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Proceedings of the 33rd World Small

Animal Veterinary Congress
Dublin, Ireland - 2008

Next WSAVA Congress :

Reprinted in IVIS with the permission of the Congress Organizers

Reprinted in IVIS with the permission of the Congress Organizers

Close this window to return to IVIS

WSAVA / FECAVA World Small Animal Congress

Medicine 18

B acteria

and inflammatory bowel disease - causes and consequences

Kenneth W. Simpson BVM&S, PhD, DipACVIM, DipECVIM-CA
Cornell University, Ithaca NY, USA

Introduction
Inflammatory bowel disease (IBD)
is the term applied to a group of
poorly understood enteropathies
that are characterized by the infil
tration of the gastrointestinal
mucosa by inflammatory cells. The
cellular infiltrate is composed of
variable populations of lymphocytes,
plasma cells, eosinophils neutro
phils 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- and IL-10.
In people with inflammatory bowel disease there is a
failure to suppress the inflammatory response, with
pro-inflammatory cytokines such as TNFa, IL-1 and
IFNproduced 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 Crohns 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 Crohns 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

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WSAVA / FECAVA World Small Animal Congress

18 Medicine

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 Crohns 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
Crohns disease and raise the possibility that antibiotics
directed against E. coli could be an effective treatment
for some patients with ileal Crohns 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 lumenal
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 mucosaassociated 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 fluroquinolones (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 Trophyrema
whippelii (the causative agent of Whipples 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 Crohns disease. Initial
investigations of GCB and Crohns-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.

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Medicine 18

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 mucosaassociated 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 fluroquinolones, rather
than immunosuppressive agents. The similarity of
E. coli strains isolated from dogs with GCB and people
with ileal Crohns disease is striking and suggests the
possibility of an IBD causing group of E. coli.
The correlation between the number of mucosaassociated 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.
References:

1.
2.
3.
4.
5.
6.
7.

Simpson KW et al. Infect. Immun., 74(8), 4778-4792 (2006).

Baumgart et al. ISME J., 1(5), 403-418 (2007).
Barnich N. Curr. Opin. Gastroenterol., 23(1), 16-20 (2007).
Van Kruiningen et al. APMIS 113:420-425 (2005).
Hostutler RA et al. J Vet Intern Med 18:499-504 (2004).
Davies DR, et al. Aust Vet J 82:58-61 (2004).
Janeczko S et al. Vet Microbiol. 128, 178-93 (2008).

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