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Keywords:

  • 16S rDNA;
  • Adherent and invasive Escherichia coli;
  • Canine;
  • Colitis;
  • Crohn's disease;
  • Enrofloxacin;
  • Fluorescent in situ hybridization

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Background: Historically, histiocytic ulcerative (HUC) (or granulomatous) colitis of Boxer dogs was considered an idiopathic immune-mediated disease with a poor prognosis. Recent reports of dramatic responses to enrofloxacin and the discovery of invasive Escherichia coli within the colonic mucosa of affected Boxer dogs support an infectious etiology.

Hypothesis: Invasive E. coli is associated with colonic inflammation in Boxer dogs with HUC, and eradication of intramucosal E. coli correlates with clinical and histologic remission.

Animals: Seven Boxer dogs with HUC.

Methods: Prospective case series. Colonic biopsies were obtained at initial evaluation in 7 dogs, and in 5 dogs after treatment with enrofloxacin. Biopsies were evaluated by standardized histopathology, and fluorescence in situ hybridization (FISH) with probes to eubacteria and E. coli.

Results: Intramucosal E. coli was present in colonic biopsies of 7/7 Boxers with HUC. Clinical response was noted in all dogs within 2 weeks of enrofloxacin (7±3.06 mg/kg q24 h, for 9.5±3.98 weeks) and was sustained in 6 dogs (median disease-free interval to date of 47 months, range 17–62). FISH was negative for E. coli in 4/5 dogs after enrofloxacin. E. coli resistant to enrofloxacin were present in the FISH-positive dog that relapsed.

Conclusions and Clinical Relevance: The correlation between clinical remission and the eradication of mucosally invasive E. coli during treatment with enrofloxacin supports the causal involvement of E. coli in the development of HUC in susceptible Boxer dogs. A poor response to enrofloxacin treatment might be due to colonization with enrofloxacin-resistant E. coli.

Abbreviation:
FISH

fluorescent in situ hybridization

HUC

histiocytic ulcerative colitis

Granulomatous colitis of Boxer dogs was first described by Van Kruiningen in a kennel of Boxer dogs in 1965.1 Some subsequent reports of this disease use the term histiocytic ulcerative colitis (HUC),2–5 and this term is more familiar to many veterinarians. The pathognomonic lesion of HUC in Boxer dogs is mucosal infiltration with large numbers of macrophages staining positively with periodic acid-Schiff (PAS), and is usually accompanied by mucosal ulceration and loss of goblet cells.1 Early reports describe a response of 6/9 dogs to chloramphenicol and the presence of intracellular bacteria within macrophages.1,6 However, these findings were poorly reproducible in subsequent studies7,8 and experimental attempts to reproduce HUC, by infecting Boxer dogs with Mycoplasma spp. isolated from colonic mucosa were unsuccessful.9 Thus HUC has until recently been regarded as an idiopathic immune-mediated disease that typically responds poorly to treatment with empirical therapies such as dietary change, antibiotics, and immunosuppressive agents.4,5

The poor response of HUC to immunosuppression led to a reappraisal of antibiotic therapy, and there are now 3 independent studies that describe dramatic improvements in clinical signs and histological lesions of Boxer dogs treated with antibiotic protocols containing enrofloxacin.10–12 These observations along with the discovery of a bacterial cause for the intestinal infiltration of PAS-positive macrophages in people with Whipple's disease13 initiated the search for an infectious agent by contemporary culture-independent methodologies. Colonic biopsies from affected Boxer dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial16S rDNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. Intramucosal Gram-negative coccobacilli were present in 100% of HUC samples from Boxer dogs but not controls, and invasive bacteria hybridized with FISH probes to Escherichia coli.12 Independent support for these findings is provided by the immunolocalization of E. coli to macrophages within the colon of 10/10 Boxer dogs with HUC.14E. coli strains isolated from affected Boxer dogs are novel in phylogeny, and have an adherent and invasive phenotype (AIEC) similar to strains isolated from people with Crohn's disease.12,15,16 These findings suggest that E. coli invasion could play a critical role in the initiation and/or progression of HUC in Boxer dogs, likely facilitated by a breed-specific susceptibility.

It is against this background that we sought to determine the temporal association of clinical and histological response to the presence or absence of intramucosal E. coli in Boxer dogs with HUC before and after treatment with enrofloxacin.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Seven Boxers (3 neutered females, 2 entire female, 1 neutered male, 1 entire male) with signs of colitis referred to MUVTH (dogs 1–5) and Cornell University Hospital for Animals (dogs 6, 7) between 2002 and 2006 were enrolled in this study. The age of onset of clinical signs ranged from 1.5 to 20 months, with a mean of 8.8 months. The age at presentation ranged from 10 to 24 months, with a mean of 17.4 months. Diagnostic evaluation included hematology, serum biochemistry, urinalysis, zinc sulfate fecal flotation, and fecal culture for enteric pathogens, including Campylobacter and Salmonella. Colonic biopsies were obtained at the time of diagnostic evaluation in all 7 dogs (endoscopic biopsies) and at variable intervals after enrofloxacina treatment in 5 dogs (endoscopic in dogs 1, 3–5; full thickness at postmortem in dog 7). For endoscopic biopsy a minimum of 7–12 colonic biopsies were collected after standard colonoscopic preparation with an oral electrolyte solutionb and pre-endoscopic enema with warm water. Enrofloxacina was prescribed at dose rates ranging from 4.8 to 12.8 mg/kg q24 h PO for a period of 4–16 weeks, equating to a mean daily dose of 7 mg/kg/d, for a mean duration of 9.5 weeks. Two of these dogs (dogs 1 and 2) are reported in a previous publication.10

Histopathology

In all dogs, hematoxylin and eosin and PAS stained paraffin-embedded formalin-fixed sections of colon were retrospectively evaluated by a veterinary pathologist (SPM) in a blinded manner to confirm the original histological diagnosis, and to assess the severity of colitis.

FISH

Colonic sections were examined by FISH in a blinded manner. Eubacterial and E. coli-specific probes targeting 16S ribosomal rDNA were used, using established methodologies.12

Colonic Mucosal Culture

Colonoscopic biopsies obtained from dogs 3–5 before enrofloxacin administration were stored in Brucella broth with 30% glycerol at −70°C before culture for Shigella, E. coli, Salmonella, Mycobacterium bovis, Mycobacterium spp., Chlamydia, and Mycosplasma. Colonic biopsies obtained from dog 7 after clinical relapse were cultured for E. coli by methods described previously.16 Antimicrobial sensitivity of E. coli was determined by disk diffusion.17

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Initial Evaluation

At the time of diagnosis, colitis was graded as severe in 4/7 dogs and moderate in 3/7, typically with large numbers of PAS + macrophages (Figs 1 and 2). Commonly observed histologic changes were neutrophilic infiltration (6 dogs; marked in dog 6, moderate in dogs 1,3, 7, and mild in dogs 4 and 5), epithelial erosion/ulceration (6 dogs; 1, 3–7), crypt hyperplasia and crypt distortion (6 dogs; 1–6). Decreased numbers of goblet cells were readily apparent in PAS-stained sections from 6/7 dogs (4 severe, dogs 1, 3, 4, 6; 2 moderate, dogs 5 and 7). Only 1 dog (dog 2) had a normal distribution and number of goblet cells.

image

Figure 1.  Colonic histopathology and fluorescence in situ hybridization (FISH) before and after treatment of histiocytic ulcerative colitis (HUC) with enrofloxacin. Colonic biopsies from dog 1 at initial diagnosis were dominated by periodic acid-Schiff (PAS)-positive macrophages (a) and invasive intramucosal bacteria that hybridized with a Cy-3-labeled probe to Escherichia coli (b: bacteria are orange, DAPI-stained nuclei are blue). After 2 weeks of enrofloxacin, mucosal infiltration with PAS-positive macrophages persists (c) but no invasive bacteria are visualized by FISH (d). Seven months after diagnosis, the infiltration of PAS-positive macrophages had resolved (though occasional PAS-positive macrophages remained), goblet cells containing abundant PAS staining mucus were apparent (e), and FISH (f) remained negative.

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image

Figure 2.  Colonic histopathology before and after treatment of histiocytic ulcerative colitis (HUC) with enrofloxacin. Hematoxylin and eosin and periodic acid-Schiff (PAS)-stained sections of endoscopic colonic biopsies from dogs 2–4 before (a, c, e) and 8–10 weeks after treatment with enrofloxacin (b, d, f). In untreated HUC (a, c, e) epithelial erosion, loss of glandular structure, and marked cellular infiltration with PAS-positive macrophages (insets) were readily apparent. After treatment with enrofloxacin (b, d, f) histological abnormalities had markedly improved in all 3 dogs, though small numbers of PAS-positive macrophages and infiltrates of lymphocytes and plasma cells were still present. An increase in PAS-positive goblet cells was observed in all 3 dogs posttreatment (insets, b, d, f).

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FISH analysis was positive for intramucosal E. coli in each of the 7 affected dogs.

Routine fecal cultures and parasitology were negative for known pathogens. Culture of colonic biopsies from dogs 3–5 was positive for E. coli, but negative for Shigella, Salmonella, M. bovis, Mycobacterium spp., Chlamydia, and Mycosplasma. E. coli isolates were sensitive to a wide range of antibiotics including enrofloxacin, gentamicin, amoxicillin–clavulanate, and cephalexin.

Clinical Response

Clinical response was evaluated in 7 Boxer dogs with HUC after treatment with enrofloxacin.a Five of these dogs had not responded to previous treatment with sulfasalazinec (4), corticosteroids (3), erythromycind (1) metronidazolee (2), amoxicillin–clavulanatef (1), and azathioprineg (1). Two dogs had received no medications before administration of enrofloxacin. A positive clinical response (cessation of tenesmus, mucoid feces, and hematochezia) was noted in 7/7 dogs within 2 weeks of administration of enrofloxacin, and was sustained in 6 dogs, with a median disease-free interval to date of 47 months (range 17–62 months). Dog 7 initially responded well to intermittent treatment with enrofloxacina (3 courses of approximately 5.0 mg/kg q24 h PO for 2–4 weeks) over a 7-month period, but became unresponsive to enrofloxacin. Culture of colonic biopsies obtained after clinical relapse yielded 2 strains of E. coli, both of which were resistant to enrofloxacin and gentamicin, and sensitive to amoxicillin–clavulanate, cefoxitin, ceftriaxone, amikacin, and kanamycin. Further treatment with amoxicillin–clavulanatef failed to elicit clinical improvement and this dog was euthanized approximately 4 weeks later because of the continued severity of clinical signs.

Histopathological Response

Blinded evaluation of biopsies obtained before and after administration of enrofloxacina revealed a marked reduction in the severity of inflammation in 4 of the 5 dogs evaluated (dogs 1, 3–5) (Figs 1 and 2). Histologic remission lagged behind clinical remission in each of the responders. In dog 1 (Fig 1) histologic evaluation of colonoscopic biopsies 2 weeks after diagnosis showed marked improvement of the initially severe, erosive HUC, but a mild infiltration of PAS + macrophages persisted. Colonoscopic biopsies collected from this dog on a 3rd occasion, 7 months after the initial diagnosis and were histologically within normal limits though rare macrophages and crypt distortion persisted (Fig 1). An impact of HUC on goblet cells was readily appreciated in PAS-stained sections from this dog, with marked increases in goblet cells and mucus at 7 months (Fig 1). In dog 3, colonic histopathology 10 weeks after diagnosis had changed from severe HUC with extensive epithelial erosions to mild, lymphoplasmacytic colitis, though small to moderate numbers of PAS-positive macrophages were observed within the lamina propria and submucosa, respectively (Fig 2). In dog 4, the initially moderate HUC with focal epithelial erosions had regressed to mild HUC with rare PAS + macrophages (predominantly in the submucosa) at repeat histological evaluation of colonic biopsies by 8 weeks after the initial diagnosis (Fig 2). The moderate HUC initially documented in dog 5 had also improved, though mild, focal PAS-positive macrophages persisted on reevaluation of colonoscopic biopsies 8 weeks later (Fig 2). In dog 7, which relapsed after an initial response to enrofloxacin,a severe granulomatous inflammation and large numbers of PAS + macrophages was present in biopsies obtained 7 months after initial evaluation. Partial resolution of epithelial erosion, a more focal distribution of inflammation, and a decrease in neutrophils were observed after 4 weeks of amoxicillin–clavulanatef but this did not correlate with clinical improvement.

FISH Posttreatment

Posttreatment FISH analysis was negative for intramucosal E. coli in 4/5 dogs (1, 3–5) at all time points evaluated after treatment with enrofloxacina (Figs 1 and 3). Intramucosal E. coli was detected in colonic mucosal biopsies from the dog (dog 7) that had become clinically unresponsive to treatment with enrofloxacin. Re-evaluation of colonic biopsies after treatment with amoxicillin–clavulanate,f showed persistence of intramucosal bacteria, though they did appear reduced in number.

image

Figure 3.  Fluorescence in situ hybridization (FISH) before and after treatment of histiocytic ulcerative colitis (HUC) with enrofloxacin. FISH of colonic biopsies from dogs 2, 3, and 4 before enrofloxacin (a, c, e) revealed multifocal intramucosal bacteria that hybridized with a Cy-3-labeled probe to Escherichia coli (bacteria are orange, DAPI-stained nuclei are blue). Many of the E. coli appeared to be within macrophages (insets a, c, e). After treatment with enrofloxacin, sections from all 3 dogs (b, d, f) were FISH negative.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

The clinical response of Boxer dogs with HUC to antibiotic regimens containing enrofloxacina strongly suggests bacterial involvement in the inflammatory process.10–12 This possibility is significantly strengthened by the recent discovery of invasive E. coli within the colonic mucosa and macrophages of affected Boxer dogs, but not dogs with other forms of colitis.12 Our observations that colonic biopsies obtained during the initial diagnostic evaluation of 7 HUC-affected Boxers contained intramucosal E. coli provides independent confirmation of these previous findings. The inclusion of dogs from Australia and the United States in the present study and dogs from the United Kingdom and United States in the previous study12 demonstrates that intramucosal colonization of E. coli in HUC is not a consequence of a geographically restricted infectious agent. In agreement with previous reports,4,5,10,11 we observed a poor clinical response in all dogs treated with various combinations of prednisolone,c sulfasalazine,d and nonenrofloxacina antimicrobials. We found that enrofloxacina (mean 7 mg/kg q24 h) for a mean duration of 9.5 weeks resulted in a positive clinical response in all dogs within 2 weeks of administration, and this was sustained in 6/7 dogs, resulting in median disease-free interval to date of 47 months (17–62 months). Sustained response correlated with the eradication of invasive intramucosal E. coli. The dog that relapsed was positive for intramucosal E. coli that was resistant to enrofloxacin.a The correlation between clinical remission and the eradication of mucosally invasive E. coli during treatment with enrofloxacina supports the causal involvement of E. coli in the development of HUC in susceptible Boxer dogs

The association of E. coli with granulomatous intestinal inflammation in Boxer dogs parallels a growing number of studies in people with Crohn's disease that report the presence of E. coli antigens and DNA within affected mucosa,16,18,19 the isolation of higher numbers of invasive E. coli from Crohn's mucosa,16,19–21 and increased circulating antibodies against E. coli OmpC.22 It is noteworthy that the E. coli strains isolated from Boxer dogs are strikingly similar to those implicated in Crohn's ileitis,12–16,20 in that they are able to invade, persist, and replicate in epithelial cells and macrophages and evoke the production of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL8.15,16,23 These characteristics are generally associated with pathogenic bacteria, and it seems likely that this emergent group of adherent and invasive E. coli (AIEC) are opportunistic pathogens able to exploit a susceptible individual rather than harmless commensals.16,19,23 This possibility is consistent with the prevailing view that inflammatory bowel disease is the result of an overexuberant inflammatory response to a subset of resident enteric bacteria in a genetically susceptible individual.24,25 Genetic polymorphisms associated with granulomatous intestinal inflammation in people involve defective intracellular processing of bacterial components such as polymorphisms in the intracytosolic muramyl dipeptide receptor NOD2, and autophagy genes such as ATG16L1, and an overactive mucosal inflammatory response (eg, IL-23r promoting loss of oral tolerance).26 Because HUC in dogs is remarkably breed specific it may be due to a heritable anomaly in Boxer dogs that confers susceptibility to invasion and persistence of AIEC within the colonic mucosa. As Boxer dogs are also prone to other granulomatous diseases such as intestinal and systemic protothecosis and cutaneous intracellular mycobacteria (canine leproid granuloma syndrome),27,28 it is possible that Boxers have a genetic defect in how they respond to granuloma-inducing microorganisms in general. Further study is needed to identify the host and bacterial factors related to invasion and persistence of E. coli in HUC susceptible Boxers.

Our finding that clinical remission in response to enrofloxacina correlates with the eradication of invasive E. coli and precedes complete histologic resolution of inflammation highlights the importance of eliminating E. coli infection in Boxers with HUC. Serial evaluation of dog 1, where the ongoing inflammation observed at 2 weeks had almost completely resolved at 7 months suggests the lag between bacterial eradication and normalization of colonic histology reflects the time taken to heal and remodel the severely damaged mucosa. These observations are similar to findings in people with Whipple's disease where regression of PAS positivity lags behind eradication of Trophyrema whipplei.29

In the present study, resistance to enrofloxacina was demonstrated in 2 E. coli strains isolated from the colonic mucosa of a dog whose clinical signs recurred after an initial response to enrofloxacin. The development of resistance to enrofloxacina has been extensively demonstrated in dogs with E. coli and other Enterobacteriaceae infections30,31 and often involves acquisition of resistance plasmids.31 Interestingly, the dog that relapsed had received several short courses of treatment with enrofloxacin (2–4 weeks) and it is possible that repeated withdrawal of enrofloxacina before complete eradication of infection facilitated recrudescence and subsequently development of enrofloxacina resistance. Short-term treatment of this dog with amoxicillin–clavulanate,g based on the results of antimicrobial susceptibility testing, resulted in a reduction in intramucosal E. coli and partial histologic improvement, but this did not translate to clinical improvement. While it is possible that a longer duration of treatment may have been effective it is important to consider that differences in the ability of antibiotics to penetrate infected macrophages may also affect outcome. Evaluation of the ability of antibiotics (at peak serum concentrations) to kill E. coli isolated from colonic Crohn's disease within J774-A1 macrophages indicates greatest efficacy of ciprofloxacin, with decreasing impact of rifampicin, tetracycline, trimethoprim, clarithromycin, and azithromycin.32 This study also found the combination of ciprofloxacin, tetracycline, and trimethoprim was more effective than ciprofloxacin alone, and that ampicillin was not effective against intracellular CD-associated E. coli. Hence, the ability to effectively penetrate and kill E. coli within macrophages may explain the positive response of HUC of Boxers to enrofloxacin, and also to chloramphenicol,1 and the negative response of the dog in the present study to amoxicillin-clavulanate.g To optimize the treatment of HUC of Boxers in the future it would seem prudent that FISH and colonic mucosal culture are performed as part of the diagnostic evaluation of Boxer dogs suspected of HUC. This would enable antimicrobial selection based on sensitivity testing of mucosal E. coli isolated from FISH positive individuals and their ability to penetrate tissues and macrophages. Based on the results of the present and previous studies,10–12 Boxer dogs with enrofloxacin-sensitive HUC should be treated for at least 6–8 weeks at doses of 5–10 mg/kg body weight q24 h.

Previous reports have documented resolution or improvement of clinical signs in some affected Boxers managed with dietary therapy and sulfasalazine.4,5 Mesalamine, but not hydrocortisone, at therapeutic concentrations has recently been shown to inhibit MAPK-dependent IL-8 release in response to adherent and invasive IBD and colon cancer E. coli isolates in vitro, and it may have some therapeutic benefit in vivo.33 However, as the dogs in this present study that received sulfasalazined failed to respond, a meaningful effect of sulfasalazined in dogs with severe HUC seems unlikely. Whether sulfasalazined has a role in treating Boxers with mild HUC or as an adjunct to enrofloxacina remains to be determined.

In conclusion, this case series serves to corroborate the association of mucosally invasive E. coli with HUC in Boxer dogs and the responsiveness of this disease to enrofloxacin.a The correlation between clinical remission and eradication of mucosally invasive E. coli supports the potential causal involvement of E. coli in the development of HUC in susceptible Boxer dogs. As clinical relapse was associated with enrofloxacina resistance, we suggest that sensitivity profiles of mucosal E. coli and the ability of antibiotics to penetrate macrophages are used to guide antimicrobial selection.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

aBaytril®, Bayer, Pymble, NSW, Australia

bColonlytely®, Dendy Pharmaceuticals, Dendy, Victoria, Australia

cSalazopyrin®, Pharmacia, Rydalmere, NSW, Australia

dErythrocin Stearate®, Abbott, Botany, NSW, Australia

eFlagyl®, Aventis, Osborne Park, WA, Australia

fClavulox®, Pfizer, West Ryde, NSW, Australia

gImuran®, Faro Pharmaceuticals, Bedminster, NJ

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References