Avian Pathology

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Pathology and diagnosis of necrotic enteritis: is it

clear-cut?

Joan A. Smyth

To cite this article: Joan A. Smyth (2016) Pathology and diagnosis of necrotic enteritis: is it clear-
cut?, Avian Pathology, 45:3, 282-287, DOI: 10.1080/03079457.2016.1158780

To link to this article: https://doi.org/10.1080/03079457.2016.1158780

Published online: 31 May 2016.

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AVIAN PATHOLOGY, 2016
VOL. 45, NO. 3, 282–287
http://dx.doi.org/10.1080/03079457.2016.1158780

REVIEW

Pathology and diagnosis of necrotic enteritis: is it clear-cut?

Joan A. Smyth
Department of Pathobiology and Veterinary Science, University of Connecticut, USA

ABSTRACT ARTICLE HISTORY

The ability to correctly recognize the disease necrotic enteritis (NE) is important not only to Received 21 December 2015
those involved in control and treatment of the disease at farm level, but it is also critically Revised 17 January 2016
important to the search for virulence factors, since a fundamental part of that process is the Accepted 25 January 2016
correct assignation of strains of Clostridium perfringens with respect to virulence. Thus,
KEYWORDS
diagnosticians and investigators need to be able to correctly recognize the lesions of NE. To Necrotic enteritis;
do this, they must be able to distinguish NE lesions from (1) other enteric diseases such as Clostridium perfringens;
coccidiosis or viral enteritis, (2) normal features of the intestine, such as the small raised, pathology; histopathology;
sometimes red, foci that represent gut-associated lymphoid tissue, (3) autolytic change which autolysis; intestine;
may be mistaken for lesions, especially at the microscopical level, by the inexperienced. diagnosis; poultry; chicken
Errors in diagnosis of NE due to C. perfringens or failure to culture affected areas in which the
bacteria of interest with respect to NE are definitively found, might explain some of the early
apparently conflicting results with respect to the role of netB in NE. This paper describes at
the gross, microscopical and bacteriological level, important features of the intestine of
normal poultry and those with NE due to C. perfringens, as well as the common interpretative
pitfalls that can lead both to underdiagnosis and overdiagnosis of NE, and to incorrect
determination of the virulence of individual C. perfringens strains.

Introduction of death and which is commonly mistaken for patho-

logical change.
The disease, necrotic enteritis (NE) of poultry caused
The purpose of this review is to make investigators
by Clostridium perfringens, was first reported in 1961
aware of the considerations necessary in reaching a
(Parish, 1961) but has been well controlled for many
correct diagnosis of NE, and to understand how
years by the use of the so-called “antibiotic growth pro-
this can affect conclusions of scientific research,
moters” (AGPs) in feedstuffs (Van Immerseel et al.,
and also to assist beginning investigators in the
2004; McDevitt et al., 2006). In recent years, there
interpretation of necropsy findings in the intestine
has been intense research interest in this disease, as
of poultry.
part of the effort to find alternate control measures
because the use of AGPs has been restricted or elimi-
nated by legislation in many countries (Bywater, Diagnosis of C. perfringens disease in birds
2005; Thompson et al., 2006). Critical to the successful
Unless a disease has a particular pathognomonic indi-
interpretation of all of the scientific studies is accuracy
cator, definitive diagnosis of disease is based on mul-
of diagnosis of the disease, so that researchers (1) are
tiple criteria such as the nature of any pathological
sure that the C. perfringens isolates they are working
findings, microbiological findings and so on. In the
with are truly from NE lesions, or not, and (2) interpret
case of enteric diseases caused by C. perfringens,
correctly the disease outcome of either field- or labora-
interpretation of pathological and microbiological
tory-based bird studies designed to examine virulence
findings presents particular challenges given that
factors or the effects of intervention strategies such as
C. perfringens is carried in the intestine of normal
vaccines.
birds, and that the clostridia (including
Definitive diagnosis of the clostridial enteric diseases
C. perfringens) are major causes of post-mortem putre-
is fraught with difficulties, even for experienced veter-
faction. Putrefaction of tissues hinders the ability to
inary pathologists, especially for (1) those not familiar
recognize lesions and poses significant interpretive
with this disease and/or the other common enteric dis- problems that can result in incorrect diagnosis: either
eases of poultry such as coccidiosis, which can have a failure to recognize lesions that are present, or conver-
wide range of presentations (2) those without knowl- sely, interpreting autolytic change as pathological
edge of the effects of and appearance of autolytic change. In birds dead from clostridial disease, the clos-
change of the intestine especially at the microscopical tridia are usually present in large numbers before
level, change which starts to develop within minutes death, consequently the rate of putrefaction is

CONTACT Joan A. Smyth joan.smyth@uconn.edu Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269, USA
© 2016 Houghton Trust Ltd

accelerated and interpretive difficulties increased. To
assist interpretation, many investigators have exam-
ined intestinal content for the presence of the relevant
clostridial toxins, surmising that presence of detectable
levels of these toxins supports diagnosis. However, it is
known that some clostridial toxins are degraded by
intestinal proteases (Popoff & Stlies, 2005; Rioseco
et al., 2012), and furthermore toxins can be present
in the intestine of healthy birds and/or be elaborated
post-mortem. For example, C. perfringens alpha toxin
was detected in the small intestine of some clinically
normal chickens (McCourt et al., 2005; Coursodon
et al., 2010). To further confound interpretation of
intestinal toxin levels, it is known that method of sto-
rage of the sample pre-testing also influences detect-
ability (Niilo, 1980; Rioseco et al., 2012). All of these
diagnostic challenges are considerations relevant to
diagnosis of NE in poultry, and furthermore, in the
case of poultry, it is not known yet which toxin
would be the relevant one to test for, and whether its
presence/absence would have diagnostic meaning.
Diagnosis of NE in poultry
For diagnosis of NE in poultry caused by C. perfringens,
identification of the characteristic intestinal lesions
together with cultural or immunohistochemical confir-
mation of the presence of C. perfringens is essential,
since other bacteria including other clostridial species
can produce lesions of virtually identical gross and his-
tologic appearance, for example, Clostridium colinum
(Songer & Uzal, 2013) and Clostridium sordellii
(Rimoldi et al., 2015). However, the recovery of
C. perfringens from the small intestine, even from an
abnormal small intestine, does not warrant a diagnosis
of NE (see below).
Significance of recovery of C. perfringens
from poultry small intestine
C. perfringens may be recovered from normal small
intestine and from intestine affected by diseases other
than NE, and even within an individual bird, these
are frequently of multiple types as shown by pulsed
field gel electrophoresis (PFGE) (Nauerby et al.,
2003). In a study of broiler chickens dead from diseases
other than NE, C. perfringens were recovered from the
small intestine of 56% of 91 birds (Smyth & Martin,
unpublished observation). Thus, isolation of
C. perfringens from the small intestine does not war-
rant a diagnosis of NE. However, the recovery of
C. perfringens together with the characteristic pathol-
ogy of NE, enables diagnosis.
Isolation of netB-positive strains of C. perfringens in
pure, or almost pure, culture from the small intestine of
chickens should raise strong suspicion of NE, and if no
NE lesions were detected by the prosector, the intestine
AVIAN PATHOLOGY 283
should be evaluated histologically. Demonstration of
clonality of the C. perfringens isolates should similarly
raise suspicion of NE.
Accurate recognition of lesions of NE
To be able to accurately recognize lesions of NE due to
C. perfringens, it is important to be familiar with the
appearance of the normal intestine in both freshly
dead poultry and poultry that have been dead for a
period of hours or days. The intestine is one of the
most difficult organs to interpret during a necropsy
examination due to the effects of autolysis, which can
present with many and varied appearances. At the
microscopical level, alterations due to autolysis are evi-
dent within minutes of death, causing interpretive dif-
ficulty, and may be misdiagnosed as pathological
change by the inexperienced.
Normal intestine, including autolytic and
artefactual changes
Macroscopical (gross) appearance: The normal intes-
tine of a freshly dead chicken or turkey is thick walled
by comparison to the intestine of similarly sized mam-
mals, and has good smooth muscle tone. In chickens
dissected immediately after death, the duodenum is
normally quite red in colour due to its rich blood
supply and this may be mistaken for pathology. This
red colouration rapidly dissipates, especially if samples
are harvested immediately after death. Smooth muscle
tone and contraction are apparent for some minutes
after death, such that intestine opened along its length
immediately after death will tend to evert, and may
even form a cylinder with the mucosal surface outer-
most. The mucosal surface of the intestine is pale
cream-pink, and is slightly texturized. It is soft,
especially in the duodenum, and it is easy to introduce
artefactual depressions and fissures with dissection
instruments. There are no circular or longitudinal
folds, rather the surface is even. There may be
occasional red and/or raised foci (0.5–1 cm) (Figure 1
(a)) which represent gut-associated lymphoid tissue
(GALT). The intestinal wall becomes thinner as the
time interval from death increases, due to autolysis,
and the accumulation of gas which distends and
stretches the wall. Smooth muscle tone is frequently
lost with increased time interval after death. The intes-
tine may acquire green discolouration due to decompo-
sition. This may have a patchy distribution.
Microscopical (histological) appearance: histological
examination shows that most of the thickness of the
avian intestine is due to a very well developed mucosal
layer. The avian intestine has very long villi compared
to those of the mammal (Figure 1(b)). The villi are
longest in the duodenum and become progressively
shorter moving distally along the intestine. In
284 J. A. SMYTH

Figure 1. (a) Longitudinally opened section of ileum from a normal broiler chicken. Good smooth muscle tone is evidenced by the
curling of the tissue at the edges. There is a raised reddened focus, which represents GALT. The edge of any such reddened focus
should always be carefully checked for evidence of necrosis, such as that seen later in this figure. (b) Longitudinal histological sec-
tion of proximal to mid-jejunum. The villi are very long, and account for most of the thickness of the intestinal wall. (c) Histological
section of duodenum. The surface epithelium of the villi is separating from the lamina propria, there are detached individualized
and clusters of epithelial cells and amorphous eosinophilic material (arrows) in the lumen. These changes are all the result of auto-
lysis; the sample was collected from a normal chicken within 5 to 10 minutes of euthanasia and fixed immediately. (d) Longitudin-
ally opened section of jejunum from a chicken with naturally occurring NE. Diffusely, the mucosa has a tan granular appearance
(necrosis); the necrotic tissue is firmly adherent. (e) Longitudinally opened section of jejunum, chicken. In some areas the mucosa
is very moist, soft and tan-coloured. There are also areas where this fibrinonecrotic material has sloughed from the surface exposing
a relatively smooth glistening surface, which histological evaluation showed was re-epithelialized. (f) Multifocal necrosis, ulceration
and mucosal sloughing. Necrotic mucosa is tan-discoloured (black arrows). The ulcers have a red base e.g. at the top right corner,
while re-epithelialized craters have a smooth glistening base (white arrow). Necrosis is evident at the margins of both the craters
and ulcers. (g) Histological section of jejunum, NE. The superficial part of the mucosa is markedly eosinophilic (coagulative necrosis)
and is separated from viable mucosa by an intense zone of heterophilic inflammation (arrows). The necrotic coagulum is coated by a
thick layer of bacteria (C. perfringens) (stained blue). (h) Histological section of a thin-walled area of jejunum. There is marked
reduction in villus length in the right half of the section, and complete loss of villi in the left half. Crypts are elongated.

longitudinal section, the villi have the classic thin slen-
der fingerlike profiles, but in transverse section, the
profiles are broader (reflecting the fact that the villi,
especially in the jejunum and ileum, are leaf-shaped),
and the surface epithelium may have a multilayered
appearance especially if the section has been cut
slightly tangentially. This multilayered appearance of
the epithelium is artefactual, and should not be inter-
preted as epithelial hyperplasia. While it is very easy
and therefore tempting to process transverse sections
for microscopical examination, longitudinal sections
are better.
The microscopical appearance of the intestine alters
very rapidly (within minutes) of death. Changes occur-
ring after death have been systematically studied in calves
(Pearson & Logan, 1978), sheep, rabbits and rats (Fell,
1961) and the present author confirms that virtually iden-
tical autolytic changes also occur in chickens and turkeys
(unpublished observations). The development of sube-
pithelial spaces were the earliest detectable changes, and
these were seen as early as three minutes after severance
of the carotid arteries of a disease challenged calf. It is
noteworthy that these changes occurred more quickly
in the disease challenged calf than in a healthy control
calf. Autolytic changes were most severe in the proximal
small intestine and were marked by seven minutes, and
included denuded villi, detached strips of epithelium
and dissociated epithelial cells. Figure 1(c) illustrates
autolytic change in chicken duodenum. Even where
mucosal biopsies are surgically collected (from humans),
artefactual separation of the villus surface epithelium
from the lamina propria or focally denuded epithelium
are not unusual findings, and are attributed to the biopsy
procedures (Gramlich & Petras, 2007). Similarly, rough
handling of intestine during post-mortem sampling can
accentuate the artefacts that will occur and fracture the
villi. The mucosal surface should not be touched during
collection procedures. Autolytic changes occur most
rapidly in the proximal small intestine, especially the duo-
denum; the distal small intestine appears to be more
resistant. In trying to interpret mucosal changes at the
microscopical level, lack of inflammatory change, for
example, fibrin accumulation or heterophilic infiltrate/
exudate are evidence that such changes are artefactual.
It is critically important to collect (and examine!) samples
from control birds collected and processed under exactly
the same conditions. It is this author’s experience that,
even using a standardized approach, there can be
between-bird variation in the degree of autolytic and arte-
factual change, so it is important that multiple control
birds are evaluated before drawing conclusions on the
findings in the test subjects.
Intestine with NE due to C. perfringens
The gross lesions of NE due to C. perfringens vary in
both extent and severity and may be focal, multifocal
AVIAN PATHOLOGY 285
to coalescing, or in severe cases may be diffuse, affect-
ing the entire small intestine. While lesions are most
commonly found in the proximal part of the jejunum
(between the distal end of the duodenum and Meckel’s
diverticulum), any part of the small intestine as well as
the caecum and/or colorectum may be affected. Necro-
tizing and fibrinonecrotizing lesions can be recognized
as tan discolouration of the mucosa (Figure 1(d–f)). In
some cases, the mucosa becomes thickened due to
build-up of adherent fibrin and necrotic debris, and
has a marked coarsely granular texture and may be
moderately firm and adherent (Figure 1(d)) or soft
and moist (Figure 1(e)). Ulcers may be recognized as
depressed foci with a roughened red exposed surface
(Figure 1(f)), and there may be some limited haemor-
rhage at the margins or in the lumen. In birds with
NE, all crater-like lesions are commonly classified as
ulcers, when in fact many of these represent areas of
mucosal thinning where necrotic mucosa that has
been underrun by new epithelium, has sloughed
(Figure 1(f)). Such areas are characterized by having
a smooth, often glistening, surface which is depressed
compared to the surrounding mucosa. Often the rim
of mucosa surrounding these re-epithelialized craters
and ulcers is necrotic, again evidenced by tan dis-
colouration (Figure 1(f)). Ulcers and re-epithelialized
craters are often visible from the serosal surface.
Sloughing of large areas of mucosa together with loss
of smooth muscle tone result in a thin flaccid intestinal
wall. Foul-smelling gas and sloughed necrotic material
may accumulate in the intestinal lumen. Frequently,
abnormally thick dark-green coloured bile discolours
the mucosal surface or luminal content in the duode-
num and proximal jejunum.
Birds dead from NE undergo rapid decomposition,
and this decomposition can mask the presence of
lesions, especially if there has been ante-mortem thin-
ning of the wall. Not only does the decomposition
make the intestine friable and difficult to open, it can
also cause necrotic mucosa to detach and fragment
very easily, and even to liquefy in some cases. This frag-
mented and or liquefying content may be mistaken for
food particles or chyme. Careful handling and close
inspection, including gentle washing to expose the
mucosal surface are often needed. If the prosector is
not vigilant, lesions may be overlooked.
Histologically, lesions of NE have a very character-
istic appearance, but as mentioned previously, this
appearance is not pathognomonic for C. perfringens;
other clostridia can produce very similar pathology.
Lesions can affect scattered single villi, or one or
more clusters of villi, or may affect all the villi in a sec-
tion. There is usually a variably thick superficial zone of
intensely eosinophilic necrotic material which is coated
by a thick mat of clostridia and which is sharply demar-
cated from healthy tissue by an intense zone of predo-
minantly heterophilic infiltrate (Figure 1(g)).
286 J. A. SMYTH
Sometimes necrotic lamina propria and bacteria are
found underlying relatively normal epithelium. Large
clumps of bacteria may also be found within the necro-
tic coagulum. The re-epithelialized craters previously
described at the gross level, are evident as areas with
markedly reduced villus height (Figure 1(h)) and
increased crypt depth.
Changes such as separation of epithelium from the
lamina propria, denuded villi, strips of detached epi-
thelial cells and dissociated cells must be interpreted
with caution (see the section describing histologic fea-
tures of autolysis and Figure 1(c), features which can be
erroneously interpreted as pathology and/or evidence
of NE).
Importance of accurate diagnosis of NE to
interpretation of research findings
As may be recognized from the preceding paragraphs,
misidentification of either other intestinal diseases or
autolytic change as NE, will lead to isolates wrongly
identified as “NE-associated C. perfringens strains”.
This author had acquired several “NE-associated
C. perfringens strains”, but not all of these produced
NE in a disease model (Smyth & Martin 2010). This
result was at first surprising and suggested complexities
in disease development not fully addressed by disease
modelling systems. However, examination of the netB
status of the isolates showed that the “NE-associated
C. perfringens strains” that produced disease in the
NE model were netB positive, while those that did
not produce disease in the NE model were netB nega-
tive. While this on one hand provided strong support
for the importance of netB to disease-producing ability,
the apparent recovery of netB negative isolates from
naturally occurring cases of disease does not support
an absolute requirement for netB in disease-producing
strains. However, the clear separation of outcomes
according to netB status was noteworthy, and
prompted the author to examine closely the history
of the netB negative NE-associated C. perfringens
strains. For one of the isolates, gross images and histo-
logic sections of the source bird were available for
examination, and were found to be consistent with a
diagnosis of coccidiosis due to Eimeria necatrix and
not NE, that is, there had been a misdiagnosis, and
therefore the isolate had been wrongly classified. This
finding, illustrates the importance of correct original
diagnosis to the conclusions that may be drawn from
research findings.
Site of sampling for isolation of causal
bacterium is critically important
C. perfringens are present in very large numbers in NE
lesions (Figure 1(g)) and the organism within the
lesions are usually of one clonal type (Nauerby et al.,
2003; Gholamiandekhordi et al., 2006), which may be
explained at least in part, by the recent finding that
netB-positive NE strains exclusively produce the bac-
teriocin, perfrin (Timbermont et al., 2014). However,
C. perfringens can also be recovered from the normal
small intestine, especially if the bird has been dead
for a while and there is time for post-mortem prolifer-
ation of the organisms. C. perfringens strains recovered
from individual birds that do not have NE are fre-
quently of multiple PFGE types (Nauerby et al.,
2003), and these strain types are usually different to
those found in NE lesions. Obviously, the intralesional
organisms are the ones that investigators need to iso-
late, so it is important to specifically swab lesions.
However, lesions may be focal or regional, and the
intestine is over 1 m long in broiler chickens over
two weeks of age, so if the lesions are not correctly
identified at the time of necropsy, it is entirely possible
that the swab site may be distant to the lesion and
therefore, that any isolates recovered may very well
not be those responsible for the lesions! This type of
sampling error may lead to results that are hard to
explain, for example some of the early conflicting
data with respect to the role of netB. Among the earlier
described netB negative isolates of C. perfringens
reported to have been recovered from cases of NE,
but which failed to cause NE in an experimental
model (Smyth & Martin 2010), was an isolate later dis-
covered to have been made from a chicken with focal
NE lesions that had only been detected during histo-
logical evaluation. Since the NE lesions were not recog-
nized at the time of necropsy, it is highly likely that the
isolate was recovered from a non-lesioned area and
therefore not a true “NE isolate”. This sort of sampling
error, among other causes, might explain some of the
other netB negative NE isolates that have been
recorded.
Concluding remarks
To reach an accurate diagnosis of NE or not-NE, diag-
nosticians and investigators must thoroughly inspect
the intestine for evidence of the characteristic lesions
and demonstrate the presence of C. perfringens in the
lesions. The main pitfalls to reaching the correct diag-
nosis are (1) erroneously interpreting normal features
and autolytic changes (especially at the microscopical
level) as NE pathology, (2) not performing a detailed
enough examination of the intestine especially in
birds where autolysis makes tissue handling difficult,
and overlooking NE pathology (3) misidentifying the
pathology of other diseases as NE (e.g. coccidiosis
due to E. necatrix and (4) diagnosing NE because
C. perfringens was present in small intestine. Such
errors can lead to isolates of C. perfringens being
wrongly identified as “NE strains” and/or “non-NE
strains” which will add difficulty to elucidating

virulence attributes. To reduce the difficulty in reach-
ing a correct diagnosis, birds should be examined and
sampled as soon as possible after death. If dead birds
cannot be examined within a few hours of discovery,
they should be refrigerated (not frozen) until they
can be examined.
Disclosure statement
No potential conflict of interest was reported by the author.
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