Avian Pathology

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

NetB and necrotic enteritis: the hole movable

story

Julian I. Rood, Anthony L. Keyburn & Robert J. Moore

To cite this article: Julian I. Rood, Anthony L. Keyburn & Robert J. Moore (2016) NetB
and necrotic enteritis: the hole movable story, Avian Pathology, 45:3, 295-301, DOI:
10.1080/03079457.2016.1158781

To link to this article: https://doi.org/10.1080/03079457.2016.1158781

Published online: 31 May 2016.

Submit your article to this journal

Article views: 3570

View related articles

View Crossmark data

Citing articles: 20 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=cavp20
AVIAN PATHOLOGY, 2016
VOL. 45, NO. 3, 295–301
http://dx.doi.org/10.1080/03079457.2016.1158781

REVIEW

NetB and necrotic enteritis: the hole movable story

Julian I. Rooda,b , Anthony L. Keyburnb,c and Robert J. Moorea,b,d
a
Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC,
Australia; bPoultry Cooperative Research Centre, University of New England, Armidale, NSW, Australia; cAustralian Animal Health Laboratory,
CSIRO, Geelong, VIC, Australia; dSchool of Science, RMIT University, Bundoora, VIC, Australia

ABSTRACT ARTICLE HISTORY

Clostridium perfringens is the primary causative agent of avian necrotic enteritis. Our Received 19 January 2016
understanding of the pathogenesis of this economically important disease has been Accepted 8 February 2016
enhanced by the discovery of C. perfringens NetB toxin, which belongs to the α-haemolysin
KEYWORDS
family of β-pore-forming toxins. In a chicken disease model, the analysis of an isogenic set of Clostridium perfringens;
strains comprising the wild type, a netB mutant, and its complemented derivative, fulfilled necrotic enteritis; NetB
molecular Koch’s postulates and revealed that NetB was essential for disease. These results toxin; vaccine; conjugative
were consistent with epidemiological surveys, which generally found that there was a higher plasmid
prevalence of netB carriage in C. perfringens isolates from diseased poultry compared to
healthy birds. The netB gene has been shown to be located on large conjugative plasmids
that are closely related to other toxin plasmids from C. perfringens, which has potential
implications for the epidemiology of necrotic enteritis infections. The crystal structures of
both monomeric NetB and the heptameric NetB pore have been determined, the latter
revealed a central pore diameter of approximately 26 Å. Finally, it has been shown that
vaccine preparations that include NetB can protect chickens against disease and a series of
single amino acid substitution derivatives of NetB that have potential value for vaccine
formulations have been isolated and analysed. It is likely that NetB will be an important
antigen to include in an effective, commercially viable, necrotic enteritis vaccine.

Introduction suffering from necrotic enteritis (Keyburn et al., 2008).

It was previously thought that α-toxin was the major
Carriage of the netB gene distinguishes virulent strains
toxin involved in necrotic enteritis (Al-Sheikhly &
of Clostridium perfringens that are capable of inducing
Truscott, 1977a, b; Fukata et al., 1988). Almost all
necrotic enteritis in poultry from strains that do not
C. perfringens strains produce α-toxin, a major extra-
cause this syndrome. NetB was discovered following
cellular toxin that has been shown to be essential for
the clear demonstration that α-toxin, previously
clostridial myonecrosis (Awad et al., 1995). Earlier
thought to be the principal virulence factor, was not
work indicated that secreted products from
essential for pathogenesis (Keyburn et al., 2006). A
C. perfringens were able to cause lesions typical of
search for an alternative toxin quickly identified NetB
necrotic enteritis in chickens and since α-toxin was
and subsequently genetic studies, biochemical and bio-
known to be a major secreted component it was
physical analysis, strain surveys and vaccination studies
inferred that it was responsible (Al-Sheikhly &
have revealed the importance of this toxin in the patho-
Truscott, 1977b). The limitation of this interpretation
genesis of necrotic enteritis. NetB toxin fulfils molecu-
is that it does not take into account other secreted
lar Koch’s postulates (Falkow, 1988) and therefore has
toxins that the bacteria may have produced. While
been demonstrated to be an essential virulence factor in
C. perfringens isolates are toxinotyped by the presence
the development of necrotic enteritis (Keyburn et al.,
of four major toxins (α, β, ε and ι toxins) various strains
2008). In this review, we will briefly describe the discov-
can also produce other so-called minor toxins such as
ery and initial analysis of NetB and subsequent studies
CPE, β2 toxin, perfringolysin O (θ-toxin) and collagen-
on its genetics, its structure and its potential role in the
ase (κ-toxin); the term minor toxin does not refer to the
next generation of necrotic enteritis vaccines.
importance or level of production of these toxins, but
rather to the fact that they are not part of the toxino-
typing scheme (Uzal et al., 2014). In addition, spon-
Identification and analysis of NetB
taneous α-toxin mutants of a virulent avian
NetB was discovered in an Australian strain of C. perfringens isolate were found to have lost their abil-
C. perfringens type A that was isolated from a chicken ity to cause disease in a chicken challenge model

CONTACT Julian I. Rood julian.rood@monash.edu

© 2016 Houghton Trust Ltd
296 J. I. ROOD ET AL.
(Thompson et al., 2006). While these data suggested
that α-toxin may be important for virulence the
authors noted that additional mutations could have
occurred and therefore a clear link between α-toxin
and virulence could not be established. No comple-
mentation studies were done to clarify the role of α-
toxin. Another inconsistency in the assumptions
regarding the role of α-toxin in necrotic enteritis is
the extent of heterophil, lymphocyte and plasma cell
infiltration in infected tissues (Al-Sheikhly & Truscott,
1977b; Shane et al., 1985; Gazdzinski & Julian, 1992).
In clostridial myonecrosis (gas gangrene), a disease pri-
marily mediated by the α-toxin of C. perfringens, there
is marked leukostasis and lack of inflammatory infil-
trate in tissues infected by C. perfringens cells (Stevens
et al., 1997; Flores-Diaz & Alape-Giron, 2003). In
necrotic enteritis, there is typically an extensive
immune cell infiltration, indicating quite a different
mechanism of pathogenesis compared to classical α-
toxin-induced disease. Finally, we showed that a
defined α-toxin null mutant of the chicken necrotic
enteritis strain EHE-NE18 was still virulent in a
chicken necrotic enteritis disease model (Keyburn
et al., 2006). In that study, the α-toxin null mutants
were capable of causing as much disease as the isogenic
wild-type strain, indicating that α-toxin is not an essen-
tial virulence factor in avian necrotic enteritis.
Subsequently, we used a dual biochemical and geno-
mics approach to identify a new toxin that is almost
exclusively found in virulent avian C. perfringens iso-
lates (Keyburn et al., 2008). Culture supernatants
from the previous EHE-NE18-derived α-toxin mutant
were tested against various mammalian and chicken
cell lines to determine whether any novel cytotoxic fac-
tors could be identified. Chicken primary hepatocellu-
lar carcinoma epithelial Leghorn male hepatoma
(LMH) cells were found to be sensitive to an unknown
protein in the supernatant of both the α-toxin null
mutant and the wild-type strain, indicating that the
cytopathic effect was not due to α-toxin. Consequently,
LMH cytotoxicity was used to follow this toxic activity
during the purification process. At the same time, we
determined the sequence of the parent EHE-NE18 gen-
ome. The N-terminal sequence of the purified toxin
protein was determined and used to search the genome
sequence. Bioinformatic analysis identified a putative
toxin gene that encoded a 323 amino acid protein,
including a 30 amino acid secretion signal sequence.
Since this protein had similarity to C. perfringens β-
toxin (38% identity), it was designated necrotic enteri-
tis toxin, β-like, NetB (Keyburn et al., 2008).
The role of NetB in disease was determined by con-
structing a structural gene (netB) mutant in strain
EHE-NE18 and assessing its virulence in a chicken dis-
ease model (Keyburn et al., 2008). Virulence testing of
an isogenic series of strains consisting of the wild-type,
the netB mutant, and the netB mutant complemented
with the wild-type netB gene, revealed that the develop-
ment of necrotic enteritis in chickens was dependent on
the ability to produce functional NetB. Importantly, the
gross lesions observed in the bird model were consistent
with necrotic enteritis lesions previously reported from
experimental and field cases of the disease (Helmboldt
& Bryant, 1971; Long et al., 1974; Keyburn et al., 2006).
Epidemiology of netB carriage
Surveys of C. perfringens isolates from chickens (Table
1) have generally found a higher prevalence of netB
carriage in strains from diseased birds compared to
strains from healthy birds, although a Danish survey
found a higher netB incidence in isolates from healthy
birds (Abildgaard et al., 2010). The isolation of netB-
positive strains from healthy birds indicates that simple
infection is not sufficient to induce disease. The need
for predisposing factors to be present in order for dis-
ease to develop is well recognized (Williams, 2005; Sho-
jadoost et al., 2012; Moore, 2016). The common
finding that a proportion of C. perfringens strains iso-
lated from diseased birds do not carry the netB gene
has sometimes been suggested to indicate that NetB
is not an essential virulence factor or that other factors
can cause virulence in its absence (Drigo et al., 2009;
Martin & Smyth, 2009). The validity of this conclusion
cannot be ruled out in all cases, but when netB-negative
strains have been tested for virulence, they have not
been found to reproduce disease whereas netB-positive
strains can readily induce disease (Keyburn et al., 2010;
Smyth & Martin, 2010). These findings are consistent
across a range of strains using different disease induc-
tion models. It is likely that the netB-negative strains
isolated from diseased birds are either pathogenic
strains that have lost the NetB plasmid during isolation
and culturing or are non-pathogenic strains that co-
infect diseased birds. There is one published report of
disease caused by experimental inoculation of netB-
negative strains (Cooper & Songer, 2010), but the
interpretation of this outcome is uncertain as it is not
clear whether the causative bacteria were re-isolated
from the lesions and tested for the presence of netB,
an essential step since disease may have been caused
by other environmentally derived strains. Within our
laboratory, we have always found that in the rare
instances where lesions are found in birds inoculated
with netB-negative strains, the strains isolated from
these lesions always carry the netB gene. Presumably,
the lesions are caused by pre-existing infections, bac-
teria carried by the birds into the facility or new strains
introduced in feed or from other contaminated sources.
The movable story
Sequence analysis of seven avian necrotic enteritis iso-
lates revealed the presence of three loci that were

Table 1. Prevalence of netB in C. perfringens toxinotype A isolates from the gastrointestinal tract of chickens.
Necrotic enteritis-diseased chickens
Origin netB incidencea
% netB positive
Australia 31/44 70
Canada 39/41 95
Denmark 12/25 48
Iran 19/36 53
Italy 16/30 53
Netherlands 43/45b
96
Sweden 31/34 91
USA 17/20 85
USA 7/12 58
a
Number of isolates netB positive/number of isolates tested.
b
Toxinotype C strains were also found; 108 of 115 isolates carried the netB gene. Not all isolates were from birds with overt necrotic enteritis.
c
Isolates from healthy birds were not investigated.
d
The number of healthy isolates tested was not given.
conserved and appeared to be preferentially associated
with C. perfringens isolates from cases of necrotic
enteritis (Lepp et al., 2010). The major plasmid-deter-
mined locus, the 42-kb NELoc-1 region, included the
netB gene and was located on an ca. 85-kb plasmid
that also carried known C. perfringens conjugation
genes. This study, and subsequent studies in our lab-
oratories (Bannam et al., 2011), revealed the complex-
ity of the plasmid profiles of these strains, which was
consistent with that of other C. perfringens strains
from animals (Li et al., 2013). Five of the North Amer-
ican necrotic enteritis isolates were examined in detail
and shown to carry from two to five large (45–90 kb)
plasmids, most of which carried the C. perfringens
transfer clostridial plasmid (tcp) conjugation locus
(Lepp et al., 2010). Although no conjugation studies
were carried out in this study, these data suggested
that all of these plasmids, including the NetB plasmids,
were conjugative. Direct evidence that these plasmids
were conjugative was obtained from detailed studies
(Bannam et al., 2011) on the Australian isolate EHE-
NE18, the strain from which NetB originally was ident-
ified (Keyburn et al., 2008).
The complexity of the plasmid profiles in these
strains, with individual strains carrying multiple plas-
mids with large regions of almost identical sequences,
created problems for next-generation sequencing
approaches to the determination of the sequences of
these plasmids (Lepp et al., 2010; Bannam et al.,
2011; Parreira et al., 2012). Subsequently, a combi-
nation of bacterial genetics and bioinformatics
approaches was used to determine the individual
sequences of the plasmids in strain EHE-NE18. The
results showed that it carried three large plasmids,
each of which was independently conjugative and car-
ried the tcp locus (Bannam et al., 2011). In this strain,
NetB was encoded on an 82-kb plasmid, pJIR3535,
which was closely related to the 70-kb β2-toxin-encod-
ing plasmid pJIR3844 and the 49-kb tetracycline resist-
ance plasmid pJIR3537 from the same strain.
Subsequent studies involved the determination of the
complete sequence of another NetB plasmid, pNETB-
NE10 (81.7 kb), which had 99.1% identity to
AVIAN PATHOLOGY 297
Healthy chickens
netB incidencea % netB positive Reference
2/55 4 Keyburn et al. (2010)
7/20 35 Chalmers et al. (2008)
14/23 61 Abildgaard et al. (2010)
0/43 0 Tolooe et al. (2011)
4/22 18 Drigo et al. (2009)
c
Allaart et al. (2012)
d
25 Johansson et al. (2010)
10/31 32 Hibberd et al. (2011)
6/79 8 Martin and Smyth (2009)
pJIR3535; PCR analysis revealed that the NELoc-1
region from 11 NetB plasmids was highly conserved
(Parreira et al., 2012).
The conjugation results have significant impli-
cations for the epidemiology of necrotic enteritis.
They imply that exogenous C. perfringens strains that
carry a NetB plasmid do not need to be able to colonize
the avian gastrointestinal tract to lead to a disease out-
break. The conjugative transfer of the NetB plasmid to
an endogenous C. perfringens strain that has already
colonized may be sufficient to cause disease under
the appropriate predisposing conditions, provided
that relevant chromosomal genes are present. A similar
scenario has been suggested for C. perfringens entero-
toxin-dependent non-foodborne outbreaks of diar-
rhoea in humans (Brynestad et al., 2001).
The hole story
At least 35% of the known protein toxins produced by
bacteria belong to the pore-forming group of mem-
brane damaging toxins (Alouf, 2001). These toxins
form pores that disrupt the phospholipid membrane
bilayer of both human and animal cells, causing
changes to host signal transduction pathways and to
an influx of ions (i.e. Na+, Cl—, Ca2+, etc.) that may
eventually lead to osmotic cell lysis. Many of these tox-
ins have been demonstrated to contribute to bacterial
virulence and to play a key role in the pathogenesis
of human and animal infections (Los et al., 2013;
Popoff, 2014).
NetB induced morphological changes in LMH cells,
resulting in significant rounding and cell lysis. These
morphological changes and subsequent cellular lactate
dehydrogenase release were able to be blocked by poly-
ethylene glycol (PEG)1000 and PEG1500, indicating
that these changes were caused by the toxin-dependent
formation of pores in the plasma membrane. Osmotic
stabilizers such as PEG can inhibit the lysis of target
cells if they are unable to pass through the pore gener-
ated by a pore-forming toxin (Ballard et al., 1993).
Based on the estimated Stokes radii of various PEG
molecules (Scherrer & Gerhardt, 1971), the results
298 J. I. ROOD ET AL.
Figure 1. The crystal structure of NetB. (a) Structure of the NetB monomer. Reproduced from (Uzal et al., 2014) with permission
from Future Microbiology as agreed by Future Medicine Ltd. (b) Structure of the NetB pore. The β-sandwich domain is shown
in green, the rim domain in blue and the stem domain in yellow. Reproduced from Savva et al. (2013) with permission of the
authors.
suggested that NetB formed a hydrophilic pore with a
functional diameter of 1.6–1.8 nm in the cell mem-
brane. Subsequent determination of the crystal struc-
ture of the NetB pore complex revealed an internal
pore diameter of ca. 26 Å (Savva et al., 2013), which
was consistent with this initial finding.
The structure of the NetB monomer has been deter-
mined to a resolution of 1.8 Å (Yan et al., 2013), which
revealed that it had the β-sandwich, latch, rim and pre-
stem domains that are typical of proteins that belong to
the α-haemolysin family of β-pore-forming toxins
(Figure 1(a)). It has an almost identical structure to
δ-toxin from C. perfringens despite the fact that these
toxins have very different amino acid sequences.
NetB has a four amino acid deletion in the rim domain,
in a region that in other β-pore-forming toxins is
responsible for lipid binding, suggesting that NetB
has a different cell surface target. Finally, analysis of
the ability of NetB to form pores in planar phospholi-
pid bilayers revealed that the NetB pore channels have
a preference for cations over anions (Yan et al., 2013).
The structure of the NetB pore, without the first 20
N-terminal amino acids, has been solved to a resol-
ution of 3.9 Å after solubilization and purification of
the complex from lipid vesicles (Savva et al., 2013).
Like the monomer, the pore structure shows similarity
to staphylococcal α-haemolysin (Figure 1(b)). It com-
prises seven NetB monomers in a ring structure, with
the predicted hydrophobic transmembrane domain
spanning residues I121 to V146 (numbering from the
N-terminal amino acid of the secreted toxin). Lipo-
some studies showed that oligomerization of NetB
was enhanced by cholesterol, which was postulated to
play an important role in pore formation.
Both site-directed mutagenesis and random muta-
genesis have been used to isolate single amino acid sub-
stitution derivatives of NetB that have reduced biological
activity (Savva et al., 2013; Yan et al., 2013; Fernandes da
Costa et al., 2014). Mutations were obtained in the rim
domain, presumably affecting membrane binding, and
in the β-sandwich domain, affecting oligomerization.
These derivatives shed light on NetB structure–function
relationships and are likely to be invaluable for sub-
sequent studies that utilize NetB derivatives for the
development of new necrotic enteritis vaccines.
Vaccination studies demonstrate the
protective efficacy of NetB
Several groups have shown that vaccination with NetB
induces an immune response that delivers a degree of
protection from the development of necrotic enteritis.
Protection has been shown both in directly vaccinated
birds (Jang et al., 2012; Fernandes da Costa et al.,
2013, 2016; Keyburn et al., 2013b; Jiang et al., 2015)
and in chicks derived from vaccinated hens (Keyburn
et al., 2013a). It is difficult to compare these studies as
they each used different vaccination schedules, different
challenge models and different scoring procedures, but
in general it appears that protection levels of 70–80%
can be achieved using recombinant NetB, native NetB,
or toxoids and bacterin vaccines containing NetB at
levels sufficient to induce strong immune responses.

Previous attempts to vaccinate against necrotic
enteritis have used a variety of other recombinant anti-
gens (Kulkarni et al., 2008; Zekarias et al., 2008; Cooper
et al., 2009; Jiang et al., 2009; Jang et al., 2012), attenu-
ated strains (Thompson et al., 2006) and conventional
clostridial toxoid vaccines (Lovland et al., 2004; Crouch
et al., 2010; Lanckriet et al., 2010) and have produced
varying degrees of protection. The challenge for necro-
tic enteritis vaccine development is to provide an effi-
cacious vaccine that is safe, affordable and is
compatible with the general management practises
applied to broiler flocks. It appears that NetB will be
an important antigen to include in an effective com-
mercially viable vaccine.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Research in our laboratories was supported by grants from
the Australian Research Council and the Poultry Coopera-
tive Research Centre, established and supported under the
Australian Government’s Cooperative Research Centres
Program.
ORCID
Julian I. Rood http://orcid.org/0000-0003-2126-7209
Robert J. Moore http://orcid.org/0000-0002-0776-
5861
References
Abildgaard, L., Sondergaard, T.E., Engberg, R.M., Schramm,
A. & Hojberg, O. (2010). In vitro production of necrotic
enteritis toxin B, NetB, by netB-positive and netB-negative
Clostridium perfringens originating from healthy and dis-
eased broiler chickens. Veterinary Microbiology, 144, 231–
235.
Allaart, J.G., de Bruijn, N.D., van Asten, A.J.A.M., Fabri, T.H.
F. & Gröne, A. (2012). NetB-producing and beta2-produ-
cing Clostridium perfringens associated with subclinical
necrotic enteritis in laying hens in the Netherlands.
Avian Pathology, 41, 541–546.
Alouf, J.E. (2001). Pore-forming bacterial protein toxins: an
overview. Current Topics in Microbiology and
Immunology, 257, 1–14.
Al-Sheikhly, F. & Truscott, R.B. (1977a). The pathology of
necrotic enteritis of chickens following infusion of broth
cultures of Clostridium perfringens into the duodenum.
Avian Diseases, 21, 230–240.
Al-Sheikhly, F. & Truscott, R.B. (1977b). The pathology of
necrotic enteritis of chickens following infusion of crude
toxins of Clostridium perfringens into the duodenum.
Avian Diseases, 21, 241–255.
Awad, M.M., Bryant, A.E., Stevens, D.L. & Rood, J.I. (1995).
Virulence studies on chromosomal alpha-toxin and theta-
toxin mutants constructed by allelic exchange provide
genetic evidence for the essential role of alpha-toxin in
AVIAN PATHOLOGY 299
Clostridium perfringens-mediated gas gangrene.
Molecular Microbiology, 15, 191–202.
Ballard, J., Sokolov, Y., Yuan, W.L., Kagan, B.L. & Tweten, R.
K. (1993). Activation and mechanism of Clostridium sep-
ticum alpha toxin. Molecular Microbiology, 10, 627–634.
Bannam, T.L., Yan, X.X., Harrison, P.F., Seemann, T.,
Keyburn, A.L., Stubenrauch, C., Weeramantri, L.H.,
Cheung, J.K., McClane, B.A., Boyce, J.D., Moore, R.J. &
Rood, J.I. (2011). Necrotic enteritis-derived Clostridium
perfringens strain with three closely related independently
conjugative toxin and antibiotic resistance plasmids.
mBio, 2, e00190–11.
Brynestad, S., Sarker, M.R., McClane, B.A., Granum, P.E. &
Rood, J.I. (2001). Enterotoxin plasmid from Clostridium
perfringens is conjugative. Infection and Immunity, 69,
3483–3487.
Chalmers, G., Bruce, H.L., Hunter, D.B., Parreira, V.R.,
Kulkarni, R.R., Jiang, Y.-F., Prescott, J.F. & Boerlin, P.
(2008). Multilocus sequence typing analysis of
Clostridium perfringens isolates from necrotic enteritis
outbreaks in broiler chicken populations. Journal of
Clinical Microbiology, 46, 3957–3964.
Cooper, K.K. & Songer, J.G. (2010). Virulence of Clostridium
perfringens in an experimental model of poultry necrotic
enteritis. Veterinary Microbiology, 142, 323–328.
Cooper, K.K., Trinh, H.T. & Songer, J.G. (2009). Immunization
with recombinant alpha toxin partially protects broiler
chicks against experimental challenge with Clostridium per-
fringens. Veterinary Microbiology, 133, 92–97.
Crouch, C.F., Withanage, G.S.K., Haas, V.d., Etoré, F. &
Francis, M.J. (2010). Safety and efficacy of a maternal vac-
cine for the passive protection of broiler chicks against
necrotic enteritis. Avian Pathology, 39, 489–497.
Drigo, I., Agnoletti, F., Bacchin, C., Guolo, A., Cocchi, M.,
Bonci, M. & Bano, L. (2009). Diffusion of Clostridium per-
fringens NetB positive strains in healthy and diseased
chickens. Italian Journal of Animal Science, 8, 761–764.
Falkow, S. (1988). Molecular Koch’s postulates applied to
microbial pathogenicity. Clinical Infectious Diseases, 10
(Suppl. 2), S274–S276.
Fernandes da Costa, S.P., Mot, D., Bokori-Brown, M., Savva,
C.G., Basak, A.K., Van Immerseel, F. & Titball, R.W.
(2013). Protection against avian necrotic enteritis after
immunisation with NetB genetic or formaldehyde toxoids.
Vaccine, 31, 4003–4008.
Fernandes da Costa, S.P., Mot, D., Geeraerrts, S., Bokori-
Brown, M., Van Immerseel, F. & Titball, R.W. (2016).
Variable protection against experimental broiler necrotic
enteritis after immunization with the C-terminal fragment
of Clostridium perfringens alpha-toxin and a non-toxic
NetB variant. Avian Pathology, 45, 381–388.
Fernandes da Costa, S.P., Savva, C.G., Bokori-Brown, M.,
Naylor, C.E., Moss, D.S., Basak, A.K. & Titball, R.W.
(2014). Identification of a key residue for oligomerisation
and pore-formation of Clostridium perfringens NetB.
Toxins, 6, 1049–1061.
Flores-Diaz, M. & Alape-Giron, A. (2003). Role of
Clostridium perfringens phospholipase C in the pathogen-
esis of gas gangrene. Toxicon, 42, 979–986.
Fukata, T., Hadate, Y., Baba, E., Uemura, T. & Arakawa, A.
(1988). Influence of Clostridium perfringens and its toxin in
germ-free chickens. Research in Veterinary Science, 44, 68–70.
Gazdzinski, P. & Julian, R.J. (1992). Necrotic enteritis in tur-
keys. Avian Diseases, 36, 792–798.
Helmboldt, C.F. & Bryant, E.S. (1971). The pathology of
necrotic enteritis in domestic fowl. Avian Diseases, 15,
775–780.
300 J. I. ROOD ET AL.
Hibberd, M.C., Neumann, A.P., Rehberger, T.G. & Siragusa,
G.R. (2011). Multilocus sequence typing subtypes of poul-
try Clostridium perfringens isolates demonstrate disease
niche partitioning. Journal of Clinical Microbiology, 49,
1556–1567.
Jang, S.I., Lillehoj, H.S., Lee, S.H., Lee, K.W., Lillehoj, E.P.,
Hong, Y.H., An, D.J., Jeong, W., Chun, J.E., Bertrand, F.,
Dupuis, L., Deville, S. & Arous, J.B. (2012). Vaccination
with Clostridium perfringens recombinant proteins in
combination with Montanide ISA 71 VG adjuvant
increases protection against experimental necrotic enteri-
tis in commercial broiler chickens. Vaccine, 30, 5401–
5406.
Jiang, Y., Mo, H., Willingham, C., Wang, S., Park, J-y., Kong,
W., Roland, K.L. & Curtiss, R. (2015). Protection against
necrotic enteritis in broiler chickens by regulated delayed
lysis Salmonella vaccines. Avian Diseases, 59, 475–485.
Jiang, Y.-F., Kulkarni, R.R., Parreira, V.R. & Prescott, J.F.
(2009). Immunization of broiler chickens against
Clostridium perfringens-induced necrotic enteritis using
purified recombinant immunogenic proteins. Avian
Diseases, 53, 409–415.
Johansson, A., Aspán, A., Kaldhusdal, M. & Engström, B.E.
(2010). Genetic diversity and prevalence of netB in
Clostridium perfringens isolated from a broiler flock
affected by mild necrotic enteritis. Veterinary
Microbiology, 144, 87–92.
Keyburn, A.L., Boyce, J.D., Vaz, P., Bannam, T.L., Ford, M.
E., Parker, D., Di Rubbo, A., Rood, J.I. & Moore, R.J.
(2008). NetB, a new toxin that is associated with avian
necrotic enteritis caused by Clostridium perfringens.
PLoS Pathogens, 4, e26.
Keyburn, A.L., Portela, R.W., Ford, M.E., Bannam, T.L., Yan,
X.X., Rood, J.I. & Moore, R.J. (2013a). Maternal immuniz-
ation with vaccines containing recombinant NetB toxin
partially protects progeny chickens from necrotic enteri-
tis. Veterinary Research, 44, 108.
Keyburn, A.L., Portela, R.W., Sproat, K., Ford, M.E.,
Bannam, T.L., Yan, X., Rood, J.I. & Moore, R.J. (2013b).
Vaccination with recombinant NetB toxin partially pro-
tects broiler chickens from necrotic enteritis. Veterinary
Research, 44, 54.
Keyburn, A.L., Sheedy, S.A., Ford, M.E., Williamson, M.M.,
Awad, M.M., Rood, J.I. & Moore, R.J. (2006). Alpha-
toxin of Clostridium perfringens is not an essential viru-
lence factor in necrotic enteritis in chickens. Infection
and Immunity, 74, 6496–6500.
Keyburn, A.L., Yan, X.X., Bannam, T.L., Van Immerseel, F.,
Rood, J.I. & Moore, R.J. (2010). Association between avian
necrotic enteritis and Clostridium perfringens strains
expressing NetB toxin. Veterinary Research, 41, 21.
Kulkarni, R.R., Parreira, V.R., Sharif, S. & Prescott, J.F.
(2008). Oral immunization of broiler chickens against
necrotic enteritis with an attenuated Salmonella vaccine
vector expressing Clostridium perfringens antigens.
Vaccine, 26, 4194–4203.
Lanckriet, A., Timbermont, L., Eeckhaut, V., Haesebrouck,
F., Ducatelle, R. & Van Immerseel, F. (2010). Variable
protection after vaccination of broiler chickens against
necrotic enteritis using supernatants of different
Clostridium perfringens strains. Vaccine, 28, 5920–5923.
Lepp, D., Roxas, B., Parreira, V.R., Marri, P.R., Rosey, E.L.,
Gong, J., Songer, J.G., Vedantam, G. & Prescott, J.F.
(2010). Identification of novel pathogenicity loci in
Clostridium perfringens strains that cause avian necrotic
enteritis. PLoS One, 5, e10795.
Li, J., Adams, V., Bannam, T.L., Miyamoto, K., Garcia, J.P.,
Uzal, F.A., Rood, J.I. & McClane, B.A. (2013). Toxin plas-
mids of Clostridium perfringens. Microbiology and
Molecular Biology Reviews, 77, 208–233.
Long, J.R., Pettit, J.R. & Barnum, D.A. (1974). Necrotic enteritis
in broiler chickens. II. Pathology and proposed pathogenesis.
Canadian Journal of Comparative Medicine, 38, 467–474.
Los, F.C., Randis, T.M., Aroian, R.V. & Ratner, A.J. (2013).
Role of pore-forming toxins in bacterial infectious dis-
eases. Microbiology and Molecular Biology Reviews, 77,
173–207.
Lovland, A., Kaldhusdal, M., Redhead, K., Skjerve, E. &
Lillehaug, A. (2004). Maternal vaccination against subcli-
nical necrotic enteritis in broilers. Avian Pathology, 33,
81–90.
Martin, T.G. & Smyth, J.A. (2009). Prevalence of netB among
some clinical isolates of Clostridium perfringens from ani-
mals in the United States. Veterinary Microbiology, 136,
202–205.
Moore, R.J. (2016). Necrotic enteritis predisposing factors in
broiler chickens. Avian Pathology, 45, 275–281.
Parreira, V.R., Costa, M., Eikmeyer, F., Blom, J. & Prescott, J.F.
(2012). Sequence of two plasmids from Clostridium perfrin-
gens chicken necrotic enteritis isolates and comparison with
C. perfringens conjugative plasmids. PLoS One, 7, e49753.
Popoff, M.R. (2014). Clostridial pore-forming toxins: power-
ful virulence factors. Anaerobe, 30, 220–238.
Savva, C.G., Fernandes da Costa, S.P., Bokori-Brown, M.,
Naylor, C.E., Cole, A.R., Moss, D.S., Titball, R.W. & Basak,
A.K. (2013). Molecular architecture and functional analysis
of NetB, a pore-forming toxin from Clostridium perfringens.
Journal of Biological Chemistry, 288, 3512–3522.
Scherrer, R. & Gerhardt, P. (1971). Molecular sieving by the
Bacillus megaterium cell wall and protoplast. Journal of
Bacteriology, 107, 718–735.
Shane, S.M., Gyimah, J.E., Harrington, K.S. & Snider, T.G.
3rd. (1985). Etiology and pathogenesis of necrotic enteri-
tis. Veterinary Research Communications, 9, 269–287.
Shojadoost, B., Vince, A.R. & Prescott, J.F. (2012). The suc-
cessful experimental induction of necrotic enteritis in
chickens by Clostridium perfringens: a critical review.
Veterinary Research, 43, 74.
Smyth, J.A. & Martin, T.G. (2010). Disease producing capa-
bility of netB positive isolates of C. perfringens recovered
from normal chickens and a cow, and netB positive and
negative isolates from chickens with necrotic enteritis.
Veterinary Microbiology, 146, 76–84.
Stevens, D.L., Tweten, R., Awad, M.M., Rood, J.I. & Bryant,
A.E. (1997). Clostridial gas gangrene: evidence that α and
θ toxins differentially modulate the immune response and
induce acute tissue necrosis. Journal of Infectious Diseases,
176, 189–195.
Thompson, D.R., Parreira, V.R., Kulkarni, R.R. & Prescott, J.
F. (2006). Live attenuated vaccine-based control of necro-
tic enteritis of broiler chickens. Veterinary Microbiology,
113, 25–34.
Tolooe, A., Shojadoost, B., Peighambar, S.M. & Tamaddon,
Y. (2011). Prevalence of netB gene among Clostridium per-
fringens isolates obtained from healthy and diseased chick-
ens. Journal of Animal and Veterinary Advances, 10, 106–
110.
Uzal, F.A., Freedman, J.C., Shrestha, A., Theoret, J.R., Garcia,
J., Awad, M.M., Adams, V., Moore, R.J., Rood, J.I. &
McClane, B.A. (2014). Towards an understanding of the
role of Clostridium perfringens toxins in human and ani-
mal disease. Future Microbiology, 9, 361–377.

Williams, R.B. (2005). Intercurrent coccidiosis and necrotic
enteritis of chickens: rational, integrated disease manage-
ment by maintenance of gut integrity. Avian Pathology,
34, 159–180.
Yan, X., Porter, C.J., Hardy, S.P., Steer, D., Smith, A.I.,
Quinsey, N.S., Hughes, V., Cheung, J.K., Keyburn, A.L.,
Kaldhusdal, M., Moore, R.J., Bannam, T.L., Whisstock, J.
C. & Rood, J.I. (2013). Structural and functional analysis
AVIAN PATHOLOGY 301
of the pore-forming toxin NetB from Clostridium perfrin-
gens. mBio, 4, e00019–e00013.
Zekarias, B., Mo, H. & Curtiss, R. (2008). Recombinant atte-
nuated Salmonella enterica serovar Typhimurium expres-
sing the carboxy-terminal domain of alpha toxin from
Clostridium perfringens induces protective responses
against necrotic enteritis in chickens. Clinical and
Vaccine Immunology, 15, 805–816.