Kenneth W. Simpson, BVM&S, PhD, DACVIM, DECVIM-CA
Inflammatory bowel disease (IBD) is the term applied to a group of poorly understood enteropathies that are characterized by the infiltration of the gastrointestinal mucosa by inflammatory cells. The cellular infiltrate is composed of variable populations of lymphocytes, plasma cells, eosinophils neutrophils and macrophages, and is often distributed throughout the gastrointestinal tract. The infiltrate is variably accompanied by changes in the mucosal architecture such as villous atrophy, fusion, fibrosis and lymphangiectasia. IBD is likely the leading diagnosis in dogs and cats presented for the investigation of chronic gastrointestinal causes of vomiting, diarrhea, weight loss and anorexia. However, little is known about its etiology, and treatment is usually based on empirical combinations of diet, antimicrobials and immunosuppression. This presentation will focus on the role of bacteria in canine and feline inflammatory bowel disease.
Evidence to Support a Role for Intestinal Bacteria in IBD
Recent research on the immunological environment in the gastrointestinal tract reveals a complex interplay between luminal constituents (e.g., dietary, bacterial microflora), the epithelium, immune effector cells (e.g., lymphocytes and macrophages) and soluble mediators such as chemokines and cytokines. In health, this system functions to avoid active inflammation by antigen exclusion and the induction of immune tolerance and the cytokine profile is dominated by the production of the immunomodulatory cytokines (cytokines are soluble mediators of inflammation) TGF-β and IL-10.
In people with inflammatory bowel disease there is a failure to suppress the inflammatory response, with pro-inflammatory cytokines such as TNFα, IL-1 and IFNγ produced in excess. Mechanistic studies in rodents with engineered genetic susceptibility to IBD, e.g., -/-IL-10, -/-IL-2 mice and HLA-B27 transgenic rats, indicate that the enteric microflora is required for inflammation, with inflammation observed in conventionally housed but not germ-free conditions. In these models, bacterial species regarded as part of the normal intestinal flora, such as Bacteroides and Enterobacteriaceae, particularly E. coli, have been associated with inflammation. Hence, it has been proposed that IBD is a consequence of an overly aggressive immune or inflammatory response to a subset of commensal enteric bacteria in genetically predisposed individuals.
Evidence implicating the resident enteric microflora in the pathogenesis of spontaneous IBD in people is provided by the clinical responses of Crohn's disease (CD) to fecal stream diversion and antimicrobials, the increased circulating and intraluminal humoral, T cell responses to enteric commensal bacteria, and imbalances in the ratio of beneficial to harmful bacteria (termed 'dysbiosis') observed in CD. Complementing this is the discovery of mutations in genes regulating microflora sensing ability (NOD2/CARD15 and TLR-4) of patients with CD. This provides mechanisms to explain individual susceptibility to the resident microflora that, in the presence of the enteric microflora and appropriate triggers, may lead to up-regulated mucosal cytokine production, delayed bacterial clearance and increased bacterial translocation, thereby promoting and perpetuating intestinal inflammation.
A diverse spectrum of pathogenic bacteria, including Listeria, Streptococcus, Enterococcus, Enterobacteriaceae, Bacteroides, Clostridium, Yersinia and Mycobacterium avium paratuberculosis (MAP), have been implicated in the development of inflammation, but no single agent has emerged as a consistent cause. In the absence of a defined cause, therapy is symptomatic and supportive, and achieves remission rather than cure.
Recent advances in molecular microbiology have led to a new awareness of the diversity and complexity of the enteric flora. Culture-independent analyses of bacterial 16S rDNA libraries reveal that only 30% of the fecal flora appears cultivable, and there is significant variation in the flora in different gastrointestinal segments and luminal contents versus the mucosa of healthy individuals. Analysis of 16S rDNA libraries constructed from endoscopic biopsies of the ileum from patients with Crohn's ileitis (a disease phenotype that occurs in approximately 70% of individuals with CD), demonstrates selective enrichment of ileal mucosa in E. coli and relative depletion in Clostridiales compared to both patients with CD restricted to the colon, and to healthy individuals. PCR of ileal DNA yielded no evidence of MAP, Listeria or Shigella, and confirmed the increase in E. coli. Similarly, 16S rDNA library-based evaluation of surgically resected CD ileum shows an increase in Proteobacteria (this phylum includes Enterobacteriaceae), decrease in Firmicutes (this phylum contains Clostridiales), and the absence of DNA from MAP in CD mucosa. Microscopy with a probe restricted to E. coli/Shigella reveals that the ileal mucosa of CD patients harbors significantly more E. coli than normal ileum, and mucosally invasive E. coli are only detected in CD mucosa. Furthermore, the number of E. coli visualized in ileal biopsies, but not bacterial colonization in general, correlates with histological and endoscopic disease activity, which suggests E. coli could be specifically involved in the inflammatory process.
Detailed investigation of the E. coli strains isolated from patients with ileal Crohn's disease show that they are able to act similarly to pathogenic bacteria such as Salmonella in cell culture, but lack known factors to explain their pathogenic behavior. These findings suggest that E. coli could be causally related to ileal Crohn's disease and raise the possibility that antibiotics directed against E. coli could be an effective treatment for some patients with ileal Crohn's disease.
Do Intestinal Bacteria Have a Role in Canine and Feline IBD?
The role of bacteria in IBD in dogs has largely focused on the identification of potential pathogens, or their toxins, in feces (e.g., Clostridium spp.), or the number and type of bacteria in duodenal juice (e.g., German Shepherd dogs).Clinical responses of dogs with chronic enteropathies to antimicrobial agents have been widely attributed to 'idiopathic small intestinal bacterial overgrowth'. However, recent research has shown that the numbers of bacteria in duodenal juice do not correlate with the clinical response to antimicrobials, diet or immunosuppression. These observations help to dispel the notion of 'idiopathic small intestinal bacterial overgrowth' as a major cause of enteropathy in dogs, but do not explain the basis of 'antibiotic responsive enteropathy' in dogs.
Investigation of the role of intestinal bacteria in feline IBD is restricted to the identification of potential pathogens in feces, and quantitative analysis of bacteria in duodenal juice. The dominant cultivable species in duodenal juice of healthy cats are: Clostridium, Bacteroides, Streptococcus and Enterobacteriaceae, including E. coli. Compared with healthy cats, cats with clinical signs of gastrointestinal tract disease have significantly fewer luminal microaerophilic bacteria, whereas total, anaerobic, and aerobic bacterial counts are not significantly different. Knowledge of the mucosa-associated flora is limited to 4 cats, with the bacteria cultured from scraped mucosa similar to those reported for duodenal juice. The spatial distribution of mucosa-associated bacteria and their relationship to mucosal inflammation has not been specifically addressed in healthy cats or cats with gastrointestinal disease.
It is against this background that we have applied contemporary culture-independent microbiological methods to examine the relationship of mucosa-associated bacteria to mucosal inflammation in boxer dogs with histiocytic ulcerative colitis (HUC; also known as granulomatous colitis of boxer dogs) and cats with IBD.
Histiocytic Ulcerative / Granulomatous Colitis of Boxer Dogs (GCB)
In contrast to the widely accepted view that GCB is an incurable immune mediated disease, the original description by Van Kruningen (1965) describes a favorable outcome in six of nine dogs treated with chloramphenicol. The results of three recent studies provide clear evidence of clinical and histological remission in 12 Boxer dogs and one English Bulldog treated with antibiotic regimens containing fluoroquinolones (enrofloxacin at dose of around 5 mg/kg PO qd for 30-60 days). Treatment with enrofloxacin, alone or in combination with metronidazole and/or amoxicillin, was generally reported to induce resolution of clinical signs within two weeks. Approximately one third of dogs remained free of clinical signs during a 5 to 14 month follow-up after discontinuation of treatment.
Given the dramatic response to antibiotics, recent studies have explored the possibility that an uncharacterized infectious agent such as Tropheryma whippelii (the causative agent of Whipple's disease) or an abnormal mucosa-associated flora is involved in the etiopathogenesis of GCB. One of these studies used a combination of culture-independent molecular techniques (16SrDNA sequencing and fluorescence in situ hybridization) to examine the mucosa-associated bacterial flora of colonic biopsies from healthy dogs, dogs with lymphoplasmacytic colitis and boxer dogs with GCB. Those investigators demonstrated selective intramucosal colonization of GCB biopsies by E. coli. Another study described the immunolocalization of E. coli, Lawsonia intracellularis, Campylobacter and Salmonella to macrophages in the colon of 10/10, 3/10, 2/10 and 1/10 boxer dogs with granulomatous colitis respectively (dogs without colitis or other forms of colitis were not examined). These findings strongly suggest that GCB is a consequence of mucosal colonization by luminal E.coli in a susceptible individual (i.e., an undefined breed specific abnormality in boxer dogs).
Interestingly, the E. coli strains isolated from the colonic mucosa of dogs with GCB adhered to, invaded and persisted in cultured epithelial cells to the same degree as E. coli strains associated with Crohn's disease. Initial investigations of GCB and Crohn's-associated E. coli indicate they are more similar in phylogeny and virulence gene profiles to extraintestinal pathogenic E. coli (e.g., uropathogenic E. coli), than diarrheagenic E. coli and point to the association of E. coli that resemble extraintestinal pathogenic strains in genotype with chronic intestinal inflammation.
Feline Inflammatory Bowel Disease
Endoscopic biopsies from 17 cats presented for investigation of vomiting, diarrhea, and anorexia and/or weight loss and 10 clinically normal colony cats were included in the study for a total of 27 cases.
The number of mucosa-associated Enterobacteriaceae was higher in cats with signs of gastrointestinal disease than healthy cats. Total numbers of mucosal bacteria were strongly associated with changes in mucosal architecture and the density of cellular infiltrates, particularly macrophages. Enterobacteriaceae spp., E. coli and Clostridium spp. were associated with significant changes in mucosal architecture (principally atrophy and fusion), upregulation of cytokines (particularly IL-8), and the number of clinical signs exhibited by the affected cats.
The Importance of Mucosa-Associated Bacteria in IBD in Dogs and Cats
These initial studies illustrate the importance of mucosa-associated bacteria in IBD in dogs and cats:
The presence of invasive E. coli in the colonic mucosa of dogs with GCB likely explains the dramatic responses observed to treatment with fluoroquinolones, rather than immunosuppressive agents. The similarity of E. coli strains isolated from dogs with GCB and people with ileal Crohn's disease is striking and suggests the possibility of an IBD causing group of E. coli.
The correlation between the number of mucosa-associated bacteria and the levels of mucosal atrophy and IL-8 in cats with IBD supports the need for clinical trials to evaluate therapeutic manipulation of the mucosa-associated flora. In this respect, the potential of antibiotics, probiotics and prebiotics to modulate or displace mucosa-associated, adherent or invasive bacteria, and dampen the local and systemic inflammatory response is exciting.
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