Parasitol Res (2014) 113:41514161

DOI 10.1007/s00436-014-4087-2

ORIGINAL PAPER

Preparation and initial application of monoclonal antibodies

that recognize Eimeria tenella microneme proteins 1 and 2
Qing Liu & Zhengtao Chen & Wenyan Shi & Hui Sun &
Jie Zhang & Hongmei Li & Yihong Xiao & Fangkun Wang &
Xiaomin Zhao

Received: 26 January 2014 / Accepted: 18 August 2014 / Published online: 28 August 2014
# Springer-Verlag Berlin Heidelberg 2014

Abstract Microneme proteins (MICs) of Eimeria species are

critical for motility of the parasite, identification and binding
of host cell-surface proteins, invasion of host cells, and intracellular survival. The microneme protein 1 (EtMIC1) and 2
(EtMIC2) from Eimeria tenella have a putative function in
parasite adhesion to the host cell to initiate an invasion process. Previous studies indicated that the EtMIC1 and EtMIC2
proteins form a complex that play roles during attachment to
and penetration of the host cell. Numerous studies demonstrated that both the EtMIC1 and EtMIC2 are important
microneme proteins which are abundantly expressed in sporozoites and schizogony stages. But the expression of EtMIC1
and EtMIC2 in the gametogony stage is unknown. To investigate the precise roles of EtMIC1 and EtMIC2 in hostparasite interactions and expressions in the gametogony stage
of E. tenella, we generated five mouse monoclonal antibodies
(MAbs) which recognize the EtMIC1 and EtMIC2 proteins
and investigated expressions of EtMIC1 and EtMIC2 proteins
in later endogenous developmental stages, particularly
Qing Liu and Zhengtao Chen contributed equally to this work.
Q. Liu : Z. Chen : W. Shi : H. Sun : J. Zhang : H. Li :
F. Wang (*) : X. Zhao (*)
Department of Preventive Veterinary Medicine, College of
Veterinary Medicine, Shandong Agricultural University, 61 Daizong
Street, Taian City, Shandong Province 271018, China
e-mail: wangfangkun1980@163.com
e-mail: xmzhao66@163.com
Q. Liu : Z. Chen : W. Shi : H. Sun : J. Zhang : H. Li : F. Wang :
X. Zhao
Shandong Provincial Key Laboratory of Animal Biotechnology and
Disease Control and Prevention, Shandong Agricultural University,
61 Daizong Street, Taian City, Shandong Province 271018, China
Y. Xiao
Department of Basic Veterinary Medicine, College of Veterinary
Medicine, Shandong Agricultural University, 61 Daizong Street,
Taian City, Shandong Province 271018, China

focused on the gametogony phase using the specific antiEtMIC1 and anti-EtMIC2 MAbs produced in this work. Our
results showed that both EtMIC1 and EtMIC2 proteins are
expressed in all developmental stages including the
gametogony stage. To our knowledge, this is the first report
that the EtMIC1 and EtMIC2 proteins are expressed in the
gametogony stage of E. tenella.
Keywords Eimeria tenella . EtMIC1 . EtMIC2 . MAbs .
Gametogony phase

Introduction
Eimeria tenella, one of seven Eimeria species infecting
chickens, is an obligate intracellular coccidian (phylum
Apicomplexa) parasite which causes a severe form of coccidiosis, an enteric disease that is a major welfare and economic
problem for the poultry industry (Dubremetz et al. 1998; Allen
and Fetterer 2002; Wallach 2010; Cowper et al. 2012). Annual
loss due to coccidiosis is estimated in excess of 500 million
worldwide, which includes the cost of prophylactic in-feed
medication, alternative treatment (if the medication fails), and
losses due to mortality, etc. (Shirley et al. 2007).
Eimeria infection involves multiple stages of attachment
to, invasion, and rapid development in intestinal epithelial
cells of the host. Microneme proteins (MICs) are critical for
motility of the parasite, identification, and binding of host cellsurface proteins, invasion of host cells (Carruthers and Tomley
2008; Cowper et al. 2012), and intracellular survival (Tomavo
et al. 2013). MICs also contribute to parasite gliding motility
and egress from host cells after intracellular replication
(Carruthers and Tomley 2008; Kafsack et al. 2009; Ryan
et al. 2000). Micronemes from sporozoites of E. tenella contain around ten abundant proteins (Kawazoe et al. 1992;
Bumstead and Tomley 2000). One of them, EtMIC1, is a

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member of the thrombospondin-related anonymous protein

(TRAP) family of proteins containing one integrin-insertion
(I) domain, five thrombospondin type I modules (TSP), and
conserved transmembrane and C-terminal regions (Tomley
et al. 1991; Lew et al. 2003). Previous studies revealed that
the EtMIC1 could induce high antibody response and strong
cell-mediated immune response, which might be an effective
antigen for vaccine (Subramanian et al. 2008; Sathish et al.
2012). A second protein, EtMIC2, which is secreted from the
host-parasite interface and intimately involved in host cell
invasion, has been found within the microneme organelles
of E. tenella sporozoites and merozoites (Tomley et al.
1996). Immunization of chickens with the EtMIC2 gene or
recombinant EtMIC2 protein provided partial immunity protection observed against experimental avian coccidiosis
(Dalloul and Lillehoj 2005; Zhang et al. 2012; Shi et al.
2014; Sun et al. 2014a).
Previous studies have shown that EtMIC1 and EtMIC2 are
both important microneme proteins, that are expressed in
invasive stages (Bumstead and Tomley 2000; Ryan et al.
2000; Sasai et al. 2008). There was a high level of synchronicity in the detection of the EtMIC1 and EtMIC2 proteins
during sporulation (Wan et al. 1999; Ryan et al. 2000; Liu
et al. 2009; Zhou et al. 2010). But the expression of EtMIC1
and EtMIC2 in later endogenous (gametogony) developmental stages is unknown.
In the present study, we generated five mouse monoclonal
antibodies (MAbs) that recognize EtMIC1 and EtMIC2 proteins, respectively and investigated the dynamic expression of
EtMIC1 and EtMIC2 in endogenous stages of E. tenella in
chickens experimentally infected with E. tenella. Our results
showed both EtMIC1 and EtMIC2 proteins are expressed in all
developmental stages including the gametogony stage. To our
knowledge, this is the first report that the EtMIC1 and EtMIC2
proteins are expressed in E. tenella gametogony stage.

Materials and methods

Parasites, animal, and cell lines
E. tenella wild type strain SD-01 was isolated, identified, and
stored in our laboratory (Sun et al. 2014a; Wang et al. 2014).
E. tenella is maintained by passage through coccidia-free 2week-old chicks, and the parasites were propagated at least
every 6 months as previously described (Fetterer and Barfield
2003). Unsporulated oocysts were obtained from the cecal
contents of the chickens 7 days post-infection (PI).
Sporulated oocysts were stored in 2.5 % potassium
dichromate at 4 C.
New-hatched Hy-Line Variety Brown layer chickens
(Dongyue poultry, Taian, China) were reared under
coccidian-free conditions. BALB/c female mice aged 4

Parasitol Res (2014) 113:41514161

5 weeks were purchased from Vital River Laboratories

(VRL, Beijing, China). All studies involving animals were
performed in compliance with state and institutional animal
care guidelines.
SP-20 myeloma cells were purchased from ATCC
(Manassas, VA) and maintained in Dulbeccos Modification
of Eagles Medium (DMEM) (Grand Island, USA) supplemented with 10 % fetal bovine serum (FBS, GIBCO, USA).
Generation of anti-EtMIC1 and anti-EtMIC2 MAb
hybridomas
Protein preparation
The EtMIC2 gene (GenBank ID: KC333870) was cloned and
identified from E. tenella wild type strain SD-01 using primers
EtMIC2F (5-ATGGCTCGAGCGTTGTCGCTG-3) and
EtMIC2R (5-TCAGGATGACTGTTGAGTGTC-3), and
the EtMIC2 protein used for generating anti-EtMIC2 MAbs
was expressed and purified using Pichia expression system
following the method described by Zhang et al. (2014).
The fragment of the EtMIC1 gene (GenBank ID:
KF791866) encoding TSP 24 domains of the EtMIC2 protein was cloned from E. tenella wild type strain SD-01 using
primers P4 (5-CCCGCTAGCCCAGTGTGCAAAACGGA
T-3) and P5 (5-CCCGTCGACGCATGGAACTTCATTGC
A-3), and identified by Chen et al. (unpublished data, manuscript submitted). The EtMIC1 polypeptide used for generating anti-EtMIC1 MAbs was expressed and purified using
Pichia expression system following the method described by
Zhang et al. (2014).
Mouse immunization and hybridoma generation
The mice used for producing anti-EtMIC1 or anti-EtMIC2
antibodies were immunized by intraperitoneally injecting the
EtMIC1 or EtMIC2 protein (50 g/mouse) suspended in
Freunds complete adjuvant (Sigma, USA), followed by two
additional immunizations at the same dose with Freunds
incomplete adjuvant (Sigma, USA) at 2-week intervals.
After the immunoreactivity against the EtMIC1 or EtMIC2
was validated, the final boost (100 g/mouse) was given
without adjuvant 1 month after the second immunization.
Four days after the last boost immunization, mice were
euthanized, their spleens removed, and spleen cells isolated
using standard techniques. The spleen cells were fused with
SP-20 myeloma cells to generate hybridomas following the
method described by Hadji-Ghasemi et al. (2003). The hybridoma cells were suspended in 100 ml of hypoxanthineaminopterin-thymidine (HAT) medium selection medium
(Sigma, USA) supplemented with 10 % FBS and 10 % mouse
peritoneal cavity macrophages (MPCM). Then, the

Parasitol Res (2014) 113:41514161

hybridoma cells were dispensed into five 96-well tissue culture plates, and incubated for 10 to 14 days at 37 C in 5 %
CO2.
To screen for positive hybridoma clones, the purified
EtMIC1 or EtMIC2 protein (2 g/well) was used to coat 96well plates with coating buffer (0.2 mol/L Na2CO3/NaHCO3,
pH 9.6) overnight at 4 C. After washing thrice with washing
buffer (phosphate buffered saline with 0.05 % Tween-20
(PBST)), plates were then blocked with phosphate buffered
saline (PBS) containing 2.5 % nonfat milk powder (Amresco,
USA) overnight at 4 C. The 100 l hybridoma supernatant
was added to each well of the plates and incubated for 1 h at
room temperature (RT). Plates were washed thrice and an
HRP-conjugated goat anti-mouse IgG (Promega, USA) in a
1:2,000 dilution was added and incubated for 1 h at RT. After
washing thrice, freshly prepared TMB (Amresco, USA) substrate solution (0.05 mg/ml) was added into the wells. After
30 min incubation at 37 C, the reaction was stopped by
adding 50 l of stopping solution (3 M sulfuric acid). Then,
absorbance was measured at 450 nm using a microplate reader
(Bio-Rad, USA).
The hybridoma clones with strong reactivity with the
EtMIC1 or EtMIC2 were re-cloned twice by limited dilution,
and their reactivity was re-confirmed by ELISA. Subcloned
hybridoma cells were cultured in the OPTI-MEM medium
(GIBCO, USA) containing 10 % FBS and weaned gradually
to the serum-free medium (Hybridoma-SFM medium,
GIBCO, USA). Hybridomas were then expanded and the
supernatant was harvested twice a week. Immunoglobulin
classes and subclasses of MAbs were determined using an
IsoQuick Mouse Monoclonal Isotyping Kit (Sigma, USA)
following the manufacturers instruction.
Antibody identification
ELISA was used for detecting the MAb specificity. EtMIC3
and EtAMA1 proteins were employed as control proteins. The
indirect fluorescent antibody test (IFAT) and Western immunoblotting assays were performed to identify MAbs for their
specific EtMIC1 or EtMIC2 protein recognitions.
Immunofluorescent staining of E. tenella sporozoites
Sporozoites of E. tenella were obtained from sporulated oocysts following the method described by Sasai et al. (2008).
Freshly excysted sporozoites were incubated in DMEM containing 10 % FBS at 41 C for 1 h. About 10 l sporozoite
suspension was smeared on each slide and air-dried. The
sporozoites on the slides were fixed with 4 % polysorbate
(Labb et al. 2005) for 10 min at RT. The slides were washed
thrice using PBS (pH 7.4) and then blocked with PBS containing 2 % (w/v) bovine serum albumin (BSA) for 30 min. To
each slide, 50 l of the diluted MAbs were added and

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incubated for 1 h at RT in a humid chamber. After two washes

with PBS, the FITC-conjugated goat anti-mouse IgG (H+L)
antibody (Cowin Biotech, China) in 1:2,000 dilution was
added to each slide to cover the sporozoites and incubated
for 60 min at RT in a humid chamber. After two washes with
PBS, the slides were observed using a fluorescence microscope (TE-2000-S, Nikon, Japan). The slides stained without
MAbs or with the anti-His-tag MAb as the primary antibody
were used as controls.
Immunofluorescent staining of yeast surface displayed
EtMIC1 and EtMIC2 proteins
Yeast surface displays with EtMIC1 and EtMIC2 proteins
were used to further test the specificity of anti-EtMIC1 and
anti-EtMIC2 MAbs following the method described by Sun
et al. (2014b). Briefly, the EtMIC1 and EtMIC2 genes were
cloned into a yeast expression vector. The vector containing
the EtMIC1 or EtMIC2 gene was transformed into yeast
(Saccharomyces cerevisiae) cells. Yeasts transformed with
the EtMIC1 or EtMIC2 expression vector were cultured in
the SD medium (0.67 % yeast nitrogen base without amino
acids and ammonium sulfate, 2 % glucose, 0.5 % casein acids
hydrolysate) at 30 C for 24 h for displays of the EtMIC1 or
EtMIC2 protein on the yeast surface. Immunostaining was
carried out as follows: yeast cells displaying the EtMIC1 or
EtMIC2 protein were incubated for 1 h with the MAbs (anti
EtMIC1 or EtMIC2 proteins) at a dilution of 1:100 in a total
volume of 300 l on a rotator at RT. Then, yeast cells were
washed with PBS (pH 7.4). The cell pellets were re-suspended
with secondary antibody solutions (FITC-conjugated goat
anti-mouse IgG (H+L) (Cowin Biotech, China)) at a dilution
of 1:100 in a total volume of 300 l, and incubated for 1 h at
RT. Finally, the yeast cells were washed twice with PBS and
observed with a fluorescence microscope (TE-2000-S, Nikon,
Japan). The yeast transformed with an empty plasmid was
stained exactly the same way and used as negative controls.
Yeast cells labeled without primary antibodies, yeast cells
displaying the EtMIC1 labeled with the anti-EtMIC2 (9-B9)
MAb, and yeast cells displaying the EtMIC2 labeled with the
anti-EtMIC1 (1-H2) MAb were used as non-specific binding
MAb controls.
Western immunoblotting
For the sporozoite protein sample preparation, sporozoites at a
density of 5107/ml (in PBS) were lysed by repeated freezing
and thawing. Soluble extracts of sporozoites were prepared
using tissue lysate buffer (7 M Urea, 2 M Thiourea, 100 mM
DTT, 4 % CHAPS, 0.5 mM EDTA, 40 mM Tris, 2 %(v/v) NP40). After sonication in an ice bath, sporozoite proteins were
extracted from lysates by centrifugation at 10,000g for 30 min.
Soluble protein extracts were separated by 12 % SDS-PAGE

4154

and then transferred onto PVDF membranes (Millipore,

USA) at 150 mA for 2 h (Wang et al. 2014). The
membranes were incubated with anti-EtMIC1 or antiEtMIC2 MAbs at RT for 1 h. After three 10-min washings in TBST buffer, the membranes were incubated
with an HRP-conjugated goat anti-mouse IgG (H +L)
(Cowin Biotech, China) at 37 C for 1 h and washed
three times for 10 min with PBST buffer. The peroxidase activity was revealed with the enhanced HRP-DAB
chromogenic substrate kit (TIANGEN, China). The soluble protein extract from uninfected ceca was used as
negative controls. The anti-His-tag MAb was used as
irrelevant MAb controls.

Detection of expression of EtMIC1 and EtMIC2 proteins

in the different endogenous developmental stages
of E. tenella
For the tissue section slide preparation, the chickens
were infected orally with 5103 sporulated E. tenella
oocysts and three chickens were sacrificed every 6 h
from 90 h to 150 h PI. The cecum was removed from
the sacrificed chickens, fixed in 4 % polysorbate, embedded in paraffin wax, and sectioned at 5 m thick.
Slides were digested by 0.25 % tryptase.
For the cecal mucosa smear slide preparation, the
cecal mucosa scraped from sacrificed chickens was diluted with PBS (pH 7.4) and smeared on glass slides.
After air-dried, the slides were fixed with 2 %
polysorbate.
The slides prepared above were blocked with PBS
containing 2 % (w/v) BSA for 30 min. Then, the slides
were incubated with anti-EtMIC1 or anti-EtMIC2 MAbs
for 1 h at RT in a humid chamber. Then, the slides
were incubated in a humid chamber with 1:50 diluted
FITC-conjugated goat anti-mouse IgG antibodies at RT
for 1 h. The slides were observed under a fluorescence
microscope (TE-2000-S, Nikon, Japan). The uninfected
cecal tissue was used as a mock control. Samples labeled without primary antibodies served as negative
controls. The anti-His-tag MAb was used as an irrelevant MAb control.
To verify immunofluorescence staining results, the
Western blot analysis was performed. For protein sample preparation of different endogenous developmental
stages of E. tenella, the cecum from E. tenella-infected
chickens sacrificed at different time points was removed
and soluble protein extracts of the host tissue containing
various developmental stages of E. tenella were prepared using tissue lysate buffer (7 M Urea, 2 M
Thiourea, 100 mM DTT, 4 % CHAPS, 0.5 mM
EDTA, 40 mM Tris, 2 % (v/v) NP-40). The protein

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separation on SDS-PAGE and Western blot analysis

were carried out as described above.

Results
Generation of anti-EtMIC1 and anti-EtMIC2 MAbs
Five mouse MAbs were initially selected on the basis of their
specific binding activity against the recombinant E. tenella
microneme protein EtMIC1 or EtMIC2, as shown in Table 1.
MAbs 1-A1, 1-C4, and 1-H2 were positive reacted with the
EtMIC1 protein, and MAbs 9-B9 and 4-C1 recognized the
EtMIC2 protein. All of the MAbs did not bind the unrelated
antigen EtMIC3 or EtAMA1 that was used as a negative
control. Isotypes of MAbs were also shown in Table 1. All
five MAbs were an IgG1 subclass of antibodies that expressed
light chain. Antibody titer values of the harvested supernatant were measured by a checkerboard pattern with several
coating conjugate concentrations and several antibody dilutions after 15 min incubation with TMB. For convenience,
only the data from a coating antigen concentration of 0.2 g
per well and an antibody dilution of 1:100 were shown in
Table 2.
Identification of anti-EtMIC1 and anti-EtMIC2 MAbs
To test the specificity of five MAbs, the yeast surface display
technique was used. The yeast S. cerevisiae was transformed
with expression vectors carrying either EtMIC1 or EtMIC2
gene. The EtMIC1 or EtMIC2 protein was expressed and
displayed on the yeast surface as designed (Sun et al. 2014b)
when the yeast was cultured. Five MAbs and an FITCconjugated goat anti-mouse secondary antibody were used
to detect EtMIC1 and EtMIC2 proteins displayed on the yeast
cell surface using immunofluorescent labeling assays. The
results showed the strong immunofluorescence on the cell
Table 1 Isotypes and reactivity of EtMIC1/2 monoclonal antibodies
MAbs

1-A1
1-C4
1-H2
9-B9
4-C11
a

Isotype

IgG1-
IgG1-
IgG1-
IgG1-
IgG1-

Reactivity in ELISAa
EtMIC1

EtMIC2

EtMIC3

AMA1

+
+
+

+
+

Optical density values greater than twice the value of negative control
wells (wells coated with 1 % BSA) were considered reactive (+). Optical
density values less than or equal to twice the value of the negative controls
were considered nonreactive ()

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Table 2 Titer values of MAbs (absorbance at 450 nm)

MAbs

EtMIC1

EtMIC2

(1:100)

1-A1

1-C4

1-H2

9-B9

4-C11

Titer

0.5820.01

0.7260.02

0.5790.02

0.6730.03

0.6120.01

Absorbencies were measured by a checkerboard pattern with several coating conjugate concentrations and several antibody dilutions, and measured after
a 15-min incubation with TMB at 37 C

surface of yeasts with each MAb immuno-stained its corresponding protein (Fig. 1ae) and no immunofluorescence on
the cell surface of yeasts transformed with the empty vector
(Fig. 1f). No immunofluorescence was detected on yeast cells
displaying the EtMIC1 labeled with the anti-EtMIC2 (9-B9)
MAb or yeast cells displaying the EtMIC2 labeled with the
anti-EtMIC1 (1-H2) MAb (Fig. 1gh).
The five MAbs were further tested by immunofluorescent
labeling the EtMIC1 or EtMIC2 protein extracted from sporozoites. Freshly excysted sporozoites were incubated together with the five MAbs generated in this work, respectively for
detecting the specificity of each monoclonal antibody. The
specific reaction and recognition were detected by an FITC or
Fig. 1 The yeast surfacedisplayed EtMIC1 and EtMIC2
proteins were
immunofluorescent-labeled either
with anti-EtMIC1 MAbs (a, b,
and c) or with anti-EtMIC2 MAbs
(d and e). ac 1-A1, 1-C4, and 1H2 antibodies. de 9-B9 and 4C11 antibodies. f Yeast cells
displayed the EtMIC1 labeled
without primary antibody as a
control. g Yeast cells displayed
the EtMIC1 labeled with the antiEtMIC2 (9-B9) MAb as a nonspecific binding MAb control. h
Yeast cells displayed the EtMIC2
labeled with the anti-EtMIC1 (1H2) MAb as a non-specific
binding MAb control

TRITC conjugated secondary antibody as described in

Materials and methods. The results showed that all MAbs
clearly stained a small region of the apical tip of E. tenella
sporozoites. The representative-stained sporozoites are shown
in Fig. 2ae. Control reactions which only used an FITC or
TRITC conjugated goat anti-mouse IgG were all negative
(Fig. 2fg). Sporozoites labeled with an irrelevant anti-Histag MAb as the primary antibody were all negative (Fig. 2hi).
The specificity of the five MAbs produced in this work was
confirmed by Western blot assays. The sporozoite protein
sample preparation and Western blot analyses were conducted
as described in Materials and methods. Results showed that
each MAb specifically recognized its corresponding protein

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Fig. 2 Immunofluorescence
staining of E. tenella sporozoites
with anti-EtMIC1 MAbs (a, b,
and c) or with anti-EtMIC2 MAbs
(d and e). After incubation with
FITC- or TRITC-conjugated
antibodies, slides were observed
by fluorescence microscopy. ac
1-A1, 1-C4, and 1-H2 antibodies.
de 9-B9 and 4-C11 antibodies.
fg Cells labeled without primary
antibodies as controls. hi Using
anti-His-tag MAbs as irrelevant
MAbs controls

Fig. 3 Western blot detection of EtMIC1 and EtMIC2 proteins in the

E. tenella sporozoites with MAbs, respectively. Reactivity with MAbs
against soluble proteins extracted from E. tenella sporozoites was detected on lanes A13, B12, and C1. Uninfected ceca soluble proteins were

used as negative controls on A4, B3, and C2. Lanes A14 were probed
with anti-EtMIC1 MAbs 1-A1, 1-C4, 1-H2, and 1-H2, respectively; lanes
B13 with anti-EtMIC2 MAbs 9-B9, 4-C11, and 4-C11; lanes C12 with
the anti-His-tag MAb used as an irrelevant MAb control

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Fig. 4 Immunofluorescence staining of different developmental stages

of E. tenella with anti-EtMIC1 (1-H2) and anti-EtMIC2 (9-B9) MAbs.
Anti-EtMIC1 (a, c, e, and g) and anti-EtMIC2 (b, d, f, and h) antibodies
specifically stained the E. tenella schizonts (schizonts and merozoites) in
cecal tissue during 90 to 132 h PI. Anti-EtMIC1 (i, k, and m) and antiEtMIC2 (j, l, and n) antibodies specifically stained the E. tenella

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gametophyte stage in cecal tissue at 138, 146, and 150 h PI. Ka, La,
Ma, and Na were enlarged images of ln. Uninfected ceca tissue (i and j)
were labeled with anti-EtMIC1 or anti-EtMIC2 antibodies, respectively.
Mock samples labeled without primary antibodies as controls, Sz schizonts, Mz merozoites, Gt gametophytes, Zt zygote

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Fig. 4 (continued)

on the membrane transferred with sporozoite protein extracts.

The relative molecular weights of the EtMIC1 and the
EtMIC2 of E. tenella sporozoites recognized on the Western
blot are approximately 100 and 50 kDa, respectively (Fig. 3),
which is consistent with previous reports for EtMIC1 (Sathish
et al. 2012) and EtMIC2 proteins from sporozoites (Sasai et al.
2008).
Expression of EtMIC1 and EtMIC2 proteins in endogenous
developmental stages
The dynamic expressions of EtMIC1 and EtMIC2 proteins in
the endogenous developmental stages especially in the later
endogenous developmental stages were studied using MAbs
generated in this work. The expressed EtMIC1 and EtMIC2
proteins in different developmental stages of E. tenella were
immunofluorescent-labeled using specific MAbs and confirmed using Western blot analyses. The results revealed that
schizonts and merozoites were clearly labeled by both specific
anti-EtMIC1 and anti-EtMIC2 MAbs from 90 to 132 h
(Fig. 4ah), and some in 138 h PI (Fig. 4ij). Expressions of
both EtMIC1 and EtMIC2 proteins were detected in gametocytes at 138, 144, and 150 h PI (Fig. 4in) and zygotes at 144
and 150 h PI (Fig. 4kn) in the cecal tissue of the infected
chickens. Figure 4KaNa are enlarged images of Fig. 4kn to
clearly show EtMIC1 and EtMIC2 proteins expressed in
gametocytes and zygotes. The strength of the fluorescence-

labeled gametocytes and zygotes was much weaker than that

of schizonts and merozoites (Fig. 4ij), which suggests that
both EtMIC1 and EtMIC2 proteins are expressed in less
abundance in the gametogony phase (sexual reproduction)
than those in the schizogony phase. Control reactions using
only a FITC-conjugated goat anti-mouse IgG and staining of
the uninfected ceca showed negative results (Fig. 4op).
Using the anti-His-tag MAb as irrelevant MAb controls also
showed negative results (data not shown). The EtMIC1 and
EtMIC2 proteins expressed in different developmental stages
of E. tenella were confirmed by Western blot analyses (Fig. 5).

Discussion
E. tenella is an Apicomplexan parasite whose members possess an apical complex containing an assortment of unique
secretory organelles (rhoptries, micronemes, and dense granules). Apical micronemes are important organelles containing
more than ten proteins that are critical for motility of
E. tenella, for recognizing and binding of host cell-surface
proteins, and for invasion of host cells (Carruthers and Tomley
2008; Cowper et al. 2012). Regulated secretion of proteins
from apical micronemes is required for host cell invasion by
providing adhesive protein complexes that bind receptors on
the host cell surface. The EtMIC1 protein is a soluble

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Fig. 5 Detections of EtMIC1 and

EtMIC2 proteins during different
development stages by Western
blot analyses. a anti-EtMIC2
MAbs. b anti-EtMIC1 MAbs. c
the anti-His-tag MAb was used as
irrelevant MAb controls. A1, B1,
and C1 uninfected ceca soluble
extract proteins. A24, B24, and
C24 ceca soluble extract
proteins in 120, 132, and 144 h PI

transmembrane protein which belongs to the TRAP family of

proteins (Tomley et al. 1991). Apart from its role in invasion,
the EtMIC1 is possibly also involved in the movement and
escape of the merozoite from infected cells. The EtMIC2 is
secreted from the host-parasite interface and has a putative
function in parasite adhesion to the host cell to initiate the
invasion process (Tomley et al. 1996; Bumstead and Tomley
2000). Several studies indicated that the EtMIC1 forms a
complex with the EtMIC2, and the EtMIC1-2 complex is
presumably mobilized from micronemes to the parasite surface during attachment and is redistributed towards the posterior end of the parasite during penetration of the host cell
(Huynh et al. 2004; Morahan et al. 2009). Previous studies
suggested that both EtMIC1 and EtMIC2 proteins have good
antigenic properties and are promising vaccine candidates
(Dalloul and Lillehoj 2005; Subramanian et al. 2008;
Sathish et al. 2012; Zhang et al. 2012; Shi et al. 2014; Sun
et al. 2014a). To investigate the precise roles of EtMIC1 and
EtMIC2 proteins in host-parasite interactions, we generated
three MAbs (1A1, 1-C4, and 1-H2) against the recombinant
EtMIC1 protein and two (9-B9 and 4-C11) against the
EtMIC2 in the present study. The specificity of the five
MAbs was tested by their specific reactions with EtMIC1
and EtMIC2 native proteins expressed on sporozoites of
E. tenella and heterologously displayed on the yeast surface
by immunofluorescent labeling assays, and confirmed by
Western blot analyses. These MAbs would be useful tools
for the detailed investigation of the characterization of
EtMIC1- and EtMIC2-related proteins in Eimeria species.
E. tenella has a complicated life cycle including an exogenous phase in the environment during which oocysts excreted from the chicken undergo sporulation and become infective, and an endogenous phase in chicken cecal epithelial cells
during which two discrete and expansive rounds of asexual
reproduction (schizogony) are followed by sexual differentiation (gametogony), fertilization and, finally, shedding of

unsporulated oocysts. Previous studies have shown that both

EtMIC1 and EtMIC2 are important microneme proteins
which are abundantly expressed in sporozoites and schizogony stages (Tomley et al. 1996; Bumstead and Tomley 2000;
Ryan et al. 2000; Sasai et al. 2008). However, their expression
in gametogony phase has never been reported. Ferguson et al.
(2000) reported that the MIC4 or the MIC4-like protein of
Toxoplasma gondii is present within the macrogamete and is
associated with the developing oocyst wall. Using
transmission electron microscopy, Paperna and Smallridge
(2001) observed the presence of micronemes in the anterior
end of the gametocyte in Hemelivia stellata (Apicomplexa:
Haemogregarinidae), a species that is somewhat distantly
related to Eimeria species. Lal et al. (2009) identified three
micronemal proteins (chitinase, CTRP, SOAP) in
Plasmodium ookinete. In the present study, we investigated
expressions of EtMIC1 and EtMIC2 proteins in later endogenous developmental stages, particularly focused on the
gametogony phase using the specific anti-EtMIC1 and antiEtMIC2 MAbs produced in this work. Our results showed that
schizonts, merozoites, gametocytes, and zygotes were all specifically immuno-labeled well with specific anti-EtMIC1 and
anti-EtMIC2 MAbs. Schizonts and merozoites were most
observed in 90 to 132 h PI, some still in 138 h PI.
Gametocytes appeared in 138 h PI but most of them were
observed in 144 to 150 h PI. Some zygotes were also observed
in 144 to 150 h PI. The time profiles and morphological
features of endogenous developmental stages of E. tenella
observed in this study are consistent with reports by Sasai
et al. (2008) and Constantinoiu et al. (2008) for schizonts and
merozoites, and reports by Kefu et al. (2006), Constantinoiu
et al. (2008), and Belli et al. (2009) for gametocytes and
zygotes. The results clearly showed that both EtMIC1 and
EtMIC2 proteins were expressed in gametocytes and zygotes
of E. tenella. EtMIC1 and EtMIC2 proteins were observed in
wall-forming bodies of mature macro-gametocytes and the

4160

wall of zygotes. Expression levels of the two proteins in the

gametogony phase seemed less than those in the schizogony
phase detected by the immunofluorescent labeling.
Numerous studies reported that microneme proteins of
Apicomplexan parasites were expressed in their invasive
stages and crucial to cell invasion and intracellular survival
of most Apicomplexan parasites (Tomley et al. 1996;
Carruthers and Tomley 2008; Cowper et al. 2012; Tomavo
et al. 2013). The new finding from the current study that
EtMIC1 and EtMIC2 proteins are also expressed in gametocytes and zygotes, stages that do not invade host cells, suggests that these two important microneme proteins may play
roles additional to those involved in invasion processes.
In order to identify a newly produced MAb, it is necessary
to test if the MAb is able to specifically recognize its corresponding native protein rather than the one expressed in vitro
to generate the MAb. In the case of low expressed proteins, to
obtain the native expressed protein may be a very difficult
task. In the present study, we tried a yeast surface display
technology to identify anti-EtMIC1 and anti-EtMIC2 MAbs.
Genes encoding EtMIC1 and EtMIC2 proteins were cloned in
yeast expression plasmids, respectively. After transformation
of the plasmids carrying the EtMIC1 or EtMIC2 gene into
yeast cells, the EtMIC1 or EtMIC2 protein was expressed and
displayed on the yeast surface. Because EtMIC1 and EtMIC2
proteins displayed on the yeast surface were expressed from
their encoding genes by yeast expression system, they would
be similar to their natively expressed ones. Therefore, the
yeast surface displayed protein would be an excellent substitute of its native one to identify its corresponding MAb. The
method is simple, quick, and reliable to identify MAbs.
Acknowledgments This work was supported by the grant from the
National Natural Science Foundation of China to XZ (No. 31172314) and
the grant from Shandong Province Science and Technology Development
Program to XZ (No. 2013GNC11017).
Conflict of interest The authors declare that they have no conflict of
interest.

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