLUSIONS: The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.