Detection of invasive E. coli in dogs with granulomatous colitis using IHC

119712
2022
VDIXXX10.1177/10406387221119712Immunohistochemistry for detection of invasive E. coliIshii et al.
Brief Report
Detection of invasive Escherichia coli
in dogs with granulomatous colitis
using immunohistochemistry
Journal of Veterinary Diagnostic Investigation
2022, Vol. 34(6) 990­–994
© 2022 The Author(s)
Article reuse guidelines:
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https://doi.org/10.1177/10406387221119712
DOI: 10.1177/10406387221119712
jvdi.sagepub.com
Patricia Eri Ishii,1 Jan S. Suchodolski, Ricardo Duarte,
Ana R. C. Pereira, Jonathan A. Lidbury, Joerg M. Steiner,
Paula R. Giaretta
Abstract. Granulomatous colitis in dogs can be associated with infection of the colonic mucosa by invasive strains of
Escherichia coli. To date, fluorescence in situ hybridization (FISH) is the gold-standard method to assess intramucosal and
intracellular bacterial invasion. However, FISH requires expensive fluorescence microscopy equipment and is therefore not
widely available. We investigated the use of immunohistochemistry (IHC) as an alternative method to detect invasive E. coli in
dogs with granulomatous colitis. Archived paraffin-embedded blocks were selected from 26 dogs with colitis, in which FISH
had been performed by an outside laboratory. Using a polyclonal antibody, IHC for E. coli was performed on sections cut from
the same blocks, and the presence of invasive E. coli was recorded. All 11 specimens in which FISH had detected E. coli were
also positive on IHC, with strong immunolabeling in the cytoplasm of macrophages and extracellularly in the lamina propria;
all 15 specimens that were negative for invasive bacteria on FISH were also negative on IHC. We found that IHC is a sensitive
technique for the detection of invasive E. coli in dogs with granulomatous colitis.
Keywords: colitis; dogs; Escherichia coli; granulomatous colitis; histiocytic colitis.
Escherichia coli–associated granulomatous colitis (GC),
also known as histiocytic ulcerative colitis, was first
described in Boxer dogs in 1965.19 Since then, French Bulldogs have also been reported to develop the disease, and a
few other single-case reports have described the condition in
a Staffordshire Terrier, Doberman Pinscher, Rottweiler, Mastiff, Beagle, and Alaskan Malamute.2,11,17 The condition has
also been reported, but is considered rare, in cats.9
Clinically, dogs with GC have clinical signs consistent
with large bowel diarrhea, such as increased frequency of
defecation, mucoid stools, hematochezia, and tenesmus.11,16
Weight loss and hypoalbuminemia are often reported in dogs
with severe forms of GC.4,16 Macroscopic findings include
irregular thickening and ulceration of the colonic mucosa.16
Microscopic lesions are characterized by infiltration of the
lamina propria and submucosa by macrophages with abundant eosinophilic cytoplasm, which is positive on periodic
acid–Schiff (PAS) stain, as well as by erosion or ulceration of
the colonic epithelium.4,16
Adherent-invasive strains of E. coli (AIEC) were identified
as the cause of GC in 2006.16 Eradication of invasive E. coli
with fluoroquinolone antimicrobials is correlated with clinical
remission of GC.4,7,11,12 Resistance of E. coli to antimicrobials
is significantly associated with a poor clinical outcome; antimicrobial susceptibility profiling guided treatment is associated with a positive long-term outcome of GC.10
Fluorescence in situ hybridization (FISH) is a molecular
approach used to detect and visualize a specific DNA or
RNA sequence with fluorescence microscopy.8,13 FISH is
considered the gold-standard method to assess colonic tissue
samples for intramucosal and/or intracellular E. coli invasion
in dogs.16 However, FISH requires expensive fluorescence
microscopy equipment and on-site interpretation by professionals with specific training, and therefore, it is not widely
available as a diagnostic tool.13 Immunohistochemistry
(IHC) is an in situ technique that allows the detection of antigens in tissue sections through specific antibodies.14 Wholeslide images can be shared digitally allowing remote
interpretation. IHC has been used widely for the diagnosis of
infectious diseases in fixed tissues, including a case of GC in
a dog.2
Gastrointestinal Laboratory, Department of Small Animal Clinical
Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas
A&M University, College Station, TX, USA (Ishii, Suchodolski, Lidbury,
Steiner, Giaretta); Gastrovet, Sao Paulo, Brazil (Duarte); School of
Veterinary Medicine and Animal Science of University of Sao Paulo, Sao
Paulo, Brazil (Pereira).
1
Corresponding author: Patricia Eri Ishii, Gastrointestinal Laboratory,
Texas A&M University, 4474 TAMU, College Station, TX 77843, USA.
[email protected]
Immunohistochemistry for detection of invasive E. coli
Our objective was to evaluate an immunoperoxidase IHC
assay using a polyclonal anti–E. coli antibody as an alternative method to detect enteroinvasive E. coli in formalinfixed, paraffin-embedded (FFPE) colonic samples from dogs
with GC.
We used archived FFPE tissue blocks from endoscopic
biopsy samples submitted to the histopathology service of the
Gastrointestinal Laboratory at Texas A&M University (College Station, TX, USA) for diagnostic purposes. The records
were reviewed, and canine cases with ≥5 colonic endoscopic
biopsies in which FISH had been performed at an external
laboratory (Simpson Laboratory, Cornell University, Ithaca,
NY, USA) were selected and divided into a control and a GC
group. At the external laboratory, samples were evaluated and
interpreted by a veterinary professional with expertise in
FISH. The control group consisted of dogs with colitis, negative PAS stain, and a negative FISH report for a universal
eubacterial probe (EUB 338; ACTCCTACGGGAGCAGC).16
The GC group consisted of dogs with granulomatous colitis
with macrophages positive on PAS stain and positive FISH for
both the EUB 338 and E. coli/Shigella probe (E. coli 16S
rRNA, GCAAAGGTATTAACTTTACTCCC).16
Stains (H&E, PAS) and IHC and FISH for E. coli were
performed on colonic sections of the blocks that had been
submitted for FISH and evaluated by a board-certified anatomic veterinary pathologist blinded to the FISH results.
Slides stained with H&E were digitalized for evaluation and
scoring of lesions according to the grading system (0 = normal, 1 = mild lesions, 2 = moderate lesions, 3 = severe lesions)
developed by the World Small Animal Veterinary Association Gastrointestinal Standardization Group.1,5 Scores were
assigned for each parameter (i.e., surface epithelial injury,
crypt dilation, fibrosis, goblet cell count, intraepithelial lymphocytes, and lamina propria infiltrates of lymphocytes
and/or plasma cells, eosinophils, neutrophils, and/or
macrophages).1,5
For IHC, 4-µm thick sections were mounted on positively
charged slides and dried overnight at 40°C. Before the assay,
mounted sections were routinely deparaffinized and rehydrated. Heat-induced antigen retrieval was performed in a
pressure cooker (Retriever for antigen unmasking; Electron
Microscopy Sciences) by heating the slides to 121°C for
20 min in 1× citrate buffer, pH 4.5 (R-Buffer C 10×; Electron
Microscopy Sciences) followed by endogenous peroxidase
blocking with 3% hydrogen peroxide for 10 min and UV protein block buffer (UltraVision protein block; Richard-Allan
Scientific) for 15 min. Tissue sections were then incubated
for 1 h at room temperature with a polyclonal anti–E. coli
antibody against O and K antigenic serotypes (Anti–E. coli,
1001; ViroStat) produced in rabbits and diluted at 1:200 with
antibody diluent (Antibody diluent reagent solution; Life
Technologies).
Intestinal samples from a piglet with colibacillosis confirmed by bacterial culture and histopathology were used
as positive controls. For negative controls, the primary
991
antibody was replaced with rabbit antiserum (Rabbit IgG
isotype control; Invitrogen) diluted to the same concentration as the E. coli antibody in consecutive sections from each
tissue. After incubation, slides were washed 3 times for 5 min
each in 1× Tris-buffered saline with Tween 20 (TBST), and
signal detection was performed using a horseradish peroxidase–linked polymer (UltraVision LP detection system;
Epredia), followed by incubation for 2 min with a red peroxidase substrate chromogen (ImmPACT NovaRED; Vector). Slides were counterstained with Mayer hematoxylin,
dehydrated, and mounted with a xylene-based non-aqueous
mounting medium.
Slides were scanned at 40× with a slide scanner (NanoZoomer 2.0 HT; Hamamatsu Photonics) and viewed with
NDP.view software (Hamamatsu Photonics) by a board-certified anatomic veterinary pathologist blinded to previous
FISH results. Samples with clusters of immunolabeling in
the cytoplasm of macrophages in the lamina propria were
considered positive for enteroinvasive E. coli infection.
Samples with immunolabeled bacteria on the mucosal surface, in the lumen of the crypts, or individualized extracellular bacteria in the lamina propria interpreted as sectioning
carryover were considered negative. Areas with tissue folding or crushing artifacts were not considered for evaluation.
Cross-reactivity of the antibody was assessed for Helicobacter spp., a genus of commensal bacteria frequently found
in the canine colonic crypts,15 as well as the enteropathogenic bacteria Clostridium perfringens, Campylobacter sp.,
and Salmonella enterica. Samples included stomach from a
dog with Helicobacter spp. infection confirmed by specific
FISH, colon from a primate with campylobacteriosis, jejunum from a dog with clostridiosis, colon from a horse with
salmonellosis, and colon and mesenteric lymph node from a
pig with salmonellosis. The case of C. perfringens infection
was confirmed by histopathology, positive culture, and
detection of C. perfringens enterotoxin in intestinal contents.
The diagnosis of campylobacteriosis and salmonellosis
were based on histopathology findings and positive bacterial
culture.
FISH for E. coli was repeated in-house following the
same protocol used by the external laboratory,16 utilizing
appropriate negative and positive controls. Samples with
clusters of fluorescent labeling in the lamina propria were
considered positive; samples with labeling restricted to
the mucosal surface or lumen of crypts were considered
negative.
The control group was comprised of 15 dogs with colitis
(4 intact males, 4 castrated males, 1 intact female, 6 spayed
females; median age: 5 y, age range: 1–12 y). The most common breeds were Schnauzers (n = 2) and Boston Terriers
(n = 2). The inflammatory infiltrate in the colon was neutrophilic (n = 2), lymphoplasmacytic (n = 2), or mixed (n = 11),
which variably included macrophages, eosinophils, neutrophils, lymphocytes, and plasma cells. The median cumulative histologic score was 6 (range: 1–12).
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Ishii et al.
Figure 1. Canine Escherichia coli–associated granulomatous colitis (GC; A, B) and canine idiopathic colitis (C, D). A. An abundant
infiltrate of enlarged macrophages in the lamina propria in GC. H&E. B. The cytoplasm of macrophages in GC is diffusely positive with a
periodic–acid Schiff stain. C. Canine idiopathic colitis with a few immunolabeled bacteria on the mucosal surface. Immunohistochemistry
(IHC) for E. coli. D. Two labeled bacteria (arrows) in a colonic crypt in canine idiopathic colitis, with no immunolabeling in the lamina
propria. IHC for E. coli.
The GC group included 11 dogs (4 intact males, 4 castrated males, 2 intact females, 1 spayed female; median age:
1 y, age range: 0.5–5 y). Breeds included French Bulldog
(n = 6), Boxer (n = 4), and Rottweiler (n = 1). Histologic findings in all cases consisted of histiocytic inflammation in the
lamina propria and/or submucosa (Fig. 1A) with a variable
infiltrate of neutrophils, plasma cells, and lymphocytes, and
erosion of the superficial epithelium. Infiltrating macrophages had abundant eosinophilic cytoplasm that was PAS
positive in all cases (Fig. 1B). The median cumulative histologic score was 8 (range: 4–12).
Immunolabeling for E. coli was not seen in the lamina
propria or intracellularly in any of the 15 control cases,
which were negative for invasive bacteria on FISH using the
EUB probe at the external laboratory and in-house FISH for
E. coli. Occasional bacteria immunolabeled as E. coli were
seen on the mucosal surface with the intestinal contents (Fig.
1C) and in the lumen of the crypts (Fig. 1D), similar to the
FISH technique.
All 11 GC cases in which FISH had detected invasive E.
coli were positive on IHC for E. coli (Fig. 2A). Strong red
clusters of immunolabeling were seen in the cytoplasm of
macrophages (Fig. 2B) located in the lamina propria and/or
submucosa along with extracellular immunolabeling in the
lamina propria (Fig. 2C). As expected, the positive control
for E. coli demonstrated strong immunolabeling. Our inhouse FISH for E. coli detected invasive bacteria consistently in all 11 cases of GC (Fig. 2D).
The primary antibody for E. coli did not cross-react with
Helicobacter spp. or C. perfringens. However, cross-reactivity occurred with Campylobacter sp. and S. enterica, with
labeled bacteria located extracellularly in the lamina propria
Immunohistochemistry for detection of invasive E. coli
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Figure 2. Canine Escherichia coli–associated granulomatous colitis. A. Immunohistochemistry (IHC) for E. coli with strong red
immunolabeling in the colonic lamina propria. B. Macrophages in the colonic lamina propria contain immunolabeled E. coli antigen within
the cytoplasm. IHC for E. coli. C. Intracellular and extracellular distribution of immunolabeling in the lamina propria. IHC for E. coli. D.
Fluorescence in situ hybridization for E. coli in the same case with labeling in the lamina propria in red, tissue autofluorescence in green,
and nucleic DNA labeled by DAPI in blue.
and on the mucosal surface of colonic lesions. In the case of
mesenteric lymphadenitis in salmonellosis cases, labeled
bacteria were seen extracellularly and intrahistiocytic within
the sinuses.
Extraintestinal E. coli strains able to adhere to and invade
colonic enterocytes, identified as AIEC, cause GC in predisposed dogs.16 In Boxer dogs and French Bulldogs with GC,
genetic susceptibility is linked to a region encoding the
CD48/SLAM family of genes on chromosome 38, which has
been associated with human inflammatory bowel disease.6
The diagnosis of GC typically relies on characteristic histopathologic findings of PAS-positive macrophages in colonic
biopsies, and FISH, to date, is considered the gold-standard
method to demonstrate invasive E. coli within the mucosa.16
We were able to detect E. coli antigen with IHC in all cases
that were positive on FISH. In a reported case in which FISH
was not available, the same polyclonal antibody was used to
label invasive E. coli in a dog with GC caused by a strain
within the EcoR group B2.2
FISH is considered a specific and sensitive technique to
detect invasive E. coli, and probes are known to cross-react with
Shigella spp.16 Disadvantages of FISH include the requirements
of a costly fluorescence microscope and an on-site pathologist
to read and interpret the slides immediately after the assay if
fluorescence image slide scanners are not available. The fluorophore signals fade over time and usually exhibit low contrast for
differentiation from tissue autofluorescence. An alternative to
FISH is chromogenic in situ hybridization (CISH) in which the
same probes used for FISH are applied, with the advantage of a
stable signal that can be read with a regular light microscope.20
However, sensitive CISH usually requires proprietary technologies with expensive reagents and time-consuming protocols.
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Ishii et al.
Our IHC protocol allowed clear localization of strongly
labeled bacterial antigen within the cytoplasm of macrophages in sections counterstained with hematoxylin. Other
advantages of IHC include the ability to scan and store the
slides for longer periods of time with preservation of the
immunolabeling, reasonable reagent costs, assay automation, and accessible whole-slide scanning for remote interpretation by a pathologist and second-opinion consultation.
Invasive bacteria were not identified in the control group of
idiopathic colitis that had similar lesion severity assessed by
the histologic scores.
A significant limitation of the polyclonal antibody that
we used was cross-reaction with Campylobacter spp. and
S. enterica. However, campylobacteriosis and salmonellosis are uncommon causes of enterocolitis in dogs and are
not associated with histiocytic or granulomatous inflammation in the colon.3,18 Therefore, our IHC assay needs to
be interpreted in the context of characteristic granulomatous inflammation with PAS-positive macrophages, and it
is not recommended for other manifestations of enteric
colibacillosis in dogs. It is important to note that most
strains of E. coli are commensal bacteria that colonize the
intestine of healthy humans and animals,16 and mere isolation of E. coli from the feces or identification of the bacteria in the intestinal lumen are insufficient to make a
diagnosis.
Acknowledgments
We thank Dr. Randi Gold for her technical assistance with FISH
and Kelly Mallet for her support with histopathology. We also
thank Dr. Carolyn Hodo for providing a sample from a case of campylobacteriosis.
Declaration of conflicting interests
Patricia Eri Ishii, Paula R. Giaretta, Jonathan A. Lidbury, Jan S.
Suchodolski, and Joerg M. Steiner are employed by the Gastrointestinal Laboratory at Texas A&M University, which offers laboratory tests, including gastrointestinal histopathology services, on a
fee-for-service basis.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article
ORCID iD
Patricia Eri Ishii
https://orcid.org/0000-0002-9569-6404
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