Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20

Clostridium perfringens in poultry: an emerging

threat for animal and public health
Filip Van Immerseel , Jeroen De Buck , Frank Pasmans , Gerard
Huyghebaert , Freddy Haesebrouck & Richard Ducatelle
To cite this article: Filip Van Immerseel , Jeroen De Buck , Frank Pasmans , Gerard
Huyghebaert , Freddy Haesebrouck & Richard Ducatelle (2004) Clostridium perfringens in
poultry: an emerging threat for animal and public health, Avian Pathology, 33:6, 537-549, DOI:
10.1080/03079450400013162
To link to this article: http://dx.doi.org/10.1080/03079450400013162

Published online: 19 Oct 2010.

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Avian Pathology (December 2004) 33(6), 537 /549

REVIEW ARTICLE

Clostridium perfringens in poultry: an emerging

threat for animal and public health
Filip Van Immerseel1*, Jeroen De Buck1, Frank Pasmans1, Gerard
Huyghebaert2, Freddy Haesebrouck1 and Richard Ducatelle1
1

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Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent
University, Salisburylaan 133, B-9820 Merelbeke, Belgium, and 2Ministry of the Flemish Community,
Agricultural Research Centre, Department of Animal Nutrition and Husbandry, Scheldeweg 68, B-9090
Melle, Belgium

The incidence of Clostridium perfringens-associated necrotic enteritis in poultry has increased in countries
that stopped using antibiotic growth promoters. Necrotic enteritis and the subclinical form of C. perfringens
infection in poultry are caused by C. perfringens type A, producing the alpha toxin, and to a lesser extent
type C, producing both alpha toxin and beta toxin. Some strains of C. perfringens type A produce an
enterotoxin at the moment of sporulation and are responsible for foodborne disease in humans. The mechanisms
of colonization of the avian small intestinal tract and the factors involved in toxin production are largely
unknown. It is generally accepted, however, that predisposing factors are required for these bacteria to colonize
and cause disease in poultry. The best known predisposing factor is mucosal damage, caused by coccidiosis.
Diets with high levels of indigestible, water-soluble non-starch polysaccharides, known to increase the viscosity
of the intestinal contents, also predispose to necrotic enteritis. Standardized models are being developed for the
reproduction of colonization of poultry by C. perfringens and the C. perfringens-associated necrotic enteritis.
One such model is a combined infection with Eimeria species and C. perfringens. Few tools and strategies
are available for prevention and control of C. perfringens in poultry. Vaccination against the pathogen and
the use of probiotic and prebiotic products has been suggested, but are not available for practical use in the field
at the present time. The most cost-effective control will probably be achieved by balancing the composition
of the feed.

Introduction
In Europe, antimicrobial growth promoters, used
to increase weight gain in broiler chickens, will be
banned from poultry feed due to the risk of
spreading of antibiotic resistance (Bedford, 2000).
The ban of growth-promoting antibiotics in broiler
feed is a factor that will inevitably change the
bacterial microflora in the intestinal tract of broiler
chickens (Knarreborg et al., 2002). In Scandinavian
countries, national policies lead to the ban of
antimicrobial growth promoters years ago. This
was almost immediately followed by health problems in broiler flocks, with most remarkable an
epidemic of Clostridium perfringens infections

(Kaldhusdal & Lvland, 2000). The C. perfringens

infections in poultry may present as acute clinical
disease or subclinical disease.
The acute form of the disease leads to increased
mortality in the broiler flocks. This can account
for 1% losses per day, for several consecutive
days during the last weeks of the rearing period
(Kaldhusdal & Lvland, 2000). In the subclinical
form, damage to the intestinal mucosa caused by
C. perfringens leads to decreased digestion and
absorption, reduced weight gain and increased feed conversion ratio (Elwinger et al., 1992;
Hofshagen & Kaldhusdal, 1992; Kaldhusdal et al.,
2001; Hofacre et al., 2003). Moreover, it is shown

*To whom correspondence should be addressed. Tel:
/32 09 264 74 48. Fax:
/32 09 264 74 94. E-mail: filip.vanimmerseel@UGent.be
ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/04/06537-13 # 2004 Houghton Trust Ltd
DOI: 10.1080/03079450400013162

538 F. Van Immerseel et al .

that cholangiohepatitis occurs in the subclinical

form of C. perfringens infection and that there is
an increased number of condemnations at
processing due to these liver lesions (Lvland &
Kaldhusdal, 1999). C. perfringens in poultry
constitutes a risk for transmission to humans
through the food chain. C. perfringens is one of
the most frequently isolated bacterial pathogens
in foodborne disease outbreaks in humans, after
some other pathogens such as Campylobacter and
Salmonella (Buzby & Roberts, 1997). Disease outbreaks due to C. perfringens can be traced back
to different sources, one of which is poultry
(Schiemann, 1977; Regan et al., 1995; Hook et al.,
1996).

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C. perfringens Virulence Factors

C. perfringens is a Gram-positive anaerobic sporeforming bacterium, able to produce various
toxins and enzymes responsible for the associated
lesions and symptoms. C. perfringens strains
are classified into five toxinotypes (A, B, C, D
and E), based on the production of four major
toxins (a, b, o and i) (Songer, 1996; Petit et al.,
1999). Necrotic enteritis and the subclinical form
of C. perfringens infection in poultry are caused
by C. perfringens type A, and to a lesser extent
type C (Songer & Meer, 1996; Engstrom et al.,
2003). C. perfringens type A strains produce the
chromosomal-encoded alpha-toxin, while C. perfringens type C strains produce alpha toxin
together with beta toxin (Petit et al., 1999). Some
strains of C. perfringens type A produce an
enterotoxin at the moment of sporulation, causing
disease in humans (Ridell et al., 1998; Sarker et al.,
1999). The mechanism of action of the enterotoxin
will be discussed further in the text. Toxinotypes B,
D and E do not play a role in poultry disease
and will not be discussed here. The mechanism
of action of C. perfringens toxins is reviewed by
Petit et al. (1999).
Alpha toxin is a phospholipase C sphingomyelinase that hydrolyzes phospholipids and promotes
membrane disorganization (Naylor et al., 1998;
Titball et al., 2000). Hydrolysis of lecithin results in
the formation of diacylglycerol, resulting in activation of protein kinase C, and subsequent stimulation of the arachidonic acid cascade. This induces
the synthesis of inflammatory mediators, such as
leukotrienes, thromboxane, platelet-agglutinating
factor and prostacyclin (Titball, 1993; Bunting
et al., 1997). These mediators cause blood vessel
contraction, platelet aggregation and myocardial
dysfunction, leading to acute death.
The beta toxin induces hemorrhagic necrosis
of the intestinal mucosa (Lawrence & Cooke,
1980; Gilbert et al., 1997). Although the exact
mechanism of action is not yet known, beta 1 toxin

is most likely also a membrane-damaging toxin.

The beta toxin has high homology with alpha
toxin, gamma toxin and leukocidin of Staphylococcus aureus, forming pores in eukaryotic cell
membranes (Hunter et al., 1993). Moreover, the
beta toxin can form a multimeric complex on
human umbilical vein endothelial cells, suggesting
that it can oligomerize, typical for pore-forming
toxins (Steinthorsdottir et al., 2000). Shatursky
et al. (2000) showed that the beta toxin forms
sodium and potassium selective channels in lipid
bilayers.
Recently, the newly discovered beta 2 toxin
has been associated with porcine, equine and
bovine gastro-enteritis (Bueschel et al., 2003;
Bacciarini et al., 2003; Waters et al., 2003). This
toxin has also been demonstrated in avian C.
perfringens type A strains (Bueschel et al., 2003),
but its exact role in pathogenesis needs to be
further elucidated. Very little sequence homology
exists with other clostridial toxins, in spite of
having the same biological activities as the beta
toxin (Gilbert et al., 1997).

Epidemiology of C. perfringens in Poultry

The incidence of C. perfringens in the intestinal
tract and in processed meat of poultry is high.
When the intestinal contents of broiler chickens are
analyzed for the presence of C. perfringens,
approximately 75% to 95% of the animals are
found positive (Shane et al., 1984; Miwa et al.,
1997; Craven et al., 2001a,b). When poultry meat is
analyzed for C. perfringens, high percentages of
positive meat samples are reported, in some cases
up to 84% (Bean et al., 1996; Miwa et al., 1998;
Craven et al., 2001a). It is suggested that colonization of poultry by C. perfringens is a very early
event in the life of the animals, and can be
transmitted within the integrated broiler chicken
operation, starting from the hatchery (Craven
et al., 2001a,b, 2003). C. perfringens is found
indeed in eggshell fragments, chicken fluff
and paper pads in the hatchery (Craven et al.,
2001b). In a follow-up study of 16 broiler flocks,
paper pads beneath chicks that were transported
from the hatchery to the rearing house were
contaminated in 15 of the 16 flocks, again illustrating the importance of the hatcheries in the contamination. C. perfringens was recovered from
broiler carcasses after chilling in 13 of the flocks
(Craven et al., 2001a). Ribotyping of isolates from
the paper pads, isolates during the grow-out phase
and carcass isolates indicate that at least some of
the C. perfringens contamination found on processed broiler carcasses can originate in the breeder
operation and can be transmitted through the
hatchery and grow-out operations (Craven et al.,
2003). C. perfringens is also widespread in the

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C. perfringens in poultry 539

environment, such as soil and water (Davies &

Wray, 1996; Thakur & Grover, 2001; Desmarais
et al., 2002). It is also shown that intestinal
droppings of wild birds contain high numbers of
C. perfringens and that free-living birds can suffer
from necrotic enteritis (Craven et al., 2000; Asaoka
et al., 2003). In environmental samples collected on
poultry farms, the highest incidences of C. perfringens are detected in wall swabs (53%), fan swabs
(46%), fly strips (43%), dirt outside the entrance
(43%) and swabs of boots (29%) (Craven et al.,
2001a). All the afore-described studies indicate that
C. perfringens is a common intestinal inhabitant,
but what can be questioned is the significance
without further typing of the strains for toxin
production.
Recently, 279 isolates from 25 Danish farms and
53 isolates from 10 Swedish farms were analyzed by
polymerase chain reaction and all isolates were
shown to be C. perfringens type A. Both healthy
and diseased flocks were analyzed in these studies.
Pulsed-field gel electrophoresis showed that healthy
chickens carried several different C. perfringens
clones within a flock and even within individual
birds, while flocks suffering from necrotic enteritis
only carried one or two clones (Engstrom et al.,
2003; Nauerby et al., 2003). The reasons for this
selective proliferation of these clones are not
known, and it has not yet been possible to relate
any subtype of C. perfringens to the development
of necrotic enteritis.

Predisposing Factors for Necrotic Enteritis in

Poultry
C. perfringens is taken up from the environment.
Environmental sources include contaminated
feed, water or any part of the broiler production
plant and the environment (Craven et al., 2001a,b,
2003). The presence of C. perfringens in the
intestinal tract of broiler chickens or inoculation
of the animals with high doses of C. perfringens,
however, does not lead per se to the development
of necrotic enteritis (Shane et al., 1984; Cowen et
al., 1987; Kaldhusdal et al., 1999; Craven et al.,
2001a; LaRagione & Woodward, 2003). One or
several predisposing factors may be required to
elicit the clinical signs and lesions of necrotic
enteritis.
The most important known predisposing factor
is intestinal damage caused by coccidial pathogens.
Intestinal damage will result in release of plasma
proteins into the lumen of the intestinal tract.
Since the minimal requirements for growth of C.
perfringens include more than 11 amino acids,
besides many growth factors and vitamins (Boyd
et al., 1948; Petit et al., 1999), leaking of plasma
to the intestinal lumen can provide a necessary
growth substrate for extensive proliferation of these

bacteria. Especially Eimeria species that colonize

the small intestine, such as Eimeria maxima and
Eimeria acervulina, are known to predispose to
necrotic enteritis (Al-Sheikly & Al-Saieg, 1979;
Hofacre et al., 1998, 2003; Jackson et al., 2003).
When broiler chickens were orally inoculated
with sporulated oocysts of Eimeria necatrix or
Eimeria acervulina at about 2 weeks of age and
fed C. perfringens-contaminated feed, the percentage of mortality was 28% and 53%, respectively.
E. acervulina or E. necatrix infection alone,
or feeding C. perfringens-contaminated feed
without Eimeria infection, did not result in
mortality (Al-Sheikly & Al-Saieg, 1979). Also
in the field, coccidial pathogens are mostly found
in outbreaks of necrotic enteritis (Helmboldt &
Bryant, 1971; Long et al., 1974; Broussard et al.,
1986). It is documented in numerous studies
that coccidial vaccines and coccidiostatic drugs
are able to prevent C. perfringens-associated necrotic enteritis (Vissienon et al., 2000; Engberg et
al., 2000; Jackson et al., 2003; Williams et al.,
2003). Removal of coccidiostats from poultry feed
is therefore also a predisposing factor for the
development of necrotic enteritis (Elwinger et al.,
1992).
It is proven that diet strongly influences the
incidence of necrotic enteritis in broilers. Diets with
high levels of indigestible, water-soluble non-starch
polysaccharides, known to increase the viscosity of
the intestinal contents, predispose to necrotic
enteritis. This is the case for diets rich in rye, wheat
and barley relative to diets rich in corn (Branton
et al., 1987; Kaldhusdal & Hofshagen, 1992;
Riddell & Kong, 1992; Kocher, 2003). Also, diets
rich in fish meal predispose to necrotic enteritis
(Kocher, 2003). More data concerning the effects of
feed composition on C. perfringens and the development of necrotic enteritis are discussed further in
the text.
The use of most antimicrobial growth promoters
leads to a dramatic decrease in the incidence of
necrotic enteritis in poultry, relative to nonmedicated animals (discussed extensively further
in the text).
This short list of well-known predisposing
factors is probably far from complete. Even when
taking into account the known predisposing
factors, it is still difficult to reproduce the symptoms and lesions of necrotic enteritis experimentally.

Clinical Signs and Lesions of C. perfringens

Infections in Poultry
Clinical necrotic enteritis
The prominent feature of necrotic enteritis is acute
death, with mortality rates that can reach 50%

540 F. Van Immerseel et al .

(Wijewanta & Seneviratna, 1971; Riddell & Kong,

1992). Clinical signs include depression, dehydration, somnolence, ruffled feathers, diarrhoea
and decreased feed consumption (Helmboldt &
Bryant, 1971; Al-Sheikly & Truscott, 1976a;
Al-Sheikly & Al-Saieg, 1979; Gadzinski & Julian,
1992).
Most evident macroscopical lesions can be
seen in the small intestine, but lesions can also
be detected in other organs, such as the caeca,
liver and kidney. The duodenum, jejunum and ileum,
and sometimes also the caeca, are thin walled,
friable, dilated and filled with gas (Broussard
et al., 1986). Mucosal surfaces are covered with a
grey brown to yellow green diphteric membrane
or pseudomembrane (Gazdinski & Julian, 1992).
Lesions in the small intestine are described in
detail by Al-Sheikly & Truscott (1976a, 1976b,
1976c). These authors infected chickens intraduodenally with broth cultures and toxins of
C. perfringens to reproduce necrotic enteritis. Both
types of inocula resulted in similar lesions. One hour
post-inoculation, a slight oedema and dilation of
vessels is seen, with some sloughed epithelium visible
in the intestinal lumen. Large numbers of Grampositive bacteria are detected attached to the
epithelial surface and among the sloughed
epithelium. At 3 h after inoculation, the intestine
has a greyish and thickened mucosa. At this
time, a marked oedema is detected resulting in
detachment of the epithelial layer from the
lamina propria, especially at the apex of the villi.
Early stages of degeneration of the villi tips
and increases in the amount of sloughed epithelium
and fibrinous exudates are apparent at this stage
of the infection. Five hours post-inoculation, coagulation necrosis of the epithelial layer and lamina
propria at the villus tip is detected. Blood vessels
are congested and/or occluded with hyaline
thrombi. Large numbers of Gram-positive bacteria
colonize the necrotic tissues, and villi are shortened.
Mononuclear cells and heterophils infiltrate the
lamina propria. Histological lesions at 8 and 12 h
post-inoculation are characterized by massive necrosis of the villi, with necrotic zones reaching
the crypts. Fibrin and cellular debris are present in
the intestinal lumen at this stage. Animals that
are able to survive, start regeneration of the exposed surfaces by formation of short and blunt villi
(Al-Sheikly & Truscott, 1976a,b,c). Histopathological lesions in field cases of necrotic enteritis are
similar as in the afore-described experimental animals (Helmboldt & Bryant, 1971; Broussard et al.,
1986; Gazdinski & Julian, 1992).
In animals with necrotic enteritis, lesions
may also be found in other organs. At 12 h
after inoculation of broth cultures or toxins
of C. perfringens, blood vessels in the liver,
spleen, heart and kidney are frequently congested,
especially in animals that are moribund (Al-Sheikly

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& Truscott, 1976a,b). This is accompanied by

changes in erythrocyte morphology, starting at
1 h post-inoculation of the toxin and most prominent just before death of the chickens. Erythrocytes
become rounded or irregular and many are
lysed (Al-Sheikly & Truscott, 1976b). In animals
that are found acutely dead at young age there
may be a greenish discoloration and enlargement
with necrotic foci in the liver (Eleazer & Harrell,
1976). Finally, necrosis of the follicular lymphocytes in the bursa of Fabricius is also described
(Frame & Bickford, 1986; Gazdinski & Julian,
1992).

The Subclinical Form of C. perfringens Infection

Besides the clinical from of necrotic enteritis
already described, a mild subclinical form has
also been described in the field, leading to decreases
in performance (Kaldhusdal & Hofshagen, 1992;
Lvland & Kaldhusdal, 2001). In these cases
only focal necrosis of the intestinal mucosa is
found. Subclinical focal lesions can be reproduced
experimentally when broth cultures of C. perfringens are centrifuged and the bacteria resuspended in
phosphate-buffered saline before inoculation, resulting in a minor necrosis in the duodenum and,
less frequently, in the ileum (Al-Sheikly & Truscott,
1976c). This is in contrast with inoculation of the
broth culture or the supernatant from the broth
culture that results in severe necrotic enteritis
(Al-Sheikly & Truscott, 1976a,b).
Subclinical C. perfringens infection also is associated with hepatitis or cholangiohepatitis. The
livers are greatly enlarged with a pale reticular
pattern, sometimes with pale and stellate foci
(Lvland & Kaldhusdal, 1999). Histopathological
lesions are bile duct hyperplasia, fibrinoid
necrosis, cholangitis and occasionally focal granulomatous inflammation (Randall et al., 1983, 1986;
Onderka et al., 1990; Lvland & Kaldhusdal, 1999;
Sasaki et al., 2000). Onderka et al. (1990) have
reproduced cholangiohepatitis after inoculation
of C. perfringens bacteria in the bile duct. The
gall bladder in inoculated birds is oedematous
and distended with flocculent material. Livers
become enlarged and tan-coloured with red and
white foci 2 weeks post-inoculation. C. perfringens
is isolated from the gall bladder and liver (Onderka
et al., 1990). It is proposed by Lvland &
Kaldhusdal (1999) that in cases of subclinical
necrotic enteritis, due to the high number of
C. perfringens bacteria residing in the small intestine, and the intestinal damage, the bacteria can
reach the biliary ducts and portal blood stream to
reach the liver.

C. perfringens in poultry 541

Prevention and Control of C. perfringens Infections

in Poultry

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Vaccination against C. perfringens

Vaccination can induce/elicit antibody titres against
clostridia and clostridial toxins in mice, lambs,
piglets, goats and calves (Troxel et al., 1997; de la
Rosa et al., 1997; Uzal et al., 1998; Springer &
Selbitz, 1999; Schoepe et al., 2001). Recently, a
fusion protein carrying antigenic determinants
of the alpha toxin, beta toxin and enterotoxin
was constructed, inducing antibodies against
each of the three separate components, when
injected in mice (Belyi & Varfolomeeva, 2003).
Necrotic enteritis in different mammalian species,
including humans, can be prevented by vaccination
(Lawrence et al., 1990; Uzal & Kelly, 1998;
Springer & Selbitz, 1999). Extrapolating from
data derived from the aforementioned studies
with other farm animals, vaccination could be a
helpful tool in preventing necrotic enteritis in
poultry. It is known indeed that flocks with high
titres of maternal antibodies against alpha toxin had
lower mortality during the production period than
flocks with low titres (Heier et al., 2001). Lvland et
al. (2004) have recently experimented in broilers
with candidate vaccines based on C. perfringens
type A and type C toxoids, with promising results in
respect of protection against subclinical necrotic
enteritis.

cases precludes the use of anticoccidial drugs, of

which some (i.e. the ionophores) also act bactericidally on clostridia (Williams, 2002). Another
possibility is that live attenuated vaccine strains
can induce a certain degree of mucosal damage,
known to be a predisposing factor for the development of necrotic enteritis (Williams, 2002).
In a study by Ernk & Bedrnk (2001), six
successive flocks in the same poultry house were
studied in which flocks 1, 2, 5 and 6 were given
coccidiostatic drugs, while flocks 3 and 4 were
given an attenuated vaccine. From the time the
coccidiostat was replaced by the attenuated vaccine
(flock 3), necrotic enteritis was observed. After
replacing the vaccine by the coccidiostatic drugs
again, necrotic enteritis disappeared (flock 6).
In the study of Waldenstedt et al. (1999), vaccinated unmedicated birds had a higher caecal
count of C. perfringens than unvaccinated medicated bird and a lower slaughter weight. The
vaccinated groups had higher numbers of
C. perfringens in the intestinal tract and experienced an outbreak of necrotic enteritis. It is not
clear whether this was due to the mucosal damage
caused by the attenuated coccidial vaccine or to
the lack of coccidiostatic antibacterial drugs in the
feed, or else to the damage caused by coccidial
field infections in vaccinated groups. Probably also
the level of attenuation of the vaccines could play
a role.

Control of coccidiosis

Growth-promoting antibiotics

Vaccination against coccidiosis may indirectly prevent losses from Clostridium, since the presence of
Eimeria species is a predisposing factor for the
development of necrotic enteritis (as described
earlier). It is not yet known whether coccidial
infection per se or only severe mucosal damage
caused by these parasites allows the clostridial
infection to initiate necrotic enteritis (Williams,
2002). Two different lines of thinking can be put
forward in this regard. First, if only severe coccidiosis should predispose to necrotic enteritis,
probably anticoccidial vaccination with attenuated
vaccine strains will protect chickens against necrotic enteritis by preventing the predisposing clinical
coccidiosis. When unvaccinated, E. maxima-infected animals were challenged intracloacally
with C. perfringens, necrotic enteritis lesions
were severe. When the animals were immunized
with an attenuated coccidial vaccine, subsequently
infected with E. maxima and then challenged with
C. perfringens, the severity of necrotic enteritis was
reduced, indicating that the reduction of coccidial
lesions prevents necrotic enteritis (Williams et al.,
2003). Second, it has to be considered that the use
of anticoccidial vaccines could also have adverse
effects on the incidence of necrotic enteritis. This
can be due to the fact that vaccination in most

Following the European Union (EU) ban of

avoparcin in January 1997, of ardamycin in January
1998, and in December 1998 of a further four
antibiotics (bacitracin, virginiamycin, tylosin and
spiramycin), there are only four substances still
authorized as growth-promoting agents in the EU.
These are monensin, salinomycin, avilamycin and
flavophospholipol. Phasing out of these four remaining antimicrobial feed additives for use as
growth promoters is expected in the near future.
The banned growth-promoting agents all had
effects on colonization of C. perfringens in poultry
and the prevention of necrotic enteritis. Avoparcin
had strong in vitro antibacterial effects on all
poultry isolates of C. perfringens in two independent studies (Devriese et al., 1993; Watkins et al.,
1997). Avoparcin reduced the numbers of C.
perfringens in the intestinal tract of broilers and
prevented necrotic enteritis in broilers, challenged with C. perfringens type A (Prescott, 1979;
Hofshagen & Kaldhusdal, 1992; Elwinger et al.,
1998). Although many C. perfringens poultry
isolates appear to be resistant against bacitracin
(Kondo, 1988; Devriese et al., 1993; Watkins et al.,
1997), inclusion of this agent in broiler feed
reduced intestinal C. perfringens counts, the number of gut lesions, lesion scores and mortality

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542 F. Van Immerseel et al .

caused by necrotic enteritis in challenge experiments and field trials (Prescott, 1979; Stutz et al.,
1983; Stutz & Lawton, 1984; Broussard et al., 1986;
Engberg et al., 2000; Lovland et al., 2003; Brennan
et al., 2003; Jackson et al., 2003). Also multiple
studies showed that tylosin (Stutz & Lawton, 1984;
Vissiennon et al., 2000; Brennan et al., 2001;
Collier et al., 2003) and virginiamycin (George
et al., 1982; Stutz & Lawton, 1984) inclusion in
broiler feed reduced the occurrence of and mortality due to necrotic enteritis, and the intestinal
counts of C. perfringens.
The four antibiotic growth-promoting agents
that are still allowed in poultry feed in the EU
have anti-Clostridium effects in poultry, with the
exception of flavomycin. Monensin and salinomycin are ionophorous antimicrobials and also act
coccidiostatic, and will therefore also decrease the
intestinal damage caused by Eimeria infections
(McDougald et al., 1996), thus reducing the
most important predisposing factors for C. perfringens-associated necrotic enteritis. Ionophorous
antibiotics, such as monensin and salinomycin,
have very low minimal inhibitory concentrations against C. perfringens (Devriese et al., 1993;
Watkins et al., 1997). Elwinger et al. (1998) have
shown that monensin (and the ionophores
narasin and maduramycin) have anti-bacterial
effects against C. perfringens in the intestinal
tract of broilers. Salinomycin reduces the counts
of C. perfringens in the intestinal tract of broilers
and decreases the severity of lesions due to necrotic
enteritis in a challenge model (Engberg et al., 2000;
Jackson et al., 2003). Monensin, and also the
ionophorous anticoccidial narasin, decreases
mortality in chickens, intraduodenally infected
with C. perfringens, by more than 20% (Vissienon
et al., 2000). Avilamycin proved to be strongly
bactericidal against C. perfringens in vitro
(Devriese et al., 1993; Watkins et al., 1997). This
growth-promoting antibiotic also decreases mortality due to necrotic enteritis in intraduodenally
infected chickens and decreases the number of
intestinal C. perfringens bacteria in broiler chickens
(Elwinger et al., 1998; Vissienon et al., 2000).
Reports on activities of flavophospholipol on C.
perfringens and necrotic enteritis in broilers are
scarce. All isolates of C. perfringens were highly
resistant to this antibiotic, as shown in studies of
Devriese et al. (1993) and Martel et al. (2004). One
report mentions a decrease in faecal shedding
of C. perfringens in broilers at slaughter age
(Bolder et al., 1999).

Competitive exclusion products

Nurmi & Rantala (1973) originally developed the
concept of competitive exclusion. By oral administration of a suspension of alimentary tract contents
from healthy adult birds to newly hatched chicks,

an adult-type microflora is established, which

protects from Salmonella and other infections
(Nurmi et al., 1992; Nisbet, 1998; Mead, 2000).
Only few studies have been carried out concerning
the effects of competitive exclusion (CE) cultures
on necrotic enteritis. A decreased incidence of
necrotic enteritis and a decreased colonization by
C. perfringens is found in chickens treated with CE
cultures, together with an increased slaughter yield
(Elwinger et al., 1992; Craven et al., 1999).
Different CE products proved effective in reducing
the lesion score in an experimental necrotic enteritis model (Hofacre et al., 1998). Also in this study,
body weight and feed consumption was higher and
feed conversion lower in the groups of animals,
treated with CE cultures. In a field trial, CE flora
delayed intestinal proliferation of C. perfringens
and appearance of C. perfringens-associated gut
lesions. This delay was associated with improved
production performance at slaughter (Kaldhusdal
et al., 2001).

Probiotics
A probiotic is defined as a live microbial feed supplement that beneficially affects the
host animal by improving its intestinal microbial
balance (Fuller, 1999). Especially lactobacilli have
been used with success in decreasing colonization
of poultry by different pathogens, such as
Salmonella and Campylobacter (Mulder et al.,
1997; Pascual et al., 1999). Most data concerning
effects of probiotics on C. perfringens are derived
from studies in humans and other mammals. It was
shown in vitro that lactobacilli reduce the adhesion
of C. perfringens to immobilized canine mucus
(Rinkinen et al., 2003). Although some studies are
inconclusive, oral uptake of lactobacilli and bifidobacteria decrease the number of intestinal
C. perfringens bacteria and spores in humans and
mice in other studies (Gallaher et al., 1996;
Romond et al., 1998; OMahony et al., 2001;
Brigidi et al., 2001). Few studies have been
performed on the protective effects of probiotic
strains against C. perfringens in chickens. Hofacre
et al. (1998) showed that a commercial probiotic
preparation reduced gross lesions of necrotic enteritis in chickens, but the protection was far less
than that conferred by competitive exclusion flora.
Lactobacilli were successful in decreasing mortality
due to necrotic enteritis from 60% to 30% in an
experimental challenge trial, when they were given
orally at day 1 of life (Hofacre et al., 2003). In this
experiment, feed conversion was decreased in the
group that was given lactobacilli, while the weight
gain was not altered.
When 1-day-old and 20-day-old chickens
were dosed with 109 spores of a Bacillus subtilis
strain and challenged 24 h later with 105 colonyforming units of C. perfringens, colonization and

C. perfringens in poultry 543

persistence of C. perfringens was suppressed (La

Ragione 7 Woodward, 2003).
Bacteriophage
Bacteriophage or bacteriophage enzymes, active
against C. perfringens strains, can possibly be used
for controlling C. perfringens in poultry. Zimmer
et al. (2002) described the murein hydrolase
from bacteriophage f3626, which lysed all tested
C. perfringens strains in their study, but not other
Clostridia and bacteria belonging to other genera.
The possible use of bacteriophage for in vivo
control of C. perfringens in poultry still needs to
be investigated.

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Prebiotics
Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by
selectively stimulating the growth and/or activity
of one or a limited number of bacterial species
already resident in the intestine. Prebiotics are able
to serve as a substrate for one or more bacterial
species with a potentially beneficial effect on the
host (Gibson & Roberfroid, 1995). Research on the
effects of prebiotics on C. perfringens is mainly
performed in mammals and in vitro systems. When
fructo-oligosaccharides (FOS) were added to a
continuous culture model of the human intestine
or to batch cultures inoculated with human faeces
in vitro, growth of C. perfringens was enhanced
(Bello et al., 2001; McBain & MacFarlane, 2001).
In rats, cats and dogs it was shown, however, that
addition of FOS to the diet resulted in significantly
fewer C. perfringens bacteria in the intestinal tract
compared with animals fed unsupplemented diet
(Gallaher et al., 1996; Sparkes et al., 1998;
Swanson et al., 2002). Recently, Hofacre et al.
(2003) showed that neither addition of FOS or
manno-oligosaccharides to the diet of broilers had
a significant effect on mortality caused by necrotic
enteritis, on weight gain and on feed conversion in
6-week-old broilers, in an experimental challenge
experiment.
Nature and form of the feed
The composition of the feed may greatly influence
the occurrence of necrotic enteritis in chickens.
Different reports show that maize (corn) in the
chicken feed reduces the incidence and severity of
necrotic enteritis, as opposed to diets based on
wheat, rye, oats and barley (Branton et al., 1987;
Kaldhusdal & Hofshagen, 1992; Riddell & Kong,
1992). When broiler chickens were inoculated
by feeding C. perfringens-inoculated feed for 3
consecutive days, mortality ranged from 0% to
12.5% in broilers fed a maize-based diet, while
the mortality in animals fed a diet with high

amounts of wheat, rye or barley ranged from

26% to 35% (Riddell & Kong, 1992). In a similar
study mortality in broilers fed a maize-based
diet was 2.9%, while replacing one-half of
the maize by wheat resulted in 18.1% mortality
(Branton et al., 1987). In an epidemiological study
in Norway, it was shown that the ratio of wheat
plus barley to maize was positively correlated with
the incidence of necrotic enteritis (Kaldhusdal &
Skjerve, 1996).
Different hypotheses are put forward concerning
the effect of the cereal type on C. perfringens and
necrotic enteritis. Barley, wheat, rye and oats
contain high levels of indigestible water-soluble
non-starch polysaccharides, which are known to
lead to increased digesta viscosity, decreased digesta passage rate and nutrient digestibility, in
contrast to maize (Annison & Choct, 1991; Choct
et al., 1996). This is proven to lead to a serious
increase in the number of intestinal anaerobic
bacteria (Hubener et al., 2002; Kocher, 2003).
Enzymes, such as xylanase, can be used to break
down non-starch polysaccharides. Addition of
enzymes to diets based on non-maize grains can
reduce digesta viscosity and increase digesta passage rate, while significantly reducing the bacterial
population in the small intestine (Bedford &
Classen, 1992; Choct et al., 1999). Supplementing
xylanase to poultry feed reduced fermentation in
the small intestine but increased caecal fermentation (Choct et al., 1999). This has been shown to
reduce the numbers of C. perfringens in the small
intestine but does not always confer protection
against necrotic enteritis (Riddell & Kong, 1992;
Jackson et al., 2003).
Another possible explanation for the different
effects of cereal types on C. perfringens-associated
necrotic enteritis is the possible existence of antiClostridium substances in digested maize, or proliferative substances in digested wheat or barley.
Indeed, proliferation of C. perfringens in digested
maize was significant lower than proliferation in
digested wheat or barley (Annett et al., 2002).
The pathogenicity of coccidia seems to be
lower in maize-based than in wheat-based diets
(Williams, 1992). Since coccidial lesions are a
predisposing factor for necrotic enteritis, this can
also play a significant role in the protective effect
of maize.
High protein content in poultry feed seems to
increase the incidence of necrotic enteritis. An
increased risk of necrotic enteritis is often associated with animal protein ingredients such as
fishmeal or meat and bonemeal (Kocher, 2003).
Studies of the effects of vegetable protein ingredients, such as soy protein, on the incidence of
necrotic enteritis in vivo are lacking.
The dietary fat source should also be considered
as a factor when searching for the optimal poultry
feed ingredients, since animal fat (mixture of lard

544 F. Van Immerseel et al .

and tallow) leads to higher counts of C. perfringens

in the ileum, compared with vegetable oil (soy oil)
(Knarreborg et al., 2002).
Finally, also the feed form can possibly influence
the incidence of necrotic enteritis in poultry. Finely
ground feed (hammer-mill) increased mortality
associated with necrotic enteritis compared with
coarsely ground feed (roller-mill) (Branton et al.,
1987). Recently Engberg et al. (2002) found no
effects of the feed grinding on C. perfringens counts
in the intestine, but found an increased digestibility
and a decreased number of C. perfringens bacteria
in the intestine when the birds were fed pellets
instead of mash.

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Poultry Meat as a Source of C. perfringens Food

Poisoning in Humans
C. perfringens can cause two different foodborne
diseases in humans; that is, type A diarrhoea
and type C necrotic enteritis, caused by enterotoxin-positive C. perfringens type A strains and
C. perfringens type C strains, respectively. The
ability to form spores and the wide temperature
range in which C. perfringens can grow are features
that allow the bacteria to multiply and survive in
food (Brynestad & Granum, 2002). Especially food
that is heated, cooled down too slowly when
freezing, and reheated insufficiently is of great
risk (Andersson et al., 1995).
Symptoms of the self-limiting type A food
poisoning start about 6 to 24 h after consumption
of contaminated food and last about 24 h. These
are characterized by abdominal pain, nausea
and diarrhoea. Symptoms caused by C. perfringens
type A usually are relatively mild and limited to the
elderly and very young children, but occasionally
death may occur due to dehydration (Brynestad
& Granum, 2002; Tanaka et al., 2003). Since
C. perfringens food poisoning is not a reportable
disease, its frequency probably is seriously underestimated. However, in Norway in the 1990s it was
registered as the most common cause of foodpoisoning (Brynestad & Granum, 2002). The prevalence in other countries, such as Japan, the
US and the UK, is also high (Labbe, 2000;
Brynestad & Granum, 2002). In England and
Wales, C. perfringens was the second most frequently reported organism, associated with foodborne outbreaks of intestinal disease, in the 1990s
(Kessel et al., 2001).
The effects of type A food poisoning are caused
by production of the Clostridium perfringens
enterotoxin (CPE), which is produced at the
moment of sporulation (Ridell et al., 1998; Sarker
et al., 1999). The CPE appears to be a bifunctional
toxin, which first induces cellular damage via its
cytotoxic activity, resulting from plasma membrane
permeability alterations caused by formation of

CPE-containing complex that may correspond

to a pore. CPE then interacts with structural
components of the epithelial tight junctions, including certain claudins and occludin. These
interactions can affect tight junction structure
and function, thereby altering paracellular permeability (Singh et al., 2000, 2001; McClane, 2000,
2001).
Outbreaks of type A food poisoning have
been traced back to consumption of chicken
meat (Schiemann, 1977; Hook et al., 1996). It is
believed that a small number of CPE-positive
C. perfringens bacteria co-exist with a large number
of non-enterotoxigenic C. perfringens bacteria in
the chicken (Miwa et al., 1996). Therefore plating
of chicken intestinal contents or faecal material
and picking colonies for characterization will
mostly not yield CPE-positive C. perfringens
strains. When intestinal contents of chickens are
analysed by nested polymerase chain reaction after
enrichment of the samples without picking single
colonies first, CPE-positive C. perfringens are
found in 40% of the samples, whereas total C.
perfringens is found in 76% (Miwa et al., 1997).
When chicken meat is analysed with this method,
12% of the samples are positive for the enterotoxigenic C. perfringens strains, while 84% of the
chicken meat samples are positive for total C.
perfringens (Miwa et al., 1998). In the studies of
Miwa et al. (1997, 1998) porcine and bovine
intestinal samples and meat were also analysed
for the presence of CPE-positive strains. The
amount of CPE-positive samples in both the
intestinal contents and meat of the chickens is
higher than for cattle samples (26% and 2%,
respectively) and pig samples (22% and 0%, respectively). More studies using very sensitive methods
are necessary to investigate the prevalence of CPEpositive C. perfringens in poultry in order to clearly
assess the importance of chicken meat in type A
food poisoning in humans.
C. perfringens type C food poisoning (necrotic
enteritis) in humans has only been reported on very
rare occasions in the industrialized world during
the recent decades. These rare cases are almost
exclusively in diabetic people (Severin et al., 1984;
Watson et al., 1991; Clarke et al., 1994; Petrillo
et al., 2000; Gui et al., 2002). The symptoms start
with abdominal pain and bloody diarrhoea, and
are followed rapidly by necrosis of the small
intestine, caused mainly by the beta toxin, with
contributions of additional toxins from C. perfringens type C (Jolivet-Reynaud et al., 1986). The
type C food poisoning is lethal in 25% of cases
(Granum, 1990). Although C. perfringens type C
can be detected in poultry (Songer & Meer, 1996),
it is not considered to be a serious threat for
humans due to the very low prevalence of type C
food poisoning.

C. perfringens in poultry 545

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Conclusions and Future Perspectives

It is believed that C. perfringens-associated necrotic
enteritis and subclinical infection will emerge in
broiler chickens after the ban of antimicrobial
growth promoters in the EU. Not only mortality
due to necrotic enteritis, but also the subclinical
form of the infection, leading to decreased weight
gain and increased feed conversion, will have severe
economic consequences for the poultry industry.
Concerns arise on the high contamination rates of
poultry by C. perfringens and the risk of transmission to the food chain, posing public health
problems.
Little time is left to build a strategy for prevention and control of C. perfringens necrotic enteritis
and subclinical disease, since antimicrobial growth
promoters, controlling the disease until now, will be
banned in the EU in 2006. Cost-effective alternatives have to be sought and more research should
be urgently done on this matter. First of all, there is
a need for an experimental model reproducing the
disease. This model then could be applied for
testing products, which are expected to control
the disease. Currently, different models are used in
the literature, of which some fail to reproduce the
disease. Moreover, it is difficult to compare results
between different experiments due to differences in
experimental set-up. A consensus experimental
model, in which all aspects, including feed composition, are standardized, may allow further progress
in the development and evaluation of new tools for
better control of this emerging disease in poultry.
Predisposing factors, such as intestinal damage
due to coccidiosis, should be taken into account
when designing a control program. A combination
of measures that include avoiding predisposing
factors and combating the pathogen itself seems
to be the strategy of choice. Research should focus
on creating new protective measures against the
disease. Balancing the composition of feed ingredients is probably the most cost-effective prevention and control measure. In other words, a balance
between optimal performance of the animal and
contamination rate by C. perfringens should be
sought. Vaccination of poultry against C. perfringens is a seriously under-researched matter, despite
of the known protective effects in other animal
species. The use of prebiotics and probiotics has
been found effective against various pathogens in
poultry, and more efforts should be done in this
complex area. Moreover, there is a lack of information on the mechanisms of colonization and
clearance of C. perfringens in poultry and the
factors determining toxin production.

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