Immunization with recombinant alpha toxin partially protects
broiler chicks against experimental challenge with
K.K. Cooper, H.T. Trinh, J. Glenn Songer*
Department of Veterinary Science and Microbiology, University of Arizona,
1117 E. Lowell Street, Tucson, AZ 85721, United States
Received 9 April 2008; received in revised form 29 May 2008; accepted 3 June 2008
Necrotic enteritis (NE) in poultry has re-emerged as a concern for poultry producers, due in part to banning, by many
countries, of the use of antimicrobial growth promoters in feeds. This re-emergence has led to a search for alternative methods
for control of the disease, particularly vaccination. The objective of this work was to determine if vaccination of broiler chicks
with recombinant alpha toxin protected against experimental challenge. Broiler chicks werevaccinated subcutaneously at 5 and
15 days of age, followed 10 days later by challenge with Clostridium perfringens. Birds were challenged twice daily on 4
consecutive days by mixing C. perfringens cultures with feed (three parts culture: four parts feed). Non-vaccinated birds
challenged with C. perfringens developed NE at the rate of 87.8%, while only 54.9% of vaccinated birds developed lesions. In
addition, non-vaccinatedbirds hadlesionscores averaging2.37, while average scores invaccinated birds were1.35. Vaccination
produced an antibody response, with post-vaccination anti-alpha toxin IgG (IgY) titers in vaccinated birds more than 5-fold
greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers >15-fold higher than those
in non-vaccinated birds. These results suggest that alpha toxin may serve as an effective immunogen, and, as such, may play a
role in pathogenesis.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Clostridium perfringens; Poultry; Alpha toxin; Necrotic enteritis; Vaccination
Prevention of food animal diseases has commonly
been accomplishedbyadministrationof antimicrobials
in feed or water. However, there are rising concerns
about effects on human health of such routine use of
antimicrobials, particularly as this pertains to the
transfer of antimicrobial resistance from animal
pathogens to human pathogens (Hampson and
Murdoch, 2003). The European Union has banned
many antimicrobial growth promoters (AGPs), and the
bans have beenaccompanied byincreasedincidenceof
Available online at www.sciencedirect.com
Veterinary Microbiology 133 (2009) 92–97
* Corresponding author. Tel.: +1 520 621 2962;
fax: +1 520 621 6366.
E-mail address: firstname.lastname@example.org (J.G. Songer).
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
poultry necrotic enteritis (NE) (Casewell et al., 2003;
Lovland et al., 2003; Van Immerseel et al., 2004). The
annual cost of necrotic enteritis to the poultry industry
worldwide is estimated at US$ 2 billion (McReynolds
et al., 2004).
NE is most commonly caused by Clostridium
perfringens type A (Al-Sheikhly and Truscott, 1977;
Kaldhusdal et al., 2001; Lovland et al., 2004). The
only so-called major toxin produced by toxinotype A
is alpha toxin (CPA) (Hofshagen and Stenwig, 1992;
Nauerby et al., 2003; Songer, 1996; Yoo et al., 1997).
There has been considerable sentiment, although
relatively little evidence, that CPA plays an important
role in pathogenesis of NE. CPA induces mucosal
damage in chicken intestinal loops (Fernandez
Miyakawa and Uzal, 2005); crude toxin preparations
reportedly produce lesions compatible with NE in
germ-free chickens, an effect which is neutralized by
anti-CPA serum (Fukata et al., 1988; Lovland et al.,
2004). Recently, Keyburn et al. (2006) reported that
conventional birds challenged with a CPA mutant
developed lesions typical of NE, and they concluded
that CPA is not involved in pathogenesis.
A few investigators have examined the role of anti-
CPA immunity in protection against NE. Vaccination
of broiler breeder hens with type A toxoids yielded
anti-CPA antibodies which were passed to progeny
and yielded partial protection against subclinical NE
(Lovland et al., 2004). In another study, birds
inoculated with a CPA mutant were subsequently
protected against challenge with a virulent isolate
(Thompson et al., 2006). The goal of the work
described here was to investigate the value of
recombinant CPA (rCPA) as a parenteral immunogen
chicks with HIS-tagged rCPA provides partial
protection against experimental challenge.
2. Methods and materials
2.1. Birds and care
Commercial jumbo Cornish ? Rock female broiler
chicks were obtained as 1-day old hatchlings from
Murray McMurrayHatchery (WebsterCity,IA,USA).
Birds were housed in 1.5 m diameter brooders
constructed of 3 mm thick pegboard and divided into
three equal parts. Commercial wood shavings pro-
vided as bedding were replaced at 2-week intervals.
Birds were fed a commercial chick starter ration
(20% protein, Eagle Milling, Casa Grande, AZ, USA)
throughexperimental day 7.Thereafter,birdswere fed
a high protein feed (28% protein) mixed 50:50 with
menhaden fishmeal (SeaLac, Omega Protein, Hous-
ton,TX, USA).On day 24, feed was withheld for20 h,
and birds were then challenged with C. perfringens.
Water was available ab lib from galvanized steel
2.2. Production of histidine-tagged CPA and
The entire cpa gene was cloned via standard
CA, USA), generating plasmid pJGS211. This
construct was used to transform E. coli DH5a,
yielding strain JGS2445. This strain expressed cpa in
its entirety, producing CPA with an N-terminal
hexahistidine tag (HIS-CPA). JGS2445 was cultivated
in 50 ml of LB broth with 100 mg ampicillin per ml,
incubated with shaking (200 rpm) at 37 8C overnight.
LB overnight culture was diluted 1–20 in 500 ml LB
broth with ampicillin (100 mg/ml) and, incubated with
shaking (200 rpm) at 37 8C to an OD600of 0.6. It was
then induced with 2.5 ml of isopropyl-beta-D-thioga-
lactopyranoside (IPTG, Gold Biotechnology, St.
Louis, MO, USA; 500 mM) and incubated for a
further 3 h, after which cells were harvested by
centrifugation (16,270 ? g, 10 min) and re-suspended
in binding buffer [20 mM Tris, 100 mM NaCl (pH
8.0)]. Cells were disrupted in a French pressure cell
(26,890 ? g, 15 min). DNase (160 U, Promega,
Madison, WI, USA) was added to the supernatant
fluid (?40 ml) and incubated at 37 8C for 15 min, then
placed on ice. Recombinant CPAwas then purified on
TALON resin (Clontech, Mountain View, CA, USA),
according to the manufacturer’s instructions. Bound
proteins were eluted with imidazole (50 mM, pH 8.0)
fraction was electrophoresed in a 10% SDS-PAGE gel
to assess purity of the HIS-CPA, which appeared as a
band at ?48 kDa. Protein concentrations were
determined by Bradford assay (Pierce, Rockford,
K.K. Cooper et al./Veterinary Microbiology 133 (2009) 92–9793
Birds were vaccinated subcutaneously (SQ) with
20 mg of HIS-CPA, emulsified 1:1 (w/v) with a
proprietary Quil A-based adjuvant
BioLabs, Lincoln, NE, USA). Vaccinations were
administered at 5 and 15 days of age. No local or
general side effects were observed in any of the
vaccinated birds in any of the studies. Serum was
collected from birds on arrival, prior to challenge (at
24 days of age), and at necropsy (on day 29).
2.3. Challenge inoculums and protocol
C. perfringens type A challenge strain JGS4143
(netB+; Keyburn etal., 2008), obtainedfrom a chicken
with NE, was stored at ?80 8C in 50% glycerol: 50%
brain heart infusion (BHI; Difco, Detroit, MI, USA).
For inoculum preparation, JGS4143 was streaked for
isolation on BHI agar with 5% citrated bovine blood.
After incubation under anaerobic conditions (5% H2:
5%CO2:90%N2)at37 8Cfor24 h,1–2colonieswere
transferred into 10 ml cooked meat medium (CMM;
Difco) in a Hungate tube and incubated in the same
atmosphere at 37 8C for 18 h. The resulting culture
was used to inoculate 100 ml fluid thioglycollate
broth (FTG; Difco), 10 ml of which was used to
inoculate 100 ml CMM. Each culture was incubated
for 18 h at 37 8C prior to passage. Thirty-three
millilitres of the CMM culturewere usedas inoculum
for 1 l FTG medium. After 18 h incubation, this FTG
serially passed culture was prepared for each
challenge feeding(totaln = 8). Sub-culturingmethod
used for preparing inoculums has shown to be most
effective at reproducing disease, and studies in our
laboratory to simplify method have failed to produce
determined by plating serial 10-fold dilutions on
Birds were inoculated on days 25–28. High protein
feed and FTG medium culturewere mixed in a ratio of
3:4 (v/v). The mixture, which had a paste-like
consistency, was then placed in galvanized steel feed
trays and offered to birds. Trays were cleaned and any
remaining feed disposed of prior to each subsequent
feeding. Water was available ab lib throughout the
challenge period. Negative control birds were chal-
lenged with uninoculated FTG mixed with high
protein feed at the same ratio.
On day 29, birds were euthanized by CO2
asphyxiation. Necrotic intestinal lesions were scored
(0: no gross lesions; 1+: thin-walled or friable small
intestine; 2+: focal necrosis or ulceration; 3+: large
patches of necrosis; 4+: severe or extensive necrosis
typical of field cases) and segments fixed in 10%
buffered formalin for histological examination.
2.4. Detection of anti-CPA antibodies
Wells of microtitration plates (Immulon 4 HBX
microtiter plates; Thermo Scientific, Waltham, MA,
USA) were coated with HIS-CPA by addition of
100 ml of 0.06 M carbonate buffer (0.018 M Na2CO3:
0.045 M NaHCO3, pH 9.6) containing 1 ng antigen.
Plates were incubated overnight at 4 8C and washed
twice with phosphate buffered saline (pH 7.2) with
0.3% Tween 20 (ICN Biomedicals, Solon, OH, USA)
(PBS-T). Bovine serum albumin (BSA; 1%) in PBS-T
(200 ml) was then added to each well and the plate
incubated for 1 h at 4 8C. Plates were again washed
with PBS-T, and then 100 ml of 2-fold serial dilutions
(1:10–1:10,240) of test sera added. After incubation
for 1 h at 37 8C, plates were washed nine times with
PBS-T, 100 ml of goat anti-chicken IgG horseradish
peroxidase conjugate (Kirkegaard and Perry Labora-
tories (KPL), Gaithersburg, MD, USA; diluted
1:8000) were added, and plates were incubated 1 h
at 37 8C. After washing six times with PBS-T, 100 ml
of o-phenylenediamine (OPD) substrate (200 mg/ml)
(Sigma–Aldrich, St. Louis, MO, USA) was added and
the plate incubated for 30 min at room temperature in
the dark. The reaction was stopped with 50 ml of 3 M
HCl and color development allowed to continue for an
additional 15 min. Results were read spectrophoto-
metrically at 490 nm. End-point ELISA titers were
an OD490? 0.4. All assays were performed in
duplicate, and any assays performed at different times
with one dilution factor variation were repeated to
2.5. Statistical analysis
A Pearson Chi-square test was used for statistical
comparison of rates of lesion development, rates of
severe lesions and mortality across treatment groups.
K.K. Cooper et al./Veterinary Microbiology 133 (2009) 92–9794
A two-way ANOVA was used to examine differences
in average lesion scores between groups.
2.6. Institutional Animal Care and Use Committee
Studies presented here were pre-reviewed and
approved by the University of Arizona IACUC, under
protocol number 02-204.
Parenteral immunization with HIS-rCPA partially
(Table 1). Differences in the percent of birds with
gross lesions were statistically significant across
treatment groups (p < 0.0001), and the percent of
significantly different across all groups (p < 0.0095).
The differences between average lesion scores for all
three groupswere also
(p < 0.0001). The mortality rate in adjuvant only
group was higher than in vaccinates, but the difference
was not statistically significant (data not shown).
Birdshadhigh averagetiters ofanti-CPA antibodies
at hatching (average titer: 120.5 ? 3.3) (Table 2), but
these maternal antibodies decreased in titer during the
studies, as demonstrated by titers in negative (non-
vaccinated, non-challenged: 19.9 ? 2.5) and positive
controls (adjuvant only, challenged: 11.7 ? 3.7) at 29
days of age. In contrast, titers in vaccinated birds were
5-fold higher (post-vaccination,
(183.8 ? 4.5),
(305.5 ? 5.7) compared to the adjuvant only birds.
at necropsyand 15-foldhigher
Protection against poultry NE by vaccination with
CPA toxoids has been controversial. On the one hand,
Lovland et al. (2004) vaccinated hens with crude
‘‘type A’’ or ‘‘type C’’ toxoids (containing CPA) and
progeny were protected against subclinical NE.
However, this provides little information about the
role of an anti-CPA response in protection; these
toxoids contained many other antigens against which
the birds may have produced a protective immune
response. On the other hand, birds inoculated with a
CPA mutant were protected against subsequent
K.K. Cooper et al./Veterinary Microbiology 133 (2009) 92–97 95
Response of vaccinated birds and controls to challenge with Clostridium perfringens
Treatment Number of birds
with severe lesions
(score 3 or 4)a,b
Number of birds
with gross lesions
rCPA vaccination, challenged
Adjuvant only, challenged
1.31 ? 0.17
2.37 ? 0.18
0.00 ? 0.00
aComposite of three independent studies.
bAverage of 16 birds per group/study.
Anti-CPA antibody response of birds following vaccination with recombinant CPA and challenge with C. perfringens
Hatching (d = 1) Post-vaccination (d = 24) Post-challenge (d = 29)
Adjuvant only, challenged
120.5 ? 3.3 (n = 22)183.8 ? 4.5 (n = 5)
34.8 ? 1.4 (n = 5)
17.4 ? 1.8 (n = 5)
305.5 ? 5.7 (n = 28)
11.7 ? 3.7 (n = 17)
19.9 ? 2.5 (n = 33)
End-pointELISAtitersare reportedasthereciprocalofthelastdilutionwhichhadanOD490of?0.400.Vaccinationswere administered at5and
15 days of age.
challenge with a virulent isolate (Thompson et al.,
2006). Furthermore, birds challenged with a cpa-
mutant NE strain, containing an 890 base pair deletion
in cpa, developed disease at a rate and severity
comparable to that in birds challenged with the parent
strain (Keyburn et al., 2006). Thus, the role of CPA in
pathogenesis of and immunity to NE has been called
The results from this study suggest that anti-CPA
immunity provides at least partial protection against
supports the findings of others (Kulkarni et al., 2007).
They identified CPA as an immunogenic protein
secreted by chicken-virulent C. perfringens isolates.
Broilers immunized intramuscularly with histidine-
taggedrecombinant CPA and boosted with active toxin
were significantly protected againsta severe challenge.
The relatively low degree of protection in our
experimental trials may relate to differences in severity
of challenge, and may translate into better protection
against natural challenge in the field. Better responses
of immunogen, or alternate routes of delivery. In this
study, birds were vaccinated SQ, which typically
generates a strong IgG (IgY) response. IgG (IgY) has a
key role in the immune response to NE in broiler
chickens (Lovland et al., 2003, 2004), and for this
reason, we did not examine the IgA response of the
birds. Stimulationof a strong IgA responseby mucosal
immunization might provide better protection.
Maternal antibodies may have interfered with the
immune response to the vaccine. This study used
commercial birds, as specific pathogen free (SPF)
birds are not free of C. perfringens. Thus, colonization
of hens by C. perfringens likely generated maternal
anti-CPA antibodies that were passed into yolk, as
evidenced, by high anti-CPA titers in newly hatched
chicks. These maternal antibodies diminished at
different rates, as shown by varying anti-CPA titers
in the adjuvant only and non-vaccinated groups at the
post-vaccination and post-challenge stages. Interfer-
ence of maternal antibodies with vaccination might
explain the minimal 5-fold increase in anti-CPA titers
in vaccinates, as compared to birds in the adjuvant
only group post-vaccination. Further vaccine studies
with broiler chicks having low or no maternal anti-
CPA titers might increase vaccine effectiveness and
protection against experimental challenge.
It remains to rationalize the results of this work
with those of Keyburn et al. (2006), in which CPAwas
suggested to be an unnecessary attribute in pathogen-
esis of NE. It may be that other attributes are required
for establishment of infection and initiation of lesion
development, and that CPA adds to the severity of
In conclusion, vaccination of broiler chickens with
recombinant CPA stimulates the production of anti-
partial protection against experimental challenge with
pathogenesis of NE in poultry, but the extent of that
role needs further investigation.
The authors thank James Theoret and Jeremy
Coombs for technical assistance with the experimental
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