Jörg M. Steiner

Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats

This study evaluated the fecal microbiota of 12 healthy pet dogs and 12 pet cats using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing. A total of 120,406 pyrosequencing reads for bacteria (mean 5017) and 5359 sequences (one pool each for dogs and cats) for fungi were analyzed. Additionally, group-specific 16S rRNA gene clone libraries for Bifidobacterium spp. and lactic acid-producing bacteria (LAB) were constructed. The most abundant bacterial phylum was Firmicutes, followed by Bacteroidetes in dogs and Actinobacteria in cats. The most prevalent bacterial class in dogs and cats was Clostridia, dominated by the genera Clostridium (clusters XIVa and XI) and Ruminococcus. At the genus level, 85 operational taxonomic units (OTUs) were identified in dogs and 113 OTUs in cats. Seventeen LAB and eight Bifidobacterium spp. were detected in canine feces. Ascomycota was the only fungal phylum detected in cats, while Ascomycota, Basidiomycota, Glomeromycota, and Zygomycota were identified in dogs. Nacaseomyces was the most abundant fungal genus in dogs; Saccharomyces and Aspergillus were predominant in cats. At the genus level, 33 different fungal OTUs were observed in dogs and 17 OTUs in cats. In conclusion, this study revealed a highly diverse bacterial and fungal microbiota in canine and feline feces.

The Fecal Microbiome in Dogs with Acute Diarrhea and Idiopathic Inflammatory Bowel Disease

Background 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. Methodology/Principal Findings 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). Conclusions 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.

Effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis.

A randomized, controlled clinical trial investigated the effect of early enteral nutrition (EN) on intestinal permeability, intestinal protein loss, and outcome in parvoviral enteritis. Dogs were randomized into 2 groups: 15 dogs received no food until vomiting had ceased for 12 hours (mean 50 hours after admission; NPO group), and 15 dogs received early EN by nasoesophageal tube from 12 hours after admission (EEN group). All other treatments were identical. Intestinal permeability was assessed by 6‐hour urinary lactulose (L) and rhamnose (R) recoveries (%L, %R) and L/R recovery ratios. Intestinal protein loss was quantified by fecal α1‐proteinase inhibitor concentrations (α1‐PI). Median time to normalization of demeanor, appetite, vomiting, and diarrhea was 1 day shorter for the EEN group for each variable. Body weight increased insignificantly from admission in the NPO group (day 3: 2.5±2.8% day 6: 4.3±2.3% mean ± SE), whereas the EEN group exhibited significant weight gain (day 3: 8.1±2.7% day 6: 9.7 ± 2.1%). Mean urinary %L was increased, %R reduced, and L/R recovery ratios increased compared to reference values throughout the study for both groups. Percent lactulose recovery decreased in the EEN group (admission: 22.6±8.0% day 6: 17.9 ± 2.3%) and increased in the NPO group (admission: 11.0±2.6% day 6: 22.5 ± 4.6%, P= .035). Fecal α1‐PI was above reference values in both groups and declined progressively. No significant differences occurred for %R, L/R ratios, or α1‐PI between groups. Thirteen NPO dogs and all EEN dogs survived (P= .48). The EEN group showed earlier clinical improvement and significant weight gain. The significantly decreased %L in the EEN versus NPO group might reflect improved gut barrier function, which could limit bacterial or endotoxin translocation.

The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing

BackgroundRecent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing.ResultsPyrosequencing revealed a previously unrecognized species richness in the canine small intestine. Ten bacterial phyla were identified. Microbial populations were phylogenetically more similar during tylosin treatment. However, a remarkable inter-individual response was observed for specific taxa. Fusobacteria, Bacteroidales, and Moraxella tended to decrease. The proportions of Enterococcus-like organisms, Pasteurella spp., and Dietzia spp. increased significantly during tylosin administration (p < 0.05). The proportion of Escherichia coli-like organisms increased by day 28 (p = 0.04). These changes were not accompanied by any obvious clinical effects. On day 28, the phylogenetic composition of the microbiota was similar to day 0 in only 2 of 5 dogs. Bacterial diversity resembled the pre-treatment state in 3 of 5 dogs. Several bacterial taxa such as Spirochaetes, Streptomycetaceae, and Prevotellaceae failed to recover at day 28 (p < 0.05). Several bacterial groups considered to be sensitive to tylosin increased in their proportions.ConclusionTylosin may lead to prolonged effects on the composition and diversity of jejunal microbiota. However, these changes were not associated with any short-term clinical signs of gastrointestinal disease in healthy dogs. Our results illustrate the complexity of the intestinal microbiota and the challenges associated with evaluating the effect of antibiotic administration on the various bacterial groups and their potential interactions.

Evaluation of serum feline pancreatic lipase immunoreactivity and helical computed tomography versus conventional testing for the diagnosis of feline pancreatitis

Serum feline trypsinogen-like immunoreactivity (fTLI) concentrations and abdominal ultrasound have facilitated the noninvasive diagnosis of pancreatitis in cats, but low sensitivities (33% and 20-35%, respectively) have been reported. A radioimmunoassay has been validated to measure feline pancreatic lipase immunoreactivity (fPLI), but the assays sensitivity and specificity have not been established. In human beings, the sensitivity of computed tomography (CT) is high (75-90%), but in a study of 10 cats, only 2 had CT changes suggestive of pancreatitis. We prospectively evaluated these diagnostic tests in cats with and without pancreatitis. In all cats, serum was obtained for fTLI and fPLI concentrations, and pancreatic ultrasound images and biopsies were acquired. Serum fPLI concentrations (P< .0001) and ultrasound findings (P = .0073) were significantly different between healthy cats and cats with pancreatitis. Serum fTLI concentrations (P = .15) and CT measurements (P = .18) were not significantly different between the groups. The sensitivity of fTLI in cats with moderate to severe pancreatitis was 80%, and the specificity in healthy cats was 75%. Feline PLI concentrations were both sensitive in cats with moderate to severe pancreatitis (100%) and specific in the healthy cats (100%). Abdominal ultrasound was both sensitive in cats with moderate to severe pancreatitis (80%) and specific in healthy cats (88%). The high sensitivities of fPLI and abdominal ultrasound suggest that these tests should play an important role in the noninvasive diagnosis of feline pancreatitis. As suggested by a previous study, pancreatic CT is not a useful diagnostic test for feline pancreatitis.

16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

Molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease.

An association between mucosa-adherent commensal bacteria and inflammatory bowel disease (IBD) has been proposed for humans. There are no reports characterizing the mucosa-adherent duodenal microbiota in dogs with idiopathic IBD using molecular methods. The aim of this study was to investigate differences in the mucosa-adherent duodenal microbiota between dogs with idiopathic IBD and healthy dogs. Duodenal biopsy samples were collected from seven dogs with IBD and seven healthy control dogs. DNA was extracted, 16S ribosomal RNA genes were amplified and 16S rRNA gene clone libraries were constructed and compared between groups. A total of 1035 clones were selected, and based on a 98% similarity criterion, 133 unique phylotypes were identified across all dogs. These phylotypes belonged to seven bacterial phyla: Proteobacteria (52.9%), Firmicutes (26.1%), Bacteroidetes (7.7%), Actinobacteria (8.6%), Fusobacteria (4.4%), Tenericutes (0.2%) and Verrucomicrobia (0.1%). Significant differences were identified in the relative abundance of several bacterial groups between dogs with IBD and healthy dogs (p<0.001). Healthy dogs and dogs with IBD clustered according to their disease status. Dogs with IBD had a significantly higher abundance of clones belonging to Alpha-, Beta-, and Gamma-proteobacteria (p<0.0001 for all classes), and a significantly lower abundance of Clostridia (p<0.0001). Bacteria of the genera Pseudomonas, Acinetobacter, Conchiformibious, Achromobacter, Brucella, and Brevundimonas, were significantly more abundant in dogs with IBD. In conclusion, significant differences of the mucosa-adherent duodenal microbiota were observed between dogs with idiopathic IBD and healthy dogs in this study. These results warrant further investigations into the role of the intestinal microbiota in the pathophysiology of canine IBD.

Diagnosis of pancreatitis.

In summary, pancreatitis is common in dogs and cats, but it seems that most cases remain undiagnosed. Serum amylase and lipase activities are useful as a quick screening test for pancreatitis in the dog only. Serum amylase or lipase activity must be at least three to five times the upper limit of the reference range to suggest a diagnosis of pancreatitis. Furthermore, the diagnosis must be confirmed by other diagnostic modalities, and normal test results do not eliminate the possibility of pancreatitis. Abdominal ultrasound is highly specific for pancreatitis in dogs and cats but is not particularly sensitive, especially in cats. Serum cPLI concentration is highly specific for exocrine pancreatic function and is also highly sensitive for pancreatitis. Similarly, initial data would suggest that serum fPLI is the most sensitive and specific diagnostic test for feline pancreatitis. Until further data are available, however, serum fPLI should be used in conjunction with other diagnostic tests to arrive at a diagnosis of feline pancreatitis. Histopathologic evidence of pancreatitis is conclusive for a diagnosis of pancreatitis. In most cases, however, lesions are localized, and the lack of histopathologic evidence of pancreatitis does not eliminate a diagnosis of pancreatitis.

Localization of pancreatic inflammation and necrosis in dogs

Few studies of the prevalence of histologic lesions of the exocrine pancreas in dogs have been reported, and none of them systematically evaluate the localization of these lesions. The purpose of this study was to describe the anatomic localization of pancreatic inflammation, necrosis, and fibrosis in dogs presented for postmortem examination. Seventy-three pancreata from dogs presented for postmortem examination were evaluated and investigated for the presence of suppurative inflammation (SI), pancreatic necrosis (PN), and lymphocytic inflammation (LI). Sections that showed evidence of SI, PN, or LI also were evaluated for pancreatic fibrosis (F). The tendency for a preferred localization for SI, PN, and LI was evaluated by chi-square analysis. A total of 47 pancreata with histologic evidence of pancreatitis (SI, PN, or LI; F alone was not considered evidence of pancreatitis) were identified. Twenty-four (51.1%) had SI, 23 (48.9%) had PN, and 34 (72.3%) had LI. Of the 47 dogs with SI, PN, or LI, 28 (59.6%) had F. The distribution of SI, PN, and LI between the right and the left limb of the pancreas and among the 5 anatomic regions was random, based on a goodness-of-fit test. We conclude that pancreatic inflammation tends to occur in discrete areas within the pancreas rather than diffusely throughout the whole organ. These findings suggest that a single biopsy is insufficient to exclude subclinical pancreatitis and that there is no preferred site for pancreatic biopsy collection unless gross lesions are apparent.

Lipid metabolism and hyperlipidemia in dogs

Lipid metabolism in dogs can be divided into exogenous and endogenous pathways and exhibits some unique characteristics compared to other species. Hyperlipidemia is common in dogs, and can be either primary or secondary to other diseases. Secondary hyperlipidemia is the most common form and can be a result of endocrine disorders, pancreatitis, cholestasis, protein-losing nephropathy, obesity, and high fat diets. Primary hyperlipidemia is less common and usually associated with certain breeds. Hypertriglyceridemia of Miniature Schnauzers is the most common type of primary hyperlipidemia in dogs in the United States, and appears to have a genetic basis although its etiology remains unknown. Possible complications of canine hyperlipidemia include pancreatitis, liver disease, atherosclerosis, ocular disease, and seizures. Management is achieved by administration of low fat diets with or without the administration of lipid-lowering agents such as omega-3 fatty acids, gemfibrozil, and niacin.