Assessment of the canine intestinal microflora using molecular methods and serum markers

Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions

Characterization of the fecal microbiome in cats with inflammatory bowel disease or alimentary small cell lymphoma.

Feline chronic enteropathy (CE) is a common gastrointestinal disorder in cats and mainly comprises inflammatory bowel disease (IBD) and small cell lymphoma (SCL). Both IBD and SCL in cats share features with chronic enteropathies such as IBD and monomorphic epitheliotropic intestinal T-cell lymphoma in humans. The aim of this study was to characterize the fecal microbiome of 38 healthy cats and 27 cats with CE (13 cats with IBD and 14 cats with SCL). Alpha diversity indices were significantly decreased in cats with CE (OTU p = 0.003, Shannon Index p = 0.008, Phylogenetic Diversity p = 0.019). ANOSIM showed a significant difference in bacterial communities, albeit with a small effect size (P = 0.023, R = 0.073). Univariate analysis and LEfSE showed a lower abundance of facultative anaerobic taxa of the phyla Firmicutes (families Ruminococcaceae and Turicibacteraceae), Actinobacteria (genus Bifidobacterium) and Bacteroidetes (i.a. Bacteroides plebeius) in cats with CE. The facultative anaerobic taxa Enterobacteriaceae and Streptococcaceae were increased in cats with CE. No significant difference between the microbiome of cats with IBD and those with SCL was found. Cats with CE showed patterns of dysbiosis similar to those in found people with IBD

High-Dose Hydrocortisone Treatment Does Not Affect Serum C-Reactive Protein (CRP) Concentrations in Healthy Dogs

Measuring C-reactive protein (CRP) in serum is a useful surrogate marker for assessing disease progression and treatment response in dogs with autoinflammatory diseases. Affected dogs often receive high-dose glucocorticoid treatment, but the effect of such treatment alone on serum CRP concentrations is unknown. We evaluated serum CRP concentrations via immunoassay (sandwich enzyme-linked immunosorbent assay and particle-enhanced turbidimetric immunoassay) in 12 healthy beagle dogs administered high-dose hydrocortisone (8 mg/kg q12 h) per os vs. placebo over 28 days (days 0, 1, 5, and 28) in a randomized parallel study design. Serum CRP concentrations slightly decreased during treatment or placebo but without a significant association with hydrocortisone administration (p = 0.761). Compared to baseline, serum CRP concentrations were decreased by >2.7-fold (minimum critical difference) in three hydrocortisone-treated dogs and two dogs in the placebo group on day 28, whereas an increase to >2.7-fold was seen in one dog receiving placebo. These results suggest a lack of confounding effects of high-dose hydrocortisone administration on serum CRP concentrations in healthy dogs. This might also hold in dogs with autoinflammatory conditions and/or administration of other high-dose corticosteroids, suggesting that CRP presents a suitable biomarker to monitor inflammatory disease processes. However, this needs confirmation by further studies evaluating corticosteroid-induced cellular (e.g., hepatic) transcriptome and proteome changes

Serum canine pancreatic-specific lipase concentrations in dogs with naturally occurring Babesia rossi infection

Babesia rossi is the cause of a highly virulent multisystemic disease with a variable outcome, which is a reliable model of systemic inflammatory response syndrome (SIRS). The objective of this study was to determine the concentration of canine pancreatic-specific lipase (cPL) in a population of dogs with naturally acquired B. rossi infection. In addition, the associations between serum cPL and death and SIRS status were examined. An observational study recruited 87 dogs diagnosed with B. rossi infection and serum cPL concentrations were measured daily until discharge or death. The median concentration of serum cPL was 124.0 µg/L (interquartile range: 51.0 µg/L – 475.5 µg/L) on admission (n = 87) and 145.5 µg/L (62.3 µg/L – 434.0 µg/L) on day two of hospitalisation (n = 40). Twenty-four dogs (28%) had a serum cPL concentration within the diagnostic range for pancreatitis (> 400 µg/L) at admission with 13 dogs (32.5%) presenting as such on the second day of hospitalisation. The median concentration of serum cPL in dogs with SIRS was 158 µg/L (interquartile range: 52.5 µg/L – 571.5 µg/L; n = 53), which was significantly higher than in those without SIRS (75 µg/L; 50.3 µg/L – 131.8 µg/L; n = 32) (P = 0.018). This study demonstrated that an unexpectedly high number of dogs diagnosed with naturally acquired canine babesiosis had a serum cPL concentration within the diagnostic range for acute pancreatitis and a significantly higher serum cPL concentration was found in dogs that were classified as having SIRS

Novel lipoprotein density profiling in healthy dogs of various breeds, healthy miniature schnauzers, and miniature schnauzers with hyperlipidemia

BACKGROUND: Despite the importance of abnormalities in lipoprotein metabolism in clinical canine medicine, the fact that most previously used methods for lipoprotein profiling are rather laborious and time-consuming has been a major obstacle to the wide clinical application and use of lipoprotein profiling in this species. The aim of the present study was to assess the feasibility of a continuous lipoprotein density profile (CLPDP) generated within a bismuth sodium ethylenediaminetetraacetic acid (NaBiEDTA) density gradient to characterize and compare the lipoprotein profiles of healthy dogs of various breeds, healthy Miniature Schnauzers, and Miniature Schnauzers with primary hypertriacylglycerolemia. A total of 35 healthy dogs of various breeds with serum triacylglycerol (TAG) and cholesterol concentrations within their respective reference intervals were selected for use as a reference population. Thirty-one Miniature Schnauzers with serum TAG and cholesterol concentrations within their respective reference intervals and 31 Miniature Schnauzers with hypertriacylglyceridemia were also included in the study. RESULTS: The results suggest that CLPDP using NaBiEDTA provides unique diagnostic information in addition to measurements of serum TAG and cholesterol concentrations and that it is a useful screening method for dogs with suspected lipoprotein metabolism disorders. Using the detailed and continuous density distribution information provided by the CLPDP, important differences in lipoprotein profiles can be detected even among dogs that have serum TAG and cholesterol concentrations within the reference interval. Miniature Schnauzers with serum TAG and cholesterol concentrations within the reference interval had significantly different lipoprotein profiles than dogs of various other breeds. In addition, it was further established that specific lipoprotein fractions are associated with hypertriacylglyceridemia in Miniature Schnauzers. CONCLUSIONS: The results of the present study suggest that density gradient ultracentrifugation using NaBiEDTA is a useful screening method for the study of lipoprotein profiles in dogs. Therefore, this method could potentially be used for diagnostic purposes for the separation of dogs suspected of having lipoprotein abnormalities from healthy dogs

Assessment of the Variation Associated with Repeated Measurement of Gastrointestinal Transit Times and Assessment of the Effect of Oral Ranitidine on Gastrointestinal Transit Times Using a Wireless Motility Capsule System in Dogs

This study aimed to evaluate the variation associated with repeated measurement of gastrointestinal (GI) transit times and the effect of oral ranitidine on GI transit times in healthy dogs using a wireless motility capsule (WMC) system. Eight privately owned healthy adult dogs were enrolled, and one developed diarrhea and was removed from the study. For the first 3 repetitions, each dog was fed a standard meal followed by oral administration of a WMC. For the 4th repetition, each dog was given ranitidine hydrochloride (75 mg PO every 12 hours) prior to and during assessment of GI transit times. Mean between-subject coefficients of variation for gastric emptying time (GET), small and large bowel transit time (SLBTT), and total transit time (TTT) were 26.9%, 32.3%, and 19.6%, respectively. Mean within-subject coefficients of variation for GET, SLBTT, and TTT were 9.3%, 19.6%, and 15.9%, respectively. Median GET, SLBTT, and TTT without ranitidine were 719, 1,636, and 2,735 minutes, respectively. Median GET, SLBTT, and TTT with ranitidine were 757, 1,227, and 2,083 minutes, respectively. No significant differences in GI transit times were found between any of the 4 repetitions. Under these experimental conditions, no significant effects of oral ranitidine on GI transit times were observed

Long-term effects of canine parvovirus infection in dogs

Background Canine parvovirus (CPV) is the most important viral cause of acute canine enteritis leading to severe damage of the intestinal barrier. It has been speculated that dogs might develop chronic disorders after surviving CPV infection. However, no studies regarding the longterm implications of CPV infection have been published to date. The aim of this study was to evaluate whether dogs that have survived CPV infection will have an increased risk for developing chronic gastroenteritis, atopic dermatitis, or cardiac disease. Methodology/Principal findings Dogs that had been treated at the Clinic of Small Animal Medicine, LMU Munich, for CPV infection for which a follow-up of at least 12 months was available, were included in the study. Owners completed a questionnaire on the presence of chronic gastrointestinal and cutaneous signs, cardiac disease, and other potential disorders. An identical questionnaire was sent to owners of matched control dogs during the same time period. Seventy-one questionnaires of dogs with CPV infection and 67 of control dogs were analyzed. Significantly more CPV-infected dogs (30/71) compared to control dogs (8/67) had developed chronic gastrointestinal signs later in their lives (P < 0.001). No significant differences were observed regarding skin diseases (P = 1), cardiac problems (P = 0.160), or any other diseases (P = 0.173) later in life. Conclusions: Results of this study suggest that dogs that survive CPV infection have a significantly higher risk (odds ratio = 5.33) for developing a chronic gastrointestinal disease. Further prospective studies to identify the trigger for the development of chronic diarrhoea and possible targeted treatment strategies are needed

The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease.

Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders. Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI). Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes

Characterization of the Fecal Microbiome in Dogs Receiving Medical Management for Congenital Portosystemic Shunts

BackgroundThe GI microbiome has not been characterized in dogs being medically managed for congenital portosystemic shunts (CPSS).ObjectivesTo characterize the fecal microbiome in a population of dogs being medically managed for CPSS.Animals27 client-owned dogs.MethodsProspective cohort study enrollment of fecal samples was performed with follow-up data collected retrospectively. The overall fecal dysbiosis index (DI) and individual bacterial abundances were determined using real-time qPCR. Medical management, clinical findings, clinicopathologic, and outcome variables were collected, and logistic regression analyses were performed to evaluate associations between these variables and overall DI and bacterial abundances. Numerical variables were evaluated with general linear models for normality and equal variance using Shapiro-Wilk test and Levene's test, respectively.ResultsAll dogs were administered a hepatic diet and lactulose, while antibiotics were used in 22 (81.5%) and acid suppressants in 7 (25.9%). Seventeen dogs (63.0%) had a DI &gt;2. The median DI in this population was 3.02 (range 4.23–8.42), and the median DI in dogs receiving and not receiving antibiotics was 4.3 (range −4.23–8.42) and 1.52 (range −1.62–5.43), respectively. No significant association between any of the analyzed variables and the DI was identified. There was a significant association between the use of metronidazole and a larger abundance of E. coli (p = 0.024).Conclusions and Clinical ImportanceDysbiosis appears to be common in dogs that are being medically managed for CPSS, though the clinical significance remains unclear

Effect of amoxicillin‐clavulanic acid on clinical scores, intestinal microbiome, and amoxicillin‐resistant Escherichia coli in dogs with uncomplicated acute diarrhea

Background Despite limited evidence of efficacy, antibiotic treatment is still frequently prescribed in dogs with uncomplicated acute diarrhea (AD). Objective To assess whether amoxicillin‐clavulanic acid has a clinical benefit, an effect on the fecal microbiome, and the proportion of amoxicillin‐resistant Escherichia coli in dogs with AD. Animals Sixteen dogs with AD of <3 days duration. Methods Prospective, placebo‐controlled, double‐blinded study. Clinical scores were compared between client‐owned dogs randomly assigned to an antibiotic (AG) or a placebo (PG) group. The intestinal microbiome was analyzed using quantitative PCR assays. Amoxicillin‐resistant fecal E. coli were assessed semiquantitatively with microbiological methods. Results There was no difference in clinical recovery between treated dogs or controls (CADS index day 10: AG group median: 2 (range: 1‐3; CI [1.4; 2.6]); PG group median: 1.6 (range: 1‐3; CI [1.1; 2.4]); P > .99). All dogs gained normal clinical scores (CADS index ≤3) after 1 to 6 days (median 2 days) after presentation. There was no significant difference in the fecal dysbiosis index (during treatment: AG mean −2.6 (SD 3.0; CI [−5.1; 0.0]); PG mean −0.8 (SD 4.0; CI [−4.2; 2.5]; P > .99) or its bacterial taxa. The proportion of resistant fecal E. coli increased (to median: 100%; range: 35%‐100%) during treatment with amoxicillin‐clavulanic acid and was still increased (median: 10%; range 2%‐67%) 3 weeks after treatment, both of which were significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)). Conclusions and Clinical Importance Our study suggests that treatment with amoxicillin‐clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin‐resistant E. coli , which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis