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Vaccine 30 (2012) 4460–4464
Contents lists available at SciVerse ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Plant expressed Coccidial antigens as potential vaccine candidates in protecting
chicken against Coccidiosis
Kota Sathisha
, Rajan Sriramana
, B. Mohana Subramaniana
, N. Hanumantha Raoa
, Balaji Kasaa
,
Jagan Donikenia
, M. Lakshmi Narasub
, V.A. Srinivasana,∗
a
Research & Development Centre, Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Andhra Pradesh, India
b
Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpalli, Hyderabad 500072, Andhra Pradesh, India
a r t i c l e i n f o
Article history:
Received 10 March 2012
Received in revised form 14 April 2012
Accepted 21 April 2012
Available online 1 May 2012
Keywords:
Plant expressed EtMIC1 and EtMIC2
Subunit vaccine
Coccidial antigens
Eimeria microneme proteins
a b s t r a c t
Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present
study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion
proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent
and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective
efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better
serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and
EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-␥ response was not
significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results
indicate the potential use of these antigens as vaccine candidates.
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Coccidiosis is a parasitic disease caused to the chicken by proto-
zoan of genus Eimeria. The pathologic effects of virulent infection
of the chicken coccidia vary according to the species, ranging from
mild enteritis to severe hemorrhage and death [1]. Despite the use
of coccidio-stats in the continuous medication programs, the eco-
nomic loss due to coccidiosis remains high [2]. Ubiquitous use of
anticoccidial drugs has also led to the development of resistance
to anticoccidial drugs [3,4]. The European Parliament and Council
decided to phase out the anti-coccidial drugs by 2012 [5]. Vacci-
nations could offer excellent alternatives to drugs as a means of
controlling coccidiosis. Efforts to develop various types of vaccines
have been relentless over the past several decades [6–8]. Recom-
binant Eimeria encoded proteins have been expressed in various
host systems and their efficacy upon in-ovo immunization for pro-
tection against Eimeria infections determined [9–11] using virulent
challenge.
The use of microbial fermentation or cell culture systems for
production of recombinant proteins has limitations such as cost,
scalability and safety that have prompted research into alterna-
tives. Plants allow the cost-effective production of recombinant
proteins on large scale, while eliminating risks of product con-
tamination with human pathogens [12]. A number of target
∗ Corresponding author. Tel.: +91 40 23000211; fax: +91 40 2300 5958.
E-mail address: srini@indimmune.com (V.A. Srinivasan).
antigens expressed in plants were used as subunit vaccine candi-
dates [13–15]. The efficacy studies on out-bred chicken population
by administering plant produced Eimeria tenella microneme-2
(EtMIC2) protein as vaccine was reported earlier [11]. In the present
study, we have evaluated the protective efficacy of plant expressed
EtMIC1 as monovalent vaccine as well as a formulation in combi-
nation with EtMIC2 against homologous challenge in chickens.
2. Material and methods
2.1. Chicken
White Leghorn day-old, coccidiosis free, male layer chickens
(commercial breed-BV 300), were obtained from Sri Venkateswara
Hatcheries (Hyderabad, India) and reared in clean brooder cages.
2.2. Coccidial oocysts
Wild type E. tenella oocysts were isolated from an Eimeria
infected farm in India. Oocysts were propagated in 3 weeks old birds
by repeated passages [16]. Purity of oocyst suspension was assessed
by species-specific nested-PCR for ribosomal Internal Transcribed
Spacer I (ITS-I) region as described by us earlier [17].
2.3. Tobacco plant
Nicotiana tabacum, cultivar Petit Havana SR1, was cultivated in
the greenhouse. Leaves from 3 to 6 weeks old plants were used for
vacuum infiltration.
0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.04.076

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K. Sathish et al. / Vaccine 30 (2012) 4460–4464 4461
Table 1
Treatment groups.
Group Immunogen (␮g/bird) Immunization (days) Bleeding (days) Splenocyte isolation (days)
Group I (n = 34) EtMIC1 (80) 0, 7, 14, 21 0, 7, 14, 21, 28 24, 27, 30, 33
Group II (n = 14) EtMIC1 (80) + EtMIC2 (50) 0, 7, 14, 21 0, 7, 14, 21, 28 24, 27, 30, 33
Group III (n = 14) PBS 0, 7, 14, 21 0, 7, 14, 21, 28 Not done
Group IV (n = 22) PBS 0, 7, 14, 21 0, 7, 14, 21, 28 24, 27, 30, 33
2.4. Cloning of EtMIC1 gene into plant expression vector
2.4.1. Cloning
E. tenella Microneme-1 (EtMIC1) gene was amplified from a
plasmid clone containing the full-length gene sequence of EtMIC1
[16] using Proofstart polymerase (Qiagen, USA) and the following
primers.
EtMIC1 forward primer – 5 ATCGCCATGGAATGGCGCCCCTTC-
CTCGGCG 3 ,
EtMIC1 reverse primer – 5 GCGGCCGCGGATGCCCACATCTCT-
GATTGTT 3 .
The amplified product was cloned into plant expression vector,
pTRA ERH, downstream of the double 35S promoter using NcoI and
NotI restriction enzyme sites.
2.4.2. Methods of Agrobacterium transformation
Transient expression, qualitative assessment of m-RNA tran-
script using RT-PCR, and protein purification from infiltrated leaves
were performed as described previously [11].
2.5. SDS-PAGE analysis and immunoblotting
Purified recombinant EtMIC1 protein was resolved on SDS-
PAGE; the protein was also electro-blotted on to PVDF membrane
(Hybond-P; GE-Healthcare, USA). The blots were probed using
either one of the following antibodies. (1) Anti-His5 monoclonal
antibody conjugated to Horse-Radish peroxidase (1:3000 dilu-
tion; Qiagen, Germany); (2) rabbit polyclonal sera reactive against
EtMIC1 protein; (3) chicken immune sera obtained by immuniz-
ing E. coli expressed recombinant EtMIC1 protein. Un-infiltrated
tobacco leaves were used as negative control, while E. coli expressed
recombinant EtMIC1 [16] proteins were used as positive control.
2.6. Immunization and efficacy study
The immune efficacy study consisted of four treatment groups.
Birds in Group I were immunized with 80 ␮g of EtMIC1 protein as
monovalent vaccine. Birds in Group II were immunized with 80 ␮g
of EtMIC1 and 50 ␮g EtMIC2 in combination as bivalent vaccine.
Whereas, Groups III and IV were sham-immunized with phosphate
buffered saline (PBS). Antigens were administered via intramus-
cular route in thigh muscle. The immunization schedule consisted
of a primary dose adjuvanted with Freund’s complete adjuvant on
7 days old birds and two booster doses adjuvanted with Freund’s
incomplete adjuvant on 14th and 21st days. Number of birds in
each group and treatment details are listed in Table 1.
2.6.1. Humoral immune response
Birds were bled prior to each immunization and on 28th day
post primary immunization. Serum antibody titers against immu-
nized proteins were measured using an indirect ELISA using E. coli
expressed recombinant EtMIC1 and EtMIC2 proteins to assess spe-
cific antibody titers.
2.6.2. Evaluation of cell-mediated immune response in EtMIC1
immunized birds
Relative IFN-␥ expression was quantified as fold increase in
IFN-␥ mRNA with respect to uninduced naïve birds. Spleens were
collected after euthanizing birds on 3rd, 6th and 9th days post final
immunization and on 3rd day post challenge. Five birds immu-
nized with plant expressed EtMIC1 were splenectomized on each
day of sampling. Splenocytes from each bird were cultured sepa-
rately as described previously [11]. The splenocytes obtained from
the sham-immunized birds were employed as negative control. The
splenocytes were stimulated using 20 ␮g/ml of recombinant E. coli
expressed EtMIC1 protein at the time of seeding. Twenty micro-
gram per milliliter of E. coli expressed Heat shock protein (HSP) was
used for mock stimulation while 15 ␮g/ml of concanavalin A (Genie,
India) was used as positive control for the evaluation of IFN-␥
response in the splenocytes. Primers and TaqMan probes to quan-
tify the IFN␥ mRNA and 28S rRNA in the samples were designed as
described previously [11]. Total RNA extraction from splenocytes,
sequence of primers, probes and the method of Real Time PCR were
described previously [11]. The Ct value of 28S was used to normal-
ize the Ct value of IFN-␥ ( Ct) as described by Leutenegger et al.
[18]. The Ct value of sham-induced cells from each spleen was
used as calibrator for other stimulated cells from the same spleen to
calculate the value of Ct ( Ct = Cttarget − Ctcalibrator) using
the comparative Ct method [19]. Statistical analysis was performed
using the OriginPro (version-7.5) software and the difference in
mean was subjected to a Student’s t-test.
2.6.3. Challenge experiment
Nine days post final immunization; birds of Groups I–III were
inoculated orally with 10,000 sporulated E. tenella oocysts. Birds
were weighed prior to challenge and on 7th and 11th days post
challenge to determine the weight gain. The average weight gain
(percent) of birds and oocyst shed per gram of fecal matter was
determined [16]. An average of three counts per group was taken to
enumerate reduction in oocyst shedding. The percentage decrease
in oocyst output compared with the sham-immunized but chal-
lenged birds was estimated [16].
3. Results
3.1. Recombinant Agrobacterium clones containing EtMIC1
expression plasmid
A 2.1 kb size EtMIC1 gene was obtained by amplifying with gene
specific primers. Cloning of EtMIC1 gene into pTRA-ERH vector was
confirmed by observing insert release upon digestion with NcoI
and NotI restriction enzymes. Recombinant agrobacterium clones
were also screened using PCR with gene specific primers for EtMIC1
sequence, agrobacterium clones harboring EtMIC1 gene produced
a PCR amplification product of 2.1 kb size (data not shown).
3.2. Transient expression of EtMIC1 protein in Nicotiana tabacum
using Agro-infiltration technique
The full-length EtMIC1 coding sequence was amplified from the
total RNA extracted from the infiltrated leaves. The amplification

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4462 K. Sathish et al. / Vaccine 30 (2012) 4460–4464
Fig. 1. (a) SDS–PAGE gel stained with coomassie blue showing EtMIC1 protein band at 100 kDa, (b) immunoblot demonstrating plant expressed EtMIC1 protein band showing
at 100 kDa, (i) specific reactivity against Anti-His5 HRPO conjugate monoclonal antibody. (ii) reactivity with polyclonal sera raised against E. coli expressed EtMIC1 protein
in chickens. (iii) Reactivity with peptide raised sera against EtMIC1 protein. Lanes 1, 2, and 5 are with plant expressed proteins, lanes 3, and 6 are with un-infiltrated leaves
taken as negative controls and lanes 4 and 7 are with pre-stained marker (Fermentas).
reactions performed using RNA extracted from mock-infiltrated
leaves and the reverse transcriptase negative control did not pro-
duce any PCR amplification (Supplementary Fig. 1), indicating
specific amplification of EtMIC1 from the RNA samples. Upon con-
firming the presence of specific mRNA, infiltrated leaf samples
were processed to purify the His6 tagged recombinant protein.
The yield of the purified protein was found to be 25 mg/kg fresh
biomass. The purified recombinant EtMIC1 protein was character-
ized using immunoblotting by probing the affinity-purified protein
with either rabbit polyclonal antibodies against EtMIC1 protein or
anti-His5 monoclonal antibody. A protein band of approximately
100 kDa, corresponding to the expected size of EtMIC1 protein,
was detected in all the immuno-blots (Fig. 1). Protein extract from
tobacco leaves infiltrated with un-transformed agrobacterium was
used as negative control in all the above blots, which showed no
reactivity to the antibodies used.
Supplementary material related to this article found, in the
online version, at http://dx.doi.org/10.1016/j.vaccine.2012.04.076.
3.3. Serum antibody response in immunized birds
The serum samples collected from the immunized birds on days
14, 21 and 28 were analyzed for the presence of the serum IgG anti-
bodies. EtMIC1 and EtMIC2 expressed in E. coli was used to measure
specific immune response in the immunized birds. Recombinant
EtMIC1 protein induced serum IgG titers in immunized birds and
exhibited an average serum antibody titers of 466.7 (±321.5) on
14th day, 541.6 (±469) on 21st day and 1131.3 (±920.8) on 28th
day. Birds immunized in combination had induced an average
serum antibody titer of 150 (±53.4) on 14th day, 500 (±185.1) on
21st day and 1700 (±667.6) on 28th day against EtMIC1 and 162.5
(±51.7) on 14th day, 525 (±237.5) on 21st day and 1900 (±848.5)
on 28th day against EtMIC2 (Fig. 2).
3.4. Evaluation of cell-mediated immune response in EtMIC1
immunized birds
The linear fit of the Ct curve obtained for IFN-␥ and 28S at
different dilutions of the RNA had a slope value of −0.0045. The
near zero slope values indicates similar PCR amplification efficiency
of IFN-␥ and 28S mRNA therefore relative quantification method
of 2− Ct was validated for comparing the mRNA expression lev-
els using 28S as internal control. There was an average increase
Fig. 2. ELISA titers (mean ± SD) of sera from birds immunized with plant expressed
EtMIC1 as monovalent vaccine and EtMIC1 and EtMIC2 in combination as bivalent
vaccine. The assay was performed using indirect ELISA in a maxisorp plate coated
with E. coli expressed EtMIC1 or EtMIC2 protein. Antibody titers in the serum were
determined as maximum sera dilution showing OD450 greater than mean + 3 × SD
of pre-immune sera (N = 14).
in IFN-␥ mRNA expression levels in the in vitro induced spleno-
cytes cultured from spleens of birds on 3rd day post-immunization
(Fig. 3). IFN-␥ m-RNA could not be detected in the induced spleno-
cytes.
3.5. Bird challenge experiment
3.5.1. Weight gain
Birds in Groups I, II, and III were challenged with 10,000 vir-
ulent E. tenella oocysts. Weight gain was assessed on 7 and 11
days post-challenge (dpc). The percentage increase in weight in the
immunized birds compared to sham immunized–challenged birds
(Group III). It was observed that the birds immunized with EtMIC1
alone had 32% (±16) and 52% (±24) increase in weight on 7 and
11 dpc respectively. Birds immunized with combined EtMIC1 and
EtMIC2 vaccine had 34% (±6.9) and 58% (±5.9) increase in weight
gain on 7 and 11 dpc. The difference in the mean weight gain was
subjected to Student’s t-test. The mean weight gain was signifi-
cantly different in all immunized birds on 7 dpc when compared
with sham-immunized and challenged group (p < 0.05*; N = 14)
(Fig. 4).

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K. Sathish et al. / Vaccine 30 (2012) 4460–4464 4463
Fig. 3. IFN-␥ levels quantified using real time RT-PCR among EtMIC1 vaccinated
birds compared to unstimulated birds on day 3 post immunization (N = 5). Mean
value for the group is indicated by a line.
Fig. 4. Percentage change in weight gain of immunized birds compared to sham-
immunized unchallenged birds. Weight gain was calculated on 7th and 11th day
after challenge. Mean value for the group is indicated by a line. The asterisks indicate
significant change in weight gain compared to sham-immunized and challenged
birds (*p < 0.05; **p < 0.01; N = 14).
3.5.2. Oocyst count
The decrease in oocyst output was calculated by comparing the
oocyst output of sham-immunized challenged birds (Group III).
Immunization of birds with EtMIC1 protein reduced the oocyst out-
put by 68% and in combination oocyst shedding was reduced by 72%
(Fig. 5). Our results indicated that immunization of birds with the
plant expressed recombinant proteins imparted significant protec-
tion to chicken against homologous challenge.
4. Discussion
Coccidiosis vaccines could offer a promising alternative to drugs
as a means of controlling coccidiosis. Numerous vaccination strate-
gies have been attempted to manage avian coccidiosis [16,20–22].
Several recombinant sub unit coccidial antigens have been used
Fig. 5. Oocyst output in immunized birds compared to mock immunized and
challenged group. The oocyst shedding per gram of faces was determined using
McMaster counting chamber. The bar represents an average of 3 counts per group.
There was more than 60% reduction in oocyst output in the vaccinated groups com-
pared to sham-immunized challenged birds.
in experimental immunizations with varying degree of success
[16,23].
Sporozoites are the invading stage of the parasite, which har-
bors microneme organelles located at the apical tip. Several types
of microneme proteins are secreted by sporozoite that play critical
role in host invasion. Recombinant microneme antigens expressed
in various expression systems have been shown to protect the
chicken against virulent challenge when used as vaccine candidate
[16]. Our earlier work suggested that plant expressed EtMIC2 pro-
tein imparts protection against homologous challenge in chicken
[11]. We have assessed the protective efficacy of plant expressed
EtMIC1 protein, which was administered either as monovalent for-
mulation or in bivalent formulation with plant expressed EtMIC2
protein.
The utility of combination of two E. tenella microneme proteins
as potential vaccine candidate has not been explored so far. For
the protozoan disease, it is important to develop multivalent vac-
cine to prevent immune evasion. Most of the microneme proteins
are known to be associated with cell invasion or parasite motility
upon attachment to the host cell. The multiplicity of these inva-
sion proteins may indicate alternate invasion strategies adopted
by the parasite. Therefore, neutralizing single major protein associ-
ated with invasion/motility may not protect birds against a virulent
pathogen. Our results indicate that birds immunized with a biva-
lent formulation containing EtMIC1 and EtMIC2 imparted better
protection to the birds.
Plant expressed EtMIC1 was able to induce high serum anti-
body response in immunized birds. We observed that the serum
antibody titers were higher for EtMIC2 protein [11] compared to
EtMIC1 protein on 14th, 21st, and 28th days post-immunization.
Birds immunized with bivalent vaccine had shown better anti-
body response against both the antigens compared to the birds
immunized with either EtMIC1 or EtMIC2 as monovalent vaccine
indicating co-operativity between the two antigens. There was an
average increase of IFN-␥ response in birds immunized with EtMIC1
alone on 3rd day post-immunization, while no CMI response was
obtained on 6th, 9th day post-immunization and 3rd day post chal-
lenge. Birds immunized with EtMIC2 alone [11] showed better CMI
response when compared to the birds immunized with EtMIC1
alone. We do not yet know whether EtMIC1 harbors effective T-cell
epitopes as indicated by poor CMI response in EtMIC1 immunized
birds. Given that EtMIC1 immunized birds had better weight gain

6. Author's personal copy
4464 K. Sathish et al. / Vaccine 30 (2012) 4460–4464
and oocyst reduction, our data suggests predominance of humoral
immune response in conferring protective immunity in birds.
Birds immunized with vaccine formulation containing both
EtMIC1 and EtMIC2 showed increased weight gain compared to the
birds immunized either with EtMIC1 or EtMIC2 separately [11]. The
un-immunized birds challenged with sporulated oocysts showed
marginal increase in weight. This phenomenon is not unusual given
the fact that the disease is self-limiting in nature. It would be inter-
esting to see if the combined vaccine formulation is also able to
impart protection to the immunized birds in heterologous chal-
lenge. We found that the increase in percentage weight gain was
significant on 7th and 11th days post-challenge in immunized birds
compared to the sham-immunized challenged birds. We observed
that there was reduction of up to 68% of oocyst output from birds
immunized with EtMIC1 protein, 66% from EtMIC2 protein [11]
and 72% from EtMIC1 and EtMIC2 proteins given in combination in
comparison with unimmunized challenged birds. Clearly the per-
centage reduction in oocyst output was more in birds immunized
with bivalent vaccine compared to the birds immunized with either
EtMIC1 or EtMIC2 alone [11]. Reduced oocysts output would help
reducing the disease burden on farm by lowering the number of
birds exposed to the pathogen. Moreover, the sub-lethal dose of
oocyst ingestion may help in establishing immunity in the birds.
The serum antibody titers were high in birds immunized with
EtMIC2 [11], while percentage increase in weight and reduction
in oocyst output was higher in EtMIC1 vaccinated birds. As dis-
cussed previously humoral immune response in Eimeria infected
bird seems to be important in conferring protection.
In conclusion, plant expressed coccidial antigens were immuno-
genic and conferred protection against challenge with live oocysts.
Vaccination with live or attenuated parasites to control coccidiosis
has limitations such as difficulties in large-scale manufacturing and
reversion to virulent form. Plants have the ability to express simul-
taneously several heterologous genes under control conditions,
which may help to develop an effective multivalent vaccine for
coccidiosis, thus offering an economically viable alternative to the
conventional protein expression platforms. Our results are encour-
aging and hold promise in developing a cost effective sub-unit
vaccine. Further work is required to optimize the dosage regimen
and the antigen payloads and formulation that are most efficacious.
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