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DOI: 10.1177/0300985812461362
2013 50: 626 originally published online 24 September 2012 Vet Pathol
V. L. Schumacher, A. Martel, F. Pasmans, F. Van Immerseel and H. Posthaus
Experimentally Induced Clostridium Perfringens Type C Enteritis in Pigs
Endothelial Binding of Beta Toxin to Small Intestinal Mucosal Endothelial Cells in Early Stages of

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Infectious Disease Research
Endothelial Binding of Beta Toxin to
Small Intestinal Mucosal Endothelial
Cells in Early Stages of Experimentally
Induced Clostridium Perfringens Type C
Enteritis in Pigs
V. L. Schumacher
1
, A. Martel
2
, F. Pasmans
2
,
F. Van Immerseel
2
, and H. Posthaus
1
Abstract
Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C
enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we
evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis.
Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of
lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread
hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a
tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early
stages of C. perfringens type C enteritis in pigs.
Keywords
beta toxin, Clostridium perfringens type C, endothelial cell, experimental infection, immunohistochemistry, intestinal loop model,
necrotizing enteritis, porcine
Clostridium perfringens is an anaerobic, Gram-positive, spore-
forming bacterial pathogen of animals and humans.
6
Its patho-
genesis is dependent on the production of potent exotoxins.
C. perfringens type C produces 2 out of a total of 4 so-called
typing toxins, alpha- (CPA) and beta toxin (CPB), but addi-
tional toxins, such as beta-2 toxin, enterotoxin, and TpeL, can
be secreted.
4
It is the etiologic agent of necrotic enteritis (NE),
a disease causing high morbidity and mortality in suckling pig-
lets
7
but also affecting other mammalian hosts, such as calves,
goats, horses, and humans.
1,6,8
CPB has been identified as an
essential virulence factor of type C strains using 2 small labora-
tory animal models.
5,9
The exact mechanism of toxicity and the
natural target cells of CPB are, however, not yet unequivocally
defined. It was hypothesized that CPB directly causes epithelial
necrosis; however, scientific proof for a direct toxic effect of
the toxin on epithelial cells is lacking. We recently provided
evidence that small intestinal microvascular endothelial cells
could be directly targeted by CPB; however, results were based
on in vitro studies and analysis of end-stage lesions of naturally
affected piglets.
2,3
Experimental infections in rabbits and mice
indicated that species differences in the reaction to this enteric
infection exist.
5,9
The goal of this limited study was (1) to
modify an experimental porcine intestinal loop infection model
to reproduce early lesions of NE and (2) to determine CPB
binding to potential target cells in the small intestine during
early stages of NE in a naturally affected animal species.
The porcine C. perfringens type C strain JF 3721
2
, the type
C reference strain NCTC 3180, and as a control, the porcine
C. perfringens type A isolate JF 3693
2
were used. Bacteria
were grown anaerobically overnight at 37

C in TGY (3% tryp-

tic soy broth, 2% glucose, 1% yeast extract, 0.1% L-cysteine).
Overnight cultures (100 ml) were added to 10 ml of TGY and
grown anaerobically until early log phase (appr. 1.5 10
7
bac-
teria per ml determined by CFU counts from serial dilutions).
1
Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern,
Switzerland
2
Department of Pathology, Bacteriology and Avian Diseases, Faculty of
Veterinary Medicine, Ghent University, Salisburylaan, Merelbeke, Belgium
Corresponding Author:
Horst Posthaus, Institute of Animal Pathology, Vetsuisse Faculty, University of
Berne, Langassstrasse 122, 3012 Bern, Switzerland.
Email: horst.posthaus@vetsuisse.unibe.ch
Veterinary Pathology
50(4) 626-629
The Author(s) 2012
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DOI: 10.1177/0300985812461362
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Animal experiments were approved by the ethical committee of
the Faculty of Veterinary Medicine, Ghent University. Three
12-hour-old and one 6-hour-old Rattlerow Seghers Pietrain
crossbred piglets were obtained from AgriVet (Melle,
Belgium). Sows were not vaccinated against C. perfringens
type C, and no cases of NE were previously diagnosed in the
herd. Intestinal loops were constructed using the procedures
of Vandenbroucke et al.
10
Individual loops of approximately
2 cm length were inoculated with 500 ml of log phase cultures
or 500 ml sterile TGY supplemented with 200 mg/ml trypsin
inhibitor (Sigma-Aldrich). Each inoculation was performed in
duplicate loops with 1 empty loop in between. Inocula and
infection times are summarized in Table 1. In 1 animal (No.
4), a time course experiment for 610 hours was performed
by reopening the abdominal suture after 2 and 4 hours and
inoculation of additional loops. Piglets were maintained under
anesthesia no longer than 12 hours, after which they were
euthanized using T61 intravenously (0.3 ml/kg bodyweight;
Intervet, Ukkel, Belgium). After euthanasia, the small intestine
was immediately excised; individual loops were opened, fixed
in 10%buffered formalin for 12 hours, and processed routinely
for histological sectioning. Histological examinations were
performed according to a standardized grading scheme. In
short, capillary dilatation was scored on a severity scale of
15 as an average of five 400 fields, using the following
guidelines: 0 none; 1 capillaries dilated in mucosa of
necrotic villi only; 2 capillaries dilated in mucosa underlying
necrotic and nonaffected villous epithelium; 3 capillaries
dilated in mucosa and submucosa; 4 capillaries dilated in
mucosa, submucosa, and muscularis; 5 capillaries dilated
in all intestinal layers. The total necrotic area of each sample
was scored as an average of all sections on each slide as 0
no necrotic enterocytes observed; 1 few necrotic cells focally
on villous tip; 2 multifocal villous tip necrosis, less than 10%
of villi affected; 3 multifocal villous tip necrosis, 10%50%
of villi affected; 4 greater than 50%of villous tips are necro-
tic; 5 greater than 50%of villous tips are necrotic, with deep
mucosal necrosis. Immunohistochemical stainings for CPB and
as control CPA (Monoclonal Antibody 10A2 [mAb-CPB],
monoclonal antibody 6H7 1F3 [CPA], Center for Veterinary
Biologics, Ames, Iowa) were performed as described by
Miclard et al.
3
Pathological Lesions
In animal Nos. 13, macroscopic lesions, including hyperemic
and roughened mucosa, only occurred in jejunal loops
inoculated with the 2 C. perfringens type C strains (Table 1).
Macroscopic lesions in the time course experiment, using a
6-hour-old piglet, were more pronounced, and infection with
both C. perfringens type C strains induced progressive hemor-
rhagic lesions (Table 1). Histologically, mild lesions, evident
as mild villous shortening and multifocal villous tip epithelial
cell sloughing and necrosis associated with dilatation of under-
lying capillaries, occurred at 6 and 8 hours postinoculation (pi)
with the 2 C. perfringens type C isolates in animal Nos. 1 and 2
(Table 1, Fig. 1). Twelve hours pi, type C straininfected loops
showed moderate villous shortening, multifocal necrosis of
villous tip epithelial cells, and capillary dilatation in the
affected villous (Fig. 2). Thrombosis was not present in sam-
ples from animal Nos. 13. In animal No. 4, at 6 hours pi, mul-
tifocal villous tip epithelial necrosis and marked capillary
dilatation throughout the mucosa, submucosa, and muscularis
were evident. This progressed to widespread capillary dilata-
tion and hemorrhage in the lamina propria at 8 hours (Fig. 3)
and marked villous shortening, with loss of epithelial cells and
Table 1. Animals, Inocula, Infection Times, and Main Macroscopic and Histologic Lesions.
a
Animal (Age) Inoculum Infection Time, hr Macroscopic Lesions Hemorrhage Capillary Dilatation Necrotic Area
Nr. 1 (12 hr) NCTC 3180 (type C) 6 Mild hyperemia No 1 1
JF 3721 (type C) 6 Mild hyperemia No 1 1
JF 3693 (type A) 6 None No 0 0
Sterile TGY 6 None No 0 0
Nr. 2 (12 hr) NCTC 3180 (type C) 8 Mild hyperemia Yes 1 2
JF 3721 (type C) 8 Mild hyperemia Yes 1 2
JF 3693 (type A) 8 None No 1 1
Sterile TGY 8 None No 0 0
Nr. 3 (12 hr) NCTC 3180 (type C) 12 Severe hyperemia Yes 1 4
JF 3721 (type C) 12 Severe hyperemia Yes 1 4
JF 3693 (type A) 12 None No 0 0
Sterile TGY 12 None No 0 0
Nr. 4 (6 hr) NCTC 3180 (type C) 6 Focal hemorrhage Yes 4 1
JF 3721 (type C) 6 Focal hemorrhage Yes 4 2
NCTC 3180 (type C) 8 Focal extensive hemorrhage Yes 5 1
JF 3721 (type C) 8 Focal extensive hemorrhage Yes 5 1
NCTC 3180 (type C) 10 Diffuse hemorrhage Yes 5 3
JF 3721 (type C) 10 Diffuse hemorrhage Yes 5 2
Sterile TGY 10 None No 0 0
a
Numerical values represent average scores obtained from 2 duplicate loops.
Schumacher et al 627
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massive hemorrhage in the lamina propria and submucosa at 10
hours pi. Few foci of necrosis and thrombosis of small vessels
in the lamina propria were visible at the 8- and 10-hour time
points. Immunohistochemically, CPB was detected at the cyto-
plasmic membrane of endothelial cells in the lamina propria of
all loops inoculated with C. perfringens type C strains. In the
multifocal lesions present in animal Nos. 13, CPB signal was
present in areas with villous tip necrosis only (Figs. 4, 5). In
animal No. 4, CPB signal on endothelial cells increased pro-
gressively with the severity of histological lesions. At 6 hours
pi, CPB was detected at endothelial cells of affected villi only.
At 8 hours pi, CPB staining also extended to submucosal
vessels (Fig. 6). At 10 hours pi, there was diffuse staining of
vessels throughout the entire sections extending into the sub-
mucosa, including areas with an intact appearing epithelium.
CPB signal was not detected at epithelial cells in any loop
infected with type C strains. Additionally, no CPB signal was
detectable in any of the control loops.
Discussion
In summary, our results demonstrate that the porcine neonatal
jejunal loop model is suitable to induce early stage lesions of
C. perfringens type C enteritis in a naturally affected host
species and thus has potential for further research applications.
The essential role of CPB in C. perfringens type C enteritis has
recently been demonstrated by a combination of reverse genet-
ics and small laboratory animal infection models.
5,9
These
models are well suited to determine essential virulence factors
and mechanisms of C. perfringens type C enteritis. However,
susceptibility of target cells to toxins might differ among host
species, and infectious models based on naturally affected spe-
cies represent valuable additions to existing models. Albeit
based on a limited number of animals, our results indicate that
the development of early lesions of C. perfringens type C enter-
itis was associated with binding of CPB to microvascular
endothelial cells in the small intestinal mucosa. In early-stage
lesions, CPB signal at endothelial cells was only detectable
in affected jejunal villi. In advanced lesions, CPB signal was
localized to endothelial cells in all layers of the intestinal loops.
These findings suggest increased resorption of CPB from the
intestinal lumen and hematogenous distribution of CPB within
the intestinal microvasculature. Our results correspond to pre-
vious studies, where we demonstrated endothelial localization
of CPB in spontaneous lesions of NE in piglets.
3
In contrast
to end-stage lesions in naturally affected animals, we did not
observe widespread vascular necrosis in this experimental
model, suggesting that vascular necrosis and thrombosis most
Figure 1. Jejunum, animal No. 1. Jejunal loop infected with C. perfringens type C strain NCTC 3180 for 6 hours, showing villous tip necrosis with
capillary dilatation in affected areas. HE. Figure 2. Jejunum, animal No. 3. Jejunal loop infected with C. perfringens type C strain NCTC 3180 for
12 hours, showing multifocal villous tip necrosis with capillary dilatation in affected areas. HE. Figure 3. Jejunum, animal No. 4. Jejunal loop
infected with C. perfringens type C strain JF 3721 for 8 hours, showing multifocal hemorrhage in lamina propria and sloughing of epithelial lining
of villi. HE. Figure 4. Jejunum, serial section of Figure 1. Focal CPB signal localized to endothelial cell membranes (arrows) in the lamina propria
in areas of villous tip necrosis. Inset: Detail of endothelial labeling. Immunohistochemical staining with mAb-CPB, counterstain hemalaun (IHC-
CPB). Figure 5. Jejunum, serial section of Figure 3. Multifocal CPB signal present on endothelial cell membranes in the lamina propria in areas of
villous tip necrosis. Inset: Detail of endothelial labeling. IHC-CPB. Figure 6. Jejunum, serial section of Figure 5. CPB signal present on endothelial
cells (arrows) throughout lamina propria, also in areas without villous tip necrosis. Inset: Detail of endothelial labeling. IHC-CPB.
628 Veterinary Pathology 50(4)
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likely occur at later time points. Due to animal welfare reasons,
animals were euthanized by 12 hours pi; thus, end point lesions
did not develop in our experiments. Further experiments using
longer infection times would be necessary to investigate the
exact time course of pathological lesions in more detail.
In contrast to an endothelial tropism of CPB, we were
unable to demonstrate CPB binding to jejunal epithelial cells.
However, epithelial damage was clearly demonstrated in our
experiments. Because CPB was able to reach endothelial cells
in the lamina propria, an initial epithelial defect leading to tis-
sue penetration of CPB is conceivable. Whether such initial
epithelial damage is due to a direct effect of C. perfringens type
C on the epithelial cells or a consequence of the endothelial
damage cannot be resolved by our limited experiments. Addi-
tionally, as CPB could be toxic to epithelial cells at concentra-
tions below the detection level of our immunohistochemistry
protocol, our results do not exclude a direct toxic effect on the
epithelium. Further in vitro studies to determine the potential
toxic effect of CPB on porcine small intestinal epithelial cells
are currently being performed in our laboratory.
Acknowledgements
We thank Leen Timbermont, Christian Puttevils, Delphine Ameye,
and Pashk Selitaj for their support.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
This study was supported by a Ghent University Visiting Foreign
Researcher Grant and a Short International Visit Grant from the Swiss
National Science Foundation(IZK0Z3_133953). Monoclonal antibodies
were kindly provided by G. Shrinivas, Center for Veterinary Biologics,
USDA, Ames, Iowa.
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