This document summarizes a study on the effect of Clostridium difficile experimental infection on the health of weaned rabbits. Thirty rabbits were divided into three groups, with two groups infected with C. difficile either subcutaneously or orally. The orally infected group showed signs of diarrhea and bloat, while no signs were seen in the subcutaneous group. No mortalities occurred. At the end of the study, the orally infected rabbits showed liver and kidney enlargement and congestion as well as mild enteritis. The C. difficile was re-isolated from infected rabbits. The study found that C. difficile can negatively impact the health of weaned rabbits.

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Journal of Applied Sciences Research, 8(8): 4672-4677, 2012
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Corresponding Author: Wafaa A. Abd El-Ghany, Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo
University, Giza -12211, Egypt
E-mail: wafaa.ghany@yahoo.com
The Effect of Clostridium difficile Experimental Infection on the Health Status of
Weaned Rabbits
Khelfa D.G., Wafaa A. Abd El-Ghany and Heba M. Salem
Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, Giza -12211, Egypt
ABSTRACT
In this study, a trial to detect the effect of Clostridium difficile (C. difficile) experimental infection on the
health status of weaned rabbits was carried out. Thirty, 5 week old weaned New Zeeland rabbits were used.
Rabbits were kept for a week under observation for adaptation and ensuring absence of any clinical signs,
mortalities and anaerobic infections. Rabbits were randomly divided into 3 equal groups; 10 rabbits for each.
Each rabbit in group (1) was subcutaneously (S/C) inoculated with 1 ml containing 1x108
colony forming unit
(CFU) of C. difficile, while each one in group (2) was orally inoculated with 2 ml (3x1010
CFU). Group (3) was
kept as non-infected control one. During 3 weeks observation period; clinical signs, mortalities, gross lesions,
histopathological changes and C. difficile re-isolation were detected. The results indicated absence of clinical
signs in C. difficile S/C challenged rabbits, while signs of bloat and brownish diarrhea were observed in orally
challenged ones. No mortalities were recorded in rabbits of both challenged groups. Lesions observed in
sacrificed orally challenged rabbits were enlargement and congestion of liver and kidneys as well as mild degree
of enteritis. The challenging C. difficile was re-isolated from sacrificed rabbits at the end of the study. It could be
concluded that C. difficile is an organism of economic importance for newly weaned rabbits as it can badly effect
on rabbits’ health status.
Key words: Rabbits, C. difficile, Signs, Lesions, Isolation
Introduction
In the past 30 years, however, Clostridium difficile (C. difficile) has been implicated as the principal
infectious cause of diarrhea in humans, and similar clinical conditions in a variety of other mammals (Songer,
1996 and Kelly and LaMont, 1998). The relevance of C. difficile to disease in animals has also become more
obvious but differs according to species, age, environmental density of spores, administration of antibiotics and
possibly other factors (Keel and Songer, 2006). Lesions attributed to spontaneous or experimentally induced C.
difficile infection in rabbits were documented (Carman and Evans, 1984). Severe C. difficile toxin-induce rabbit
enteritis which characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea
(Cirle et al., 2012).
So, this work was planned to study the effect of C. difficile experimental infection on the health status of
weaned rabbits through detection of the developed clinical signs, mortalities, gross lesions, histopathologcal
changes in the affected tissues and C. difficile re-isolation.
Materials And Methods
Rabbits:
Thirty, 5 week old, newly weaned New Zeeland rabbits of mixed sex were obtained from a commercial
farm and then housed in thoroughly cleaned and disinfected batteries. The rabbits received ration without feed
additives. Feed and water were given to rabbits ad libitum. The rabbits were kept for one week observation
period for adaptation and for detection of any signs or mortality before experimental work. Rabbits were
vaccinated by rabbit's viral hemorrhagic diseases vaccine at 7 weeks of age (1cm/ rabbit) and also received
formalized polyvalent rabbit's pasteurellosis vaccine at 8 weeks old (1cm/ rabbit).

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Clostridium difficile strain:
Identified field strain of C. difficile that isolated from apparently healthy, diseased and dead weaned rabbits
were kindly obtained from Poultry Diseases Department, Faculty of Veterinary Medicine, Cairo University,
Egypt.
Preparation of C. difficile inoculum used in experimental infection:
Inoculum of C. difficile was prepared by plate count technique according to Mostafa, (1992) as follow:
For subcutaneous (S/C) route:
 Concentration of the inoculum was 1x108
colony forming unit (CFU).
 Dose of the inoculum was 1 ml/ each rabbit.
For oral route:
 Concentration of the inoculum was 3x1010
(CFU).
 Dose of the inoculum was 2 ml/ each rabbit.
Experimental design:
Thirty, 5 week old, newly weaned New Zeeland rabbits were used in experimental infection. Rectal swabs
were collected from purchased rabbits at arrival as well as feed and water samples were examined to ensure
their freedom of anaerobic infections. Thirty rabbits were randomly divided into 3 equal groups, 10 rabbits for
each.
The experimental design including the groups, routes of inoculation and concentration and dose of
inoculum are summarized in Table (1).
Table 1: The experimental design of different groups:
Group
Number
No. of
Rabbits/group
Clostridium difficile
challenge
Routes of
inoculation
Concentration of
inoculum
Dose of
inoculum/rabbit
1 10 + Subcutaneously 1x108
CFU 1 ml
2 10 + Orally 3x1010
CFU 2 ml
3 10 - - - -
Measured parameters:
Clinical signs and mortalities:
Rabbits were kept for three weeks observation period post challenge. Clinical signs and mortalities were
observed daily till the end of the study.
Post-mortem lesions:
Any dead rabbits during observation period were subjected to post-mortem examination for detection of
lesions.
Histopathological examination:
Tissue specimens including liver, kidneys and small and large intestines were collected from sacrificed
rabbits of all groups at the end of the work for histopathological examination (Banchroft et al., 1996).
Bacterial re-isolation:
Rectal swabs from living rabbits as well as liver and intestinal samples from sacrificed rabbits were
collected for C. difficile re-isolation (Smith and Holdman, 1968).
Results And Discussion
Clostridium difficile is a Gram-positive, anaerobic and spore-forming bacillus commonly associated with
diarrhea and colitis in humans and other mammals (Songer, 1996).
This study was designed to study the effect of C. difficile experimental infection on the health status of
weaned rabbits regarding clinical signs, mortalities, gross lesions, histopathological changes in the affected
tissues and C. difficile re-isolation.

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No clinical signs, mortalities or lesions were observed in non-infected control rabbits group along the whole
experimental period.
No clinical signs were seen in rabbits challenged with C. difficile after S/C inoculation, while signs of
severe brownish diarrhea (Fig. 1) and bloat (Fig. 2) were detected in some animals of orally infected rabbits.
Similarly, Prescott, (1977) and Keel and Songer, (2006) referred to the role of C. difficile in induction of
enteritis in rabbits.
Rabbits challenged with C. difficile either in oral or S/C route showed no mortalities along the observation
period.
At the end of the study, C. difficile S/C challenged sacrificed rabbits revealed no gross lesions. However,
orally challenged sacrificed ones showed enlargement and congestion of the liver (Fig. 3) and kidneys (Fig. 4),
mild degree of enteritis (Fig. 5) with un-digested feed particles mixed with slimy exudates in the small intestine
but the large intestine contained watery brownish contents (Fig. 6) with offensive odour. Comparable results
were reported by Rehg and Shoung, (1981) and Mitchell et al., (1986) who considered C. difficile is a cause of
cecitis in rabbits, also, Perkins et al., (1995) found that spontaneous C. difficile associated disease in rabbits is
principally associated with lesions in the small intestine, especially the ileum, causing mucosal necrosis.
Contrary results were obtained by Eglow et al., (1992) and Keel and Songer, (2006) who observed absence of
both clinical signs and accordingly the lesions caused by C. difficile in neonate rabbits. Essential virulence
factors of C. difficile are large exotoxins, toxin A (TcdA) does not affect ileal explants from 5-dayold rabbits,
even at dosages that cause severe lesions in ileal explants from adults. A prominent hypothesis to explain the
resistance of such neonates is that they lack the proper toxin receptors until later in life (Borriello and Wilcox,
1998). Binding of TcdA to ileal brush borders is decreased in neonatal rabbits, but maximal binding is observed
in 90-day-old rabbits (Eglow et al., 1992).
The histopathological examination of scarified rabbits at the end of experimental trial revealed that there
was no histopathological alteration observed in the tissue specimen collected from control group as there was
normal histological structure of liver, kidney, small intestine and large intestine, Group of rabbits orally
challenged with C. difficile showed congestion in the central vein associated with ballooning and degeneration
in the hepatocytes (Fig. 7.A), vacuolization in the lining endothelium of the glomerular tuft associated with
degeneration in the lining epithelium of the renal tubules (Fig. 7.B), fusion in the villi of small intestine with
inflammatory cells infiltration in the lamina propria (Fig. 7.C) and massive number of inflammatory cells
infiltration was detected in the lamina propria associated with oedema in the sub-mucosal layer of large intestine
(Fig. 7.D). Group of rabbits exposed to C. difficile S/C experimental infection revealed dilatation in the portal
vein associated with degenerative change in the hepatocytes (Fig. 8.A), vacuolization in the lining endothelium
of the glomerular tuft associated with degeneration in the lining epithelium of the renal tubules (Fig. 8.B),
diffuse goblet cells formation in the lining mucosal epithelial cells of small intestine associated with
inflammatory cells infiltration in the lamina propria (Fig. 8.C) and massive number of inflammatory cells
infiltration was detected in the lamina propria of large intestine (Fig. 8.D). Generally, in all C.difficile
experimentally inoculated groups, the microscopical alterations in liver, kidneys and intestines are nearly similar
to that recorded by Mitchell et al., (1986).
The results of C. difficile re-isolation from either rectal swabs or liver and intestinal samples showed no re-
isolation of any C. difficile organisms from control non infected rabbits, while C.difficile was re-isolated from
challenged groups, where it appeared on blood agar as glossy, grey and circular colonies with rough edges and
no haemolysis (Fig. 9) and a characteristic farm yard smell odour.
Conclusion:
From the obtained abovementioned results, it could be concluded that C. difficile is an organism of
economic importance for newly weaned rabbits as it can badly effect on rabbits’ health status. Further
researches may be needed to explain the pathogenesis of C. difficile infections and the mechanisms of rabbit’s
colonization as they are points of particular importance. Such information could improve animal welfare and
livestock revenues.

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Fig. 1: Fig. 2:
Fig. 1: A rabbit orally infected with C. difficile with signs of severe brownish diarrhea.
Fig. 2: A rabbit orally infected with C. difficile with signs of bloat.
Fig. 3: A liver of rabbit orally infected with C. difficile showed enlargement and congestion.
Fig. 4: A kidneys of rabbit orally infected with C. difficile showed enlargement and congestion.
Fig. 5: Small intestine of rabbit orally infected with C. difficile showed enteritis.
Fig. 6: Large intestine of rabbit orally infected with C. difficile showed watery brownish contents.

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Fig. 7: Histopathological findings of group of rabbits orally challenged with C. difficile sacrificed at the end of the
experimental period, (A) liver showing congestion in the central vein (cv) with ballooning degeneration in the
hepatocytes (d). H&E X64 (B) kidney showing vacuolization in the lining endothelium of the glomerular tuft (g)
with tubular degeneration (d) H&E X80 (C) small intestine showing fusion of the villi with inflammatory cells
infiltration (m) in the lamina propria H&E X40 (D) large intestine showing inflammatory cells infiltration in the
lamina propria (m) with oedema in the submucosa (o) H&E X40
Fig. 8: Histopathological findings of group of rabbits subcutaneously challenged with C. difficile at the end of the
experimental period, (A) liver showing dilatation in the portal vein (pv) and degeneration in the hepatocytes (d)
H&E X64 (B) kidney showing vacuolization in the lining endothelium of the glomerular tuft (g) with tubular
degeneration (d) H&E X64 (C) small intestine showing goblet cells formation in the lining mucosal epithelium (g)
with inflammatory cells infiltration (m) H&E X40 (D) large intestine showing massive number of inflammatory
cells infiltration (m) in the lamina propria H&E X40.
A
C
B
D
A
C
B
D

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Fig. 9: Colonies of C. difficile organism on blood agar shows glossy, grey and circular colonies with rough edges and no
haemolysis.
References
Banchroft, J.D., A. Stevens, D.R. Turner, 1996. Theory and practice of histological techniques. Fourth Ed.
Churchil Livingstone, New York, London, San Francisco, Tokyo.Borriello S.P., Wilcox M.H. 1998.
Clostridium difficile infections of the gut: theun answered questions. J. Antimicrob. Chemother., 41(Suppl
C): 67-69.
Carman, R.J., R.H. Evans, 1984. Experimental and spontaneous Clostridial enteropathies of laboratory and free
living lagomorphs. Lab. Anim. Sci., 34: 443-452.
Cirle, A.W., M.C. Gina, Li. Yuesheng, W.P. Sean, A.F. Robert, R. Jayson, B.E. Peter, L. Joel, L.G. Richard,
2012. Effects of adenosine A2A receptor activation and alanyl-glutamine in Clostridium difficile toxin
induced ileitis in rabbits and cecitis in mice. Warren et al. BMC Infectious Diseases, 12(13): 1-12.
Eglow, R., C. Pothoulakis, S. Itzkowitz, E.J. Israel, C.J. O’Keane, D. Gong, N. Gao, Y.L. Xu, W.A. Walker,
J.T. LaMont, 1992. Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is
associated with decreased toxin A receptor. J. Clin. Invest., 90: 822-829.
Keel, M.K., J.G. Songer, 2006. The Comparative Pathology of Clostridium difficile–associated disease. Vet.
Pathol., 43: 225-240.
Kelly, C.P., J.T. LaMont, 1998. Clostridium difficile infection. Annu. Rev. Med., 49: 375-390.
Mitchell, T.J., J.M. Ketley, S.C. Aslam, J. Stephen, D.W. Burdon, D.C.A. Candy, R. Daniel, 1986. Effect of
toxin A and B of Clostridium difficile on rabbit ileum and colon. Gut, 27: 78-85.
Mostafa, E.M., 1992. Studies on the incidence of Clostridial organisms in domestic rabbits. M.V.Sc. Thesis
(Microbiology), Fac. Vet. Med., Zagazig Univ.
Perkins, S.E., J.G. Fox, N.S. Taylor, D.L. Green, N.S. Lipman, 1995. Detection of Clostridium difficile toxins
from the small intestine and cecum of rabbits with naturally acquired enterotoxemia. Lab. Anim. Sci., 45:
379-384.
Prescott, J.F., 1977. Tyzzer's disease in rabbit in Britain.Vet. Rec., 100(14): 285-286.
Rehg, J.E., L.Y. Shoung, 1981. Clostridium difficile colitis in a rabbit following antibiotic therapy for
pasteurellosis. J. Am. Vet. Med. Assoc., 179: 1296.
Smith, L.D., L.V. Holdman, 1968.The pathogenic anaerobic bacteria. Charleshomaspublisher, USA. Ist
ed. pp:
201-255.
Songer, J.G., 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol.Rev., 9: 216-234.